Staged assembly of nanostructures

[0001] The present invention relates to methods for the assembly of nanostructures and assembly units for use in the construction of nanostructures.

[0002] Nanostructures are structures with individual components having one or more characteristic dimensions in the nanometer range (from about 1-100 nm). The advantages of assembling structures in which components have physical dimensions in the nanometer range have been discussed and speculated upon by scientists for over forty years. The advantages of these structures were first pointed out by Feynman (1959, There's Plenty of Room at the Bottom, An Invitation to Enter a New Field of Physics (lecture), Dec. 29, 1959, American Physical Society, California Institute of Technology, reprinted in Engineering and Science, February 1960, California Institute of Technology, Pasadena, Calif.) and greatly expanded on by Drexler (1986, Engines of Creation, Garden City, N.Y.: Anchor Press/Doubleday). These scientists envisioned enormous utility in the creation of architectures with very small characteristic dimensions. The potential applications of nanotechnology are pervasive and the expected impact on society is huge (e.g., 2000, Nanotechnology Research Directions: IWGN Workshop Report; Vision for Nanotechnology R & D in the Next Decade; eds. M. C. Roco, R. S. Williams and P. Alivisatos, Kluwer Academic Publishers). It is predicted that there will be a vast number of potential applications for nanoscale devices and structures including electronic and photonic components; medical sensors; novel materials; biocompatible devices; nanoelectronics and nanocircuits; and computer technology.

[0003] Assembly of nanostructures presents significant problems, however, because their individual components or subunits are very small. Manipulation of individual components necessary in the fabrication of nanostructures, even when possible, is slow and tedious. Manipulation becomes particularly problematic when considering the assembly of complex nanostructures that are made up of a large number of components. Two methods of assembling components from the bottom up have been proposed: (i) creation of nanoscale assemblers that manipulate and place components individually and (ii) self-assembly of individual components that are designed to interact with one another in only one way to create, through interactions with other components, complete nanostructures.

[0004] The term assemblers was coined by Drexler to indicate molecular machines capable of translating molecular instructions into three-dimensional structures by analogy to the role of the ribosome in protein synthesis. Development of the initial stages leading to an assembler has proven difficult, and a practical implementation of a working assembler may be overly difficult, if not impossible, due to inherent limitations (Smalley, 2001, Of Chemistry, Love and Nanobots, Scientific American 285(3):76-77). As for self-assembling nanostructures, practical implementations have been thwarted by the need to design components that both have the desired functionalities and exhibit the necessary interactions with neighboring components needed to achieve the self-assembly process.

[0005] There is a huge gap between the popular vision of computer nanochips self-assembling by the billions out of a solution of molecular components, and the real, pragmatic problems involved in assembling complex nanodevices. This gap reflects the difference between a crystal and a true device, as diagramed in FIG. 1. Crystal assembly from solution is a result of a huge number of identical subunits interacting in identical ways. Crystal size is not readily controllable, and there is nothing to distinguish unique positions within the crystal. A periodic array of nanoparticles or nanocomponents with spacing in the nanometer range can provide only a very limited number of the potential functions envisioned for nanostructures. Non-periodic nanodevice architectures can provide a much broader range of functions needed for the advancement of nanotechnology.

[0006] A nanodevice should be of well-defined size and shape with each position in the device distinguishable from all others. Self-assembly can only be utilized for the synthesis of a nanodevice of a thousand components if the uniqueness of each component position can be encoded through the design and synthesis of a thousand distinct components. Each component is designed to interact tightly, specifically, and uniquely with its neighbors, and to be incapable of interacting with components other than its neighbors; and each harbors a functionality distinct to its position within the device. Such uniqueness of component position places significant constraints on the design of components of nanostructures, and raises problems that have not yet been solved for a real system.

[0007] The design and fabrication of many joining pairs that interact with highly specific and non-cross-reacting interactions represents a challenge at least as great as the design of the functional elements themselves. These problems led Whitesides and Love, when they analyzed the advantages and disadvantages of bottom-up methods of nanofabrication such as self-assembly, to state that these methods cannot produce designed, interconnected patterns and are not well suited for building electronic devices (Whitesides and Love, 2001, The Art of Building Small, Scientific American, 285(3): 39-47). The present invention provides for the assembly of complex nanostructures using a method that circumvents this difficult problem.

[0008] Construction of nanostructures in which components are tied together through the interactions of molecules that are biologically programmed for molecular recognition, such as the complementary base-pairing of DNA and RNA (Niemeyer, 2000, Self-assembled nanostructures based on DNA: towards the development of nanobiotechnology, Curr. Opin. Chem. Biol. 4: 609-18) provides one potential solution to these problems. A huge diversity of complementary pairs exist that do not cross-react and that can be synthesized with existing technology. Such programmed molecular building blocks have the drawback, however, that a large number of distinct components must be designed and synthesized to make true self-assembly of a nanostructure possible.

[0009] Several approaches currently exist for assembling nanostructures. All these approaches, as discussed below, have their drawback. For example, U.S. Pat. No. 5,864,013 (Goldberg, Materials for the production of nanometer structures and use thereof, issued Jan. 26, 1999), U.S. Pat. No. 5,877,279 (Goldberg, Materials for the production of nanometer structures and use thereof, issued Mar. 2, 1999), PCT WO 96/11947(A1) (Goldberg, Materials for the production of nanometer structures and use thereof, published Apr. 25, 1996), and PCT WO 00/77196(A1), Goldberg, Gene and protein sequences of phage T4 gene 35, published Dec. 21, 2000) disclose that proteins can be used as components of nanostructures that are engineered from constituents of the long tail fibers of T-even bacteriophages. Phage tail fiber proteins exhibit several characteristics that make them attractive for construction of nanocomponents: (i) they are mechanically rigid; (ii) highly resilient physically; (iii) they are very long and thin; (iv) their length can be increased or decreased using standard cloning techniques; (v) they form strong, rigid bonds to one another; (vi) these bonds are highly specific; (vii) additional functional groups or binding sites may be added at points along the rods that do not disrupt the structural rigidity of the rods, using standard directed mutagenesis and cloning techniques or other specific covalent or non-covalent modification procedures.

[0010] Nanofabrication based on T-even bacteriophage tail fiber proteins depends on their modular nature, with their terminal binding domains well-defined and separate from their intervening rigid structural elements. This arrangement suggests a general system for building by self-assembly. The ability to exchange the order of the joining members by cutting and splicing the structural elements, while maintaining rigidity of the protein provides the flexibility for rational design of assembly units and for construction based on a controlled self-assembly using a structurally relevant biomaterial. The trimeric nature of phage tail fiber proteins (Cerritelli et al., 1996, Stoichiometry and domainal organization of the long tail-fiber of bacteriophage T4: a hinged viral adhesin, J. Mol. Biol. 260(5): 767-80), however, limits the geometry to which they can be adapted in their use in a self-assembly or staged-assembly process.

[0011] U.S. Pat. No. 5,468,851 (Seeman et al., Construction of geometrical objects from polynucleotides, issued Nov. 21, 1995) discloses another approach for assembling nanostructures. It discloses the assembly of geometrical objects from polynucleotides by nucleic acid ligation. It discloses that one, two and three dimensional structures can be synthesized or modified from polynucleotides. A core structure is expanded by cleavage of a loop with a restriction endonuclease. Another polynucleotide is ligated to the sticky ends, so that the recognition site of the restriction enzyme is not reformed. This process is repeated as many times as necessary to synthesize a desired structure. U.S. Pat. No. 5,468,851 also discloses that a geometrical object assembled from a polynucleotide could provide a useful three-dimensional scaffolding upon which enzymatic or antibody binding domains could be linked to provide high density multivalent processing sites to link to and solubilize otherwise insoluble enzymes, or to entrap, protect and deliver a variety of molecular species. The limitation of this approach, however, is that the disclosed nanostructures, made of a single, double-stranded, polynucleotide lattice, lack structural rigidity and are subject to enzymatic, chemical and photo-degradation. Furthermore, the disclosed nanostructures provide only a limited range of spatial geometries.

[0012] U.S. Pat. No. 6,072,044 (Seeman et al., Nanoconstructions of geometrical objects and lattices from antiparallel nucleic acid double crossover molecules, issued Jun. 6, 2000) discloses yet another approach for assembling nanostructures. It discloses that two and three-dimensional polynucleic acid structures, such as periodic lattices, can be constructed from an ordered array of antiparallel, double-crossover molecules assembled from single-stranded oligonucleotides or polynucleotides. The construction proceeds by the creation of staggered ends by enzyme cleavage, then ligation to form a linkage. Such antiparallel double-crossover molecules have the structural rigidity necessary to serve as building block components for two- and three-dimensional structures having high translational symmetry associated with crystals. Whereas the patent discloses the assembly of nanostructures, the disclosed method does not accommodate the non-periodic placement of functional moieties within the assembly. And while a regularly repeating nanostructure is disclosed, the nanostructure cannot achieve completely defined positions of functionality within the nanostructure.

[0013] PCT publication WO 01/00876 (Mirkin et al., Nanoparticles having oligonucleotides attached thereto and uses therefore, published Jan. 4, 2001) discloses a method of synthesizing nanoparticle-oligonucleotide conjugates. The drawback of this method, however, is that although the nanoparticles are linked together with well-controlled average distances between them, the method cannot provide for controlled geometry or stoichiometry, since the DNA units that provide the specific complementary binding sites are conjugated to inorganic particles with indeterminate stoichiometry and geometry. The particles will assemble to form a nanomaterial with an indeterminate particle packing and therefore, precisely defined, three-dimensional structures cannot result from the disclosed fabrication methods.

[0014] U.S. Pat. No. 5,969,106 (Rothstein et al., Self-aligning peptides modeled on human elastin and other fibrous proteins, issued Oct. 19, 1999) discloses designs of synthetic proteins based on several naturally occurring fibrous proteins. These synthetic proteins have multiple domains, including two P-sheet joining domains and an a-helical domain to link the -sheet domains together. The patent discloses that P-sheet domains of different subunits join together by hydrophobic interactions between interfaces of the subunits, resulting in long polymeric fibers. These fibers are then formed into biocompatible coatings for prostheses. This approach does not appear to allow for forming nanostructures, however, as no method for controlling the assembly process is described that would allow ordering of the components.

[0015] PCT publication WO 98/28320 (Heller et al., Affinity based self-assembly systems and devices for photonic and electronic applications, published Jul. 2, 1998) discloses methods for fabricating nanoscale structures using the self-assembling, hybridizing properties of nucleic acids. The publication discloses that a component that has many affinity surface identities is oriented in an electric field, and then reacted with an affinity site. According to the disclosed method, nanostructures are assembled by attaching a first affinity sequence at many locations on a support, and then cross-linked with a functionalized second affinity sequence that reacts with the first sequence and that has an unhybridized overhang sequence. The self-assembling, hybridizing properties of nucleic acids can thus be used to fabricate components for building nanostructures such as octahedron and lattice nanodevices (see FIG. 3B of WO 98/28320). The drawback of such an approach, however, is that since inorganics are used to organize these structures, it would be impossible to control the geometry or stoichiometry of the interactions to produce the disclosed nanostructures.

[0016] U.S. Pat. No. 5,712,366 (McGrath et al., Fabrication of nanoscale materials using self-assembling proteins, issued Jan. 27, 1998) discloses a method of fabricating nanoscale structural materials via spontaneous organization of self-assembling proteins. The disclosed self-assembling proteins include at least one recognition sequence, i.e., a charged residue selected from the group consisting of Glu, Lys, Arg and Asp. The disclosed method comprises admixing proteins that include species of the recognition sequence that are prone to dimerization. As disclosed in U.S. Pat. No. 5,712,366, admixed proteins are caused to spontaneously organize into nanoscale structural materials via their respective recognition sequences. U.S. Pat. No. 5,712,366 discloses that in certain embodiments, the amino acid sequences used as structural components are optimized for coiled-coil formation, and designed to mimic leucine zipper protein sequences. Specificity is introduced by controlling the identity and placement of charged residues on the faces of each helix. For example, to construct self-assembling fibers, the genes for polypeptides A2 and B2 are modified by incorporating additional recognition elements at the N- or C-termini. These added elements, which are designed to react with each other and not with polypeptides A2 or B2, impose a driving force for ordered supramolecular assembly, resulting in alignment of all of the dimers in a head-to-tail orientation within an assembling fibril.

[0017] Nevertheless, a major shortcoming of the method disclosed in U.S. Pat. No. 5,712,366 is that the molecular components are designed to spontaneously recognize their nearest neighbors, and these nearest-neighbor interactions can only define a repeating pattern of units. The repeated use of identical interactions among identical units does not provide, however, for the incorporation of special units possessing specific functionalities into specifically defined positions.

[0018] U.S. Pat. No. 6,107,038 (Choudhary et al., Method of binding a plurality of chemicals on a substrate by electrophoretic self-assembly, issued Aug. 22, 2000) discloses an electrophoretic technique for moving a plurality of chemicals into distinct zones for immobilization on a solid surface. The technique includes introducing a first electrolyte and a second electrolyte into a channel, and interposing between the first and second electrolytes at least one solution containing a plurality of chemicals. Under a given electric field, the first electrolyte has anions with higher effective mobility than the chemicals and the second electrolyte has anions with lower effective mobility than the chemicals. When an electrical potential is applied across the length of the channel the plurality of chemicals in the solution are moved into spatial zones. The chemicals in the zones can then be bound to the interior surface of the channel. Chemicals so bound to the wall surface can be used as the initiator or anchor to which chemical components can be added in order to build linear structures such as arrays and electrical conducting structures.

[0019] The drawback of the method disclosed in U.S. Pat. No. 6,107,038, however, is that in order to carry out the assembly process, the nanocomponents must be physically manipulated by an electric field and introduced as a plurality of components. Construction of two-dimensional arrays and higher-order architectures thus depends on the placement of anchor molecules that are separated on the range of micrometers or more. The method does not provide for precisely controlling the distance between spatial zones or the distance between anchors. To construct higher-order arrays and geometrical architectures in a stoichiometric fashion, the distances between spatial zones and anchor molecules need to be precisely controlled, both spatially and geometrically. This method for depositing chemicals onto surfaces specifically, while controlling their positions electrophoretically, cannot be used for the construction of a three-dimensional nanostructures.

[0020] PCT publication WO 00/68248 (Yeates et al., Self assembling proteins, published Jan. 16, 2001) discloses methods of constructing a fusion protein composed of at least two oligomerization domains that are rigidly linked to each other. The disclosed fusion protein is capable of self-assembling with additional fusion proteins to produce a nanostructure such as an open cage, a closed shell, a ball, a molecular sieve, a matrix, or a carrier. The disclosed methods are limited, however, to regular structures, either finite structures with elements defined by point group symmetries, or regularly repeating structures of indeterminate length in one dimension (e.g., fiber), two dimensions (e.g., thin film) or three dimensions (e.g., crystal).

[0021] The drawback of the method disclosed in WO 00/68248 is that fusion protein units are assembled into nanostructures by self-assembly and cannot spontaneously recognize where they belong within a larger framework. The units used in the method are designed only to spontaneously recognize their nearest neighbors, and these nearest-neighbor interactions can only define a repeating pattern. As discussed hereinabove, the repeated use of identical interactions among identical units does not provide for the incorporation of special units possessing specific functionalities into specifically defined positions.

[0022] U.S. Pat. No. 5,948,897 (Sen et al., Method of binding two or more DNA double helices and products formed, issued Sep. 7, 1999) discloses a nucleic acid complex having double-stranded sections with a domain of guanine nucleotides. The disclosed domain comprises a pair of substantially uninterrupted guanine sequences that bond together. This domain can interact with other similar domains such that two nucleic acid complexes comprising these domains have the ability to bind together to form DNA superstructures. The drawbacks of such a self-assembly method for building a nanostructure, however, are that it proceeds through bonding of domains (e.g., poly-G) of double-stranded DNA to form superstructures, it does not provide for the incorporation of special units possessing specific functionalities into specifically defined positions and it does not provide a diversity of spatial geometries.

[0023] PCT publication WO 01/21646 (Woolfson et al., Protein structures and protein fibres, published Mar. 29, 2001) discloses the construction of nanoscale molecular sieves, grids, and scaffolds from peptides. WO 01/21646 disclosed the formation of protein fibers through the design of specific amino acid heptads that form alpha-helical coiled-coil structures. First and second peptide monomer units are mixed and associate via self-assembly to form a protein structure. While the publication discloses construction of longitudinal fibers, the length of the fibers formed is not controllable. Moreover, incorporation of functional moieties into the monomer units, either before or after self-assembly, is not stoichiometric or specific. Oligomerization and multimerization of the monomer units occurs upon mixing of the complementary monomer unit pair (FIG. 4D, WO 01/21646). The publication discloses that upon mixing of the monomer pairs, a number of protein fibrils of various diameters were obtained (see also Pandya et al., 2000, Sticky-end assembly of a designed peptide fiber provides insight into protein fibrillogenesis, Biochemistry 39(30): 8728-34). This suggests that the self-assembly of the heterodimeric fibers does not occur, as expected, as two monomers per building block. The major drawback the method disclosed in WO 01/21646, however, is that fusion proteins are assembled into nanostructures by self-assembly, the formation of which is not readily controllable. As in other self-assembly methods, this method results in the formation of regular repeating structures that lack units at specific or selected positions in the nanostructure.

[0024] U.S. Pat. No. 6,248,529 (Connolly et al., Method of chemically assembling nanoscale devices, issued Jul. 19, 2001) discloses the construction of nanoscale devices including electronic circuits that use DNA as a support structure. U.S. Pat. No. 6,248,529 discloses fabrication of manufacturing nanocircuits, such as transistors, diodes, and inductors, utilizing DNA as the starting scaffold and support structure. The disclosed method includes masking a region of nucleic acid with a nucleic acid binding molecule. The nucleic acid binding molecule is specific for a recognition sequence on the DNA starting scaffold and hence masks a portion of the DNA. The unmasked portion of the DNA is then coated with a material, such as conducting or semi-conducting material, whereupon removal of the nucleic acid binding molecule reveals an uncoated portion of the DNA. Upon removal of the nucleic acid binding protein, a second coating material can be applied to the uncoated regions of the nucleic acid template to form a nanoscale device, such as a circuit element. The drawback of such an approach, however, is that it cannot be used for placing nanoparticles in arbitrary, designed positions in a three-dimensional nanodevice.

[0025] PCT publication WO 01/16155 (Erlanger et al, Antibodies specific for fullerenes, issued March 2001) discloses antibodies for a wide range of fullerenes and a method for preparing electronic or chemical nanoscale devices from single-walled fullerenes or nanotubes. According to the method, an antibody, as well as fullerene, is incorporated into the disclosed nanodevice. A disadvantage of this method, however, is that the flexibility of the incorporated antibody molecule (as opposed to an antibody fragment) would make precise location of the fullerene difficult.

[0026] Hence, despite the availability of a number of different methods for the self-assembly of nanostructures, as discussed hereinabove, there is a need in the art for a method that provides for the incorporation of structural and functional units into a nanostructure at selected, specific positions. There is a further need in the art for nanostructures containing such functional and structural elements at selected positions. The present invention provides such a method and such nanostructures.

[0027] The present invention provides compositions and methods for the staged assembly of nanostructures. According to the methods of the invention, assembly of nanostructures proceeds by sequential, non-covalent, vectorial addition of specific assembly units to an initiator unit or a nanostructure intermediate during an assembly cycle, a process that is referred to herein as staged assembly. Attachment of each assembly unit is, by design, mediated by the specific, non-covalent binding of one or more pre-designated joining elements of one assembly unit to a complementary joining element present on the initiator unit or assembly intermediate. To avoid self-polymerization, each assembly unit is designed so that no joining element that is a part of the assembly unit can interact with any other joining element of that same assembly unit. Self-polymerization of the assembly unit is therefore obviated: only one assembly unit can be added to a target joining element on the initiator or nanostructure intermediate during each assembly cycle, and binding of the assembly unit to the target initiator unit or nanostructure intermediate is vectorial. The process is carried out in a massively parallel fashion such that a very large number of identical assemblies are fabricated simultaneously.

[0028] One object of the staged assembly method of the invention is to fabricate nanostructures in which: a) each assembly unit occupies a specific, predetermined location in the nanostructure; b) multiple nanostructures are assembled simultaneously; and c) all the nanostructures are identical in architecture and assembly unit order. In a preferred embodiment of the staged assembly method of the invention, an initiator unit is immobilized on a substrate and additional units are added sequentially in a procedure analogous to solid phase polymer synthesis. Only a few distinct unit-unit interactions need to be used, since the size and shape of the nanodevice will be defined by the order in which units are added. The staged assembly method of the invention requires far fewer non-cross-reacting complementary pairs of joining elements than self-assembly or auto-assembly. Since the engineering or identification of complementary and non-cross-reacting pairs of joining elements constitutes a major barrier to the design of assembly units, the use of the staged assembly method of the invention represents a significant improvement over self-assembly for bottom-up assembly of nanostructures. Each position in the nanodevice can be uniquely defined through the process of staged assembly, and units of distinct functionalities can be added at any desired position. This system enables massive parallel manufacture of complex nanodevices, and different complex nanodevices can be further self-assembled into higher order architectures in a hierarchic manner.

[0029] In one embodiment, the invention provides a method for staged assembly of a nanostructure comprising:

[0030] (a) contacting a surface-bound nanostructure intermediate comprising at least one unbound joining element with a solution comprising an assembly unit comprising a plurality of different joining elements, wherein:

[0031] (i) none of the joining elements of said plurality of different joining elements can interact with itself or with another joining element of said plurality,

[0032] (ii) a single joining element of said plurality can bind non-covalently to a single unbound joining element of the surface-bound nanostructure intermediate, and

[0033] (iii) the joining elements do not consist of or comprise T-even or T-even-like bacteriophage tail fiber proteins or binding fragments thereof;

[0034] (b) removing unbound assembly units; and

[0035] (c) repeating steps (a) and (b) to form a nanostructure.

[0036] The present invention also provides assembly units for use in the staged assembly methods of the invention disclosed herein. Assembly units of the invention may further comprise structural and/or joining elements, as well as, in certain embodiments, one or more functional elements. If an assembly unit comprises a functional element, that functional element may be attached to, or incorporated within, a joining element, or, in certain embodiments, a structural element. Such an assembly unit of the invention, which may comprise a structural element and a plurality of non-identical, non-interacting, joining elements, may be, in certain embodiments, structurally rigid. The assembly unit of the invention has well-defined recognition and binding properties, i.e., joining elements that exhibit specificity, through specific non-covalent interactions, for a complementary joining element.

[0037] In another embodiment, the invention provides a nanostructure assembly unit comprising a plurality of different joining elements, wherein:

[0038] (a) none of the joining elements of said plurality can interact with itself or with another joining element of said plurality;

[0039] (b) a single joining element of said plurality can bind non-covalently to a single unbound joining element of a surface-bound nanostructure intermediate; and

[0040] (c) the joining elements do not consist of or comprise T-even or T-even-like bacteriophage tail fiber proteins or binding fragments thereof.

[0041] The invention provides structural elements comprising antibodies or binding derivatives or binding fragments thereof, including, but not limited to, structural elements comprising: monoclonal antibodies, multispecific antibodies, Fab or F(ab)₂ antibody fragments, single-chain antibody fragments (scFvs), bispecific IgG, chimeric IgG or bispecific heterodimeric F(ab)₂ antibodies, diabodies or multimeric scFv fragments. The invention also provides structural elements comprising bacterial pilin proteins, leucine zipper-type coiled coils, or four-helix bundles.

[0042] According to the staged-assembly method of the invention, the order in which assembly units are added is determined by the desired structure and/or activity of the nanostructure. Joining elements are chosen, by design, to permit staged assembly of the desired nanostructure. Since the choice of joining elements is generally independent of the functional elements to be incorporated into the nanostructure, assembly units are designed to comprise joining elements needed to place the assembly units in the proper place within the nanostructure and the functional elements needed to confer the desired function on the nanostructure as a whole.

[0043] The invention provides joining elements that exhibit antigen-antibody interactions, including, but not limited to, joining elements comprising: recombinantly engineered antibodies or binding derivatives or binding fragments thereof, molecules that exhibit idiotope/anti-idiotope interactions, or two non-complementary idiotopes. The invention also provides joining elements comprising peptide epitopes, bacterial pilin proteins or binding derivatives or binding fragments thereof, or peptide nucleic acids (PNAs).

[0044] In certain embodiments, the invention provides methods for staged assembly of a nanostructure wherein at least one joining element comprises a binding domain of an antibody or a pilin protein or a binding derivative or binding fragment thereof. In another embodiment, the invention provides a method for staged assembly of a nanostructure wherein at least one joining element comprises a peptide nucleic acid (PNA) or binding derivative thereof.

[0045] In yet other embodiments, the invention provides a nanostructure assembly unit wherein at least one joining element comprises a binding domain of an antibody or a pilin protein or binding derivative or binding fragment thereof. In another embodiment, the invention provides a nanostructure assembly unit wherein at least one joining element comprises a peptide nucleic acid (PNA) or binding derivative thereof.

[0046] In yet another embodiment, the invention provides a nanostructure assembly unit wherein the assembly unit comprises a first structural element that is bound to a second structural element to form a stable complex, and wherein the first structural element is covalently linked to at least one joining element.

[0047] Attachment of each assembly unit to an initiator unit or nanostructure intermediate is mediated by formation of a specific, binding-pair interaction between one joining element of the assembly unit and one or more unbound complementary joining element(s) carried by the initiator unit or nanostructure intermediate. Since according to the methods of the invention, at most only one joining element of an assembly unit will associate by specific non-covalent binding interactions to any given joining element of an initiator assembly unit or nanostructure intermediate in each assembly cycle, such addition of the assembly unit to the initiator unit or nanostructure intermediate will occur in a pre-designed, vectorial manner.

[0048] The methods of the invention make possible the fabrication of highly complex architectures with only a few distinct, non-cross-reacting joining pairs. These methods permit the precise geometric and spatial positioning of individual components in the nanometer range. The staged-assembly methods of the invention make possible the mass production of multi-dimensional, non-periodic architectures in which organic and inorganic nanocomponents are placed with precision into three-dimensional constructs.

[0049] Assembly Unit: An assembly unit is an assemblage of atoms and/or molecules comprising structural elements, joining elements and/or functional elements. Preferably, an assembly unit is added to a nanostructure as a single unit through the formation of specific, non-covalent interactions.

[0050] Assembly Unit, Initiator: An initiator assembly unit is the first assembly unit incorporated into a nanostructure that is formed by the staged assembly method of the invention. It may be attached, by covalent or non-covalent interactions, to a solid substrate or other matrix as the first step in a staged assembly process. An initiator assembly unit is also known as an initiator unit.Bottom-up: Bottom-up assembly of a structure (e.g.,a nanostructure) is formation of the structure through the joining together of substructures using, for example, self-assembly or staged assembly.

[0051] Capping Unit: A capping unit is an assembly unit that comprises at most one joining element. Additional assembly units cannot be incorporated into the nanostructure through interactions with the capping unit once the capping unit has been incorporated into the nanostructure.

[0052] Cross-reactive: With respect to joining pairs, two joining pairs are said to be cross-reactive if a joining element from one pair can bind with specificity to a joining element from the other pair.

[0053] Functional Domain: A functional domain is a functional element comprising an amino acid sequence.

[0054] Functional Element: A functional element is a moiety exhibiting any desirable physical, chemical or biological property that may be built into, bound or placed by specific covalent or non-covalent interactions, at well-defined sites in a nanostructure.

[0055] Joining Element: A joining element is a portion of an assembly unit that confers binding properties on the unit, including, but not limited to: binding domain, hapten, antigen, peptide, PNA, DNA, RNA, aptamer, polymer or other moiety, or combination thereof, that can interact through specific, non-covalent interactions, with another joining element.

[0056] Joining Elements, Complementary: Complementary joining elements are two joining elements that interact with one another through specific, non-covalent interactions.

[0057] Joining Elements, Non-Complementary: Non-complementary joining elements are two joining elements that do not specifically interact with one another, nor demonstrate any tendency to specifically interact with one another.

[0058] Joining Pair: A joining pair is two complementary joining elements.

[0059] Nanocomponent: A nanocomponent is a substructure or portion of a nanostructure.

[0060] Nanomaterial: A nanomaterial is a material made up of a crystalline, partially crystalline or non-crystalline assemblage of nanoparticles.

[0061] Nanoparticle: A nanoparticle is an assemblage of atoms or molecules, bound together to form a structure with dimensions in the nanometer range (1-1000 nm). The particle may be homogeneous or heterogeneous. Nanoparticles that contain a single crystal domain are also called nanocrystals.

[0062] Nanostructure or Nanodevice: A nanostructure or nanodevice is an assemblage of atoms and/or molecules comprising structural, functional and/or joining elements, the elements having at least one characteristic length (dimension) in the nanometer range.

[0063] Nanostructure intermediate: A nanostructure intermediate is an intermediate substructure created during the assembly of a nanostructure to which additional assembly units can then be added.

[0064] Non-covalent Interaction, Specific: A specific non-covalent interaction is, for example, an interaction that occurs between an assembly unit and a nanostructure intermediate.

[0065] PNA: Peptide nucleic acid Self-assembly: Self-assembly is spontaneous organization of components into an ordered structure. Also known as auto-assembly.

[0066] Staged Assembly of a Nanostructure: Staged assembly of a nanostructure is a process for the assembly of a nanostructure wherein a series of assembly units are added in a pre-designated order, starting with an initiator unit that is typically immobilized on a solid matrix or substrate. Each step results in the creation of an intermediate substructure, referred to as the nanostructure intermediate, to which additional assembly units can then be added. An assembly step comprises (i) a linking step, wherein an assembly unit is linked to an initiator unit or nanostructure intermediate through the incubation of the matrix or substrate with attached initiator unit or nanostructure intermediate in a solution comprising the next assembly units to be added; and (ii) a removal step, e.g., a washing step, in which excess assembly units are removed from the proximity of the intermediate structure or completed nanostructure. Staged assembly continues by repeating steps (i) and (ii) until all of the assembly units are incorporated into the nanostructure according to the desired design of the nanostructure. Assembly units bind to the initiator unit or nanostructure intermediate through the formation of specific, non-covalent bonds. The joining elements of the assembly units are chosen so that they attach only at pre-designated sites on the nanostructure intermediate. The geometry of the assembly units, the structural elements, and the relative placement of joining elements and functional elements, and the sequence by which assembly units are added to the nanostructure are all designed so that functional units are placed at pre-designated positions relative to one another in the structure, thereby conferring a desired function on the completely assembled nanostructure.

[0067] Structural Domain : A structural domain is a structural element comprising a protein sequence.

[0068] Structural Element: A structural element is a portion of an assembly unit that provides a structural or geometric linkage between joining elements, thereby providing a geometric linkage between adjoining assembly units. Structural elements provide the structural framework for the nanostructure of which they are a part.

[0069] Subassembly: A subassembly is an assemblage of atoms or molecules consisting of multiple assembly units bound together and capable of being added as a whole to an assembly intermediate (e.g., a nanostructure intermediate). In many embodiments of the invention, structural elements also support the functional elements in the assembly unit.

[0070] Top-down: Top-down assembly of a structure (e.g.,a nanostructure) is formation of a structure through the processing of a larger initial structure using, for example, lithographic techniques.

[0071] FIGS. 1(A-B). A. Diagram of a crystalline array of nanoparticles (i.e., a nanomaterial) in which there are no special positions, and the size and overall geometry of the array is not completely defined. B. Diagram of a nanodevice, an assemblage of nanoparticles or assembly units in which the positioning of each unit is completely defined according to design. The extent of the structure, and the relative positions of all of the functional units, are defined precisely according to the functional requirements of the device.

[0072] FIG. 2. Staged assembly of assembly units. In practice, each step in the staged assembly will be carried out in a massively parallel fashion. In step 1, an initiator unit is immobilized on a solid substrate. In the embodiment of the invention illustrated here, the initiator unit has a single joining element. In step 2, a second assembly unit is added. The second unit has two non-complementary joining elements, so that the units will not self-associate in solution. One of the joining elements on the second assembly unit is complementary to the joining element on the initiator unit. Unbound assembly units are washed away between each step (not shown).

[0073] After incubation, the second assembly unit binds to the initiator unit, resulting in the formation of a nanostructure intermediate made up of two assembly units. In step 3, a third assembly unit is added. This unit has two non-complementary joining elements, one of which is complementary to the only unpaired joining element on the nanostructure intermediate. This unit also has a functional unit (F3).

[0074] A fourth assembly unit with functional element F4 and a fifth assembly unit with functional element F5 are added in steps 4 and 5, respectively, in a manner exactly analogous to steps 2 and 3. In each case, the choice of joining elements prevents more than one unit from being added at a time, and leads to a tightly controlled assembly of functional units in pre-designated positions.

[0075] FIG. 3. Generation of a nanostructure from subassemblies. A nanostructure can be generated through the sequential addition of subassemblies, using steps analogous to those used for the addition of individual assembly units as illustrated above in FIG. 2. The arrow indicates the addition of a subassembly to a growing nanostructure.

[0076] FIG. 4. A diagram illustrating the addition of protein units and inorganic elements to a nanostructure according to the staged assembly methods of the invention. In step 1, an initiator unit is bound to a solid substrate. In step 2, an assembly unit is bound specifically to the initiator unit. In step 3, an additional assembly unit is bound to the nanostructure undergoing assembly. This assembly unit comprises an engineered binding site specific for a particular inorganic element. In step 4, the inorganic element (depicted as a cross-hatched oval) is added to the structure and bound by the engineered binding site. Step 5 adds another assembly unit with a binding site engineered for specificity to a second type of inorganic element, and that second inorganic element (depicted as a hatched diamond) is added in step 6.

[0077] FIG. 5. Line drawing representing the a-carbon trace of an intact IgGl (Protein Data Bank (pdb) entry 1IGY) (Harris et al., 1998, Crystallographic structure of an intact IgG1 monoclonal antibody, J. Mol. Biol. 275(5): 861-72). (For a description of the Protein Data Bank (pdb), see Berman et al., 2000, The Protein Data Bank, Nucl. Acids Res. 235-42; Saqi et al, 1994, PdbMotifa tool for the automatic identification and display of motifs in protein structures, Comput. Appl. Biosci. 10(5): 545-46.) Thick lines represent the heavy chains and thin lines represent the light chains. The Fv and C_H1 domains of the Fab fragment and the C_H2 and C^H3 domains of the Fc fragment are labeled. Ball-and-stick modeling, indicated by gray arrowheads, represent disulfide cysteine bonds. Clusters of disulfide bridging interactions occur in the flexible hinge region located between the Fab and Fc fragments. These interactions may aid in dimerization and provide structural integrity of this otherwise highly flexible region in the immunoglobulin. Drawing was treated with the program SETOR (Evans, 1993, SETOR: Hardware lighted three-dimensional solid model representations of macromolecules, J. Mol. Graphics, 11: 134-38).

[0078] FIG. 6. Line drawing representing the -carbon trace of a Fab fragment that can be used as the structural element for design of an assembly unit (pdb entry 1CIC ). The heavy lines represent the heavy chain and the light lines represent the light chain. The domains of the heavy chain (V_H and C_H1) and the light chain (V_L and C_L) are labeled. Also indicated is the flexible Fab elbow or bend region connecting the variable domains and constant domains. The Fab angle of the bend varies considerably (127-176) even among members of the same antibody class.

[0079] FIGS. 7(A-B). Diagram of two diabody units, Unit 1 (A) and Unit 2 (B) and their associated genes. A. Unit 1 is an AB diabody in which the V_H and V_L domains of A define a lysozyme isotopic antibody (D1.3) and in which the V_H and V_L domains of B define a virus neutralizing idiotopic antibody (730.1.4). In order to facilitate purification of the desired diabody product, the gene encoding V_HA and V_LB includes a hexahistidine tag, whereas the gene encoding V_HB and V_LA does not. B. Unit 2 is BA diabody in which the V_H and V_L domains of B define a virus neutralizing idiotopic antibody (409.5.3) and in which the V_H and V_L domains of A define a lysozyme isotopic antibody (E5.2). In order to facilitate purification of the desired diabody product, the gene encoding V_HB and V_LA includes a hexahistidine tag, whereas the gene encoding V_HA and V_HB does not.

[0080] FIG. 8. Line drawing representing the three-dimensional structures of the -carbon trace of a diabody (pdb entry 1 LMK) (top) and a single chain Fv (scFv) antibody (pdb entry 2APA) (bottom). For the monomeric scFv structure (bottom), heavy lines represent the heavy chain and the light lines represent the light chain. For the dimeric diabody structure (top), however, the heavy lines represent both the heavy chain and light chain of one scFv, while the light lines represent both the heavy and light chain of the other scFv. scFv constructs that have the heavy-light variable domains linked together by a longer peptide linkers form stable monomers. Those with shorter linkers associate with a second scFv molecule to form a bivalent diabody as shown. Note that the immunoglobulin fold contained within both structures is very similar. scFv and diabodies, or binding derivatives or binding fragments thereof, can be used as the basic elements for the design of assembly units.

[0081] FIG. 9. Schematic representation of various IgGs including monovalent, bivalent, monospecific and bispecific antibodies. IgGs that are derived from a single cell line are homozygous for IgG. The resulting IgGs are therefore bivalent-monospecific antibodies. A hybrid hybridoma, e.g., a quadroma, arises from a fusion cell line. IgGs that are produced by hybrid hybridomas may be mixtures of heterologous bivalent-bispecific (e.g., heterologous-F(ab)₂) and homozygous bivalent-monospecific (e.g., F(ab)₂) IgG. Hybrid hybridoma heterodimers therefore represent a source of bivalent-bispecific F(ab)₂. The intact IgG molecules or binding derivative or binding fragment thereof can be used as the basic elements for the design of assembly units.

[0082] FIG. 10. Schematic representation of an IgG molecule cleaved into its component fragments, F(ab)₂ and Fc, upon limited exposure to protease. The hinge region, containing several disulfide-bond interactions, helps maintain dimerization of the Fab fragments. Subsequent exposure of the F(ab)₂ to reducing conditions disrupts the hinge disulfide bridging interactions between the fragments to yield monomeric Fab. Separate functional fragments of the IgG can be isolated (i.e., Fab fragments) for specific uses in the design of assembly units such as creating bivalent-bispecific heterologous F(ab)₂ by chemical cross-linking.

[0083] FIGS. 11(A-D). Dimerization motifs that have been developed to promote the multimerization of antigen-binding fragments that contain various specificities. Leucine zipper motifs (depicted as elongated ovals) such as Jun-Fos or GCN4 (Kostelny et al., 1992, Formation of a bispecific antibody by the use of leucine zippers, J. Immunol. 148(5): 1547-53; de Kruif et al., 1996, Leucine zipper, dimerized bivalent and bispecific scFv antibodies from a semi-synthetic antibody phage display library, J. Biol. Chem. 271(13): 7630-34), or four-helix bundle motifs (depicted as rectangles in (C) and (D)), such as Rop (Pack et al., 1993, Improved bivalent miniantibodies, with identical avidity as whole antibodies, produced by high cell density fermentation of Escherichia coli, Biotechnology (NY) 11(11): 1271-77; Muller et al., 1998, A dimeric bispecific miniantibody combines two specificities with avidity, FEBS Lett. 432(1-2): 45-49), may be employed to promote the stable dimerization of antigen-binding multimers. These dimerized antigen-binding multimers may be utilized as the structural and joining elements in assembly unit fabrication.

[0084] FIG. 12. Diagram of single-chain Fv fragments (scFv). The top half of the diagram shows monomeric, dimeric (diabody), trimeric (triabody) and tetrameric (tetrabody) associations among V_H-linker-V, scFv fragments. The bottom half of the diagram shows such associations among V_L-linker-V_H scFv fragments. These associations between scFv domains are dependent upon the length of the peptide linker joining the V_H and V_L units. Longer peptide linkers (12-15 residues) favor monomeric formation, whereas shorter linkers (0-5 residues), favor multimeric structures. The linkage order of the V_H and V_L genes also affects multimer formation, activity and stability of the resultant scFv proteins. This type of recombinant antibody represents one of the smallest functional antigen binding entities derived from an IgG and can be utilized as the structural and joining elements in assembly unit fabrication.

[0085] FIG. 13. Diagram of the structure of a P-pilus. The pilus is anchored to the outer membrane of E. coli through an N-terminal membrane anchor in paph. Most of the pilus is made up of many copies of papA. The rod is terminated by a single copy of papK that acts as an adaptor between the rod structure and a thin, distal structure called a fibrillum. The fibrillum consists of a few copies of papE, followed by a single copy of papF and a single copy of papG, which acts as the adhesin at the distal tip of the structure.

[0086] FIGS. 14 (A-B). A. Diagram of the interaction of two pilins, showing the close interaction of the N-terminal extension of one pilin (depicted in the lower right of the figure) with the groove on the surface of the other pilin (depicted in the upper left of the figure). Pilins interact through the binding of a long N-terminal extension from the pilin to the body of an adjacent pilin. This provides an extended, specific interaction with significant mechanical strength. B. Diagram of the interaction of papE with a hybrid pilin constructed from the N-terminal arm of papF spliced onto the protein body of papA. Replacing the N-terminal arm of a pilin with the N-terminal arm of a different pilin alters its binding specificity. Here, papA has had its N-terminal arm replaced by that of papF (arrow), now making it possible for the papA to interact with papE through the use of interactions normally used to stabilize the papF-papE interaction

[0087] FIG. 15. Diagram of ROP protein, a four-helix bundle.

[0088] FIG. 16. Diagram of an idiotope/anti-idiotope Fab-Fab interaction. The diagram shows the -carbon trace of two Fab fragments interacting through idiotopic/anti-idiotopic interactions (pdb entry 1CIC). The heavy lines represent the heavy chains and the light lines represent the light chains of the Fab fragments. Most of the idiotopic/anti-idiotopic protein binding interactions occur between the loops of the heavy chains contained in the complementarity determining region (CDR). In this case, the association between Fabs results in a nearly linear association.

[0089] FIG. 17. Diagram of a staged assembly of hybrid pilin subunits. The illustrated process is described in Section 6 (Example 1). The addition of hybrid pilin subunits proceeds according to the steps indicated in the diagram. Hybrid pilins are made up of the protein body of one pilin (designated in capital letters, e.g., A, H, E, K) and the N-terminal extension of another pilin (designated in lower-case letters, e.g., k, a, f, e). The positioning of the ras epitope is indicated.

[0090] FIG. 18. Comparison of PNA (peptide nucleic acid, left) and DNA (right) structure. Note that PNA has a neutral peptide or peptide-like backbone instead of a negatively-charged sugar-phosphate backbone.

[0091] FIGS. 19(A-B). Two PNA/oligopeptide units can dimerize to form a single assembly unit. Two possible configurations for an assembly unit are shown here (FIG. 19A and FIG. 19B). The PNA portion provides joining elements A and B, while the oligopeptide portion forms two coiled coil structural elements (S) stabilized by disulfide bonds at either end. One or more functional units (F), comprised of, e.g., protein segments, may also be incorporated into the assembly unit. In certain embodiments, the assembly unit can have a randomly coiled peptide that comprises a functional element, F, in the internal or center portion of the dimer (FIG. 19A) or at the end of the PNA molecule opposite the end comprising the joining element (FIG. 19B). In each of these diagrams, the N-terminal end of the PNA/oligopeptide unit is towards the left of the diagram and the C-terminal end is towards the right.

[0092] FIG. 20. Line diagram indicating the order of elements of the upper synthetic protein monomer forming the staged assembly subunit shown in FIG. 19A. The order of the elements in the corresponding lower unit would be identical except that the PNA element is at the C-terminus. This reflects the parallel arrangement of the leucine zippers aligning the two units. The functionality sequence encodes the region at which a functional element may be added to the assembly subunit. Glycines separate each element to reduce steric interference between elements. Numbers below the line indicate the typical length in residues of each element.

[0093] FIG. 21. Diagram of eleven steps of a staged assembly that utilizes four bispecific assembly units and one tetraspecific assembly unit to make a two-dimensional nanostructure. For details, see Section 8 (Example 3).

[0094] FIGS. 22(A-B). Diagram of a staged assembly that utilizes nanostructure intermediates as subassemblies. In Steps 1-3, a nanostructure intermediate is constructed, two joining elements are capped and the nanostructure intermediate is released from the solid substrate. In Step 5, the nanostructure intermediate from Step 3 is added to an assembly intermediate (shown in Step 4 attached to the solid substrate) as an intact subassembly. For details, see Section 9 (Example 4).

[0095] FIGS. 23(AA-BF). Diagram of the sequence of the 32 steps used in the staged assembly of an exemplary cubic nanostructure. The cubic nanostructure is assembled from assembly units comprising structural elements from engineered diabody and triabody fragments. The joining elements of the assembly units are the multispecific binding domains from diabodies or triabodies. Seven complementary joining pairs are used: A and A, B and B, C and C, D and D, E and E, F and F, and G and G. The numbering (1-32) indicates the assembly unit added during each step. For details, see Section 11 (Example 6).

[0096] The present invention provides compositions and methods for the staged assembly of nanostructures. According to the methods of the invention, assembly of nanostructures proceeds by sequential, non-covalent, vectorial addition of specific assembly units to an initiator unit or a nanostructure intermediate during an assembly cycle, a process that is referred to herein as staged assembly. Attachment of each assembly unit is, by design, mediated by the specific, non-covalent binding of one or more pre-designated joining elements of one assembly unit to a complementary joining element present on the initiator unit or assembly intermediate. To avoid self-polymerization, each assembly unit is designed so that no joining element that is a part of the assembly unit can interact with any other joining element of that same assembly unit. The process is carried out in a massively parallel fashion such that a very large number of identical assemblies are fabricated simultaneously.

[0097] An assembly unit is herein defined as an assemblage of atoms and/or molecules comprising structural elements, joining elements and/or functional elements. In one embodiment, an assembly unit can be added to a nanostructure as a single unit through the formation of one or more specific interactions. In another embodiment, an assembly unit that comprises two or more assembly units, i.e., a subassembly, can be added to a nanostructure. An assembly unit may comprise one or more structural elements, and may further comprise one or more functional elements and one or more joining elements. If an assembly unit comprises a functional element, that functional element may be attached to or incorporated within a joining element or, in certain embodiments, a structural element. Such an assembly unit, which may comprise a structural element and one or a plurality of non-interacting joining elements, may be, in certain embodiments, structurally rigid and have well-defined recognition and binding properties. In one aspect of the invention, each joining element in the assembly unit exhibits specificity for a complementary joining element. A functional element can, in certain embodiments, be used to provide an attachment site for a moiety with a desirable physical, chemical, or biological property. Such a moiety could be, for example, a peptide, protein (e.g., enzyme), protein domain, small molecule, inorganic nanoparticle, atom, cluster of atoms, magnetic, photonic or electronic nanoparticles, or a marker such as a radioactive molecule, chromophore, fluorophore, chemiluminescent molecule, or enzymatic marker. Such functional elements can also be used for cross-linking linear, one-dimensional nanostructures to form two-dimensional and three-dimensional nanostructures.

[0098] According to the methods of the invention, the first assembly unit (i.e., the initiator unit) has one or a plurality of joining element(s) comprising the first joining element of a joining pair, which joining element is available for binding by another assembly unit comprising the second joining element of the joining pair. In preferred embodiments, the initiator unit is attached to a solid support. Attachment of each assembly unit is, by design, mediated by the specific, non-covalent binding of a single pre-determined joining element of one assembly unit to its complementary joining element. The complementary joining element is presented by an initiator or nanostructure intermediate.

[0099] Each interaction of a joining element is designed such that the joining element of an assembly unit does not interact with any other joining element of said assembly unit. Self-polymerization of the assembly unit is thereby obviated in each assembly cycle: only one assembly unit can be added to a target joining element on the initiator unit or nanostructure intermediate, and binding of the assembly unit to the target initiator or nanostructure intermediate will be vectorial.

[0100] The invention provides structural elements comprising antibodies or binding derivatives or binding fragments thereof, including, but not limited to, structural elements comprising: monoclonal antibodies, multispecific antibodies, Fab or F(ab)₂ antibody fragments, single-chain antibody fragments (scFvs), bispecific IgG, chimeric IgG or bispecific heterodimeric F(ab)₂ antibodies, diabodies or multimeric scFv fragments. A binding derivative of an antibody or antibody fragment is a derivative that exhibits the binding specificity of the antibody, antibody fragment, single-chain antibody fragment (scFv), etc., from which the binding derivative is derived. A binding fragment of an antibody or antibody fragment is a fragment that exhibits the binding specificity of the antibody, antibody fragment, single-chain antibody fragment (scFv), etc., from which the binding fragment is derived.

[0101] The invention also provides structural elements comprising bacterial pilin proteins, leucine zipper-type coiled coils, or four-helix bundles.

[0102] The invention provides joining elements that exhibit antigen-antibody interactions, including, but not limited to, joining elements comprising: recombinantly engineered antibodies or binding derivatives or binding fragments thereof, molecules that exhibit idiotope/anti-idiotope interactions, or two non-complementary idiotopes. The invention also provides joining elements comprising peptide epitopes, bacterial pilin proteins or binding derivatives or binding fragments thereof, or peptide nucleic acids (PNAs). A binding derivative of a molecule such as a peptide epitope, pilin protein or PNA is a derivative that exhibits the binding specificity of the peptide epitope, pilin protein or PNA from which the binding derivative is derived. A binding fragment of a molecule such as a peptide epitope or pilin protein is a fragment that exhibits the binding specificity of the peptide epitope or pilin protein from which the binding fragment is derived.

[0103] The staged-assembly methods described herein make possible the mass production of nanostructures that are multi-dimensional and have non-periodic architectures, and in which organic and inorganic nanocomponents are placed with precision in designated locations. The resulting nanostructures utilize proteins to control the assembly of structures that may, in certain embodiments, incorporate organic materials or inorganic materials such as metallic, semiconducting or magnetic nanoparticles (Bruchez et al., 1998, Semiconductor nanocrystals as fluorescent biological tags, Science 281: 2013-16; Peng et al., 2000, Shape control of CdSe nanocrystals, Nature 404(6773): 59-61; Whaley et al., 2000, Selection of peptides with semiconductor binding specificity for directed nanocrystal assembly. Nature 405: 665-68). Proteins offer many advantages over other molecules for the controlled assembly of complex architectures.

[0104] The staged-assembly methods disclosed herein do not depend on the physical manipulation of individual components and thus constitute a highly efficient and economical means for the precise geometric and spatial positioning of individual components in the nanometer range. The methods of the invention make possible the fabrication of highly complex architectures with only a few distinct, non-cross-reacting joining pairs. This greatly simplifies the problem of component design.

[0105] The staged-assembly methods disclosed herein provide a practical and sensible solution for solving the complicated and intricate problem of economic, massively parallel manufacturing of highly complex nanostructures. This is in sharp contrast to the nanoconstruction of nanostructured materials by self-assembly. With self-assembly, complete control of the material architecture is precluded. Self-assembly of nanodevices is limited, since each assembly unit in the nanostructure must have its position encoded by joining elements that form specific interactions with adjacent assembly units, but that do not interact with any other assembly unit making up the nanostructure. For example, self-assembly of a device composed of 100 assembly units would require 100 or more complementary joining pairs and furthermore, the 100 joining pairs would have to be designed so that they did not cross-react with one another. The same nanostructure could be assembled by the staged assembly process as described herein, with far fewer non-cross-reacting joining pairs. In Section 11, Example 6, an example is provided of the staged assembly of a three-dimensional, cube-shaped structure made up of 32 assembly units. Self-assembly of this structure would require the use of 32 non-cross-reacting, complementary joining pairs. As disclosed in Section 11 (Example 6), staged assembly of the same structure can be accomplished with only seven non-cross-reacting complementary joining pairs.

[0106] The present invention provides methods for staged assembly that enable massively parallel synthesis of complex, non-periodic, multi-dimensional nanostructures in which organic and inorganic moieties are placed, accurately and precisely, into a pre-designed, three-dimensional architecture. Staged assembly requires that a series of units be added in a given pre-designed order to an initiator unit and/or nanostructure intermediate. Because a large number of identical initiators are used and because subunits are added to all initiators/intermediates simultaneously, staged assembly fabricates multiple identical nanostructures in a massively parallel manner. In preferred embodiments, the initiator units are bound to a solid substrate, support or matrix. Additional assembly units are added sequentially in a procedure akin to solid phase polymer synthesis. The intermediate stage(s) of the nanostructure while it is being assembled, and which comprises the bound assembly units formed on the initiator unit, is generally described as either a nanostructure intermediate or simply, a nanostructure. Addition of each assembly unit to the nanostructure intermediate undergoing assembly depends upon the nature of the joining element presented by the previously added assembly unit and is independent of subsequently added assembly units. Thus assembly units can bind only to the joining elements exposed on the nanostructure intermediate undergoing assembly; that is, the added assembly units do not self-interact and/or polymerize.

[0107] Since the joining elements of a single assembly unit are non-complementary and therefore do not interact with one another, unbound assembly units do not form dimers or polymers. An assembly unit to be added is preferably provided in molar excess over the initiator unit or nanostructure intermediate in order to drive its reaction with the intermediate to completion. Removal of unbound assembly units during staged assembly is facilitated by carrying out staged assembly using a solid-substrate-bound initiator so that unbound assembly units can be washed away in each cycle of the assembly process.

[0108] This scheme provides for assembly of complex nanostructures using relatively few non-cross-reacting, complementary joining pairs. Only a few joining pairs need to be used, since only a limited number of joining elements will be exposed on the surface of an assembly intermediate at any one step in the assembly process. Assembly units with complementary joining elements can be added and incubated against the nanostructure intermediate, causing the added assembly units to be attached to the nanostructure intermediate during an assembly cycle. Excess assembly units can then be washed away to prevent them from forming unwanted interactions with other assembly units during subsequent steps of the assembly process. Each position in the nanostructure can be uniquely defined through the process of staged assembly and distinct functional elements can be added at any desired position. The staged assembly method of the invention enables massive parallel manufacture of complex nanostructures, and different complex nanostructures can be further self-assembled into higher order architectures in a hierarchic manner.

[0109] FIG. 2 depicts an embodiment of the staged assembly method of the invention in one dimension. In step 1, an initiator unit is immobilized on a solid substrate. In step 2, an assembly unit is added to the initiator (i.e. the matrix bound initiator unit), resulting in a nanostructure intermediate composed of two units. Only a single assembly unit is added in this step, because the second assembly unit cannot interact (i.e. polymerize) with itself.

[0110] The initiator unit, or any of the assembly units subsequently added during staged assembly including the capping unit, may contain an added functional element and/or may comprise a structural unit of different length from previously added units. For example, in step 3 of FIG. 2, a third assembly unit is added that comprises a functional element. In steps 4 and 5, additional assembly units are added, each with a designed functional group. Thus in the embodiment of staged assembly depicted in FIG. 2, the third, fourth and fifth assembly units each carry a unique functional element (designated by geometric shapes protruding from the top of the assembly units in the figure).

[0111] The embodiment of staged assembly depicted in FIG. 2 requires only two non-cross-reacting, complementary joining pairs. Self-assembly of the structure, as it stands at the end of step 5, would require four non-cross-reacting, complementary joining pairs. This relatively modest improvement in number of required joining pairs becomes far greater as the size of the structure increases. For instance, for a linear structure of N units assembled by an extension of the five steps illustrated in FIG. 2, staged assembly would still require only two non-cross-reacting, complementary joining pairs, whereas self-assembly would require (N1) non-cross-reacting, complementary joining pairs.

[0112] The number of nanostructures fabricated is determined by the number of initiator units bound to the matrix while the length of each one-dimensional nanostructure is a function of the number of assembly cycles performed. If assembly units with one or more different functional elements are used, then the order of assembly will define the relative spatial orientation of each functional element relative to the other functional elements.

[0113] After each step in the method of staged assembly of the invention, excess unbound assembly units are removed from the attached nanostructure intermediate by a removal step, e.g., a washing step. The substrate-bound nanostructure intermediate may be washed with an appropriate solvent (e.g., an aqueous solution or buffer). The solvent must be able to remove subunits held by non-specific interactions without disrupting the specific, interactions of complementary joining elements. Appropriate solvents may vary as to pH, salt concentration, chemical composition, etc., as required by the assembly units being used.

[0114] A buffer used for washing the nanostructure intermediate can be, for example, a buffer used in the wash steps implemented in ELISA protocols, such as those described in Current Protocols in Immunology (see Chapter 2, Antibody Detection and Preparation, Section 2.1 Enzyme-Linked Immunosorbent Assays, John Wiley & Sons, 2001, Editors John E. Coligan, Ada M. Kruisbeek, David H. Margulies, Ethan M. Shevach, Warren Strober, Series Editor: Richard Coico).

[0115] In certain embodiments, an assembled nanostructure is capped by addition of a capping unit, which is an assembly unit that carries only a single joining element. Furthermore, if the initiator unit has been attached to the solid substrate via a cleavable bond, the nanostructure can be removed from the solid substrate and isolated. However, in some embodiments, the completed nanodevice will be functional while attached to the solid substrate and need not be removed.

[0116] The above-described steps of adding assembly units can be repeated in an iterative manner until a complete nanostructure is assembled, after which time the complete nanostructure can be released by breaking the bond immobilizing the first assembly unit from the matrix at a designed releasing moiety (e.g,. a protease site) within the initiator unit or by using a pre-designed process for release (e.g., lowering of pH). The process of staged assembly, as illustrated in FIGS. 2 and 3 is one of the simplest embodiments contemplated for staged assembly. In other embodiments, assembly units with additional joining elements can be used to create more complex assemblies. Assembly units may be added individually or, in certain embodiments, they can be added as subassemblies (FIG. 3). The result is a completely defined nanostructure with functional elements that are distributed spatially in relationship to one another to satisfy desired design parameters. The compositions and methods disclosed herein provide means for the assembly of these complex, designed nanostructures and of more complex nanodevices formed by the staged assembly of one or a plurality of nanostructures into a larger structure. Fabrication of multidimensional nanostructures can be accomplished, e.g., by incorporating precisely-spaced assembly units containing additional joining elements into individual, one-dimensional nanostructures, where those additional joining elements can be recognized and bound by a suitable cross-linking agent to attach the individual nanostructures together. In certain preferred embodiments, such cross-linking could be, e.g., an antibody or a binding derivative or a binding fragment thereof.

[0117] In some embodiments of the staged assembly method of the invention, the initiator unit is tethered to a solid support. Such tethering is not random (i.e., is not non-specific binding of protein to plastic or random biotinylation of an assembly unit followed by binding to immobilized streptavidin) but involves the binding of a specific element of the initiator unit to the matrix or substrate. The staged assembly process is a vectorial process that requires an unobstructed joining element on the initiator unit for attachment of the next assembly unit. Random binding of initiator units to substrate would, in some cases, result in the obstruction of the joining element needed for the attachment of the next assembly unit, and thus lowering the number of initiator units on which nanostructures are assembled.

[0118] In other embodiments of the staged assembly method of the invention, the initiator unit is not immobilized to a solid substrate. In this case, a removal step, e.g., a washing step, can be carried out on a nanostructure constructed on a non-immobilized or untethered initiator unit by: (1) attaching a magnetic nanoparticle to the initiator unit and separating nanostructure intermediates from non-bound assembly units by applying a magnetic field; 2) separating the larger nanostructure intermediates from unbound assembly units by centrifugation, precipitation or filtration; or 3) in those instances in which a nanostructure intermediate or assembled nanostructure is more resistant to a destructive treatment (e.g., protease treatment or chemical degradation), unbound assembly units are selectively destroyed.

[0119] Proteins have well-defined binding properties, and the technology to manipulate the intermolecular interactions of proteins is well known in the art (Hayashi et al., 1995, A single expression system for the display, purification and conjugation of single-chain antibodies, Gene 160(1): 129-30; Hayden et al., 1997, Antibody engineering, Curr. Opin. Immunol. 9(2): 201-12; Jung et al., 1999, Selection for improved protein stability by phage display, J. Mol. Biol. 294(1): 163-80, Viti et al., 2000, Design and use of phage display libraries for the selection of antibodies and enzymes, Methods Enzymol. 326: 480-505; Winter et al., 1994, Making antibodies by phage display technology, Annu. Rev. Immunol. 12: 433-55). The contemplated staged assembly of nanostructures, however, need not be limited to components composed primarily of biological molecules, e.g., proteins and nucleic acids, that have specific recognition properties. The optical, magnetic or electrical properties of inorganic atoms or molecules will be required for some embodiments of nanostructures fabricated by staged assembly.

[0120] There will be many embodiments of this invention in which components not made up of proteins will be advantageously utilized. In other embodiments, it may be possible to utilize the molecular interaction properties of proteins or nucleic acids to construct nanostructures composed of both organic and inorganic materials.

[0121] In certain embodiments, inorganic nanoparticles are added to components that are assembled into nanostructures using the staged assembly methods of the invention. This may be done using joining elements specifically selected for binding to inorganic particles. For example, Whaley and co-workers have identified peptides that bind specifically to semiconductor binding surfaces (Whaley et al., 2000, Selection of peptides with semiconductor binding specificity for directed nanocrystal assembly, Nature 405: 665-68). In one embodiment, these peptides are inserted into protein components described herein using standard cloning techniques. Staged assembly of protein constructs as disclosed herein, provides a means of distributing these binding sites in a rigid, well-defined three-dimensional array.

[0122] Once the binding sites for a particular type of inorganic nanoparticle are all in place, the inorganic nanoparticles can be added using a cycle of staged assembly analogous to that used to add proteinaceous assembly units. To accomplish this, it may be necessary, in certain embodiments to adjust the solution conditions under which the nanostructure intermediates are incubated, in order to provide for the solubility of the inorganic nanoparticles. Once an inorganic nanoparticle is added to the nanostructure intermediate, it is not possible to add further units to the inorganic nanoparticle in a controlled fashion because of the microheterogeneities intrinsic to any population of inorganic nanoparticles. These heterogeneities would render the geometry and stoichiometry of further interactions uncontrollable.

[0123] FIG. 4 is a diagram illustrating the addition of protein units and inorganic elements to a nanostructure according to the staged assembly methods of the invention. In step 1, an initiator unit is bound to a solid substrate. In step 2, an assembly unit is bound specifically to the initiator unit. In step 3, an additional assembly unit is bound to the nanostructure undergoing assembly. This assembly unit comprises an engineered binding site specific for a particular inorganic element. In step 4, the inorganic element (depicted as a cross-hatched oval) is added to the structure and bound by the engineered binding site. Step 5 adds another assembly unit with a binding site engineered for specificity to a second type of inorganic element, and that second inorganic element (depicted as a hatched diamond) is added in step 6.

[0124] The order in which assembly units are added is determined by the desired structure and/or activity that the product nanostructure, and the need to minimize the number of cross-reacting joining element pairs used in the assembly process. Hence determining the order of assembly is an integral part of the design of a nanostructure to be fabricated by staged assembly. Joining elements are chosen, by design, to permit staged assembly of the desired nanostructure. Since the choice of joining element(s) is generally independent of the functional elements to be incorporated into the nanostructure, the joining elements are mixed and matched as needed to fabricate assembly units with the necessary functional elements and joining elements that will provide for the placement of those functional elements in the desired spatial orientation.

[0125] For example, assembly units comprising two joining elements, designed using the six joining elements that make up three joining pairs, can include any of 18 pairs of the joining elements that are non-interacting. There are 21 possible pairs of joining elements, but three of these pairs are interacting (e.g. A-A) and their use in an assembly unit would lead to the self-association of identical assembly units with one another. In the example illustrated below, joining elements are denoted as A, A, B, B, C and C, where A and A, B and B, and C and C are complementary pairs of joining elements (joining pairs), i.e. they bind to each other with specificity, but not to any of the other four joining elements depicted. Six representative assembly units, each of which comprises two joining elements, wherein each joining element comprises a non-identical, non-complementary joining element, are depicted below. In this depiction, each assembly unit further comprises a unique functional element, one of a set of six, and represented as F₁ to F₆. According to these conventions, six possible assembly units can be designated as:

[0126] A-F₁-B

[0127] B-F₂-A

[0128] B-F₃-C

[0129] C-F₄-B

[0130] B-F₅-A

[0131] A-F₆-C

[0132] Staged assembly according to the methods disclosed herein can be used to assemble the following illustrative linear, one-dimensional nanostructures, in which the order and relative vectorial orientation of each assembly unit is independent of the order of the functional elements (the symbol - is used to represent the solid substrate to which the initiator is attached and a double colon represents the specific interaction between assembly units):

-A-F1-B::B-F2-A::A-F1-B::B-F2-A::A-F1-B::B-F2-A::A-F1-B::B-F2-A
-A-F1-B::B-F2-A::A-F6-C::C-F4-B::B-F2-A::A-F1-B::B-F5-A::A-F6-C
-A-F1-B::B-F2-A::A-F1-B::B-F5-A::A-F1-B::B-F2-A::A-F1-B::B-F3-C
-A-F1-B::B-F3-C::C-F4-B::B-F3-C::C-F4-B::B-F3-C::C-F4-B::B-F2-A

[0133] As is apparent from this illustration, a large number of unique assembly units can be constructed using a small number of complementary joining elements. Moreover, only a small number of complementary joining elements are required for the fabrication of a large number of unique and complex nanostructures, since only one type of assembly unit is added in each staged assembly cycle and, therefore, joining elements can be used repeatedly without rendering ambiguous the position of an assembly unit within the completed nanostructure.

[0134] In each of the cases illustrated above, only two or three joining pairs have been used. Self-assembly of any of these structures would require the use of seven non-cross-reacting joining pairs. If these linear structures were N units in extent, they would still only require two or three joining pairs, but for self-assembly, they would require (N1) non-cross-reacting, complementary joining pairs.

[0135] In another aspect of the invention, by interchanging the positions of the two joining elements of an assembly unit depicted above, the spatial position and orientation of the attached functional element will be altered within the overall structure of the nanostructure fabricated. This aspect of the invention illustrates yet another aspect of the design flexibility provided by staged assembly of nanostructures as disclosed herein.

[0136] Attachment of each assembly unit to an initiator or nanostructure intermediate is mediated by formation of a specific joining-pair interaction between one joining element of he assembly unit and one or more unbound complementary joining elements carried by the initiator or nanostructure intermediate. In many embodiments, only a single unbound complementary joining element will be present on the initiator or nanostructure intermediate. However, in other embodiments, it may be advantageous to add multiple identical assembly units to multiple sites on the assembly intermediate that comprise identical joining elements. In these embodiments, the staged assembly proceeds by the parallel addition of assembly units, but only a single unit will be attached at any one site on the intermediate, and assembly at all sites that are involved will occur in a pre-designed, vectorial manner.

[0137] Structural integrity of the nanostructure is of critical importance throughout the process of staged assembly, and the assembly units are preferably connected by non-covalent interactions. A specific non-covalent interaction is, for example, an interaction that occurs between an assembly unit and a nanostructure intermediate. The specific interaction should exhibit adequate affinity to confer stability to the complex between the assembly unit and the nanostructure intermediate sufficient to maintain the interaction stably throughout the entire staged assembly process. A specific non-covalent interaction should exhibit adequate specificity such that the added assembly unit will form stable interactions only with joining elements designed to interact with it. The interactions that occur among elements during the staged assembly process disclosed herein are preferably operationally irreversible. A binding constant that meets this requirement cannot be defined unambiguously since irreversible is a kinetic concept, and a binding constant is based on equilibrium properties. Nevertheless, interactions with Kd's of the order of 10⁷ or lower (i.e. higher affinity and similar to the Kd of a typical diabody-epitope complex) will typically act irreversibly on the time scale of interest, i.e. during staged assembly of a nanostructure.

[0138] The intermolecular interactions need not act irreversibly, however, on the timescale of the utilization of a nanostructure (i.e. its shelf life or working life expectancy). In certain embodiments, nanostructures fabricated according to the staged assembly methods disclosed herein are subsequently stabilized by chemical fixation (e.g., by fixation with paraformaldehyde or glutaraldehyde) or by cross-linking. The most common schemes for cross-linking two proteins involve the indirect coupling of an amine group on one assembly unit to a thiol group on a second assembly unit (see, e.g., Handbook of Fluorescent Probes and Research Products, Eighth Edition, Chapter 2, Molecular Probes, Inc., Eugene, Ore.; Loster et al., 1997, Analysis of protein aggregates by combination of cross-linking reactions and chromatographic separations, J. Chromatogr. B. Biomed. Sci. Appl. 699(1-2): 439-61; Phizicky et al., 1995, Protein-protein interactions: methods for detection and analysis, Microbiol. Rev. 59(1): 94-123).

[0139] In certain embodiments of the invention, the fabrication of a nanostructure by the staged assembly methods of the present invention involves joining relatively rigid and stable assembly units, using non-covalent interactions between and among assembly units. Nevertheless, the joining elements that are incorporated into useful assembly units can be rather disordered, that is, neither stable nor rigid, prior to interaction with a second joining element to form a stable, preferably rigid, joining pair. Therefore, in certain embodiments of the invention, individual assembly units may include unstable, flexible domains prior to assembly, which, after assembly, will be more rigid. In preferred embodiments, a nanostructure fabricated using the compositions and methods disclosed herein is a rigid structure.

[0140] According to the methods of the invention, analysis of the rigidity of a nanostructure, as well as the identification of any architectural flaws or defects, are carried out using methods well-known in the art, such as electron microscopy.

[0141] In another embodiment, structural rigidity can be tested by attaching one end of a completed nanostructure directly to a solid surface, i.e., without the use of a flexible tether. The other end of the nanostructure (or a terminal branch of the nanostructure, if it is a multi-branched structure) is then attached to an atomic force microscope (AFM) tip, which is movable. Force is applied to the tip in an attempt to move it. If the nanostructure is flexible, there will be an approximately proportional relationship between the force applied and tip movement as allowed by deflection of the nanostructure. In contrast, if the nanostructure is rigid, there will be little or no deflection of the nanostructure and tip movement as the level of applied force increases, up until the point at which the rigid nanostructure breaks. At that point, there will be a large movement of the AFM tip even though no further force is applied. As long as the attachment points of the two ends are stronger than the nanostructure, this method will provide a useful measurement of rigidity.

[0142] According to the present invention, each position in a nanostructure is distinguishable from all others, since each assembly unit can be designed to interact tightly, specifically, and uniquely with its neighbors. Each assembly unit can have an activity and/or characteristic that is distinct to its position within the nanostructure. Each position in the nanostructure is uniquely defined through the process of staged assembly, and through the properties of each assembly unit and/or functional element that is added at a desired position. In addition, the staged-assembly methods and assembly units disclosed herein are amenable to large scale, massively parallel, automated manufacturing processes for construction of complex nanostructures of well-defined size, shape, and function.

[0143] The methods and compositions of the present invention capitalize upon the precise dimensions, uniformity and diversity of spatial geometries that proteins are capable of that are used in the construction of the assembly units employed herein. Furthermore, as described hereinbelow, the methods of the invention are advantageous because genetic engineering techniques can be used to modify and tailor the properties of those biological materials used in the methods of the invention disclosed herein, as well as to synthesize large quantities of such materials in microorganisms.

[0144] Assembly units provided by the present invention and used in the staged assembly methods disclosed herein comprise an assemblage of atoms and/or molecules comprising structural elements, joining elements and/or functional elements. In certain embodiments, assembly units can be added to a nanostructure as a single unit through the formation of specific interactions. In other embodiments, assembly units can be added as subassemblies.

[0145] In order to participate in a staged assembly, each assembly unit, other than a capping unit, should have a minimum of two joining elements or sites at which a specific intermolecular interaction can take place. Initiator units may be considered to have a minimum of two joining elements if the element conferring immobilization to the substrate or matrix is considered a joining element. Joining elements, however, are generally considered to interact via non-covalent interactions and in many embodiments, the interaction between the initiator unit and the substrate or matrix may be covalent. Capping units need to have, at most, one joining element, so that once added to a nanostructure or nanostructure intermediate, no subsequently added assembly units can extend from the capping unit assembled to the nanostructure. Therefore, in certain embodiments, an assembly unit comprising only a single joining element can be used to cap a completed nanostructure (or to terminate one branch of a multi-branched vectorial growth network of a nanostructure), thereby preventing further additions of assembly units to a particular position within the nanostructure.

[0146] In certain embodiments, the assembly unit comprises a joining element that exhibits antigen-antibody interactions, including, but not limited to, a joining element comprising: recombinantly engineered antibody or binding derivative or binding fragment thereof, a molecule that exhibits idiotope/anti-idiotope interactions, or two non-complementary idiotopes. In other embodiments, the assembly unit comprises a joining element comprising a peptide epitope, a bacterial pilin protein or binding derivative or binding fragment thereof, or a peptide nucleic acid (PNA).

[0147] In certain embodiments, the assembly unit also comprises a structural element comprising an antibody or binding derivative or binding fragment thereof, including, but not limited to, a structural element comprising: a monoclonal antibody, a multispecific antibody, a Fab or F(ab)₂ antibody fragment, a single-chain antibody fragment (scFv), a bispecific IgG, a chimeric IgG or bispecific heterodimeric F(ab)₂ antibody, a diabody or multimeric scFv fragment. The invention also provides structural elements comprising a bacterial pilin protein, a leucine zipper-type coiled coil, or a four-helix bundle.

[0148] In certain embodiments, the assembly unit comprises a multi-domain polypeptide chain in which a flexible segment, generally an oligopeptide that may comprise two to five glycine units, is disposed between different domains in order to allow independent folding of each peptide or protein domain. The number of such glycine residues is generally determined empirically, as would be apparent to those of ordinary skill in the art. Therefore, in certain embodiments, a flexible segment is disposed between a joining element and a structural element, or between a functional domain and a joining element, structural domain or a portion thereof.

[0149] The present invention provides for the staged assembly of nanostructures that utilizes assembly units comprising recombinantly-engineered antibodies and/or portions thereof. Recombinant antibodies are among the preferred sources disclosed herein of structural elements and joining elements used for fabricating nanostructures in a staged-assembly process. In certain embodiments, structural and/or joining elements comprise binding derivatives or binding fragments of any class of immunoglobulin molecules, including IgG, IgM, IgE, IgA, IgD and any subclass thereof. In specific embodiments, structural and/or joining elements comprise modified, engineered or recombinantly-derived Fab or scFv fragments of IgG molecules.

[0150] In certain embodiment, a structural and/or joining element comprises a binding derivative or binding fragment of a protein of interest, such as an antibody or pilin protein. Derivatives of a protein of interest used in the methods of the invention, e.g., an antibody or pilin protein, can be made by altering sequences by substitutions, additions or deletions that provide for functionally equivalent molecules. Due to the degeneracy of nucleotide coding sequences, other DNA sequences that encode substantially the same amino acid sequence as the gene encoding the protein of interest may be used in the practice of the present invention. These include, but are not limited to, nucleotide sequences comprising all or portions of a gene, which is altered by the substitution of different codons that encode a functionally equivalent amino acid residue within the sequence, thus producing a silent change.

[0151] Likewise, derivatives of a protein of interest include, but are not limited to, those containing, as a primary amino acid sequence, all or part of the amino acid sequence of a protein of interest including altered sequences in which functionally equivalent amino acid residues are substituted for residues within the sequence resulting in a silent change. For example, one or more amino acid residues within the sequence can be substituted by another amino acid of a similar polarity that acts as a functional equivalent, resulting in a silent alteration. Substitutes for an amino acid within the sequence may be selected from other members of the class to which the amino acid belongs. For example, the nonpolar (hydrophobic) amino acids include alanine, leucine, isolcucine, valine, proline, phenylalanine, tryptophan and methionine. The polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine. The positively charged (basic) amino acids include arginine, lysine and histidine. The negatively charged (acidic) amino acids include aspartic acid and glutamic acid.

[0152] Derivatives or analogs of antibody or pilin proteins include but are not limited to those molecules comprising regions that are substantially homologous to the antibody or pilin protein of interest or a binding fragment thereof (e.g., in various embodiments, at least 60% or 70% or 80% or 90% or 95% identity over an amino acid sequence of identical size or when compared to an aligned sequence in which the alignment is done by a computer homology program known in the art) or whose encoding nucleic acid is capable of hybridizing to a sequence encoding the protein of interest, under highly stringent or moderately stringent conditions. Such highly or moderately stringent conditions are commonly known in the art.

[0153] By way of example and not limitation, exemplary conditions of high stringency are as follows: Prehybridization of filters containing DNA is carried out for 8 h to overnight at 65 C. in buffer composed of 6 SSC, 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.02% BSA, and 500 g/ml denatured salmon sperm DNA. Filters are hybridized for 48 h at 65 C. in prehybridization mixture containing 100 g/ml denatured salmon sperm DNA and 5-2010⁶ cpm of ³²P-labeled probe. Washing of filters is done at 37 C. for 1 h in a solution containing 2 SSC, 0.01% PVP, 0.01% Ficoll, and 0.01% BSA. This is followed by a wash in 0.1 SSC at 50 C. for 45 min before autoradiography. Other conditions of high stringency that may be used are well known in the art.

[0154] By way of example and not limitation, exemplary conditions of moderate stringency are as follows: Filters containing DNA are pretreated for 6 h at 55 C. in a solution containing 6 SSC, 5 Denhart's solution, 0.5% SDS and 100 g/ml denatured salmon sperm DNA. Hybridizations are carried out in the same solution and 5-2010⁶ cpm ³²P-labeled probe is used. Filters are incubated in hybridization mixture for 18-20 h at 55 C., and then washed twice for 30 minutes at 60 C. in a solution containing 1 SSC and 0.1% SDS. Filters are blotted dry and exposed for autoradiography. Other conditions of moderate stringency that may be used are well-known in the art.

[0155] Other conditions of high stringency that may be used are well known in the art. In general, for probes between 14 and 70 nucleotides in length the melting temperature (TM) is calculated using the formula: Tm( C.)81.516.6(logmonovalent cations (molar))0.41 (% GC)(500/N) where N is the length of the probe. If the hybridization is carried out in a solution containing formamide, the melting temperature is calculated using the equation Tm( C.)81.516.6(logmonovalent cations (molar))0.41 (% GC)(0.6 1% formamide)(500/N) where N is the length of the probe. In general, hybridization is carried out at about 20-25 degrees below Tm (for DNA-DNA hybrids) or 10-15 degrees below Tm (for RNA-DNA hybrids).

[0156] The present invention also provides for the staged assembly of nanostructures that utilizes assembly units comprising a fragment of a protein of interest, e.g., an antibody or pilin protein. In a specific embodiment of the invention, a protein consisting of or comprising a fragment of a protein of interest consists of at least 4 contiguous amino acids of the protein of interest. In other embodiments, the fragment consists of at least 5, 6, 7, 8, 9, 10, 15, 20, 35 or 50 contiguous amino acids of the protein of interest. In specific embodiments, such fragments are not larger than 35, 100, 200, 300 or 350 amino acids.

[0157] The present invention also provides for the staged assembly of nanostructures that utilizes assembly units comprising fusion proteins. The production of fusion or chimeric protein products (comprising a desired protein (e.g., an IgG), fragment, analog, or derivative joined via a peptide bond to a heterologous protein sequence (of a different protein)). Such chimeric protein products can be made by ligating the appropriate nucleic acid sequences encoding the desired amino acid sequences to each other by methods known in the art, in the proper reading frame, and expressing the chimeric product by methods commonly known in the art. Alternatively, such a chimeric product may be made by protein synthetic techniques, e.g., by use of a peptide synthesizer.

[0158] The three-dimensional structures of IgG and its binding derivatives or binding fragments, e.g., IgG, Fab, scFv, (scFv)₂ (scFv)₃), have been solved (Braden et al., 1996, Crystal structure of an Fv-Fv idiotope-anti-idiotope complex at 1.9 A resolution, J. Mol. Biol. 264(1): 137-51; Ban et al., 1994, Crystal structure of an idiotype-anti-idiotype Fab complex, Proc. Natl. Acad. Sci. U.S.A. 91(5): 1604-08; Perisic et al., 1994, Crystal structure of a diabody, a bivalent antibody fragment, Structure 2(12): 1217-26; Harris et al., 1998, Crystallographic structure of an intact IgG1 monoclonal antibody, J. Mol. Biol. 275(5): 861-72; Pei et al., 1997, The 2.0-A resolution crystal structure of a trimeric antibody fragment with noncognate V_H-V_L domain pairs shows a rearrangement of V_H CDR3, Proc. Natl. Acad. Sci. USA 94(18): 9637-42). Each IgG-derived antibody fragment preferably contains at least one monovalent and monospecific complementarity determining region (CDR) or joining element. The CDR is preferably the site contained in each structure at which the highly specific intermolecular interaction can occur between the protein components.

[0159] Recombinantly engineered antibodies meet many of the basic criteria for use in the construction of assembly units for staged-assembly of nanostructures and are preferred sources of joining elements used for fabricating such nanostructures. Not only are such recombinant antibody binding domains structurally well characterized, they also have inherent binding specificities (joining elements) necessary for assembly unit addition.

[0160] For example, the known three-dimensional structure of many recombinant engineered components can serve as a guide for design of structural modifications to the antibody fragment that will enable the insertion of peptides (for example, at the site of a surface loop) that will confer novel binding, structural or functional properties to the antibody fragment. Moreover, there is a huge diversity of intermolecular specificities, such as that involving an antibody and a specific epitope, that can be either designed and constructed, or selected from a library. Advances in recombinant antibody technology have led to the creation of multivalent, multispecific and multifunctional antibodies (Chaudhary et al., 1989, A recombinant immunotoxin consisting of two antibody variable domains fused to Pseudomonas exotoxin, Nature 339(6223): 394-97; Neuberger et al. 1984, Recombinant antibodies possessing novel effector functions, Nature 312(5995): 604-08; Wallace et al., 2001, Exogenous antigen targeted to FcgammaRI on myeloid cells is presented in association with MHC class I, J. Immunol. Methods 248(1-2): 183-94) that may be used, according to the methods of the invention, as sources of structural elements and joining elements. Such multivalent, multispecific and multifunctional antibodies can be modified by the addition of functional groups for the construction of assembly units used for the fabrication of nanostructures as described herein.

[0161] An initiator assembly unit is the first assembly unit incorporated into a nanostructure that is formed by the staged assembly method of the invention. An initiator assembly unit may be attached, in certain embodiments, by covalent or non-covalent interactions, to a solid substrate or other matrix. An initiator assembly unit is also known as an initiator unit. Staged assembly of a nanostructure begins by the non-covalent, vectorial addition of a selected assembly unit to the initiator unit. According to the methods of the invention, an assembly unit is added to the initiator unit through (i) the incubation of an initiator unit, which in some embodiments, is immobilized to a matrix or substrate, in a solution comprising the next assembly unit to be added. This incubation step is followed by (ii) a removal step, e.g., a washing step, in which excess assembly units are removed from the proximity of the initiator unit.

[0162] Assembly units bind to the initiator unit through the formation of specific, non-covalent bonds. The joining elements of the next assembly unit are chosen so that they attach only at pre-designated sites on the initiator unit. Only one assembly unit can be added to a target joining element on the initiator unit during the first staged-assembly cycle, and binding of the assembly unit to the target initiator unit is vectorial. Staged assembly continues by repeating steps (i) and (ii) until all of the desired assembly units are incorporated into the nanostructure according to the desired design of the nanostructure.

[0163] In a preferred embodiment of the staged assembly method of the invention, an initiator unit is immobilized on a substrate and additional units are added sequentially in a procedure analogous to solid phase polymer synthesis.

[0164] An initiator unit is a category of assembly unit, and therefore can comprise any of the structural, joining, and/or functional elements described hereinbelow as being comprised in an assembly unit of the invention. An initiator unit can therefore comprise any of the following molecules, or a binding derivative or binding fragment thereof: a monoclonal antibody; a multispecific antibody, a Fab or F(ab)₂ fragment, a single-chain antibody fragment (scFv); a bispecific, chimeric or bispecific heterodimeric F(ab)₂; a diabody or multimeric scFv fragment; a bacterial pilin protein, a leucine zipper-type coiled coil, a four-helix bundle, a peptide epitope, or a PNA, or any other type of assembly unit disclosed herein.

[0165] In certain embodiments, the invention provides an initiator assembly unit which comprises at least one joining element. In other embodiments, the invention provides an initiator assembly unit with two or more joining elements.

[0166] Initiator units may be tethered to a matrix in a variety of ways. The choice of tethering method will be determined by several design factors including, but not limited to: the type of initiator unit, whether the finished nanostructure must be removed from the matrix, the chemistry of the finished nanostructure, etc. Potential tethering methods include, but are not limited to, antibody binding to initiator epitopes, His tagged initiators, initiator units containing matrix binding domains (e.g., chitin-binding domain, cellulose-binding domain), antibody binding proteins (e.g., protein A or protein G) for antibody or antibody-derived initiator units, streptavidin binding of biotinylated initiators, PNA tethers, and specific covalent attachment of initiators to matrix.

[0167] In certain embodiments, an initiator unit is immobilized on a solid substrate. Initiator units may be immobilized on solid substrates using methods commonly used in the art for immobilization of antibodies or antigens. There are numerous methods well known in the art for immobilization of antibodies or antigens. These methods include non-specific adsorption onto plastic ELISA plates; biotinylation of a protein, followed by immobilization by binding onto streptavidin or avidin that has been previously adsorbed to a plastic substrate (see, e.g., Sparks et al., 1996, Screening phage-displayed random peptide libraries, in Phage Display of Peptides and Proteins, A Laboratory manual, editors, B. K. Kay, J. Winter and J. McCafferty, Academic Press, San Diego, pp. 227-53). In addition to ELISA microtiter plates, protein may be immobilized onto any number of other solid supports such as Sepharose (Dedman et al., 1993, Selection of target biological modifiers from a bacteriophage library of random peptides: the identification of novel calmodulin regulatory peptides, J. Biol. Chem. 268; 23025-30) or paramagnetic beads (Sparks et al., 1996, Screening phage-displayed random peptide libraries, in Phage Display of Peptides and Proteins, A Laboratory manual, editors, B. K. Kay, J. Winter and J. McCafferty, Academic Press, San Diego, pp. 227-53). Additional methods that may be used include immobilization by reductive amination of amine-containing biological molecules onto aldehyde-containing solid supports (Hermanson, 1996, Bioconjugate Techniques, Academic Press, San Diego, p. 186), and the use of dimethyl pimelimidate (DMP), a homobifunctional cross-linking agent that has imidoester groups on either end (Hermanson, 1996, Bioconjugate Techniques, Academic Press, San Diego, pp. 205-06). This reagent has found use in the immobilization of antibody molecules to insoluble supports containing bound protein A (e.g., Schneider et al., 1982, A one-step purification of membrane proteins using a high efficiency immunomatrix, J. Biol. Chem. 257, 10766-69).

[0168] In a specific embodiment, an initiator unit is a diabody that comprises a tethering domain (T) that recognizes and binds an immobilized antigen/hapten and an opposing domain (A) to which additional assembly units are sequentially added in a staged assembly. Antibody 8F5, which is directed against the antigenic peptide VKAETRLNPDLQPTE (SEQ ID NO: 70) derived human rhinovirus (Serotype 2) viral capsid protein Vp2, is used as the T domain (Tormo et al., 1994, Crystal structure of a human rhinovirus neutralizing antibody complexed with a peptide derived from viral capsid protein VP2, EMBO J. 13(10): 2247-56). The A domain is the same lysozyme anti-idiotopic antibody (E5.2) previously described for Diabody Unit 1. The completed initiator assembly unit therefore contains 8F5730.1.4 (TA ) as the opposing CDRs. The initiator unit is constructed and functionally characterized using the methods described herein for characterizing joining elements and/or structural elements comprising diabodies.

[0169] In order to immobilize the initiator unit onto a solid support matrix, the rhinovirus antigenic peptide may fused to the protease recognition peptide factor Xa through a short flexible linker spliced at the N termini of the Factor Xa sequence, IEGR, (Nagai and Thogersen, 1984, Generation of beta-globin by sequence-specific proteolysis of a hybrid protein produced in Escherichia coli, Nature309(5971): 810-12) and between the Factor Xa sequence and the antigenic peptide sequence. This fusion peptide may be covalently linked to CH-Sepharose 4B (Pharmacia); a sepharose derivative that has a six-carbon long spacer arm and permits coupling via primary amines. (Alternatively, Sepharose derivatives for covalent attachment via carboxyl groups may be used.) The covalently attached fusion protein will serve as a recognition epitope for the tethering domain 8F5 in the initiator unit (TA).

[0170] Once the initiator is immobilized, additional diabody units (diabody assembly units 1 and 2) may be sequentially added in a staged assembly, unidirectionally from binding domain A. Upon completion of the staged assembly, the nanostructure may be either cross-linked to the support matrix or released from the matrix upon addition of the protease Factor Xa. The protease will cleave the covalently attached antigenic /Factor Xa fusion peptide, releasing the intact nanostructure from the support matrix, since, by design, there are no Factor Xa recognition sites contained within any of the designed protein assembly units.

[0171] An alternate strategy of cleaving the peptide fusion from the solid support matrix that does not require the addition of Factor Xa, can also be implemented. This method utilizes a cleavable spacer arm attached to the sepharose matrix. The antigen peptide is covalently attached through a phenyl-ester linkage to the matrix. Once the immobilized antibody binds initiator assembly unit, the initiator assembly unit remains tethered to the support matrix until chemical cleavage of the spacer arm with imidazoleglycine buffer at pH 7.4 at which point the initiator unit/antigen complex (and associated nanostructure) are released from the support matrix.

[0172] In certain embodiments of the present invention, an assembly unit comprises a structural element. The structural element generally has a rigid structure (although in certain embodiments, described below, the structural element may be non-rigid). The structural element is preferably a defined peptide, protein or protein fragment of known size and structure that comprises at least about 50 amino acids and, generally, fewer than 2000 amino acids. Peptides, proteins and protein fragments are preferred since naturally-occurring peptides, proteins and protein fragments have well-defined structures, with structured cores that provide stable spatial relationships between and among the different faces of the protein. This property allows the structural element to maintain pre-designed geometric relationships between the joining elements and functional elements of the assembly unit, and the relative positions and stoichiometries of assembly units to which it is bound.

[0173] The use of proteins as structural elements has particular advantages over other choices such as inorganic nanoparticles. Most populations of inorganic nanoparticles are heterogeneous, making them unattractive scaffolds for the assembly of a nanostructure. In most populations, each inorganic nanoparticle is made up of a different number of atoms, with different geometric relationships between facets and crystal faces, as well as defects and impurities. A comparably sized population of proteins is, by contrast, very homogeneous, with each protein comprised of the same number of amino acids, each arranged in approximately the same way, differing in arrangement, for the most part, only through the effect of thermal fluctuations. Consequently, two proteins designed to interact with one another will always interact with the same geometry, resulting in the formation of a complex of predictable geometry and stoichiometry. This property is essential for massively parallel bottom-up assembly of nanostructures.

[0174] A structural element may be used to maintain the geometric relationships among the joining elements and functional elements of a nanostructure. As such, a rigid structural element is generally preferred for construction of nanostructures using the staged assembly methods described herein. This rigidity is typical of many proteins and may be conferred upon the protein through the properties of the secondary structural elements making up the protein, such as a-helices and -sheets.

[0175] Structural elements may be based on the structure of proteins, protein fragments or peptides whose three-dimensional structure is known or may be designed ab initio. Examples of proteins or protein fragments that may be utilized as structural elements in an assembly unit include, but are not limited to, antibody domains, diabodies, single-chain antibody variable domains, and bacterial pilins.

[0176] In some embodiments, structural elements, joining elements and functional elements may be of well-defined extent, separated, for example, by glycine linkers. In other embodiments, joining elements may involve peptides or protein segments that are integral parts of a structural element, or may comprise multiple loops at one end of a structural element, such as in the case of the complementarity determining regions (CDRs) of antibody variable domains (Kabat et al., 1983, Sequences of Proteins of Immunological Interest, U.S. Department of Health and Human Services). A CDR is a joining element that is an integral part of the variable domain of an antibody. The variable domain represents a structural element and the boundary between the structural element and the CDR making up the joining element (although well-defined in the literature on the basis of the comparisons of many antibody sequences) may not always be completely unambiguous structurally. There may not always be a well-defined boundary between a structural element and a joining element, and the boundary between these domains, although well-defined on the basis of their respective utilities, may be ambiguous spatially.

[0177] Structural elements of the present invention comprise, e.g., core structural elements of naturally-occurring proteins that are then modified to incorporate joining elements, functional elements, and/or a flexible domain (e.g., a tri-, tetra- or pentaglycine), thereby providing useful assembly units. Consequently, in certain embodiments, structures of existing proteins are analyzed to identify those portions of the protein or part thereof that can be modified without substantially affecting the rigid structure of that protein or protein part.

[0178] For example, in certain embodiments, the amino acid sequence of surface loop regions of a protein or structural element are altered with little impact on the overall folding of the protein. The amino acid sequences of a surface loop of a protein are generally preferred as amino acid positions into which the additional amino acid sequence of a joining element, a functional element, and/or a flexible domain may be inserted, with the lowest probability of disrupting the protein structure. Determining the position of surface loops in a protein is carried out by examination of the three-dimensional structure of the protein or a homolog thereof, if three-dimensional atomic coordinates are available, using, for example, a public-domain protein visualization computer program such as RASMOL (Sayle et al., 1995, RasMol: Biomolecular graphics for all, Trends Biochem. Sci. (TIBS) 20(9): 374-376; Saqi et al., 1994, PdbMotifa tool for the automatic identification and display of motifs in protein structures, Comput. Appl. Biosci. 10(5): 545-46). In this manner, amino acids included in surface loops, and the relative spatial locations of these surface loops, can be determined.

[0179] If the three-dimensional structure of the protein being engineered is not known, but that of a close homolog is known (as is the case, for example, for essentially all antibody molecules), the amino acid sequence of the molecule of interest, or a portion thereof, can be aligned with that of the molecule whose three-dimensional structure is known. This comparison (done, for example, using BLAST (Altschul et al., 1997, Gapped BLAST and PSI-BLAST: a new generation of protein database search programs, Nucleic Acids Res. 25: 3389-3402) or LALIGN (Huang and Miller, 1991, A time efficient, linear-space local similarity algorithm, Adv. Appl. Math. 12: 337-357) allows identification of all the amino acids in the protein of interest that correspond to amino acids that constitute surface loops (-turns) in the protein of known three-dimensional structure. In regions in which there is high sequence similarity between the two proteins, this identification is carried out with a high level of certainty. Once a putative loop is identified and altered according to methods disclosed herein, the resultant construct is tested to determine if it has the expected properties. This analysis is performed even in those instances where identification of the loop is highly reliable, e.g. where that determination is based upon a known three-dimensional protein structure.

[0180] Antibodies are multivalent molecules made up of polypeptide chains including light (L) chains of approximately 220 amino acids and heavy (H) chains of 450-575 amino acids. The average molecular weight for an intact IgG molecule is in the range 152-196 kD. Structural studies performed on antibodies have revealed that both the light and heavy chains contain a characteristic domain termed the immunoglobulin fold. The immunoglobulin fold is defined as a barrel-shaped sandwich consisting of two layered anti-parallel P-sheets linked together by a disulfide bond. The predominant secondary structure in an antibody is an anti-parallel -sheet with short stretches of -helix. (For review, see Padlan, 1994, Anatomy of the antibody molecule, Mol. Immunol. 31(3): 169-217; Padlan, 1996, X-ray crystallography of antibodies, Adv. Protein Chem. 49: 57-133; and references cited therein.)

[0181] The light chains contain two immunoglobulin domains, one at the N-terminal portion, which varies from antibody to antibody (V_L), and the other at the C-terminal portion, which is relatively constant (C_L). The heavy chains contain four or five immunoglobulin domains, depending upon the class of immunoglobulin. The N-terminal domain varies (V_H) and the other distal domains remain constant (C_H1, C_H2, C_H3, and, in certain cases C_H4). The units of the light and heavy chains associate through disulfide bonds as well as other non-covalent interactions to form the characteristic Y-shaped dimer composed of two light chains and two heavy chains. The antibody fragment containing the V_L chain and the V_H chain is termed the F_v fragment. The portion containing the entire light chain, as well as the variable portion and first constant domain (C_H1) of the heavy chain, is termed the Fab fragment. Interactions of the variable domains with the constant domains in Fab are not very strong, lending a degree of flexibility and positional variability to the overall structure of the molecule. There can be a large variation (from 127-176) in the angle between the Fab variable domain and the Fab constant domain. This angle is known as the Fab elbow or bend (Padlan, 1994, Anatomy of the antibody molecule. Mol. Immunol. 31(3): 169-217).

[0182] The N-terminal regions of the two Fab arms bind antigen (Mian et al., 1991, Structure, function and properties of antibody binding sites, J. Mol. Biol. 217(1): 133-51; Wilson et al., 1994, Structure of anti-peptide antibody complexes, Res. Immunol. 145(1): 73-8; Wilson et al., 1994, Antibody-antigen interactions: new structures and new conformational changes, Curr. Opin. Struct. Biol. 4(6): 857-67). The Fab arms, in turn, are connected by a flexible polypeptide to the third fragment, termed the Fc fragment, which is responsible for triggering effector functions that eliminate the antigen as well as dimerize the antigen binding sites.

[0183] The Fc portion of the IgG antibody molecule is made up of the two constant domains C_H2 and C_H3. The polypeptide segment connecting the Fab and Fc fragments is defined as the hinge and has variable length and flexibility depending upon the antibody class and isotype. This flexible hinge region provides a natural demarcation between the Fc and Fab fragments of the antibody. The hinge and the Fab elbow or bend contained in an intact IgG molecule allow for significant flexibility between the two antigen binding sites and thus permit numerous cross-linking geometries (FIGS. 5 and 6).

[0184] The proteins making up native and recombinant antibody fragments are candidates for the structural elements of nanostructures assembled by staged assembly. Antibodies used in the staged assembly methods of the invention include, but are not limited to, IgG monoclonal, humanized or chimeric antibodies. Binding derivatives or binding fragments of antibodies used in the staged assembly methods of the invention also include, but are not limited to, single chain antibodies (scFv) including monomeric ((scFv) fragments), dimeric ((scFv)₂ or diabodies), trimeric ((scFv)₃ or triabodies) and tetrameric ((scFv)₄ or tetrabodies) single chain antibodies; Fab fragments; F(ab)₂ fragments; and fragments produced by a Fab expression library (Huse et al., 1989, Generation of a large combinatorial library of the immunoglobulin repertoire in phage lambda, Science, 246, 1275-81).

[0185] General methods of antibody production and use are commonly known in the art. These methods may be used for producing structural and joining elements for use in the staged assembly methods and assembly units of the invention (see, e.g., Harlow and Lane, 1988, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York; incorporated herein by reference in its entirety).

[0186] A molecular clone of an antibody to an antigen of interest can be prepared by techniques well-known in the art. Recombinant DNA methodology may be used to construct nucleic acid sequences that encode a monoclonal antibody molecule, or antigen binding region thereof (see, e.g., Sambrook et al., 2001, Molecular Cloning, A Laboratory Manual, Third Edition, Chapters 1, 2, 3, 5, 6, 8, 9, 10, 13, 14, 15 and 18, Cold Spring Harbor Laboratory Press, N.Y.; Ausubel et al., 1989, Current Protocols in Molecular Biology, Chapters 1, 2, 3, 5, 6, 8, 10, 11, 12, 15, 16, 19, 20 and 24, Green Publishing Associates and Wiley Interscience, N.Y.; Current Protocols in Immunology, Chapters 2, 8, 9, 10, 17 and 18, John Wiley & Sons, 2001, Editors John E. Coligan, Ada M. Kruisbeek, David H. Margulies, Ethan M. Shevach, Warren Strober, Series Editor: Richard Coico).

[0187] Antibodies can be expressed in bacteria either intracellularly or extracellularly by secretion into the bacterial periplasm (Tomlinson and Holliger, 2000, Methods for generating multivalent and bispecific antibody fragments, Methods Enzymol. 326: 461-79). Intracellular expression of recombinant antibodies, however, frequently leads to the formation of insoluble aggregates of the protein, which are referred to as inclusion bodies, presumably due to the non-reducing environment of the bacterial cytoplasm, which inhibits disulfide bond formation between antibody domains. It is possible to refold the antibodies into functional proteins through solubilization of the inclusion bodies with strong denaturants followed by exposure to renaturing conditions, by methods commonly known in the art.

[0188] In order to circumvent the need for renaturation, a coding sequence for a bacterially-derived periplasmic signal sequence can be spliced at the N-terminal portion of the gene encoding the antibody to direct the recombinant protein to the bacterial periplasm. The oxidizing environment of the periplasmic space favors proper folding of the antibody domains, including disulfide bond formation. The success of these methods in producing good yields of functional antibody can depend upon the antibody type, derivation and method of overproduction (see Ward, 1992, Antibody engineering: the use of Escherichia coli as an expression host, FASEB J. 6(7): 2422-27; Ward, 1993, Antibody engineering using Escherichia coli as host, Adv. Pharmacol. 24: 1-20; Zhu et al., 1996, High level secretion of a humanized bispecific diabody from Escherichia coli, Biotechnology (NY) 14(2): 192-96; Sheets et al., 1998, Efficient construction of a large nonimmune phage antibody library: the production of high-affinity human single-chain antibodies to protein antigens, Proc. Natl. Acad. Sci. USA 95(11): 6157-62; Tomlinson et al., 2000, Methods for generating multivalent and bispecific antibody fragments, Methods Enzymol. 326: 461-79).

[0189] Antibody molecules may be purified by techniques well-known in the art, e.g., immunoabsorption or immunoaffinity chromatography, chromatographic methods such as HPLC (high performance liquid chromatography), or a combination thereof.

[0190] Monoclonal antibodies (mAbs), or binding derivatives or binding fragments thereof, may be used as structural elements according to the methods of the invention. mAbs are homogeneous populations of antibodies directed against a particular antigen. A mAb to an antigen of interest can be prepared by using any technique known in the art that provides for the production of antibody molecules. These include, e.g., the hybridoma technique originally described by Kohler and Milstein (1975, Continuous cultures of fused cells secreting antibody of predefined specificity, Nature 256: 495-97; Voet and Voet, 1990, Biochemistry, John Wiley and Sons, Inc., Chapter 34), the human B cell hybridoma technique (Kozbor et al., 1983, Immunology Today 4: 72-79; Kozbor et al., U.S. Pat. No. 4,693,975, entitled Human hybridroma sic fusion partner for production of human monoclonal antibodies, issued Sep. 15, 1987), and the EBV-hybridoma technique (Cole et al., 1985, Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96; Roder et al., 1986, The EBV-hybridoma technique, Methods Enzymol. 121: 140-67). Such antibodies may be of any immunoglobulin class including IgG, IgM, IgE, IgA, IgD and any subclass thereof. The mAbs that may be used in the methods of the invention may be synthesized by any technique commonly known in the art. For example, human monoclonal antibodies may be made by any of numerous techniques known in the art (e.g., Teng et al., 1983, Construction and testing of mousehuman heteromyelomas for human monoclonal antibody production, Proc. Natl. Acad. Sci. USA. 80: 7308-12; Cole et al., 1984, Human monoclonal antibodies, Mol. Cell. Biochem. 62(2): 109-20; Olsson et al., 1982, Immunochemical Techniques, Meth. Enzymol. 92: 3-16).

[0191] By contrast, polyclonal antibodies cannot be used as components in the present invention. Polyclonal antibodies represent a population of antibodies in which many molecules of different precise specificity exists. Although they may all bind to a particular antigen, they will bind different parts of the antigen with different geometries, a property that is inconsistent with the precise assembly of a nanostructure.

[0192] Multispecific antibodies, or binding derivatives or binding fragments thereof, may be used as structural elements for use in the staged assembly methods of the invention. Specific or specificity, as used herein, refers to the ability of an antibody to bind a defined epitope to one distinct antigen-recognition site. Bispecific antibodies, therefore, comprise two distinct antigen recognition sites, each capable of binding a different antigen. Multispecific antibodies have the ability to bind more than two different epitopes, each through the action of a distinct joining element, i.e., an antigen-recognition site.

[0193] In certain embodiments, homogeneous bispecific or multispecific mAbs can be created for use as structural elements, via immortilization of lymphocyte clones, created by fusing myeloma cells with lymphocytes raised against an antigen of interest as described above generally for the production of monoclonal antibodies. By such methods, multispecific mAbs can be produced in virtually unlimited quantities. Using methods well-known in the art, multispecific mAbs may be created that specifically target and bind a selected biological substance (see, e.g., Colcher et al., 1999, Single-chain antibodies in pancreatic cancer, Ann. NY Acad. Sci. 880: 263-80; Hudson, 1999, Recombinant antibody constructs in cancer therapy, Curr. Opin. Immunol. 11(5): 548-57; Kipriyanov et al., 1999, Bispecific tandem diabody for tumor therapy with improved antigen binding and pharmacokinetics, J. Mol. Biol. 293(1): 41-56; Segal et al., 1999, Bispecific antibodies in cancer therapy, Curr. Opin. Immunol. 11(5): 558-62; Trail et al., 1999, Monoclonal antibody drug conjugates in the treatment of cancer, Curr. Opin. Immunol. 11(5): 584-88; Hudson, 2000, Recombinant antibodies: a novel approach to cancer diagnosis and therapy, Expert Opin. Investig. Drugs 9(6): 1231-42).

[0194] In certain embodiments, a multispecific mAb for use as a structural element according to the methods of the invention may be a bispecific and/or bivalent mAb. A bispecific antibody has the ability to bind two different epitopes, each contained on a distinct antigen-recognition site. A bivalent antibody has the ability to bind to two different epitopes.

[0195] Bispecific antibodies may be created using methods well-known in the art (see, e.g., Weiner et al., 1995, Bispecific monoclonal antibody therapy of B-cell malignancy, Leuk. Lymphoma 16(3-4): 199-207; Helfrich et al., 1998, Construction and characterization of a bispecific diabody for retargeting T cells to human carcinomas, Int. J. Cancer 76(2): 232-39; Arndt et al., 1999, A bispecific diabody that mediates natural killer cell cytotoxicity against xenotransplanted human Hodgkin's tumors, Blood 94(8): 2562-8; Kipriyanov et al., 1999, Bispecific tandem diabody for tumor therapy with improved antigen binding and pharmacokinetics, J. Mol. Biol. 293(1): 41-56; Sundarapandiyan et al., 2001, Bispecific antibody-mediated destruction of Hodgkin's lymphoma cells, J. Immunol. Methods 248(1-2): 113-23).

[0196] Technologies for the production of multivalent and multispecific antibodies are well known in the art (see, e.g., Pluckthun et al., 1997, New protein engineering approaches to multivalent and bispecific antibody fragments, Immunotechnology 3(2): 83-105; Santos et al., 1998, Development of more efficacious antibodies for medical therapy and diagnosis, Prog. Nucleic Acid Res. Mol. Biol. 60: 169-94; Alt et al., 1999, Novel tetravalent and bispecific IgG-like antibody molecules combining single-chain diabodies with the immunoglobulin gammal Fc or CH3 region, FEBS Lett. 454(1-2): 90-94; Hudson et al., 1999, High avidity scFv multimers; diabodies and triabodies, J. Immunol. Methods 231(1-2): 177-89; Tomlinson et al., 2000, Methods for generating multivalent and bispecific antibody fragments, Methods Enzymol. 326: 461-79; Todorovska et al., 2001, Design and application of diabodies, triabodies and tetrabodies for cancer targeting. J. Immunol. Methods 248(1-2): 47-66). For example, genes encoding antibodies of known specificity may be rescued from hybridoma cell lines and can provide the starting material for cloning the rearranged V_L and V_H genes thorough employment of recombinant DNA technologies (Ward et al., 1989, Binding activities of a repertoire of single immunoglobulin variable domains secreted from Escherichia coli, Nature 341(6242): 544-46; Sheets et al., 1998, Efficient construction of a large nonimmune phage antibody library: the production of high-affinity human single-chain antibodies to protein antigens, Proc. Natl. Acad. Sci. USA 95(11): 6157-62). Universal DNA primers may be designed to anneal to the target V-domain genes and amplified through employment of the polymerase chain reaction. Through design of restriction sites within these primers, the resulting amplified DNA products can be cloned directly for expression in a range of different hosts including bacteria, yeast, plant and insect cells (Tomlinson et al., 2000, Methods for generating multivalent and bispecific antibody fragments, Methods Enzymol. 326: 461-79). These host cells, rather than hybridoma cell lines, can be used, for the production of recombinant engineered antibodies for use in the methods of the invention.

[0197] In certain embodiments of the invention, a structural element comprises a diabody fragment. A diabody has two CDRs, and is capable of making two highly specific, non-covalent interactions. A diabody, or a binding derivative or binding fragment thereof, may be incorporated into a nanostructure in such a way that only one of the two CDRs is used. In certain embodiments, the CDRs themselves serve as joining elements, and the body of the diabody between the two CDRs serves as a structural element.

[0198] Methods well known in the art are used for the expression, purification and characterization of diabody fragments from E. coli (Poljak, 1994, An idiotopeanti-idiotope complex and the structural basis of molecular mimicking, Proc. Natl. Acad. Sci. USA 91(5): 1599-600; Zhu et al., 1996, High level secretion of a humanized bispecific diabody from Escherichia coli, Biotechnology (NY) 14(2): 192-96; Todorovska et al., 2001, Design and application of diabodies, triabodies and tetrabodies for cancer targeting, J. Immunol. Methods 248(1-2): 47-66). Examples of a structural element comprising a diabody fragment are illustrated in FIG. 7. The diabody expression cassettes represented in FIG. 7 are designed so that the pelB signal sequence spliced to the N-terminus of the V_H domains genes coding the diabody fragments are targeted and secreted into the E. coli periplasmic space, where the oxidative environment allows proper folding of the diabody. After induction, the overexpressed diabodies fragments are harvested from the E coli periplasm according to established protocols well-known in the art.

[0199] In a preferred embodiment of the invention (FIG. 7), diabodies are engineered to add a hexahistidine tag (His6) at the C-terminus of the V_L domains to facilitate purification using an immobilized metal affinity chromatography resin (Scopes, 1994, Protein Purification, Principles and Practice, Third Edition, Springer-Verlag, London, pp. 183-85; Scopes, 1994 Protein Purification: Principles and practice (Springer Advanced texts in Chemistry), Third ed., London). Protein overexpression of diabody assembly unit-1 (FIG. 7A), for example, will contain a mixture of species including; 2 (V_HAV_LBHis₆), 2(V_HBV_LA), and (V_HBV_LA, V_HAV_LBHis₆). The number of His₆ tags determines the concentration of imidazole (20-250 mM gradient) at which each protein unit contained in the mixture will elute. Those with no hexahistidine tags will exhibit little or no affinity towards the column resin. Those with one hexahistidine tag will generally elute between 20-40 mM imidazole (bispecific diabody) and those with two hexahistidine tags will generally elute between 50 and 100 mM imidazole. Elution peaks may be detected by UV absorbance and verified with SDS-PAGE, native-PAGE or ELISA assay. Even though the purification procedure described above guards against the isolation of unwanted non-bispecific diabody byproducts, methods are employed to ensure that the isolated diabody of interest has functional bispecificity as disclosed hereinbelow.

[0200] FIG. 7A depicts an AB diabody in which the V_H and V_L domains of A define a lysozyme isotopic antibody (D1.3) and in which the V_H and V_L domains of B define a virus neutralizing idiotopic antibody (730.1.4). In order to facilitate purification of the desired diabody product, the gene encoding V_HA and V_LB includes a hexahistidine tag, whereas the gene encoding V_HB and V_LA does not. FIG. 7B depicts a BA diabody in which the V_H and V_L domains of B define a virus neutralizing idiotopic antibody (409.5.3) and in which the V_H and V_L domains of A define a lysozyme isotopic antibody (E5.2). In order to facilitate purification of the desired diabody product, the gene encoding V_HB and V_L A includes a hexahistidine tag, whereas the gene encoding V_HA and V_HB does not.

[0201] In certain embodiments, sandwich ELISA or BlAcore protocols may be implemented to determine simultaneous and dual occupancy of both antigen-binding sites (bispecificity), as well as equilibrium constants (Abraham et al., 1996, Determination of binding constants of diabodies directed against prostate-specific antigen using electrochemiluminescence-based immunoassays, J. Mol. Recognit. 9(5-6): 456-61; McGuinness et al., 1996, Phage diabody repertoires for selection of large numbers of bispecific antibody fragments, Nat. Biotechnol. 14(9): 1149-54; McCall et al., 2001, Increasing the affinity for tumor antigen enhances bispecific antibody cytotoxicity, J. Immunol. 166(10): 6112-17). In a specific embodiment in which an idiotype/anti-idiotype binding constant is determined using the BlAcore technique, one of the antibodies is dissolved in a liquid phase and the other is coupled to the solid phase. Implementation of this technique permits the determination of the association and dissociation rates (k_on and k_off respectively) for determination of the dissociation constant (Kd) (Goldbaum et al., 1997, Characterization of anti-anti-idiotypic antibodies that bind antigen and an anti-idiotype, Proc. Natl. Acad. Sci. USA 94(16): 8697-701). Other protocols that do not require recombinant antigens, but that can detect bispecificity may also be employed, and include the rosetting assay as described by Holliger et al. (1997, Retargeting serum immunoglobulin with bispecific diabodies, Nat. Biotechnol. 15(7): 632-36).

[0202] In a specific embodiment, a diabody may comprise one or more sites for the insertion of a joining element, a structural element or a functional element. Table 1 shows peptide regions contained in diabody units that may be used for the insertion of joining, structural or functional elements. A peptide region is a portion of a protein of interest, e.g., of an antibody or a binding derivative or binding fragment thereof. A peptide region is preferably exposed on the surface of the protein of interest, and is amenable to being re-engineered through the insertion of additional peptides or the alteration of its sequence or both. Table 1 summarizes the amino acids identified as -turns located on the surface of a diabody with V_H-V_L variable domain linkage (pdb entry 1LMK). Residue regions are defined within the diabody fragment from analysis of the atomic coordinates and numbered according to the residue assignments deposited under entry 1LMK pdb. Chain assignments are labeled in accord with the corresponding deposited pdb coordinates.

TABLE 1
Identified Peptide Regions Contained in Diabody Structural
Elements for the Insertion of Joining, Structural or
Functional Elements
	Domain	Secondary Structure	Residue (Chain)
	VH	-turn	Residues 13-16, 39-44,
			62-66, 73-77
			(A and C chains)
	VL	C-terminal -C	Residue 312
			(A and C chains)
	VH	C-terminal -C	Residue 1
			(A and C chains)

[0203] In certain embodiments, binding sites may be added as joining elements to a diabody to make possible structural branches, forks, T-junctions, or multidimensional architectural binding sites, in addition to the two joining elements formed by the oppositely directed CDRs. Alteration of the sequence of surface loops in proteins appears to have little impact on the overall folding of a protein, and it is frequently possible to make insertion mutants at the sites of -turns. The surface loops are the places where sequences can be added to the protein with the lowest probability of disrupting the protein structure.

[0204] Specific sites within the diabody unit have been precisely defined for insertions. For example, in certain embodiments, joining elements mays be spliced internal to, or replacing the -turn residues as disclosed herein in Table 1. Since the general three-dimensional structure of diabodies is known, and since it is possible to homology-model the three-dimensional structure of diabodies of similar sequence (Guex and Peitsch, 1997, SWISS-MODEL and the Swiss-PdbViewer: An environment for comparative protein modelling, Electrophoresis 18: 2714-23; Guex and Peitsch, 1999, Molecular modelling of proteins, Immunology News 6: 132-34; Guex et al., 1999, Protein modelling for all, TIBS 24: 364-67, the -turns located on the surface of a diabody of similar amino acid sequence to a diabody of known structure are readily identified by a sequence comparison (using, e.g., BLAST, Altschul et al., 1997, Gapped BLAST and PSI-BLAST: a new generation of protein database search programs, Nucleic Acids Res. 25: 3389-3402), followed by a visual investigation of the x-ray coordinates of the protein of similar sequence.

[0205] In one embodiment, a visual investigation of the three-dimensional structure of a diabody is performed with the molecular visualization package QUANTA (Accelrys Inc., San Diego, Calif.) run on a Silicon Graphics Workstation. The coordinates defining the three-dimensional positions of the atoms of a diabody molecule are included in the PDB entry 1LMK. Upon such an analysis, it is apparent that there are surface loops that include residues shown in Table 1, which represent sites with high potential for accepting the insertion of a peptide such as the HIV-1 V3 loop antigen. All amino acids included in surface loops of this diabody molecule can be determined from this information, and the relative spatial locations of these surface loops has also been determined. The information provided by the three-dimensional structure of the immunoglobulin being engineered (whether derived directly from X-ray crystallography, or from homology modeling based on a homologous structure) allows the identification of all the amino acids in the protein of interest that correspond to amino acids that constitute surface loops.

[0206] In a specific embodiment, DNA encoding a peptide epitope derived from the ras protein is inserted into a diabody assembly unit coding sequence at a site defined by visual investigation of the three-dimensional atomic coordinates as determined by x-ray crystallography. The ras epitope is flanked by four glycines on either side, to provide flexibility and accessibility for cognate antibody binding.

[0207] Once the diabody assembly unit/ras peptide protein fusion (represented as BA) has been expressed and purified, it is characterized for retention of diabody valency and function as well as epitope recognition by the appropriate antibody by methods such as ELISA or BlAcore analysis.

[0208] Functional elements, such as enzymes, toxins, and antigenic peptides, have already been successfully spliced to the termini of scFv fragments resulting in various multifunctional antibodies (Chaudhary et al., 1989, A recombinant immunotoxin consisting of two antibody variable domains fused to Pseudomonas exotoxin, Nature 339(6223): 394-97; Suzuki et al., 1997, Construction, bacterial expression, and characterization of hapten-specific single-chain Fv and alkaline phosphatase fusion protein, J. Biochem. (Tokyo) 122(2): 322-29; Williams et al., 2001, Numerical selection of optimal tumor imaging agents with application to engineered antibodies, Cancer Biother. Radiopharm. 16(1): 25-35). Functional elements that are made up of proteins or peptides can be fused directly into the proteinaceous portion of an assembly unit using the methods of molecular biology followed by expression of the proteins in appropriate host.

[0209] In certain embodiments of the invention, a structural element for the staged assembly of a nanostructure comprises an antibody fragment. Such a fragment includes, but is not limited to, an Fab fragment, or an F(ab)₂ fragment, which can be produced by pepsin digestion of an IgG antibody molecule, thereby releasing the Fc portion. Pepsin digestion can be followed by reducing the disulfide bridges between the resulting F(ab)₂ fragments thereby generating single Fab fragments.

[0210] Fab fragments are elongated dirigible shaped molecules that contain a monovalent and monospecific CDR at the N-terminal end of the molecule. In certain embodiments, an assembly unit is engineered from a Fab fragment by inserting a peptide epitope at the C terminal portion of the Fab fragment. Consequently, a peptide fused to the C-terminus of the Fab fragment may act as a target for another engineered Fab, to provide a highly specific and tight interaction between adjacent Fabs in a nanostructure constructed by staged assembly. The size, shape and structure of the Fab fragment (FIG. 6) make it preferred for use as a structural element because it also comprises, by virtue of its structure, a naturally occurring joining element. Electron micrographic and X-ray structural studies have revealed that the proximal portion of the Fab fragment is often linearly opposed to the distal portion (Fischmann et al., 1991, Crystallographic refinement of the three-dimensional structure of the FabD1.3-lysozyme complex at 2.5 A resolution, J. Bio. Chem 266: 12915-20; Ban et al., 1994, Crystal structure of an idiotype-anti-idiotype Fab complex, Proc. Natl. Acad. Sci. U.S.A. 91(5): 1604-8; Padlan, 1996, X-ray crystallography of antibodies, Adv. Protein Chem. 49: 57-133; Harris, Skaletsky et al., 1998). The flexible elbow bend, which is located in the middle of the fragment, allows for alternative geometries (Roux et al., 1997, Flexibility of human IgG subclasses, J. Immunol. 159(7): 3372-82; Roux et al., 1998, Comparisons of the ability of human IgG3 hinge mutants, IgM, IgE, and IgA2, to form small immune complexes: a role for flexibility and geometry, J. Immunol. 161(8): 4083-90). Alternatively, Fab expression libraries may be constructed (Huse et al., 1989, Generation of a large combinatorial library of the immunoglobulin repertoire in phage lambda, Science, 246, 1275-81) to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity.

[0211] According to the methods of the invention, staged assembly of nanostructures can employ, in certain embodiments, structural elements comprising single-chain scFv fragments. An scFv antibody is composed of a fusion peptide that links the carboxyl terminus of the Fv variable heavy chain (V_H) to the amino terminus of the Fv variable light chain (V_L) or vice versa (Freund et al., 1994, Structural and dynamic properties of the Fv fragment and the single-chain Fv fragment of an antibody in solution investigated by heteronuclear three-dimensional NMR spectroscopy, Biochemistry 33(11): 3296-303; Hudson et al., 1999, High avidity scFv multimers; diabodies and triabodies, J. Immunol. Methods 231(1-2): 177-89; Le Gall et al., 1999, Di-, tri- and tetrameric single chain Fv antibody fragments against human CD19: effect of valency on cell binding, FEBS Lett 453(1-2): 164-68; Worn et al., 2001, Stability engineering of antibody single-chain Fv fragments, J. Mol. Biol. 305(5): 989-1010).

[0212] Single-chain antibodies may also be used as structural elements for use in the staged assembly methods of the invention. Single-chain antibodies may be produced by the methods of, e.g., Ladner; (U.S. Pat. No. 4,946,778, entitled Single polypeptide chain binding molecules, issued Aug. 7, 1990); Bird (1988, Single-Chain Antigen-Binding Proteins, Science 242(4877): 423-26); Huston et al. (1988, Protein engineering of antibody binding sites: recovery of specific activity in an anti-digoxin single-chain Fv analogue produced in Escherichia coli, Proc. Natl. Acad. Sci. USA 85: 5879-83), or Ward et al., (1989, Binding activities of a repertoire of single immunoglobulin variable domains secreted from Escherichia coli, Nature 334: 544-46).

[0213] An scFv fragment is a substructure of a Fab fragment that can be visualized as a Fab fragment, cut in half at the elbow-bend, missing the terminal constant light and heavy chain domains Freund et al., 1994, Structural and dynamic properties of the Fv fragment and the single- chain Fv fragment of an antibody in solution investigated by heteronuclear three-dimensional NMR spectroscopy, Biochemistry 33(11): 3296-303; Malby et al., 1998, Three-dimensional structures of single-chain Fv-neuraminidase complexes, J. Mol. Biol. 279(4): 901-10) (FIG. 8). Rather than being elongated and dirigible shaped, as in Fab fragments, scFv are smaller and more globular shaped. While approximately half the size of a Fab fragment, a scFv fragment still contains a functional monovalent/monospecific CDR at the N-terminal portion of the molecule. The scFv represents the minimal antigen binding motif that can be expressed in E. coli.

[0214] In general, scFv fragments are monovalent, maintaining tertiary and quaternary structures similar to that found in the Fv portion of an intact antibody (FIGS. 5 and 8) (Boulot et al., 1990, Crystallization and preliminary X-ray diffraction study of the bacterially expressed Fv from the monoclonal anti-lysozyme antibody D1.3 and of its complex with the antigen, lysozyme, J. Mol. Biol. 213(4): 617-19; Braden et al., 1996, Crystal structure of an Fv-Fv idiotope-anti-idiotope complex at 1.9 A resolution, J. Mol. Biol. 264(1): 137-51; Fuchs et al., 1997, Primary structure and functional scFv antibody expression of an antibody against the human protooncogene c-myc, Hybridoma 16(3): 227-33; Hoedemaeker et al., 1997, A single chain Fv fragment of P-glycoprotein-specific monoclonal antibody C219. Design, expression, and crystal structure at 2.4 A resolution, J. Biol. Chem. 272(47): 29784-89; Malby et al., 1998, Three-dimensional structures of single-chain Fv-neuraminidase complexes, J. Mol. Biol. 279(4): 901-10). A Gly/Ser peptide linker that is, optimally, 15 amino acids in length, can be used to join the two variable fragments and help maintain favorable interactions between the V, and V_L domains (Perisic et al. 1994, Crystal structure of a diabody, a bivalent antibody fragment, Structure 2(12): 1217-26; Takemura et al., 2000, Construction of a diabody (small recombinant bispecific antibody) using a refolding system, Protein Eng. 13(8): 583-88; Worn et al., 2001, Stability engineering of antibody single-chain Fv fragments, J. Mol. Biol. 305(5): 989-1010). These Gly/Ser linkers can be used to provide flexibility and protease resistance. Furthermore, scFv antibody fragments have similar function, in terms of antigen recognition and binding, as that of intact antibodies.

[0215] The smaller size of the scFv fragment, as well as the relative positioning of the CDR, make it well-suited as a protein component to be incorporated into assembly units of the present invention for fabrication of nanostructures. One advantage of scFv over Fab fragments is that the technology for engineering and producing scFv's is more advanced (see, e.g., Ward, 1993, Antibody engineering using Escherichia coli as host, Adv. Pharmacol. 24: 1-20; Luo et al., 1996, Construction and expression of bi-functional proteins of single-chain Fv with effector domains, J. Biochem. (Tokyo) 120(2): 229-32; Wu et al., 2000, Designer genes: recombinant antibody fragments for biological imaging, Q. J. Nucl. Med. 44(3): 268-83; Worn et al., 2001, Stability engineering of antibody single-chain Fv fragments, J. Mol. Biol. 305(5): 989-1010). Using these art-known methods, specific CDRs may be created, and functional elements may be added to scFv's for use as protein components to be incorporated into assembly units useful in for staged assembly of nanostructures.

[0216] In another embodiment, a similar strategy is used to incorporate additional intermolecular binding sites on the scFv as was described above for Fab fragments. The C-terminal distal portion or -turn regions can be replaced by defined peptide epitopes such as, but not limited to those provided in Table 6, below. These peptide epitopes can replace defined -turn motifs or be directly linked to the C-terminal amino acid of the V_H or V_L heavy chain (depending upon the order of the linked heavy and light variable domains) (Table 7), by manipulation of the appropriate encoding DNA sequences using recombinant DNA procedures well known in the art. The resulting scFv fragment will contain an antigen binding recognition site on one portion of the scFv fragment and a joining element that is a peptide epitope, either replacing the defined -turn motifs, or linked at the C-terminal portion of the scFv fragment. Thus the fused peptide epitope will serve as a highly specific joining element in the formation of a joining pair between adjacent assembly units comprising scFv in a staged assembly.

[0217] In certain embodiments of the invention, a structural element comprises an antibody fragment such as a bispecific IgG fragment, chimeric IgG fragment or a bispecific heterodimeric F(ab)₂ antibody fragment. Whereas naturally occurring IgG molecules are bivalent by design, but monospecific because their CDRs are identical, IgG molecules, such as those created by hybridoma technology, can be produced that are either bivalent or bispecific, using the methods of, e.g., Suresh et al. (1986, Bispecific monoclonal antibodies from hybrid hybridomas, Methods Enzymol. 121: 210-28); Holliger et al. (1993, Engineering bispecific antibodies, Curr. Opin. Biotechnol. 4(4): 446-49); Hayden et al. (1997, Antibody engineering, Curr. Opin. Immunol. 9(2): 201-12); Carter (2001, Bispecific human IgG by design, J. Immunol. Methods 248(1-2): 7-15).

[0218] Bispecific IgGs may be created by any method known in the art, e.g., by chemical coupling methodologies or through the development of hybrid hybridoma cell lines (also referred to as hybrid hybridoma technology) (Milstein et al., 1983, Hybrid hybridomas and their use in immunohistochemistry, Nature 305(5934): 537-40) (FIG. 9).

[0219] Another approach used to obtain bispecific antibodies comprises exposing IgG to limited proteolytic digestion, where the two identical Fab fragments are released from the Fc fragment upon cleavage of the hinge polypeptide (FIG. 10). These single monovalent Fab fragments can be used alone, or chemically linked together (at the hinge cysteines) with a Fab fragment of separate origin to form a bispecific heterodimeric F(ab)₂. Chemically linked bispecific F(ab)₂ fragments have been studied and evaluated in several small-scale clinical trials (Hudson, 1999, Recombinant antibody constructs in cancer therapy, Curr. Opin. Immunol. 11(5): 548-57; Segal et al., 1999, Bispecific antibodies in cancer therapy, Curr. Opin. Immunol. 11(5): 558-62). Several other rational design strategies have been developed in order to engineer the Fe portion of heavy chains to promote the heterodimerization of bispecific antibodies. These strategies can include, for example, steric complementarity design mutations (knobs-into-holes utilizing phage display technology) as well as the design of additional inter-chain disulfide bonds and/or salt-bridge interactions between the heavy chains of the Fe fragment (Carter 2001, Bispecific human IgG by design, J. Immunol. Methods 248(1-2): 7-15). The enhanced complementarity between heavy chains of a desired bispecific antibody makes bispecific antibodies a preferred source for structural elements for use in the staged assembly of nanostructures as disclosed herein.

[0220] In one embodiment, bispecific antibodies are produced by replacing the Fc dimer-forming motif with another dimerization motif. In one non-limiting example, leucine zippers that can form heterodimers, such those found in Fos and Jun proteins, are linked to two different Fab portions of an IgG molecule by gene fusion. When expressed individually in an appropriate cell line, the fusion IgG's can be isolated as Fab-(zipper)₂ homodimers. Heterodimer formation is then achieved by reduction of the disulfide bonds within the hinge region of the homodimers to release the monomeric subunits. The resulting monomers are mixed together and placed under oxidizing conditions, resulting in bispecific heterodimers containing Fos-Jun paired leucine zipper motifs as the majority of the end products. Variations of this technique can be used to produce bispecific Fab and Fv fusion proteins (Kostelny et al., 1992, Formation of a bispecific antibody by the use of leucine zippers, J. Immunol. 148(5): 1547-53; Tso et al., 1995, Preparation of a bispecific F(ab)₂ targeted to the human IL-2 receptor, J. Hematother. 4(5): 389-94; de Kruif et al., 1996, Leucine zipper, dimerized bivalent and bispecific scFv antibodies from a semi-synthetic antibody phage display library, J. Biol. Chem. 271(13): 7630-34). Additional multimerization motifs used to promote bispecific dimer formation include, but are not limited to: transcriptional factor p53 (Rheinnecker et al., 1996, Multivalent antibody fragments with high functional affinity for a tumor-associated carbohydrate antigen, J. Immunol. 157(7): 2989-97), streptavidin (Muller et al., 1998, A dimeric bispecific miniantibody combines two specificities with avidity, FEBS Lett. 432(1-2): 45-49), or helix-bundle motifs such as Rop (Pack et al., 1993, Improved bivalent miniantibodies with identical avidity as whole antibodies produced by high cell density fermentation of Escherichia coli, Biotechnology 11: 1271-77; Dubel et al., 1995, Bifunctional and multimeric complexes of streptavidin fused to single chain antibodies (scFv), J. Immun. Methods 178: 201-09) (FIG. 11). Such antibodies are useful in the present invention as a source of a plurality of joining elements that are non-identical and that do not interact with each other.

[0221] While the above-described methodologies permit the production and isolation of bispecific antibodies, the methods also result in the creation of mixtures of IgG products, in low yields or combinations of both. Multivalent and multifunctional antibodies of high quality, quantity and purity may be created by recombinant antibody technology ((see , e.g., Morrison et al., 1989, Genetically engineered antibody molecules, Adv. Immunol. 44: 65-92; Shin et al., 1993, Hybrid antibodies, Int. Rev. Immunol. 10(2-3): 177-86; Sensel et al., 1997, Engineering novel antibody molecules, Chem. Immunol. 65: 129-58; Hudson et al., 1998, Recombinant antibody fragments, Curr. Opin. Biotechnol. 9(4): 395-402).

[0222] In other embodiments of the invention, human, humanized or chimeric (e.g., human-mouse or human-other species) monoclonal antibodies (mAbs), or binding derivatives or binding fragments thereof, may be used as structural elements for use in the staged assembly methods of the invention. Humanized antibodies are antibody molecules from non-human species having one or more complementarity determining regions (CDRs) from the non-human species and a framework region from a human immunoglobulin molecule. Humanized antibodies are also referred to as chimeric antibodies. Humanized or chimeric antibodies may be produced by methods well known in the art (see, e.g., Queen, U.S. Pat. No. 5,585,089, entitled Humanized immunoglobulins, issued Dec. 17, 1996, which is incorporated herein by reference in its entirety).

[0223] Chimeric antibodies may be used as structural elements according to the methods of the invention. A chimeric antibody is a molecule in which different portions are derived from different animal species, such as those having a variable region derived from a murine mAb and a human immunoglobulin effector or constant region. Techniques have been developed for the production of chimeric antibodies (Morrison et al., 1984, Chimeric human antibody molecules: mouse antigen-binding domains with human constant region domains, Proc. Natl. Acad. Sci. USA 81: 6851-55; Neuberger et al., 1984, Recombinant antibodies possessing novel effector functions, Nature, 312, 604-08; Takeda et al., 1985, Construction of chimaeric processed immunoglobulin genes containing mouse variable and human constant region sequences, Nature 314: 452-54) by splicing the genes from a mouse antibody molecule of appropriate antigen specificity together with genes from a human antibody molecule of appropriate biological or effector activity.

[0224] In certain embodiments of the invention, structural elements comprise diabodies or multimeric scFv fragments. scFv fragments, especially those with shortened peptide linkers, e.g. 3, 4 or 5 amino acid residues in length, form dimers ((scFv₂) or diabodies) rather than monomers in solution (Dolezal et al., 2000, ScFv multimers of the anti-neuraminidase antibody NC10: shortening of the linker in single-chain Fv fragment assembled in V(L) to V(H) orientation drives the formation of dimers, trimers, tetramers and higher molecular mass multimers, Protein Eng. 13(8): 565-74). Interchain domain interactions, rather than intrachain domain interactions, occur in order to form the stable dimeric diabody fragments (Holliger et al., 1993, Diabodies: small bivalent and bispecific antibody fragments, Proc. Natl. Acad. Sci. U.S.A. 90(14): 6444-48). A shortened peptide linker may prevent intrachain domain pairing and thus allow formation of interchain interactions that result in diabody fragment formation (Perisic et al. 1994, Crystal structure of a diabody, a bivalent antibody fragment, Structure 2(12): 1217-26).

[0225] In certain embodiments, diabodies can be used as the structural elements for the staged assembly of one-, two- and three-dimensional nanostructures. As used herein, the term diabody refers to dimeric single-chain variable antibody fragments (scFv). An scfv fragment, as described above, is composed of a fusion peptide that links the carboxyl terminus of the Fv variable heavy chain to the amino terminus of the Fv variable light chain (V_H-V_L) or vice versa (i.e. V_L-V_H) (Pluckthun et al., 1997, New protein engineering approaches to multivalent and bispecific antibody fragments, Immunotechnology 3(2): 83-105; Hudson, 1998, Recombinant antibody fragments, Curr. Opin. Biotechnol. 9(4): 395-402; Kipriyanov et al., 1999, Generation of recombinant antibodies, Mol. Biotechnol.12(2): 173-201).

[0226] In certain embodiments, a diabody or multimeric fragment is thermostable (see, e.g., Jermutus et al., 2001, Tailoring in vitro evolution for protein affinity or stability, Proc. Natl. Acad. Sci. USA 98(1): 75-80; Worn et al., 2001, Stability engineering of antibody single-chain Fv fragments, J. Mol. Biol. 305(5): 989-1010). Thermostability is a useful characteristic for structural elements utilized in the staged assembly of one- two- and three-dimensional nanostructures.

[0227] Unlike a monovalent scFv fragment, a diabody is a bivalent molecule containing two bodies that include two separate antigen-binding sites in opposition to one another and related by approximately 170 about the pseudo-two-fold axis of symmetry (parallel to the interface) (Perisic et al., 1994, Crystal structure of a diabody, a bivalent antibody fragment, Structure 2(12): 1217-26; Poljak, 1994, Production and structure of diabodies Structure 2: 1121-23; Hudson et al., 1999, High avidity scFv multimers; diabodies and triabodies, J. Immunol. Methods 231(1-2): 177-89) (FIG. 8).

[0228] A monospecific diabody contains two identical antigen-binding sites, both with specificity for the same ligand/hapten. A bispecific diabody contains two antigen-binding sites, each specific for a different ligand/hapten; that is, a bispecific diabody is derived from two different non-paired scFv fragments. The first hybrid fragment contains the V_H coding region from a first Fv antibody and a V_L coding region derived from a second Fv antibody. The resulting V_H-V_L hybrid fragment is joined together by a short Gly/Ser linker. The second hybrid fragment contains the V_L coding region from the first Fv antibody and the V_H coding region derived from the second Fv antibody.

[0229] The use of bispecific links permits the creation of bispecific antibody fragments that demonstrate bispecific affinity towards each ligand (Poljak, 1994, An idiotope-anti-idiotope complex and the structural basis of molecular mimicking, Proc. Natl. Acad. Sci. U.S.A. 91(5): 1599-1600; Kipriyanov et al., 1998, Bispecific CD3CD19 diabody for T cell-mediated lysis of malignant human B cells, Int. J. Cancer 77(5): 763-72; Arndt et al., 1999, A bispecific diabody that mediates natural killer cell cytotoxicity against xenotransplanted human Hodgkin's tumors, Blood 94(8): 2562-68; Takemura et al., 2000, Construction of a diabody (small recombinant bispecific antibody) using a refolding system, Protein Eng. 13(8): 583-88). Certain bispecific diabodies demonstrate affinities towards ligands/haptens similar to that demonstrated by whole IgG (Holliger et al., 1993, Engineering bispecific antibodies, Curr. Opin. Biotechnol. 4(4): 446-49; Yagi et al., 1994, Superantigen-like properties of an antibody bispecific for MHC class II molecules and the V beta domain of the T cell antigen receptor, J. Immunol. 152(8): 3833-41).

[0230] Diabodies exhibit several properties that make them particularly attractive for use the in staged assembly methods of the invention: (i) they are structures containing oppositely directed antigen binding sites; (ii) the geometrical opposition of the two antigen-binding sites optimizes the potential for building linear nanostructures or linear extensions of nanostructures; (iii) they have a well-defined size, shape, structure and stoichiometry; (iv) they have structural rigidity and well-defined recognition and binding properties; (v) binding motifs exhibiting specificity for a very broad range of organic and inorganic moieties can be identified and incorporated into a diabody structure (vi) their X-ray structure has been solved (FIG. 8) and can serve as a blueprint for identifying positions at which it is possible to add functional groups or binding sites; (vii) diabodies form strong intermolecular bonds to one another; (viii) the intermolecular bonds are highly specific; (ix) the immunoglobulin fold provides a structured protein core (structural element) and a stable spatial relationship among the different faces of the protein; (x) loops in which additional binding sites may be inserted are readily identified through an examination of the three-dimensional structure of a diabody (Zhu et al., 1996, High level secretion of a humanized bispecific diabody from Escherichia coli, Biotechnology (NY) 14(2): 192-96; Hudson et al., 1999, High avidity scFv multimers; diabodies and triabodies, J. Immunol. Methods 231(1-2): 177-89). Taken together, these properties are advantageous for using diabodies as structural elements for constructing complex, multidimensional nanostructures.

[0231] scFv fragments can also associate into multivalent multimers (Hudson et al., 1999, High avidity scFv multimers; diabodies and triabodies, J. Immunol. Methods 231(1-2): 177-89; Power et al., 2000, Synthesis of high avidity antibody fragments (scFv multimers) for cancer imaging, J. Immunol. Methods 242(1-2): 193-204; Todorovska et al., 2001, Design and application of diabodies, triabodies and tetrabodies for cancer targeting, J. Immunol. Methods 248(1-2): 47-66) (FIG. 12). Multimer formation is dependent upon the length of the linker used to associate the variable domains (V-domain) together, as well as the V-domain composition and orientation (V_H-V_L versus V_L-V_H). Reducing the linker length below three residues usually favors trimer or triabody formation, e.g., scFv)₃. Tetrabody formation, e.g., (scFv)₄ also has been reported in at least two instances where the linker length was 0 residues in length and the V-domain orientation was V_L-V_H (Todorovska et al., 2001, Design and application of diabodies, triabodies and tetrabodies for cancer targeting, J. Immunol. Methods 248(1-2): 47-66).

[0232] An antibody variable domain may functionally comprise both a structural element and a joining element in an assembly unit for staged assembly. Like structural elements, the extent of a joining element may not always be completely defined. For example, the -sheet structure of an antibody variable domain maintains the geometric relationship between the CDR and the other parts of the molecule. But it is also important for maintaining the structural relationships between the loops of the CDR that provide the binding affinity and specificity of the complementary partner of the joining pair. Consequently, an antibody variable domain may functionally comprise both a structural element and a joining element in an assembly unit. Thus, although antibody molecules and binding fragments of antibodies are preferred elements of joining elements, they may also provide structural framework for many embodiments, and as described above, for an assembly unit.

[0233] In certain embodiments of the invention, structural elements comprise bacterial pilin proteins, or binding derivatives or binding fragments thereof. Pilins are the protein units making up bacterial adhesion pili. Bacterial adhesion pili (P-pili) are formed by the polymerization of pilins (see, e.g., Bullitt and Makowski, 1995, Structural polymorphism of bacterial adhesion pili, Nature 373: 164-67; Bullitt and Makowski, 1998, Bacterial adhesion pili are heterologous assemblies of similar units, Biophysics J. 74: 623-32). Pili units may be assembled in vitro (see, e.g., Bullitt et al., 1996, Development of pilus organelle sub-assemblies in vitro depends on chaperone uncapping of a beta zipper, Proc. Nat. Acad. Sci. USA 93: 12890-95).

[0234] P-pili expressed on the surface of E. coli are helical filaments 6.8 nm in diameter, with an ellipsoidal central cavity 2.5 nm1.5 nm that winds about the helical axis, connecting to radial channels that extend to the surface of the pili (Hultgren and Normark, 1991, Biogenesis of the bacterial pilus, Curr. Opin. Genet. Dev. 1: 313-18; Hultgren et al., 1993, Pilus and nonpilus bacterial adhesins: assembly and function in cell recognition, Cell 73(5): 887-901; Bullitt and Makowski, 1995, Structural polymorphism of bacterial adhesion pili, Nature 373: 164-67). Each pilus comprises approximately 1000 copies of the major pilin, PapA, and one or a few copies of the minor pilins, PapH, PapK, PapE, PapF, and PapG. In the PapA-containing coiled rod region of the helix, there are 3.29 subunits per turn of the helix, with a 7.6 A rise per subunit (Bullitt and Makowski, 1995, Structural polymorphism of bacterial adhesion pili, Nature 373: 164-167). The fibrillae at the distal tip of the pilus is made up of four distinct but homologous pilins (FIG. 13). The distal end of papA will interact with the proximal end of papA or papK. The proximal end of papK will interact only with papA; its distal end only with papE; and so on as required by its remarkable architecture. These specific interactions are summarized in Table 2 below.

TABLE 2
Pilin-Pilin Protein Interactions
	Pilin	End	Interacts with Pilin
	papA	Proximal	papA and papH
	papA	Distal	papA and papK
	pap H	Proximal	none
	pap H	Distal	papA
	papK	Proximal	papA
	papK	Distal	papE
	papE	Proximal	papH and papE
	papE	Distal	papE and papF
	papF	Proximal	papE
	papF	Distal	papG
	papG	Proximal	papF
	papG	Distal	Does not interact with the
			N-terminal extension of any
			papA, papH, papE, papF, or
			papG

[0235] The interaction between pilin proteins is mediated by the N-terminal extension of each pilin protein that binds to the immediately adjacent pilin protein in P-pili, yielding an extended intermolecular interface that provides significant mechanical strength to the pilus (Sauer et al., 1999, Structural basis of chaperone function and pilus biogenesis, Science 285: 1058-61; Choudhury et al., 1999, X-ray structure of the FimC-FimH chaperone-adhesin complex from uropathogenic Escherichia coli, Science 285: 1061-66). The affinity and specificity of this binding are determined by the interaction between the extended N-terminal arm and the groove on the adjacent pilin protein (FIG. 14A). Consequently, replacement of the N-terminal arm of one pilin with that from another (forming a hybrid or chimeric pilin protein) provides a means of altering the specificity of binding of a pilin unit, and provides a means of designing protein units that may be used as structural and/or joining elements in staged assembly, i.e., where the N-terminus and body of one pilin do not interact to form dimers and polymers (FIG. 14B) (see also Section 5.6.6 below). A hybrid or chimeric pilin protein comprises the pilin amino terminal extension of a first pilin protein and the pilin protein body of a second pilin protein and lacks the pilin protein body of the first pilin protein and the pilin amino terminal extension of the second pilin protein, wherein the amino terminal extension of the first pilin protein does not bind to the pilin protein body of the second pilin protein.

[0236] A comparison of the sequences of all the pilins that make up a P-pilus indicate that the region that links the N-terminal extension with the body of that pilin protein is highly conserved among pilins and that the position for fusing heterologous pilin parts is well-defined based on that homology.

[0237] Functionality may be added to the pilin subunits at positions identified as being (i) on the surface of the subunits; (ii) unimportant to the interaction of the subunits with one another and (iii) unimportant for the stability of the subunits themselves. It has been shown that in many proteins, large loop insertions are tolerated and many redesigns have generated proteins that successfully fold to stable, active structures. Some redesigns have been entirely the choice of the investigators, whereas others have incorporated a randomization and selection step to identify optimal sequences (Regan, 1999, Protein redesign, Current Opinion in Structural Biology 9: 494-99). One region amenable to reengineering is a surface loop on papA comprised of gly107-ala108-gly109. This loop satisfies the criteria that must be met by a position where a heterologous peptide may be successfully inserted.

[0238] Pilin proteins may be expressed and purified by methods commonly known in the art (e.g., Bullitt and Makowski, 1995, Structural polymorphism of bacterial adhesion pili, Nature 373: 164-67; Bullitt et al., 1996, Development of pilus organelle sub-assemblies in vitro depends on chaperone uncapping of a beta zipper, Proc. Natl. Acad. Sci. USA 93: 12890-95).

[0239] In certain embodiments, the invention encompasses structural elements comprising leucine zipper-type coiled coils for use in the construction of nanostructures using the staged assembly methods of the invention. Leucine zippers are well-known, a-helical protein structures (Oas et al., 1994, Springs and hinges: dynamic coiled coils and discontinuities, TIBS 19: 51-54; Branden et al., 1999, Introduction to Protein Structure 2and ed., Garland Publishing, Inc., New York) that are involved in the oligomerization of proteins or protein monomers into dimeric, trimeric, and tetrameric structures, depending on the exact sequence of the leucine zipper domain (Harbury et al., 1993, A switch between two-, three-, and four-stranded coiled coils in GCN4 leucine zipper mutants, Science 262: 1401-07). While only dimers are disclosed herein for simplicity, it would be apparent to one of ordinary skill in the art that trimeric and tetrameric units may also be used for the construction of assembly units for use in staged assembly of nanostructures according to the methods disclosed herein. In certain embodiments, trimeric and tetrameric units could be especially useful for incorporation of functional elements that, e.g., require two or more chemical moieties for proper activity, for example, the incorporation of two cysteine moieties for binding of gold particles. Several non-limiting examples of leucine-zipper domains are provided in Table 3 below.

[0240] Table 3 shows canonical leucine zippers and high stability dimerization sequences. The top line shows register of the repeat unit. Residues in the a and d positions are generally hydrophobic and control the oligomerization. Residues in the e and g positions are generally charged and create salt bridges to stabilize the oligomerization.

TABLE 3
Canonical Leucine Zippers and High Stability
Dimerization Sequences
		abcdefgabcdefgabcdefgabcdefgabcdefg
	GCN4	MKQLEDKVEELLSKNYHLENEVARLKKL
		(SEQ ID NO: 1)
	c-Fos	TDTLQAETDQLEDEKYALQTEIANLLKE
		(SEQ ID NO: 2)
	c-Jun	AARLEEKVKTLKAQNYELASTANMLREQ
		(SEQ ID NO: 3)
	C/EBPb	VLETQHKNERLTAEVEQLQKKLSTLSREFKQLRNL
		(SEQ ID NO: 4)
	ATF4	CKELTGENEALEKKADSLKERIQYLAKEIEEVKDL
		(SEQ ID NO: 5)
	c-myc	CCGVQAEEQKLTSEEDLLRKRREQLKHKLEQLX
		(SEQ ID NO: 6)
	Max	CGGMRRKNDTHQQDIDDLKRQNALLEQQVRALX
		(SEQ ID NO: 7)
	CREB	VKSLENRVAVLENQNKTLIEELKALKDLYSHK
		(SEQ ID NO: 8)
	PAP1	VVTLKELHSSTTLENDQLRQKVRQLEEELRILK
		(SEQ ID NO: 9)

[0241] Many naturally occurring leucine zippers may be used according to the methods of the invention, including those found in the yeast transcription factor GCN4 and in the mammalian Fos, Jun and Myc oncogenes. Additional proteins containing leucine zippers and other coiled coil-type oligomerization sequences can be identified by searching public protein databases such as SWISS-PROT/TrEMBL (Bairoch and Apweiler, 2000, The SWISS-PROT protein sequence database and its supplement TrEMBL in 2000, Nucl. Acids Res. 28: 45-48). Table 4 shows the results of such a search, using the keywords coiled coil and dimer.In Table 4, the common names of genes are listed, as well as their SWISS-PROT accession numbers, sequence description and sequence. The SWISS-PROT accession number is a unique identifier for a sequence record. An accession number applies to the complete record and is usually a combination of a letter(s) and numbers, such as a single letter followed by five digits (e.g., Q12345) or a combination of six letters and digits (e.g., Q1Z2F3). The coiled coil sequences are underlined.

TABLE 4
Examples of Proteins Containing Coiled Coil Dimerization Sequences
That Can Be Used for Structural Elements of Assembly Units
Sequence	Accession	Sequence
ID	number	description	Sequence
SWISS_PR0	O54931	A-kinase anchor	MEIGVSVAECKSVPGVTSTPHSKDHSSPFYSPS
OT:	O54932	protein 2	HNGLLADHHESLDNDVAREIQYLDEVLEANCCD
AKA2_MOU	O54933	(Protein kinase	SSVDGTYNGISSPEPGAAILVSSLGSPAHSVTE
SE		A anchoring	AEPTEKASGRQVPPHIELSRIPSDRMAEGERAN
		protein	GHSTDQPQDLLGNSLQAPASPSSSTSSHCSSRD
		2)(PRKA2)	GEFTLTTLKKEAKFELRAFHEDKKPSKLFEEDE
		(AKAP expressed	REKEQFCVRKVRPSEEMIELEKERRELIRSQAV
		in kidney and	KKNPGIAAKWWNPPQEKTIEEQLDEEHLESHRR
		lung) (AKAP-KL)	YKERKEKRAQQEQLQLQQQQQQQLQQQQLQQQQ
			LQQQQLQQQLQQQQLSTSQPCTAPAAHKHLDGI
			EHTKEDVVTEQIDFSAARKQFQLMENSRQTLAK
			GQSTPRLFSIKPYYKPLGSIHSDKPPTILRPAT
			VGGTLEDGGTQAAKEQKAPCVSESQSAGAGPAN
			AATQGKEGPYSEPSKRGPLSKLWAEDGEFTSAR
			AVLTVVKDEDHGILDQFSRSVNVSLTQEELDSG
			LDELSVRSQDTTVLETLSNDFSMDNISDSGASN
			ETTSALQENSLADFSLPQTPQTDNPSEGREGVS
			KSFSDHGFYSPSSTLGDSPSVDDPLEYQAGLLV
			QNAIQQAIAEQVDKAEAHTSKEGSEQQEPEATV
			EEAGSQTPGSEKPQGMFAPPQVSSPVQEKRDIL
			PKNLPAEDRALREKGPSQPPTAAQPSGPVNMEE
			TRPEGGYFSKYSEAAELRSTASLLATQESDVMV
			GPFKLRSRKQRTLSMIEEEIRAAQEREEELKRQ
			RQVRQSTPSPRAKNAPSLPSRTTCYKTAPGKIE
			KVKPPPSPTTEGPSLQPDLAPEEAAGTQRPKNL
			MQTLMEDYETHKSKRRERMDDSSYTSKLLSCKV
			TSEVLEATRVNRRKSASGLALGGRDLR
			(SEQ ID NO: 10)
SWISS_PR	Q99996	A-kinase anchor	MEDEERQKKLEAGKAKIEELSLAFLVRQLAQFR
OT:	Q9UQQ4	protein 9	QRKAQSDGQSPSKKQKKKRKTSSSKHDVSAHHD
AKA9_HUM	Q9UQH3	(Protein kinase	LNIDQSQCNEMYINSSQRVESTVIPESTIMRTL
AN	Q9YGY2	A anchoring	HSGEITSHEQGFSVELESEISTTADDCSSEVNG
	O14869	protein	CSFVMRTGKPTNLLREEEFGVDDSYSEQGAQDS
	O43355	9)(PRKA9) (A-	PTHLEMMESELAGKQHEIEELNRELEEMRVTYG
	O94895	kinase anchor	TEGLQQLQEFEAAIKQRDGIITQLTANLQQARR
	Q9YGB8	protein 450	EKDETMREFLELTEQSQKLQIQEQQLQASETLR
		kDa) (AKAP 450)	NSTHSSTAADLLQAKQQILTHQQQLEEQDHLLE
		(A-kinase	DYQKKKEDFTMQTSFLQEKIKVYEMEQDKKVEN
		anchorprotein	SNKEEIQEKETTIEELNTKTIEEEKKTLELKDK
		350 kDa) (AKAP	LTTADKLLGELQEQIVQKNQEIKNMKLELTNSK
		350) (hgAKAP	QKERQSSEEIKQLMGTVEELQKRNHKDSQFETD
		350) (AKAP 120	IVQRMEQETQRKLEQLRAELDEMYGQQIVQMKQ
		like	ELTRQHMAQMEEMKTRHKGSMENALRSYSNITV
		protein)(Hyper	NEDQIKLMNVAINSLNIKLQDTNSQKEKLKEEL
		ion protein)	GLILEEKCALQRQLEDLVEELSFSREQIQRARQ
		(Yotiao	TIAEQESKLNEAHKSLSTVEDLKAEIVSASESR
		protein)	KELELKHEAEVTNYKIKLEMLEKEKNAVLDRMA
		(Centrosome-	ESQEAELERLRTQLLESHEEELSKLKEDLEIRH
		and golgi-	RINIEKLKDNLGIHYKQQIDCLQNEMSQKTETM
		localizedPKN-	QFEKDNLITKQNQLILEISKLKDLQQSLVNSKS
		associated	EEMTLQINELQKETEILRQEEKEKGTLEQEVQE
		protein) (CG-	LQLKTELLEKQMKEKENDLQEKFAQLEAENSIL
		NAP)	KDEKKTLEDMLKIHTPVSQEERLIFLDSIKSKS
			KDSVWEKEIEILIEENEDLKQQCIQLNEEIEKQ
			RNTFSEAEKNFEVNYQELQEEYACLLKVKDDLE
			DSKNKQELEYKSKLKALNEELHLQRINPTTVKM
			KSSVFDEDKTFVAETLEMGEVVEKDTTELMEKL
			EVTKREKLELSQRLSDLSEQLKQKHGEISFLNE
			EVKSLKQEKEQVSLRCRELEIIINHNRAENVQS
			CDTQVSSLLDGVVTMTSRGAEGSVSKVNKSFGE
			ESKIMVEDKVSFENMTVGEESKQEQLILDHLPS
			VTKESSLRATQPSENDKLQKELNVLKSEQNDLR
			LQMEAQRICLSLVYSTHVDQVREYMENEKDKAL
			CSLKEELIFAQEEKIKELQKIHQLELQTMKTQE
			TGDEGKPLHLLIGKLQKAVSEECSYFLQTLCSV
			LGEYYTPALKCEVNAEDKENSGDYISENEDPEL
			QDYRYEVQDFQENMHTLLNKVTEEYNKLLVLQT
			RLSKIWGQQTDGMKLEFGEENLPKEETEFLSIH
			SQMTNLEDIDVNHKSKLSSLQDLEKTKLEEQVQ
			ELESLISSLQQQLKETEQNYEAEIHCLQKRLQA
			VSESTVPPSLPVDSVVITESDAQRTMYPGSCVK
			KNIDGTIEESGEFGVKEETNIVKLLEKQYQEQL
			EEEVAKVIVSMSIAFAQQTELSRISGCKENTAS
			SKQAHAVCQQEQHYENEMKLSQDQIGFQTFETV
			DVKFKEEEKPLSKELGEHGKEILLSNSDPHDIP
			ESKDCVLTISEEMFSKDKTFIVRQSIHDEISVS
			SMDASRQLMLNEEQLEDMRQELVRQYQEHQQAT
			QRSSIDNENLVSERERVLLEELEALKQLSLAGR
			EKLCCELRNSSTQTQNGNENQGEVEEQTFREKE
			LDRKPEDVPPEILSNERYALQKANNRLLKILLE
			VVKTTAAVEETIGRHVLGILDRSSKSQSSASLI
			WRSEAEASVKSCVHEEHTRVTDESIPSYSGSDM
			PRNDINMWSKVTEEGTELSQRLVRSGFAGTEID
			PENEELMLNISSRLQAAVEKLLEAISETSSQLE
			HAKVTQTELMRESERQKQEATESLKCQEELRER
			LHEESRAREQLAVELSKAEGVIDGYADEKTLFE
			RQIQEKTDIIDRLEQELLCASNRLQELEAEQQQ
			IQEERELLSRQKEAMKAEAGPVEQQLLQETEKL
			MKEKLEVQCQAEKVRDDLQKQVKALEIDVEEQV
			SRFIELEQEKNTELMDLRQQNQALEKQLEKMRK
			FLDEQAIDREHERDVFQQEIQKLEQQLKVVPRF
			QPISEHQTREVEQLANHLKEKTDKCSELLLSKE
			QLQRDIQERNEEIEKLEFRVRELEQALLVSADT
			FQKVEDRKHFGAVEAKPELSLEVQLQAERDAID
			RKEKEITNLEEQLEQFREELENKNEEVQQLHMQ
			LEIQKKESTTRLQELEQENKLFKDDMEKLGLAI
			KESDAMSTQDQHVLEGKFAQIIQEKEVEIDQLN
			EQVTKLQQQLKITTDNKVIEEKNELIRDLETQI
			ECLMSDQECVKRNREEEIEQLNEVIEKLQQELA
			NIGQKTSMNAHSLSEEADSLKHQLDVVIAEKLA
			LEQQVETANEEMTFMKNVLKETNFKMNQLTQEL
			FSLKRERESVEKIQSIPENSVNVAIDHLSKDKP
			ELEVVLTEDALKSLENQTYFKSFEENGKGSIIN
			LETRLLQLESTVSAKDLELTQCYKQIKDMQEQG
			QFETEMLQKKIVNLQKIVEEKVAAALVSQIQLE
			AVQEYAKFCQDNQTISSEPERTNIQNLNQLRED
			ELGSDISALTLRISELESQVVEMHTSLILEKEQ
			VEIAEKNVLEKEKKLLELQKLLEGNEKKQREKE
			KKRSPQDVEVLKTTTELFHSNEESGFFNELEAL
			RAESVATKAELASYKEKAEKLQEELLVKETNMT
			SLQKDLSQVRDHLAEAKEKLSILEKEDETEVQE
			SKKACMFEPLPIKLSKSIASQTDGTLKISSSNQ
			TPQILVKNAGIQINLQSECSSEEVTEIISQFTE
			KIEKMQELHAAEILDMESRHISETETLKREHYV
			AVQLLKEECGTLKAVIQCLRSKEVFGEYNMCES
			TLCDSGSDWGQGIYLTHSQGFDIASEGRGEESE
			SATDSFPKKIKGLLRAVHNEGMQVLSLTESPYS
			DGEDHSIQQVSEPWLEERKAYINTISSLKDLIT
			KMQLQREAEVYDSSQSHESFSDWRGELLLALQQ
			VFLEERSVLLAAFRTELTALGTTDAVGLLNCLE
			QRIQEQGVEYQAAMECLQKADRRSLLSEIQALH
			AQMNGRKITLKREQESEKPSQELLEYNIQQKQS
			QMLEMQVELSSMKDRATELQEQLSSEKMVVAEL
			KSELAQTKLELETTLKAQHKHLKELEAFRLEVK
			DRTDEVHLLNDTLASEQKKSRELQWALEKEKAK
			LGRSEERDKEELEDLKFSLESQKQRNLQLNLLL
			EQQKQLLNESQQKTESQRMLYDAQLSEEQGRNL
			ELQVLLESEKVRIREMSSTLDRERELHAQLQSS
			DGTGQSRPPLPSEDLLKELQKQLEEKHSRIVEL
			LNETEKYKLDSLQTRQQMSKDRQVHRKTLQTEQ
			EANTEGQKKMHELQSKVEDLQRQLEEKRQQVYK
			LDLEGQRLQGIMQEFQKQELEREEKRESRRILY
			QNLNEPTTWSLTSDRTRNWVLQQKIEGETKESN
			YAKLIEMNGGGTGCNHELEMIRQKLQCVASKLQ
			VLPQKASERLQFETADDEDFIWVQENIDEIILQ
			LQKLTGQQGEEPSLVSPSTSCGSLTERLLRQNA
			ELTGHISQLTEEKNDLRNMVMKLEEQIRWYRQT
			GAGRDNSSRFSLNGGANIEAIIASEKEVWNREK
			LTLQKSLKRAEAEVYKLKAELRNDSLLQTLSPD
			SEHVTLKRIYGKYLRAESFRKALIYQKKYLLLL
			LGGFQECEDATLALLARMGGQPAFTDLEVITNR
			PKGFTRFRSAVRVSIAISRMKFLVRRWHRVTGS
			VSININRDGFGLNQGAEKTDSFYHSSGGLELYG
			EPRHTTYRSRSDLDYIRSPLPFQNRYPGTPADE
			NPGSLACSQLQNYDPDRALTDYITRLEALQRRL
			GTIQSGSTTQFHAGMRR
			(SEQ ID NO: 11)
SWISS_PR	Q28628	A-kinase anchor	REKLEVQCQAEKVRDDLQKQVKALEIDVEEQVC
OT:		protein 9	RFIELEQEKNAELMDLRQQNQALEKQLEKMRKM
AKA9_RAB		(Protein kinase	DLRQQNQALEKQLEKMRKFLDEQAIDREHERDV
IT		A anchoring	FQQEIQKLEQQLKLVPRFQPISEHOQREVEQLT
		protein	NHLKEKTDKCSELLLSKEQLQRDVOQRNEEIEK
		9)(PRKA9) (A-	LECRVRELEQALLSVQTLSKRWRTRNSFGAVEP
		kinase anchor	KAELCLEVQLQAERDAIDRKEKEITNLEEQLEQ
		protein 120	FREELENKNEEVQQLHMQLEIQKKESTTRLQEL
		kDa) (AKAP 120)	EQENKLFKDEMEKLGFAIKESDAVSPQDQQVLF
		(Fragment)	GKFAQIIHEKEVEIDRLNEQIIKLQQQLKITTD
			NKVIEEKNELIRDLEAQIECLMSDQERVRKNRE
			EEIEQLNEVIEKLQQELANIDQKTSVDPSSLSE
			EADSLKHQLDKVIAEKLALEHQVETTNEEMAVT
			KNVLKETNFKMNQLTQELCSLKREREKMERIQS
			VPEKSVNMSVGDLSKDKPEMDLIPTEDALAQLE
			TQTQLRSSEESSKVSLSSLETKLLQLESTVSTK
			DLELTQCYKQIQDMREQGRSETEMLQTKIVSLQ
			KVLEEKVAAALVSQVQLEAVQEYVKLCADKPAV
			SSDPARTEVPGLSQLAGNTMESDVSALTWRISE
			LESQLVEMHSSLISEKEQVEIAEKNALEKEKKL
			QELQKLVQDSETKQRERERQSRLHGDLGVLEST
			TSEESGVFGELEALRAESAAPKGELANYKELAE
			KLQEELLVKETNMASLPKELSHVRDQLTEAEDK
			LSHFSEKEDKTEVQEHGTICILEPCPGQIGESF
			ASQTEGAVQVNSHTQTPQIPVRSVGIQTHSQSD
			SSPEEVAEIISRFTEKIEQMRELHAAEILDMES
			RHISETETLKREHCIAVQLLTEECASLKSLIQG
			LRMPEGSSVPSLTHSNAYQTREVGSSDSGSDWG
			QGIYLTQSQGFDTASEARGEEGETSTDSFPKKI
			KGLLRAVHNEGMQVLSLTEGPCGDGEDYPGHQL
			SESWLEERRAYLSTISSLKDFITKMQVQREVEV
			YDSSQSHENISDWRGELLLALQQVFLRERSVLL
			AAFKTELTALGTRDAAGLLNCLEQRIPRTEY
			(SEQ ID NO: 12)
SWISS_PR	Q94981	Ariadne-1	MDSDNDNDFCDNVDSGNVSSGDDGDDDFGMEVD
OT:		protein (Ani-1)	LPSSADRQMDQDDYQYKVLTTDEIVQHQREIID
ARI1_DRO			EANLLLKLPTPTTRILLNHFKWDKEKLLEKYED
ME			DNTDEEFKCAHVINPENATEAIKQKTSRSQCEE
			CEICFSQLPPDSMAGLECGHRFCMPCWHEYLST
			KIVAEGLGQTTSCAAHGCDILVDDVTVANLVTD
			ARVRVKYQQLTTNSFVECNQLLRWCPSVDCTYA
			VKVPYAEPRRVHCKCGHVECFACGENWHDPVKC
			RWLKKWIKKCDDDSETSNWIAANTKECPRCSVT
			IEKDGGCNHMVCKNQNCKNEFCWVCLGSWEPHG
			SSWYNCNRYDEDEAKTARDAQEKLRSSLARYLH
			YYNRYMNHMQSMKFENKLYASVKQKMEEMQQHN
			MSWIEVQELKKAVDILCQCRQTLMYTYVFAYYL
			KKNNQSMIFEDNQKDLESATEMLSEYLERDITS
			ENLADIKQKVQDKYRYCEKRCSVLLKHVHEGYD
			KEWWEYTE
			(SEQ ID NO: 13)
SWISS_PR	Q9UBS5	Gamma-	MLLLLLLAPLPLRPPGAGGAQTPNATSSGCQII
OT:	Q95375	aminobutyric	HPPWEGGIRYRGLTRDQVKAINELPVDYEIEYV
GBR1_HUM	Q9UQQ0	acid type B	CRGEREVVGPKVRKCLANGSWTDMDTPSRCVRI
AN	Q96022	receptor,	CSKSYLTLENGKVFLTGGDLPALDGARVDERCD
	Q95975	subunit 1	PDFHLVGSSRSICSQGQWSTPKPHCQVNRTPHS
	Q95468	precursor	ERRAVYIGALEPMSGGWPGGQACQPAVEMALED
		(GABA-	VNSRRDILPDYELKLIHHDSKCDPGQATKYLYE
		Breceptor 1)	LLYNDPIKIILMPGCSSVSTLVAEAARMWNLIV
		(GABA-B-R1)	LSYGSSSPALSNRQRFPTFFRTHPSATLHNPTR
		(Gb1)	VKLFEKWGWKKIATIQQTTEVPTSTLDDLEERV
			KEAGIEITERQSFPSDPAVPVKNLKRQDARIIV
			GLFYETEARKVFCEVYKERLFGKKYVWELIGWY
			ADNWFKIYDPSINCTVDEMTEAVEGHITTEIVM
			LNPANTRSISNMTSQEEVEKLTKRLKRHPEETG
			GEQEAPLAYDAIWALALALNKTSGGGGRSGVRL
			EDPNYNNQTITDQIYRAMNSSSPEGVSGHVVFD
			ASGSRMAWTLIEQLQGGSYKKIGYYDSTKDDLS
			WSKTDKWIGGSPPADQTLVIKTERFLSQKLEIS
			VSVLSSLGIVLAVVCLSFNIYNSHVRYIQNSQP
			NLNNLTAVGGSLALAAVEPLGLDGYHIGRNQPP
			FVCQARLWLLGLGFSLGYGSMPTKIWWVHTVFT
			KKEEKKEWRKTLEPWKLYATVGLLVGMDVLTLA
			IWQIVDPLHRTIETFAKEEPKEDIDVSILPQLE
			HCSSRKMNTWLGIFYGYKGLLLLLGIFLAYETK
			SVSTEKINDHRAVGMAIYNVAVLCLITAPVTMI
			LSSQQDAAFAFASLAIVFSSYITLVVLFVPKMR
			RLITRGEWQSEAQDTMKTGSSTNNNEEEKSRLL
			EKENRELEKIIAEKEERVSELRHQLQSRQQLRS
			RRHPPTPPEPSGGLPRGPPEPPDRLSCDGSRVR
			LLYK
			(SEQ ID NO: 14)
SWISS_PR	P03069	General control	MSEYQPSLFALNPMGFSPLDGSKSTNENVSAST
OT:	P03068	protein GCN4	STAKPMVGQLIFDKFIKTEEDPIIKQDTPSNLD
GCN4_YEA		(Amino acid	FDFALPQTATAPDAKTVLPIPELDDAVVESFFS
ST		biosynthesis	SSTDSTPMFEYENLEDNSKEWTSLFDNDIPVTT
		regulatoryprot	DDVSLADKAIESTEEVSLVPSNLEVSTTSPLPT
		ein)	PVLEDAKLTQTRKVKKPNSVVKKSHHVGKDDES
			RLDHLGVVAYNRKQRSIPLSPIVPESSDPAALK
			RARNTEAARRSRARKLQRMKQLEDKVEELLSKN
			YHLENEVARLKKLVGER
			(SEQ ID NO: 15)
SWISS_PR	O60282	Kinesin heavy	MADPAECSIKVMCRFRPLNEAETLRGDKFIPKF
OT:	O95079	chain isoform	KGDSTVVIGQGKPYVFDRVLPPNTTQEQVYNAC
KF5C_HUM		SC (Kinesin	AKQIVKDVLEGYNGTIFAYGQTSSGKTHTMEGK
AN		heavy chain	LHDPQLMGTIPRIAHDIFDHIYSMDENLEFHIK
		neuron-	VSYFEIYLDKIRDLLDVSKTNLAVHEDKNRVPY
		specific2)	VKGCTERFVSSPEEVMDVIDEGKANRHVAVTNM
			NEHSSRSHSIFLINIKQENVETEKKLSGKLYLV
			DLAGSEKVSKTGAEGAVLDEAKNINKSLSALCN
			VISALAEGTKTHVPYRDSKMTRILQDSLGGNCR
			TTIVICCSPSVENEAETKSTLMFGQRAKTIKNT
			VSVNLELTAEEWKKKYEKEKEKNKTLKNVIQHL
			EMELNRWRNGEAVPEDEQISAKDQKNLEPCDNT
			PIIDNIAPVVAGISTEEKEKYDEEISSLYRQLD
			DKDDEINQQSQLAEKLKQQMLDQDELLASTRRD
			YEKIQEELTRLQIENEAAKDEVKEVLQALEELA
			VNYDQKSQEVEDKTRANEOQTDELAQKTTTLTT
			TQRELSQLQELSNHQKKRATEILNLLLKDLGEI
			GGIIGTNDVKTLADVNCVIEEEFTMARLYISKM
			KSEVKSLVNRSKQLESAQMDSNRKMNASERELA
			ACQLLISQHEAKIKSLTDYMQNMEQKRRQLEES
			QDSLSEELAKLRAQEKMHEVSFQDKEKEHLTRL
			QDAEEMKKALEQQMESHREAHQKQLSRLRDEIE
			EKQKIIDEIRDLNQKLQLEQEKLSSDYNKLKIE
			DQEREMKLEKLLLLNDKREQAREDLKGLEETVS
			RELQTLHNLRKLFVQDLTTRVKKSVELDNDDGG
			GSAAQKQKISFLENNLEQLTKVHKQLVRDNADL
			RCELPKLEKRLRATAERVKALESALKEAKENAM
			RDRKRYQQEVDRIKEAVRAKNMARRAHSAQIAK
			PIRPGHYPASSPTAVHAIRGGGGSSSNSTHYQK
			(SEQ ID NO: 16)
SWISS_PR	P28738	Kinesin heavy	MADPAECSIKVMCRFRPLNEAEILRGDKFIPKF
OT:	Q9Z2F8	chain isoform	KGEETVVIGQGKPYVFDRVLPPNTTQEQVYNAC
KF5C_MOU		5C (Kinesin	AKQIVKDVLEGYNGTIFAYGQTSSGKTHTMEGK
SE		heavy chain	LHDPQLMGIIPRIAHDIFDHIYSMDENLEFHIK
		neuron-	VSYFEIYLDKIRDLLDVSKTNLAVHEDKNRVPY
		specific2)	VKGCTERFVSSPEEVMDVIDEGKANRHVAVTNM
			NEHSSRSHSIFLINIKQENVETEKKLSGKLYLV
			DLAGSEKVSKTGAEGAVLDEAKNINKSLSALGN
			VISALAEGTKTHVPYRDSKMTRILQDSLGGNCR
			TTIVICCSPSVFNEAETKSTLMFGQRAKTIKNT
			VSVNLELTAEEWKKKYEKEKSKNKALKSVLQHL
			EMELNRWRNGEAVPEDEQISAKDHKSLEPCDNT
			PIIDNITPVVDGISAEKEKYDEEITSLYRQLDD
			KDDEINQQSQLAEKLKQQMLDQDELLASTRRDY
			EKIQEELTRLQIENEAAKDEVKEVLQALEELAV
			NYDQKSQSVSDKTRANSQLTDSLAQKTTTLTTT
			QRSLSQLQELSNHQKKRATEILNLLLKDLGEIG
			GIIGTNDVKTLADVNGVIEEEFTMARLYISKMK
			SEVKSLVNRSKQLESAQMDSNRKMNASERELAA
			CQLLISQHEAKIKSLTDYMQNMEQKRRQLEESQ
			DSLSEELAKLRAQEKMHEVSFQDKEKEHLTRLQ
			DAEEVKKALEQQMESHREAHQKQLSRLRDETEE
			KQRIIDEIRDLNQKLQLEQERLSSDYNKLKIED
			QEREVKLEKLLLLNDKREQAREDLKGLEETVSI
			ELQTLHNLRKLFVQDLTTRVKKSVELDSDDCCC
			SAAQKQKISFLENNLEQLTKVHKQLVRDNADLR
			CELPKLEKRLRATAERVKALESALKEAKENAMR
			DRKRYQQEVDRIKEAVRAKNMARRAHSAQIAKP
			IRPQHYPASSPTAVHAVRGGGGGSSNSTHYQK
			(SEQ ID NO: 17)
SWISS_PR	P34540	Kinesin heavy	MEPRTDGAECGVQVFCRIRPLNKTEEKNADRFL
OT:		chain	PKFPSEDSISLGGKVYVFDKVFKPNTTQEQVYK
KTNH_CAE			GAAYHIVQDVLSGYNGTVFAYGQTSSGKTHTME
EL			CVIGDNGLSGIIPRIVADIFNHIYSMDENLQFH
			IKVSYYEIYNEKIRDLLDPEKVNLSIHEDKNRV
			PYVKGATERFVGGPDEVLQAIEDGKSNRMVAVT
			NMNEHSSRSHSVFLITVKQEHQTTKKQLTGKLY
			LVDLAGSEKVSKTGAQGTVLEEAKNINKSLTAL
			GIVISALAEGTKSHVPYRDSKLTRILQESLGGN
			SRTTVIICASPSHFNEAETKSTLLFGARAKTIK
			NVVQINEELTAEEWKRRYEKEKEKNTRLAALLQ
			AAALELSRWRAGESVSEVEWVNLSDSAQMAVSE
			VSGGSTPLMERSIAPAPPMLTSTTGPITDEEKK
			KYEEERVKLYQQLDEKDDEIQKVSQELEKLRQQ
			VLLQEEALGTMRENEELIREENNRFQKEAEDKQ
			QEGKEMMTALEEIAVNLDVRQAECEKLKRELEV
			VQEDNQSLEDRMNQATSLLNAHLDECGPKIRHF
			KEGIYNVIREFNIADIASQNDQLPDHDLLNHVR
			IGVSKLFSEYSAAKESSTAAEHDAEAKLAADVA
			RVESGQDAGRMKQLLVKDQAAKEIKPLTDRVNM
			ELTTLKNLKKEFMRVLVARCQANQDTEGEDSLS
			GPAQKQRIQPLENNLDKLTKVHKQLVRDNADLR
			VELPKMEARLRGREDRIKILETALRDSKQRSQA
			ERKKYOQEVERTKEAVRORNMRRMNAPQIVKPI
			RPGQVYTSPSAGMSQGAPNGSNA
			(SEQ ID NO: 18)
SWISS_PR	P17210	Kinesin heavy	MSAEREIPAEDSIKVVCRFRPLNDSEEKAGSKF
OT:	Q9V7L9	chain	VVKFPNNVEENCISIAGKVYLFDKVFKPNASQE
KINH_DRO			KVYNEAAKSIVTDVLAGYNGTIFAYGQTSSGKT
ME			HTMEGVIGDSVKQGIIPRIVNDIFNHIYAMEVN
			LEFHIKVSYYEIYMDKIRDLLDVSKVNLSVHED
			KNRVPYVKGATERFVSSPEDVFEVIEEGKSNRH
			IAVTNMNEHSSRSHSVFLINVKQENLENQKKLS
			GKLYLVDLAGSEKVSKTGAEGTVLDEAKNINKS
			LSALGNVISALADGNKTHIPYRDSKLTRILQES
			LGGNARTTIVICCSPASFNESETKSTLDFGRRA
			KTVKNVVCVNEELTAEEWKRRYEKEKEKNARLK
			GKVEKLEIELARWRAGETVKAEEQINMEDLMEA
			STPNLEVEAAQTAAAEAALAAQRTALANMSASV
			AVNEQARLATECERLYQQLDDKDEEINQQSQYA
			EQLKEQVMEQEELIANARREYETLQSEMARIQQ
			ENESAKEEVKEVLQALEELAVNYDQKSQEIDNK
			NKDIDALNEELQQKQSVFNAASTELQQLKDMSS
			HQKKRITEMLTNLLRDLGEVGQAIAPGESSIDL
			KMSALAGTDASKVEEDFTMARLFISKMKTEAKN
			IAQRCSNMETQQADSNKKISEYEKDLGEYRLLI
			SQHEARMKSLQESMREAENKKRTLEEQIDSLRE
			ECAKLKAAEHVSAVNAEEKQRAEELRSMEDSQM
			DELREAHTRQVSELRDEIAAKQHEMDEMKDVHQ
			KLLLAHQQMTADYEKVRQEDAEKSSELQNIILT
			NERREQARKDLKGLEDTVAKELQTLHNLRKLFV
			QDLQQRIRKNVVNEESEEDGGSLAQKQKISFLE
			NNLDQLTKVHKQLVRDNADLRCELPKLEKRLRC
			TMERVKALETALKEAKEGAMRDRKRYQYEVDRI
			KEAVRQKHLGRRGPQAQIAKPIRSGQGAIAIRG
			GGAVGGPSPLAQVNPVNS
			(SEQ ID NO: 19)
SWISS_PR	P33176	Kinesin heavy	MADLAECNIKVMCRFRPLNESEVNRGDKYIAKF
OT:		chain	QGEDTVVTASKPYAPDRVFQSSTSQEQVYNDCA
KINH_HUM		(Ubiquitous	KKIVKDVLEGYNGTIFAYGQTSSGKTHTMEGKL
AN		kinesin heavy	HDPEGMGIIPRIVQDIFNYIYSMDENLEEHIKV
		chain) (UKHC)	SYFETYLDKIRDLLDVSKTNLSVHEDKNRVPYV
			KGCTERFVCSPDEVMDTIDEGKSNRHVAVTNMN
			EHSSRSHSIFLINVKQENTQTEQKLSGKLYLVD
			LAGSEKVSKTGAEGAVLDEAKNINKSLSALGNV
			ISALAEGSTYVPYRDSKMTRILQDSLGGNCRTT
			IVICCSPSSYNESETKSTLLFGQRAKTIKNTVC
			VNVELTAEQWKKKYEKEKEKNKILRNTIQWLEN
			ELNRWRNGETVPIDEQFDKEKANLEAFTVDKDI
			TLTNDKPATAIGVIGNFTDAERRKCEEEIAKLY
			KQLDDKDEEINQQSQLVEKLKTQMLDQEELLAS
			TRRDQDNMQAELNRLQAENDASKEEVKEVLQAL
			EELAVNYDQKSQEVEDKTKEYELLSDELNQKSA
			TLASIDAELQKLKEMTNHQKKRAAEMMASLLKD
			LAEIGIAVGNNDVKQPEGTGMIDEEFTVARLYI
			SKMKSEVKTMVKRCKQLESTQTESNKKMEENEK
			ELAACQLRISQHEAKIKSLTEYLQNVEQKKRQL
			EESVDALSEELVQLRAQEKVHEMEKEHLNKVQT
			ANEVKQAVEQQIQSHRETHQKQISSLRDEVEAK
			AKLITDLQDQNQKMMLEQERLRVEHEKLKATDQ
			EKSRKLHELTVMQDRREQARQDLKGLEETVAKE
			LQTLHNLRKLFVQDLATRVKKSAEIDSDDTGGS
			AAQKQKISFLENNLEQLTKVHKQLVRDNADLRC
			ELPKLEKRLRATAERVKALESALKEAKENASRD
			RKRYQQEVDRIKEAVRSKNMARRGHSAQIAKPI
			RPGQHPAASPTHPSAIRGGGAFVQNSQPVAVRG
			GGGKQV
			(SEQ ID NO: 20)
SWISS_PR	P21613	Kinesin heavy	MDVASECNIKVICRVRPLNEAEERAGSKFILKF
OT:		chain	PTDDSISIAGKVFVFDKVLKPNVSQEYVYNVGA
KINH_LOL			KPIVADVLSGCNGTIFAYGQTSSGKTHTMEGVL
PE			DKPSMHGIIPRIVQDIFNYTYGMDENLEPHIKI
			SYYEIYLDKIRDLLDVTKTNLAVHEDKNRVPFV
			KGATERPVSSPEEVMEVIDEGKNNRHVAVTNMN
			EHSSRSHSVFLTNVKQENVETQKKLSGKLYLVD
			LAGSEKVSKTGAEGAVLDEAKNINKSLSALGNV
			ISALADGNKSHVPYRDSKLTRILQESLGGNART
			TMVICCSPASYNESETKSTLLPGQRAKTIKNVV
			SVNEELTADEWKRRYEKEKERVTKLKATMAKLE
			AELQRWRTGQAVSVEEQVDLKEDVPAESPATST
			TSLAGGLIASMNEGDRTQLEEERLKLYQQLDDK
			DDEINNQSQLIEKLKEQMMEQEDLTAQSRRDYE
			NLQQDMSRIQADNESAKDEVKEVLQALEELAMN
			YDQKSQEVEDKNKENENLSEELNQKLSTLNSLQ
			NELDQLKDSSMHHRKRVTDMMINLLKDLGDTCT
			IVGGNAAETKPTAGSGEKIEEEFTVARLYISKM
			KSEVKTLVSRNNQLENTQQDNFKKIETHEKDLS
			NCKLLTQQHEAKMASLQEAIKDSENKKRMLEDN
			VDSLNEEYAKLKAQEQMHLAALSEREKETSQAS
			ETREVLEKQMEMHREQHQKQLQSLRDEISEKQA
			TVDNLKDDNQRLSLALEKLQADYDKLKQEEVEK
			AAKLADLSLQTDRREQAKQDLKGLEETVAKELQ
			TLHNLRKLFVQDLQNKVKKSCSKTEEEDEDTGG
			NAAQKQKISFLENNLEQLTKVHKQLVRDNADLR
			CELPKLEKRLRATMERVKSLESALKDAKEGAMR
			DRKRYQHEVDRIKEAVRQKNLARRGHAAQIAKP
			IRPGQHQSVSPAQAAAIRGGGGLSQNGPMTTST
			PTRMAPESKA
			(SEQ ID NO: 21)
SWISS_PR	Q61768	Kinesin heavy	MADPAECNIKVMCRPRPLNESEVNRGDKYVAKF
OT:	Q08711	chain	QGEDTVVIASKPYAPDRVFQSSTSQEQVYNDCA
KTNH_MOU	Q61580	(Ubiquitous	KKIVKDVLEGYNGTIFAYGQTSSGKTHTMEGKL
SE		kinesin heavy	HDPEGMGIIPRIVQDIFNYTYSMDENLEFHIKV
		chain) (UKHC)Z	SYFEIYLDKTRDLLDVSKTNLSVHEDKNRVPYV
			KGCTERFVCSPDEVMDTIDEGKSNRHVAVTNMN
			EHSSRSHSIFLINVKQENTQTEQKLSGKLYLVD
			LAGSEKVSKTGAEGAVLDEAKNINKSLSALGNV
			ISALAEGSTYVPYRDSKMTRILQDSLGGNCRTT
			IVICCSPSSYNESETKSTLLFGQRAKTIKNTVC
			VNVELTAEOWKKKYEKEKEKNKTLRNTIQWLEN
			ELNRWRNGETVPIDEQFDKEKANLEAFTADKDI
			AITSDKGAAAVGMAGSFTDAERRKCEEELAKLY
			KQLDDKDEEINQQSQLVEKLKTQMLDQEELLAS
			TRRDQDNMQAELNRLQAENDASKEEVKEVLQAL
			EELAVNYDQKSQEVEDKTKEYELLTDEFNQKSA
			TLASIDAELQKLKEMTNHQKKRAAEMMASLLKD
			LAEIGIAVGNNDVKQPEGTGMIDEEFTVARLYI
			SKMKSEVKTMVKRCKQLESTQTESNKKMEENEK
			ELAACQLRISQHEAKIKSLTEYLQNDEQKKRQL
			EESLDSLGEELVQLRAQEKVHEMEKEHLNKVQT
			ANEVKQAVEQQIQSHRETHQKQISSLRDEVEAK
			EKLITDLQDQNQKMVLETERLRVEHERLKATDQ
			EKSRKLHELTVMQDRREQARQDLKGLEETVAKE
			LQTLHNLRKLFVQDLATRVKKSAEVDSDDTGGS
			AAQKQKISFLENNLEQLTKVHKQLVRDNADLRC
			ELPKLEFRLRATAERVKALESALKEAKENASRD
			RKRYQQEVDRIKEAVRSKNMARRGHSAQIAKPI
			RPGQHPAASPTHPGTVRGGGSFVQNNQPVGLRG
			GGGKQS
			(SEQ ID NO: 22)
SWISS_PR	P48467	Kinesin heavy	MSSSANSIKVVARERPQNRVEIESGGQPIVTEQ
OT:		chain	GPDTCTVDSKEAQGSFTFDRVEDMSCKQSDIFD
KINH_NEU			FSIKPTVDDILNGYNGTVFAYGQTGAGKSYTMM
CR			GTSIDDPDGRGVIPRIVEQIFTSILSSAANIEY
			TVRVSYMEIYMERIRDLLAPQNDNLPVHEEKNR
			GVYVKGLLEIYVSSVQEVYEVMRRGGNARAVAA
			TNMNQESSRSHSIFVTTTTQKNVETGSAKSGQL
			FLVDLAGSEKVGKTGASGQTLEEAKKINKSLSA
			LGMVINALTDGKSSHVPYRDSKLTRILQESLGG
			NSRTTLIINCSPSSYNDAETLSTLFEGMRAKSI
			KNKAKVNAELSPAELKWMLAKAKTQITSFENYI
			VNLESEVQVWRGGETVPKEKWVPPLELAITPSK
			SASTTARPSTPSRLLPESRAETPAISDRAGTPS
			LPLDKDEREEFLRRENELQDQIAEKESIAAAAE
			RQLRETKEELIALKDHDSKLGKENERLISESNE
			FKMQLERLAEENKEAQITIDGLKDANSELTAEL
			DEVKQQMLDMKMSAKETSAVLDEKEKKKAEKMA
			KMMAGFDLSGDVFSDNERAVADAIAQLDALEEI
			SSAGDAIPPEDIKALREKLVETQCFVRQAFLSS
			FSAASSDAEARKRAELEARLEALQQEHEELLSR
			NLTEADKEEVKALLAKSLSDKSAVQVELVEQLK
			ADIALKNSETEHLKALVDDLQRRVKAGGAGVAM
			ANGKTVQQQLAEFDVMKKSLMRDLQNRCERVVE
			LEISLDETREQYNNVLRSSNNRAQQKKMAFLER
			NLEQLTQVQRQLVEQNSALKKEVAIAERKLMAR
			NERIQSLESLLQESQEKMAQANHKFEVQLAAVK
			DRLEAAKAGSTRGLGTDAGLGGFSIGSRIAKPL
			RGGGDAVAGATATNPTIATLQQNPPENKRSSWF
			FQKS
			(SEQ ID NO: 23)
SWISS_PR	P35978	Kinesin heavy	MADPAECNIKVVCRVRPMNATEQNTSHICTKFI
OT:		chain	SEEQVQIGGKLNMFDRIFKPNTTQEEVYNKAAR
KINH_STR			QIVKDVLDGYNGTIFAYGQTSSGKTFTMEGVMG
PU			NPQYMGIIPRIVQDIFNHIYQMDESLEFHIKVS
			YFEIYMDRIRDLLDVSKTNLSVHEDKNRVPFVK
			GATERFASSPEEVMDVIEEGKSNRHIAVTNMNE
			HSSRSHSIFLIQVKQENMETKKKLSGKLYLVDL
			AGSEKVSKTGAEGTVLDEAKNINKSLSALGNVI
			SALADGKKSHIPYRDSKMTRILQESLGGNARTT
			IVICCSPSSFNESESKSTLMFGQRAKTIKNTVT
			VNMELTAEEWRNRYEKEKEKNGRLKAQLLILEN
			ELQRWRAGESVPVKEQGNKNDEILKEMMKPKQM
			TVHVSEEEKNKWEEEKVKLYEQLDEKDSEIDNQ
			SRLTEKLKQQMLEQEELLSSMQRDYELLQSQMG
			RLEAENAAAKEEAKEVLQALEEMAVNYDEKSKE
			VEDKNRMNETLSEEVNEKMTALHTTSTELQKLQ
			ELEQHQRRRITEMMASLLKDLGEIGTALCGNAA
			DMKPNVENIEKVDEEFTMARLFVSKMKTEVKTM
			SQRCKILEASNAENETKIRTSEDELDSCRMTIQ
			QHEAKMKSLSENIRETEGKKRHLEDSLDMLNEE
			IVKLRAAEEIRLTDQEDKKREEEDKMQSATEMQ
			ASMSEQMESHRDAHQKQLANLRTEINEKEHQME
			ELKDVNQRMTLQHEKLQLDYEKLKIEEAEKAAK
			LRELSQQFDRREQAKQDLKGLEETVAKELQTLH
			NLRKLFVSDLQNRVKKALEGGDRDDDSGGSQAC
			KQKISFLENNLEQLTKVHKQLVRDNADLRCELP
			KLERRLRATSERVKALEMSLKETKEGAMRDRKR
			YQQEVDRIREAVRQRNFAKRGSSAQIAKAIRAG
			HPPPSPGGSTGIRGGGYSGIRGGGSPVIRPPSH
			GSPEPISHNNSFEKSLNPNDAENMEKKANKRLP
			KLPPGGNKLTESDIAAMKARSKARNNTPGKAPL
			TTSGEQGS
			(SEQ ID NO: 24)
SWISS_PR	043093	Kinesin heavy	MSGNNIKVVCRFRPQNSLEIREGGTPIIDIDPE
OT:KINH		chain (Synkin)	GTQLELKGKEFKGNFNFDKVFGMNTAQKDVFDY
SYNRA			SIKTIVDDVTAGYNGTVFAYGQTGSGKTFTMMG
			ADIDDEKTKGIIPRIVEQIFDSIMASPSNLEFT
			VKVSYMEIYMEKVRDLLNPSSENLPIHEDKTKG
			VYVKGLLEVYVGSTDEVYEVMRRGSNNRVVAYT
			NMNAESSRSHSIVMFTITQKNVDTGAAKSGKLY
			LVDLAGSEKVGKTGASGQTLEEAKKINKSLTAL
			GMVINALTDGKSSHVPYRDSKLTRILQESLGGN
			SRTTLIINCSPSSYNEAETLSTLRFGARAKSIK
			NKAKVNADLSPAELKALLKKVKSEAVTYQTYIA
			ALEGEVNVWRTGGTVPEGKWVTMDKVSKGDFAG
			LPPAPGFKSPVSDEGSRPATPVPTLEKDEREEF
			IKRENELMDQISEKETELTNREKLLESLREEMG
			YYKEQEQSVTKENQQMTSELSELRLQLQKVSYE
			SKENAITVDSLKEANQDLMAELEELKKNLSEMR
			QAHKDATDSDKEKRKAEKMAQMMSGFDPSGILN
			DKERQIRNALSKLDGEQQQTLTVEDLVSLRREL
			AESKMLVEQHTKTISDLSADKDAMEAKKIELEG
			RLGALEKEYEELLDKTIAEEEANMQNADVDNLS
			ALKTKLEAQYAEKKEVQQKEIDDLKRELDRKQS
			GHEKLSAAMTDLRAANDQLQAALSEQPFQAPQD
			NSDMTEKEKDIERTRKSMAQQLADFEVMKKALM
			RDLQNRCEKVVELEMSLDETREQYNNVLRASNN
			KAQQKKMAFLERNLEQLTNVQKQLVEQNASLKK
			EVALAERKLIARNERIQSLETLLHNAQDKLLNQ
			NKKFEQQLATVRERLEQARSQKSQNSLAALNES
			RIAKPLRGNGAAIDNGSDDGSLPTSPTDKRDKR
			SSWMPGFMNSR
			(SEQ ID NO: 25)
SWISS_PR	Q12840	Neuronal	MAETNNECSIKVLCRFRPLNQAEILRGDKFIPI
OT:		kinesin heavy	FQGDDSVVIGGKPYVFDRVFPPNTTQEQVYHAC
KINN_HUM		chain (NKHC)	AMQIVKDVLAGYNGTIFAYGQTSSGKTHTMEGK
AN		(Kinesin heavy	LHDPQLMGIIPRIARDIFNHIYSMDENLEPHIK
		chain isoform	VSYFEIYLDKIRDLLDVTKTNLSVHEDKNRVPF
		5A)(Kinesin	VKGCTERFVSSPEEILDVIDEGKSNRHVAVTNM
		heavy chain	NEHSSRSHSIFLINIKQENMETEQKLSGKLYLV
		neuron-specific	DLAGSEKVSKTGAEGAVLDEAKNINKSLSALGN
		1)	VISALAEGTKSYVPYRDSKMTRILQDSLGGNCR
			TTMFICCSPSSYNDAETKSTLMEGQRAKTIKNT
			ASVNLELTAEQWKKKYEKEKEKTKAQKETIAKL
			EAELSRWRNGENVPETERLAGEEAALGAELCEE
			TPVNDNSSIVVRIAPEERQKYEEEIRRLYKQLD
			DKDDEINQQSQLIEKLKQQMLDQEELLVSTRGD
			NEKVQRELSHLQSENDAAKDEVKEVLQALEELA
			VNYDQKSQEVEEKSQQNQLLVDELSQKVATMLS
			LESELQRLQEVSGHQRKRIAEVLNGLMKDLSEF
			SVIVGNGEIKLPVEISGAIEEEFTVARLYTSKI
			KSEVKSVVKRCRQLENLQVECHRKMEVTGRELS
			SCQLLISQHEAKIRSLTEYMQSVELKKRHLEES
			YDSLSDELAKLQAQETVHEVALKDKEPDTQDAD
			EVKKALELQMESHREAHHRQLARLRDEINEKQK
			TIDELKDLNQKLQLELEKLQADYEKLKSEEHEK
			STKLQELTFLYERHEQSKQDLKGLEETVARELQ
			TLHNLRKLFVQDVTTRVKKSAEMEPEDSGGIHS
			QKQKISPLENNLEQLTKVHKQLVRDNADLRCEL
			PKLEKRLRATAERVKALEGALKEAKEGAMKDKR
			RYQQEVDRIKEAVRYKSSGKRAHSAQIAKPVRP
			GHYPASSPTNPYGTRSPECISYTNSLFQNYQNL
			YLQATPSSTSDMYFANSCTSSGATSSGCPLASY
			QKANMDNCNATDINDNRSDLPCGYEAEDQAKLF
			PLHQETAAS
			(SEQ ID NO: 26)
SWISS_PR	P33175	Neuronal	MAETNNECSIKVLCRFRPLNQAEILRGDKFIPI
OT:KINN	Q9Z2F9	kinesin heavy	FQGDDSVIIGGKPYVFDRVFPPNTTQEQVYHAC
MOUSE		chain (NKHC)	AMQIVKDVLAGYNGTIFAYGQTSSGKTHTMEGK
		(Kinesin heavy	LHDPQLMGIIPRIARDIENHIYSMDENLEPHIK
		chain isoform	VSYFEIYLDKIRDLLDVTKTNLSVHEDKNRVPF
		5A)	VKGCTERFVSSPEEILDVIDEGKSNRHVAVTNM
		(Kinesin heavy	NEHSSRSHSIPLINIKQENVETEQKLSGKLYLV
		chain	DLAGSEKVSKTGAEGAVLDEAKNINKSLSALGN
		neuron-specific	VISALAEGTKSYVPYRDTKMTRILQDSLGGNCR
		1)	TTMFICCSPSSYNDAETKSTLMFGQRAKTIKNT
			ASVNLELTAEQWKKKYEKEKEKTKAQKETIANV
			EAELSRWRNGENVPETERLAGEDSALGAELCEE
			TPVNDNSSIVVRIAPEERQKYEEEIRRLYKQLD
			DKDDEINQQSQLIEKLKQQMLDQEELLVSTRGD
			NEKVQRELSHLQSENDAAKDEVKEVLQALEELA
			VNYDQKSQEVEEKSQQNQLLVDELSQKVATMLS
			LESELQRLQEVSGHQRKRIAEVLNGLMRDLSEF
			SVIVGNGEIKLPVEISGAIEEEFTVARLYISKI
			KSEVKSVVKRCRQLENLQVECHRKMEVTGRELS
			SCQLLISQHEAKIRSLTEYMQTVELKKRHLEES
			YDSLSDELARLQAHETVHEVALKDKEPDTQDAE
			EVKKALELQMENHREAHHRQLARLRDEINEKQK
			TIDELKDLNQKLQLELEKLQADYERLKNEENEK
			SAKLQELTFLYERHEQSKQDLKGLEETVARELQ
			TLHNLRKLFVQDVTTRVKKSAEMEPEDSGGIHS
			QKQKISFLENNLEQLTKVHKQLVRDNADLRCEL
			PKLEKRLRATAERVKALEFALKEAKEGAMKDKR
			RYQQEVDRIKEAVRYKSSGKRGHSAQIAKPVRP
			GHYPASSPTNPYGTRSPECISYTNNLFQNYQNL
			HLQAAPSSTSDMYFASSGRTSVAPLASYQKANM
			DNGNATDINDNRSDLPCGYEAEDQAKLEPLHQE
			TAAS
			(SEQ ID NO: 27)
SWISS_PR	P28742	Kinesin-like	MARSSLPNRRTAQFEANKRRTIAHAPSPSLSNG
OT:		protein KIP1	MHTLTPPTCNNGAATSDSNIHVYVRCRSRNKRE
KIP1_YEA			TEEKSSVVISTLGPQGKEIILSNGSHQSYSSSK
ST			KTYQFDQVFGAESDQETVFNATAKNYIKEMLHG
			YNCTIFAYGQTGTGKTYTMSGDTNILGDVQSTD
			NLLLGEHAGIIPRVLVDLFKELSSLNKEYSVKT
			SFLELYNENLKDLLSDSEDDDPAVNDPKRQIRT
			FDNNNNNSSIMVKGMQEIFINSAHEGLNLLMQG
			SLKRKVAATKCNDLSSRSHTVFTITTNIVEQDS
			KDHGQNKNFVKIGKLNLVDLAGSENINRSGAEN
			KRAQEAGLINKSLLTLGRVINALVDHSNHIPYR
			ESKLTRLLQDSLGGMTKTCIIATISPAKISMEE
			TASTLEYATRAKSIKNTPQVNQSLSKDTCLKDY
			IQEIEKLRNDLKNSRNKQGIFITQDQLDLYESN
			SILIDEQNLKIHNLREQIKKFKENYLNQLDINN
			LLQSEKEKLIAIIQNFNVDFSNFYSEIQKIHHT
			NLELMNEVIQQRDFSLENSQKQYNTNQNMQLKI
			SQQVLQTLNTLQGSLNNYNSKCSEVIKGVTEEL
			TRNVNTHKAKHDSTLKSLLNITTNLLMNQMNEL
			VRSISTSLEIFQSDSTSHYRKDLNEIYQSHQQF
			LKNLQNDTKSCLDSIGSSILTSINEISQNCTTN
			LNSMNVLIENQQSGSSKLIKEQDLEIKKLKNDL
			INERRISNQFNQQLAEMKRYFQDHVSRTRSEFH
			DELNKCIDNLKDKQSKLDQDIWQKTASIFNETD
			IVVNKIHSDSIASLAHNAENTLKTVSQNNESFT
			NDLISLSRGMNMDISSKLRSLPINEFLNKISQT
			ICETCGDDNTIASNPVLTSIKKFQNIICSDIAL
			TNEKIMSLIDETQSQIETISNENNINLIAINEN
			FNSLCNFILTDYDENIMQISKTQDEVLSEHCEK
			LQSLKILGMDIFTAHSIEKPLHEHTRPEASVIK
			ALPLLDYPKQFQIYRDAENKSKDDTSNSRTCIP
			NLSTNENFPLSQFSPKTPVPVPDQPLPKVLIPK
			SINSAKSNRSKTLPNTEGTGRESQNNLKRRFTT
			EPILKGEETENNDTLQNKKLHQ
			(SEQ ID NO: 28)
SWISS_PR	P28743	Kinesin-like	MIQKMSPSLRRPSTRSSSGSSNIPQSPSVRSTS
OT:		protein KIP2	SFSNLTRNSIRSTSNSGSQSISASSTRSNSPLR
KIP2_YEA			SVSAKSDPFLHPGRIRIRRSDSINNNSRKNDTY
ST			TGSITVTIRPKPRSVGTSRDHVGLKSPRYSQPR
			SNSHHGSNTFVRDPWFITNDKTIVHEEIGEFKF
			DHVEASHCTNLEVYERTSKPMIDKLLMGFNATI
			FAYGMTGSGKTFTMSGNEQELGLIPLSVSYLET
			NIMEQSMNGDKKEDVIISYLEIYNERIYDLLES
			GLEESGSRISTPSRLYMSKSNSNGLGVELKIRD
			DSQYGVKVIGLTERRCESSEELLRWIAVGDKSR
			KIGETDYNARSSRSHAIVLIRLTSTNVKNGTSR
			SSTLSLCDLAGSERATGQQERRKEGSFINKSLL
			ALGTVISKLSADKMNSVGSNIPSPSASGSSSSS
			GNATNNGTSPSNHIPYRDSKLTRLLQPALSGDS
			IVTTICTVDTRNDAAAETMNTLRFASRAKNVAL
			HVSKKSIISNGNNDGDKDRTIELLRRQLEEQRR
			MISELKNRSNIGEPLTKSSNESTYKDIKATGND
			GDPNLALMRAENRVLKYKLENCEKLLDKDVVDL
			QDSEIMEIVEMLPFEVGTLLETKFQGLESQIRQ
			YRKYTQKLEDKIMALEKSGHTAMSLTGCDGTEV
			IELQKMLERKDKMIEALQSAKRLRDRALKPLIN
			TQQSPHPVVDNDK
			(SEQ ID NO: 29)
SWISS_PR	P46822	Kinesin light	MSNMSQDDVTTGLRTVQQGLEALREEHSTISNT
OT:	Q18088	chain (KLC)	LETSVKGVKEDEAPLPKQKLSQINDNLDKLVCG
KLC_CAEE			VDETSLMLMVFQLTQGMDAQHQKYQAQRRRLCQ
L			ENAWLRDELSSTQIKLQQSEQMVAQLEEENKHL
			KYMASIKQLDDGTQSDTKTSVDVGPQPVTNETL
			QELGFGPEDEEDMNASQFNQPTPANQMAASANV
			GYEIPARLRTLHNLVIQYASQGRYEVAVPLCKQ
			ALEDLEKTSGHDHPDVATMLNILALVYRDQNKY
			KEAANLLNEALSIREKCLGESHPAVAATLNNLA
			VLFGKRGKFKDAEPLCKRALEIREKVLGDDHPD
			VAKQLNNLALLCQNQGKYEEVEKYYKRALEIYE
			SKLGPDDPNVAKTKNNLSSAYLKQGKYKEAEEL
			YKQILTRAHEREFGQISGENKPIWQIAEEREEN
			KHKGEGATANEQAGWAKAAKVDSPTVTTTLKNL
			GALYRRQGKYEAAETLEDVALRAKKQHEPLRSG
			AMGGIDEMSQSMMASTIGGSRNSMTTSTSQTGL
			KNKLMNALGFNS
			(SEQ ID NO: 30)
SWISS_PR	P46824	Kinesin light	MTQMSQDEIITNTKTVLQGLEALRVEHVSIMNG
OT:	Q9VU05	chain (KLC)	IAEVQKDNEKSDMLRKNIENIELGLSEAQVMMA
KLC_DROM			LTSHLQNIEAEKHKLKTQVRRLHQENAWLRDEL
E			ANTQQKFQASEQLVAQLEEEKKHLEFMASVKKY
			DENQEQDDACDKSRTDPVVELFPDEENEDRHNM
			SPTPPSQFANQTSGYEIPARLRTLHNLVIQYAS
			QGRYEVAVPLCKQALEDLERTSGHDRPDVATML
			NILALVYRDQNKYKEAANLLNDALSIRGKTLGE
			NHPAVAATLNNLAVLYGKRGKYKDAEPLCKRAL
			EIREKVLGKDHPDVAKQLNNLALLCQNQGKYDE
			VEKYYQRALDIYESKLGPDDPNVAKTKNNLAGC
			YLKQGRYTEAEILYKQVLTRAHEREFGAIDSKN
			KPIWQVAEEREEHKFDNRENTPYGEYCGWHKAA
			KVDSPTVTTTLKNLGALYRRQCMFEAAETLEDC
			AMRSKKEAYDLAKQTKLSQLLTSNEKRRSKAIK
			EDLDFSEEKNAKP
			(SEQ ID NO: 31)
SWISS_PR	P46825	Kinesin light	MEVTQTVKSYRIKKIEEIGKMTALSQEEIISNT
OT:		chain (KLC)	KTVIQGLDTLKNEHNQILNSLLTSMKTIRKENG
KLC_LOLP			DTNLVEEKANILKKSVDSIELGLGEAQVMMALA
E			NHLQHTEAEKQKLRAQVRRLCQENAWLRDELAN
			TQQKLQMSEQKVATIEEEKKHLEFMNEMKKYDT
			NEAQVNEEKESEQSSLDLGFPDDDDDGGQPEVL
			SPTQPSAMAQAASGGCEIPARLRTLHNLVIQYA
			SQGRYEVAVPLCKQALEDLEKTSGHDHPDVATM
			LNILALVYRDQGKYKEAANLLNDALGIREKTLG
			PDHPAVAATLNNLAVLYGKRGKYKDAEPLCKRA
			LVIREKVLGKDHPDVAKQLNNLALLCQNQGKYE
			EVERYYQRALEIYQKELGPDDPNVAKTKNNLAS
			AYLKQGKYKQAEILYKEVLTRAHEKEFGKVDDD
			NKPIWMQAEEREENKAKYKDGAPQPDYGSWLKA
			VKVDSPTVTTTLKNLGALYRRQGKYEAAETLEE
			CALRSRKSALEVVRQTKISDVLGSDFSKGQSPK
			DRKRSNSRDRNRRDSMDSVSYEKSGDGDEHEKS
			KLHVGTSHKQ
			(SEQ ID NO: 32)
SWISS_PR	Q05090	Kinesin light	MSGSKLSTPNNSGGGQGNLSQEQIITGTREVIK
OT:	Q05089	chain (KLC)	GLEQLKNEHNDILNSLYQSLKMLKKDTPGDSNL
KLC_STRP	Q05088		VEEKTDIIEKSLESLELGLGEAKVMMALGHHLN
U	Q04801		MVEAEKQKLRAQVRRLVQENTWLRDELAATQQK
			LQTSEQNLADLEVKYKHLEYMNSIKKYDEDRTP
			DEEASSSDPLDLGFPEDDDGGQADESYPQPQTG
			SGSVSAAAGGYEIPARLRTLHNLVIQYASQSRY
			EVAVPLCKQALEDLEKTSGHDHPDVATMLNILA
			LVYRDQNKYKEAGNLLHDALAIREKTLGPDHPA
			VAATLNNLAVLYGKRGKYKEAEPLCKRALEIRE
			KVLGKDHPDVAKQLNNLALLCQNQGKYEEVEWY
			YQRALEIYEKKLGPDDPNVAKTKNNLAAAYLKQ
			GKYKAAETLYKQVLTRAHEREFGLSADDKDNKP
			IWMQAEEREEKGKFKDNAPYGDYGGWHKAAKVD
			SRSRSSPTVTTTLKNLGALYRRQGKYDAAEILE
			ECAMKSRRNALDMVRETKVRELLGQDLSTDVPR
			SEAMAKERHHRRSSGTPRHGSTESVSYEKTDGS
			EEVSIGVAWKAKRKAKDRSRSIPAGYVEIPRSP
			PHVLVENGDGKLRRSGSLSKLRASVRRSSTKLL
			NKLKGRESDDDGGMKRASSMSVLPSRGNDESTP
			APIQLSQRGRVGSHDNLSSRRQSGNF
			(SEQ ID NO: 33)
SWISS_PR	Q42401	Matrilin-3	MRRALGTLGCCLALLLPLLPAARGVPHRHRRQP
OT:		precursor	LGSGLGRHGAADTACKNRPLDLVFIIDSSRSVR
MTN3_CHI			PEEFEKVKIELSKMIDTLDVGERTTRVAVMNYA
CK			STVKVEFPLRTYFDKASMKEAVSRIQPLSAGTM
			TGLAIQAAMDEVFTEEMGTRPANFNIPKVVIIV
			TDGRPQDQVENVAANARTAGIEIYAVGVGRADM
			QSLRIMASEPLDEHVFYVETYGVIEKLTSKFRE
			TFCAANTCALGTHDCEQVCVSNDGSYLCDCYEG
			YTLNPDKRTCSAVDVCAPGRHECDQICVSNNGS
			YVCECFEGYTLNPDKKTCSAMDVCAPGRHDCAQ
			VCRRNGGSYSCDCFEGFTLNPDKKTCSAVDVCA
			PGRHDCEQVCVRDDLFYTCDCYQGYVLNPDKKT
			CSRATTSSLVTDEEACKCEAIAALQDSVTSRLE
			ALSTKLDEVSQKLQAYQDRQQVV
			(SEQ ID NO: 34)
SWISS_PR	O15232	Matrilin-3	MPRPAPARRLPGLLLLLWPLLLLPSAAPDPVAR
OT:		precursor	PGFRRLETRGPGGSPGRRPSPAAPDGAPASGTS
MTN3_HUM			EPGRARGAGVCKSRPLDLVFIIDSSRSVRPLEF
AN			TKVKTFVSRIIDTLDIGPADTRVAVVNYASTVK
			IEFQLQAYTDKQSLKQAVGRITPLSTGTMSGLA
			IQTAMDEAFTVEAGAREPSSNIPKVAIIVTDGR
			PQDQVNEVAARAQASGIELYAVGVDRADMASLK
			MMASEPLEEHVFYVETYGVIEKLSSRPQETFCA
			LDPCVLGTHQCQHVCISDGEGKHHCECSQGYTL
			NADKKTCSALDRCALNTHGCEHICVNDRSGSYH
			CECYEGYTLNEDRKTCSAQDKCALGTHGCQHIC
			VNDRTGSHHCECYEGYTLNADKKTCSVRDKCAL
			GSHGCQHICVSDGAASYHCDCYPGYTLNEDKKT
			CSATEEARRLVSTEDACGCEATLAFQDKVSSYL
			QRLNTKLDDILEKLKINEYGQIHR
			(SEQ ID NO: 35)
SWISS_PR	O35701	Matrilin-3	MLLSAPLRHLPGLLLLLWPLLLLPSLAAPGRLA
OT:	Q9JHM0	precursor	RASVRRLGTRVPGGSPGHLSALATSTRAPYSGG
MTN3_MOU			RGAGVCKSRPLDLVFIIDSSRSVRPLEFTKVKT
SE			FVSRIIDTLDIGATDTRVAVVNYASTVKIEPQL
			NTYSDKQALKQAVARITPLSTGTMSGLAIQTAM
			EEAFTVEAGARGPMSNIPKVAIIVTDGRPQDQV
			NEVAARARASGIELYAVGVDRADMESLKMMASK
			PLEEHVFYVETYGVIEKLSARFQETFCALDQCM
			LGTHQCQHVCVSDGDGKHHCECSQGYTLNADGK
			TCSAIDKCALSTHGCEQICINDRNGSYHCECYG
			GYALNADRRTCAALDKCASGTHGCQHICVNDGA
			GSHHCECFEGYTLNADKKTCSVRNKCALGTHGC
			QHICVSDGAVAYHCDCFPGYTLNDDKKTCSDIE
			EARSLISIEDACGCGATLAFQEKVSSHLQKLNT
			KLDNILKKLKVTEYGQVHR
			(SEQ ID NO: 36)
SWISS_PR	Q24167	Similar protein	MVSLTDTIEAAAEKQKQSQAVVTNTSASSSSCS
OT:	Q9VAA5		SSESSSPPSSSVGSPSPGAPKTNLTASGKPKEK
SIMA_DRO			RRNNEKRKEKSRDAARCRRSKETEIFMELSAAL
ME			PLKTDDVNQLDKASVMRTTIAELKIREMLQFVP
			SLRDCNDDTKQDIETAEDQQEVKPKLEVGTEDW
			LNGAEARELLKQTMDGELLVLSHEGDITYVSEN
			VVEYLGITKIDTLGQQIWEYSHQCDHAEIKEAL
			SLKRELAQKVKDEPQQNSGVSTHHRDLFVRLKC
			TLTSRGRSINIKSASYKVIHITGHLVVNAKGER
			LLMAIGRPIPHPSNIEIPLGTSTELTKHSLDMR
			FTYVDDKMHDLLGYSPKDLLDTSLFSCQHGADS
			ERLMATFKSVLSKGQGETSRYRFLGKYGGYCWI
			LSQATIVYDKLKPQSVVCVNYVISNLENKHEIY
			SLAQQTAASEQKEQHHQAAETEKEPEKAADPEI
			IAQETKETVNTPIHTSELQAKPLQLESEKAEKT
			IEETKTIATIPPVTATSTADQIKQLPESNPYKQ
			ILQAELLIKRENHSPGPRTITAQLLSGSSSGLR
			PEEKRPKSVTASVLRPSPAPPLTPPPTAVLCKK
			TPLGVEPNLPPTTTATAAIISSSNQQLQIAQQT
			QLQNPQQPAQDMSKGFCSLFADDGRGLTMLKEE
			PDDLSHHLASTNCIQLDEMTPESDMLVGLMGTC
			LLPEDINSLDSTTCSTTASGQHYQSPSSSSTSA
			PSNTSSSNNSYANSPLSPLTPNSTATASNPSHQ
			QQQQHHNQQQQQQQQQQHHPQHHDNSNSSSNID
			PLFNYREESNDTSCSQHLHSPSITSKSPEDSSL
			PSLCSPNSLTQEDDFSEEAFAMRAPYIPIDDDM
			PLLTETDLMWCPPEDLQTMVPKEIDAIQQQLQQ
			LQQQHHQQYAGNTGYQQQQQQPQLQQQHFSNSL
			CSSPASTVSSLSPSPVQQHHQQQQAAVFTSDSS
			ELAALLCGSGNGTLSILAGSGVTVAEECNERLQ
			QHQQQQQQTSGNEERTFQQLQQELQLQEEQQQR
			QQQQQQQQQQQQQQQLLSLNIECKKEKYDVQMG
			GSLCHPMEDAEENDYSKDSANLDCWDLIQMQVV
			DTEPVSPNAASPTPCKVSAIQLLQQQQQLQQQQ
			QQQQNIILNAVPLITIQNNKELMQQQQQQQQQQ
			QQEQLQQPAIKLLNGASIAPVNTKATIRLVESK
			PPTTTQSRMAKVNLVPQQQQHGNKRHLNSATGA
			GNPVESKRLKSGTLCLDVQSPQLLQQLIGKDPA
			QQQTQAAKRAGSERWQLSAESKQQKQQQQQSNS
			VLKNLLVSGRDDDDSEAMITDEDNSLVQPIPLG
			KYGLPLHCHTSTSSVLRDYHNNPLISGTNFQLS
			PVPGGSDSSGGDGETGSVVSLDDSVPPGLTACD
			TDASSDSGIDENSLMDGASGSPRKRLSSTSNST
			NQAESAPPALDVETPVTQKSVEEEFEGGGSGSN
			APSRKTSISFLDSSNPLLHTPAMMDLVNDDYIM
			GEGGFEFSDNQLEQVLGWPEIA
			(SEQ ID NO: 37)
SWISS_PR	P23497	Nuclear	MAGGGGDLSTRRLNECISPVANEMNHLPAHSHD
OT:	Q13343	autoantigen Sp-	LQRMPTEDQGVDDRLLYDIVFKHEKRNKVEISN
SP10_HUM	O75450	100 (Speckled	AIKKTFPFLEGLRDRDLITNKMFEDSQDSCRNL
AN	Q9UE32	100 kDa)	VPVQRVVYNVLSELEKTFNLPVLEALFSDVNMQ
		(Nuclear dot-	EYPDLIHIYKGFENVIHDKLPLQESEEEEREER
		associatedSp10	SGLQLSLEQGTGENSFRSLTWPPSGSPSHAGTT
		0 protein)	PPENGLSEHPCETEQINAKRKDTTSDKDDSLGS
		(Lysp100b)	QQTNEQCAQKAEPTESCEQIAVQVNNGDAGREM
			PCPLPCDEESPEAELHNHGIQINSCSVRLVDIK
			KEKPFSNSKVECQAQARTHHNQASDIIVISSED
			SEGSTDVDEPLEVFISAPRSEPVINNDNPLESN
			DEKEGQEATCSRPQIVPEPMDFRKLSTFRESFK
			KRVIGQDHDFSESSEEEAPAEASSGALRSKHGE
			KAPMTSRSTSTWRIPSRKRRFSSSDFSDLSNGE
			ELQETCSSSLRRGSGSQPQEPENKKCSCVMCFP
			KGVPRSQEARTESSQASDMMDTMDVENNSTLEK
			HSGKRRKKRRHRSKVNGLQRGRKKDRPRKHLTL
			NNKVQKKRWQQRGRKANTRPLKRRRKRGPRIPK
			DENINEKQSELPVTCGEVKGTLYKERFKQGTSK
			KCIQSEDKKWFTPREFEIEGDRGASKNWKLSIR
			CGGYTLKVLMENKPLPEPPSTRKKRILESHNNT
			LVDPCEEHKKKNPDASVKFSEPLKKCSETWKTI
			FAKEKGKFEDMAKADKAHYEREMKTYIPPKGEK
			KKKFKDPNAPKRPPLAFFLPCSEYRPKIKGEHP
			GLSIDDVVKKLAGMWNNTAAADKQFYEKKAAKL
			KEKYKKDIAAYRAKGKPNSAKKRVVKAEKSKKK
			KEEEEDEEDEQEEENEEDDDK
			(SEQ ID NO: 38)
SWISS_PR	P04267	Tropomyosin 1,	MEAIKKKMQMLKLDKENAIDRAEQAEADKKQAE
OT:		smooth muscle	DRCKQLEEEQQGLQKKLKGTEDEVEKYSESVKE
TPM1_CR1		Gizzard beta-	AQEKLEQAEKKATDAEAEVASLNRRIQLVEEEL
CR		tropomyosin)	DRAQERLATALQKLEEAEKAADESERGMKVIEN
		(Smooth-	RAMKDEEKMELQEMQLKEAKHTAEEADRKYEEV
		musclealpha-	ARKLVVLEGELERSEERAEVAESRVRQLEEELR
		tropomyosin)	TMDQSLKSLIASEEEYSTKEDKYEEEIKLLGEK
		(Tropomyosin	LKEAETRAEPAERSVAKLEKTIDDLEESLASAK
		beta chain,	EENVGIHQVLDQTLLELNNL
		smooth muscle)	(SEQ ID NO: 39)
SWISS_PR	P09493	Tropomyosin	MDAIKKKMQMLKLDKENALDRAEQAEADKKAAE
OT:		alpha chain,	DRSKQLEDELVSLQKKLKGTEDELDKYSEALKD
TPM1_HUM		skeletal muscle	AQERLELAEKKATDAEADVASLNRRIQLVEEEL
AN		(Tropomyosin 1,	DRAQERLATALQKLEEAEKAADESERGMKVTES
		skeletal	RAQKDEEKMEIQEIQLKEAKHIAEDADRKYEEV
		muscle)	ARKLVIIESDLERAEERAELSEGKCAELEEELK
			TVTNNLKSLEAQAEKYSQKEDRYEEEIKVLSDK
			LKEAETRAEFAERSVTKLEKSIDDLEDELYAQK
			LKYRAISEELDHALNDMTSI
			(SEQ ID NO: 40)
SWISS_PR	P04268	Tropomyosin 2,	MDAIKKRMQMLKLDKENALDRAEQAEADKKAAE
OT:		smooth muscle	ERSKQLEDDIVQLEKQLRVTEDSRDQVLEELHK
TPM2_CRI		(Gizzard gamma-	SEDSLLSAEENAAKAESEVASLNRRTQLVEEEL
CK		tropomyosin)	DRAQERLATALQKLEEAEKAADESERCMKVIEN
		(Smooth-muscle	RAQKDEEKMEIQEIQLKEAKHIAEEADRKYEEV
		beta-	ARKLVILEGDLERAEERAELSESKCAELEEELK
		tropomyosin	LVTNEAKSLEAQAEKYSQKEDKYEEEIKVLTDK
			LKEAETRAEFAERSVTKLEKSIQDLEERVAHAK
			EENLNMHQMLDQTLLELNNM
			(SEQ ID NO: 41)
SWISS_PR	P19353	Tropomyosin	MAGISSIDAVKKKIQSLQQVADEAEERAEHLQR
QT:		beta 3,	EADAERQARERAEAEVASLNRRIQLVEEELDRA
TPM3C_RI		fibroblast	QERLATALQKLEEAEKAADESERGMKVIENRAM
CK			KDEEKMELQEMQLKEARHTAEEADRKYEEVARK
			LVVLEGELERSEERAEVAESRVRQLEEELRTMD
			QSLKSLIASEEEYSTKEDKYEEEIKLLGEKLKE
			AETRAEFAERSVAKLEKTIDDLEESLASAKEEN
			VGIHQVLDQTLLELNNL
			(SEQ ID NO: 42)
SWISS_PR	P06753	Tropomyosin	MEAIKKKMQMLKLDKENALDRAEQAEAEQKQAE
OT:		alpha chain,	ERSKQLEDELAAMQKKLKGTEDELDKYSEALKD
TPM3_HUM		skeletal muscle	AQEKLELAEKKAADAEAEVASLNRRIQLVEEEL
AN		type	DRAQERLATALQKLEEAEKAADESERGMKVIEN
		(Tropomyosin 3,	RALKDEEKMELQEIQLKEAKHIAEEADRKYEEV
		skeletal	ARKLVIIEGDLERTEERAELAESKCSELEEELK
		muscle)	NVTNNLKSLEAQAEKYSQKEDKYEEEIKILTDK
			LKEAETRAEFAERSVAKLEKTIDDLEDELYAQK
			LKYKAISEELDHALNDMTSI
			(SEQ ID NO: 43)
SWISS_PR	P49438	Tropomyosin	MAALSSLEAVRKKIRSLQEQADAAEERAGKLQR
OT:		alpha chain,	EVDQERALREEAESEVASLNRRIQLVEEELDRA
TPM5_CHI		major brain	QERLATALQKLEEAEKAADESERGMKVIENRAQ
CK		isoform	KDEEKMEIQEIQLKEAKHIAEEADRKYEEVARK
			LVIIEGDLERAEERAELSESKCAELEEELKTVT
			NNLKSLEAQAEKYSQKEDKYEEEIKVLTDKLKE
			AETRAEFAERSVTKLEKSIDDLEDQLYQQLEQN
			SRLTNELKLALNED
			(SEQ ID NO: 44)
SWISS_PR	P49439	Tropomyosin	MDAIKKKMQMLKLDKENALDRAEQAEADKKAAE
OT:		alpha chain,	ERSKQLEDELVALQKKLKGTEDELDKYSESLKD
TPM6_CHI		minor brain	AQEILELADKKATDAESEVASLNRRIQLVEEEL
CK		isoform	DRAQERLATALQKLEEAEKAADESERGMKVIEN
			RAQKDEEKMEIQEIQLKEAKHIAEEADRKYEEV
			ARKLVIIEGDLERAEERAELSESKCAELEEELK
			TVTNNLKSLEAQAEKYSQKEDKYEEEIKVLTDK
			LKEAETRAEFAERSVTKLEKSIDDLEDQLYQQL
			EQNSRLTNELKLALNED
			(SEQ ID NO: 45)
SWISS_PR	P13104	Tropomyosin	MDAIKKKMQMLKLDKENALDRAEQAETDKKAAE
OT:		alpha chain,	ERSKQLEDDLVALQKKLKATEDELDKYSEALKD
TPMA_BRA		skeletal muscle	AQEKLELAEKKATDAEGDVASLNRRIQLVEEEL
RE			DRAQERLATALQKLEEAEKAADESERGMKVIEN
			RALKDEEKMELQEIQLKEAKHIAEEADRKYEEV
			ARKLVIVEGELERTEERAELNECKCSELEEELK
			TVTNNMKSLEAQAEKYSAKEDKYEEEIKVLTDK
			LKEAETRAEFAERSVAKLEKTIDDLEDELYAQK
			LKYKAISEELDHALNDMTSI
			(SEQ ID NO: 46)
SWISS_PR	P02559	Tropomyosin	MDAIKKKMQMLKLDKENALDRAEQAEADKKAAE
OT:	P18442	alpha chain,	ERSKQLEDELVALQKKLKGTEDELDKYSESLKD
TPMA_COT		skeletal muscle	AQEKLELADKKATDAESEVASLNRRIQLVEEEL
JA			DRAQERLATALQKLEEAEKAADESERGMKVIEN
			RAQKDEEKMEIQEIQLKEAKHIAEEADRKYEEV
			ARKLVIIEGDLERAEERAELSESKCAELEEELK
			TVTNNLKSLEAQAEKYSQKEDKYEEEIKVLTDK
			LKEAETRAEFAERSVTKLEKSTDDLEDELYAQK
			LKYKAISEELDHALNDMTSI
			(SEQ ID NO: 47)
SWISS_PR	P02558	Tropomyosin	MDAIKKKMQMLKLDKENALDRAEQAEADKKAAE
OT:	P46902	alpha chain,	DRSKQLEDELVSLQKKLKGTEDELDKYSEALKD
TPMA_MOU	P99034	skeletal and	AQEKLELAEKKATDAEADVASLNRRIQLVEEEL
SE		cardiac muscle	DRAQERLATALQKLEEAEKAADESERGMKVIES
			RAQKDEEKMEIQEIQLKEAKHIAEDADRKYEEV
			ARKLVIIESDLERAEERAELSEGKCAELEEELK
			TVTNNLKSLEAQAEKYSQKEDKYEEEIKVLSDK
			LKEAETRAEFAERSVTKLEKSIDDLEDELYAQK
			LKYKAISEELDHALNDMTSI
			(SEQ ID NO: 48)
SWISS_PR	P13105	Tropomyosin	MDAIKKKMQMLKLDKENALDRAEQAEADKKGAE
OT:		alpha chain,	DKSKQLEDELVAMQKKMKGTEDELDKYSEALKD
TPMA_RAN		skeletal muscle	AQEKLELAEKKATDAEADVASLNRRIQLVEEEL
TE			DRAQERLATALQKLEEAEKAADESERGMKVIEN
			RALKDEEKIELQEIQLKEAKHIAEEADRKYEEV
			ARKLVIIEGDLERAEERAELSESKCAELEEELK
			TVTNNLKSLEAQAEKYSQKEDKYEEEIKVLTDK
			LKEAETRAEFAERTVAKLEKSIDDLEDELYAQK
			LKYKAISEELDHALNDMTSI
			(SEQ ID NO: 49)
SWISS_PR	P04692	Tropomyosin	MDAIKKKMQMLKLDKENALDRAEQAEADKKAAE
OT:		alpha chain,	DRSKQLEDELVSLQKKLKGTEDELDKYSEALKD
TPMA_RAT		skeletal muscle	AQEKLELAEKKATDAEADVASLNRRIQLVEEEL
			DRAQERLATALQKLEEAEKAADESERGMKVIES
			RAQKDEEKMEIQEIQLKEAKHIAEDADRKYEEV
			ARKLVIISSDLERAEERAELSEGKCAELEEELK
			TVTNNLKSLEAQAEKYSQKEDKYEEEIKVLSDK
			LKEAETRAEFAERSVTKLEKSIDDLEDELYAQK
			LKYKAISEELDHALKDMTSI
			(SEQ ID NO: 50)
SWISS_PR	Q01173	Tropomyasin	MDAIKKKMQMLKLDKENALDRAEQAEADKKGAE
OT:		alpha chain,	DKSKQLEDELVALQKKLKGTEDELDKYSEALKD
TPMA_XEN		skeletal muscle	AQEKLELSDKKATDAEGDVASLNRRIQLVEEEL
LA			DRAQERLSTALQKLEEAEKAADESERGMKVIEN
			RALEDEERMELCEIQLKEAKHIAEEADRKYEEV
			ARKLVIIEGDLERAEERAELSESKCAELEEELK
			TVTNNLKSLEAQAEKYSQKEDKYEEEIKVLTDK
			LKEAETRAEPAERTVAKLEKSIDDLEDELYAQK
			LKYKAISEELDHALNDMTSI
			(SEQ ID NO: 51)
SWISS_PR	P19352	Tropomyosin	MEAIKKKMQMLKLDKENAIDRAEQAEADKKQAE
OT:		beta chain,	DRCKQLEEEQQGLQKKLKGTEDEVEKYSESVKE
TPMB_CHI		skeletal muscle	AQEKLEQAEKKATDAEAEVASLNRRIQLVEEEL
CK			DRAQERLATALQKLEEAEKAADESERGMKVIEN
			RAMKDEEKMELQEMQLKEAKHIAEEADRKYEEV
			ARKLVVLEGELERSEERAEVAESKCGDLEEELK
			IVTNNLKSLEAQADKYSTKEDKYEEEIKLLGEK
			LKEAETRAEFAERSVAKLEKTIDDLEDEVYAQK
			MKYKAISEELDNALNDITSL
			(SEQ ID NO: 52)
SWISS_PR	P07951	Tropomyosin	MDAIKKKMQMLKLDKENAIDRAEQAEADKKQAE
OT:		beta chain,	DRQKQLEEEQQALQKKLKGTEDEVEKYSESVKE
TPMB_HUM		skeletal muscle	AQEKLEQAEKKATDAEADVASLNRRIQLVEEEL
AN		(Tropomyosin 2,	DRAQERLATALQKLEEAEKAADESERGMKVIEN
		skeletal	RAMKDEEKMELQEMQLKEAKHIAEDSDRKYEEV
		muscle)	ARKLVILEGELERSEEPAEVAESKCGDLEEELK
			IVTNNLKSLEAQADKYSTKEDKYEEEIKLLEEK
			LKEAETRAEFAERSVAKLEKTIDDLEDEVYAQK
			MKYKAISEELDNALNDITSL
			(SEQ ID NO: 53)
SWISS_PR	P02560	Tropomyosin	MDAIKKKMQMLKLDKENAIDRAEQAEADKKQAE
OT:		beta chain,	DRCKQLEEEQQALQKKLKGTEDEVEKYSESVKD
TPMB_MOU		skeletal muscle	AQEKLEQAEKKATDAEADVASLNRRIQLVEEEL
SE			DRAQERLATALQKLEEAEKAADESERGMKVIEN
			RAMKDEEKMELQEMQLKEAKHIAEDSDRKYEEV
			ARKLVILEGELERSEERAEVAESKCGDLEEELK
			IVTNNLKSLEAQADKYSTKEDKYEEEIKLLEEK
			LKEAETRAEPAERSVAKLEKTIDDLEDEVYAQK
			MKYKAISEELDNALNDITSL
			(SEQ ID NO: 54)
SWISS_PR	P42639	Tropomyosin	MDAIKKKMQMLKLDKENALDRADEAEADKKAAE
CT:		alpha chain,	DRSKQLEDELVSLQKKLKATEDELDKYSEALKD
TPMC_PIG		cardiac muscle	AQEKLELAEKKATDAEADVASLNRRIQLFEEEL
			DRAQERLATALQKLEEAEKAADESERGMKVIES
			RAQKDEEKMEIQEIQLKEAKHIAEDADRKYEEV
			ARKLVIIESDLERAEERAELSEGKCAELEEELK
			TVTNNLKSLEAQAEKYSQKEDKYEEEIKVLSDK
			LKEAETRAEFAERSVTKLEKSIDDLEDELYAQK
			LKYKAISEELDHALNDMTSI
			(SEQ ID NO: 55)
SWISS_PR	P18441	Tropomyosin	MDAIKKKMQMLKLDKENALDRAEQAEADKKAAE
CT:		alpha chain,	ERSKQLEDELVALQKKLKGTEDELDKYSESLKD
TPMF_CHI		fibroblast	AQEKLELADKKATDAESEVASLNRRIQLVEEEL
CK		isoform Fl	DRAQERLATALQKLEEAEKAADESERGMKVIEN
			RAQKDEEKMEIQEIQLKEAKHIAEEADRKYEEV
			ARKLVIIEGDLERAEERAELSESQVRQLEEQLR
			IMDQTLKALMAAEDKYSQKEDKYEEEIKVLTDK
			LKEAETRAEFAERSVTKLEKSIDDLEEKVAHAK
			EENLNMHQMLDQTLLELNNM
			(SEQ ID NO: 56)
SWISS_PR	P08942	Tropomyosin	MDAIKKKMQMLKLDKENALDRAEQAEADKKAAE
CT:		alpha chain,	ERSKQLEDELVALQKKLKGTEDELDKYSESLKD
TPMG_COT		fibroblast	AQEKLELADKKATDAESEVASLNRRIQLVEEEL
JA		isoform F2	DRAQERLATALQKLEEAEKAADESERGMKVIEN
			RAQKDEEKMEIQEIQLKEAKHIAEEADRKYEEV
			ARKLVIIEGDLERAEERAELSESKQAELEEELK
			TVTNNLKSLEAQAEKYSQKEDKYEEEIKVLTDK
			LKEAETRAEFAERSVTKLEKSIDDLEEKVAHAK
			EENLNMHQMLDQTLLELNNM
			(SEQ ID NO: 57)
SWISS_PR	Q01174	Tropomyosin	MAGITSLEAVKRKIKCLQDQADEAEERAEKLQR
OT:		alpha chain,	ERDMERKLREAAEGDVASLNRRIQLVEEELDRA
TPMN_XEN		non-muscle	QERLSTALQKLEEAEKAADESERGMKVIENRAL
LA			KDEEKMELQEIQLKEAKHIAEEADRKYEEVARK
			LVIIEGDLERAEERAELSESHYRQLEDQQRIMD
			QTLKTLIASEEKYSQKEDKYEEEIKVLTDKLKE
			AETRAEFAERTVAKLEKSIDDLEEKVAHAKEEN
			LNMHQMLDQTLLELNNM
			(SEQ ID NO 58)
SWISS_PR	P49436	Tropomyosin	MDAIKKKMQMLKLDKENALDRAEQAEADKKAAE
OT:		alpha chain,	ERSKQLEDDIVQLEKQLRVTEDSRDQVLEELHK
TPMS_CHI		smooth muscle	SEDSLLFAEENAAKAESEVASLNRRIQLVEEEL
CK			DRAQERLATALQKLEEAEKAADESERGMKVIEN
			RAQKDEEKMEIQEIQLKEAKHIAEEADRKYEEV
			ARKLVIIEGDLERAEERAELSESKCAELEEELK
			TVTNNLKSLEAQAEKYSQKEDKYEEEIKVLTDK
			LKEAETRAEFAERSVTKLEKSIDDLEEKVAHAK
			EENLNMHQMLDQTLLELNNM
			(SEQ ID NO: 59)
SWISS_PR	P49437	Tropomyosin	MDAIKKKMQMLKLDKENALDRAEQAEADKKAAE
OT:		alpha chain,	ERSKQLEDDIVQLEKQLRVTEDSRDQVLEELHK
TPMS_COT		smooth muscle	SEDSLLSAEEIAAKAESEVASLNRRIQLVEEEL
JA			DRAQERLATALQKLEEAEKAADESERGMKVIEN
			RAQKDEEKMEIQEIQLKEAKHIAEEADRKYEEV
			ARKLVIIEGDLERAEERAELSESKCAELEEELK
			TVTNNLKSLEAQAEKYSQKEDKYEEEIKVLTDK
			LKEAETRAEFAERSVTKLEKSIDDLEEKVAHAK
			EENLNMHQMLDQTLLELNNM
			(SEQ ID NO: 60)
SWISS_PR	P10469	Tropomyosin	CRLRIFLRTASSEHLHERKLRETAEADVASLNR
OT:		alpha chain,	RIQLVEEELDRAQERLATVLQKLEEAEKAADES
TPMS_HUM		smooth muscle	ERGMKVIESRAQKDEEKMEIQEIQLKEAKHIAE
AN		(Tropomyosin 1,	DADRKYEEVARKLVIIESDLERAEERAELSEGQ
		smooth muscle)	VRQLEEQLRIMDSDLESINAAEDKYSQKEDRYE
		(Fragment)	EEIKVLSDKLKEAETRAEFAERSVTKLEKSIDD
			LEEKVAHAKEENLSMHQMLDQTLLELNNM
			(SEQ ID NO: 61)
SWISS_PR	P06469	Tropomyosin	MDAIKKKMQMLKLDKENALDRAEQAEADKKAAE
OT:		alpha chain,	DRSKQLEEDISAKEKLLRASEDERDRVLEELHK
TPMS_RAT		smooth muscle	AEDSLLAADETAAKAEADVASLNRRIQLVEEEL
			DRAQERLATALQKLEEAEKAADESERGMKVIES
			RAQKDEEKMEIQEIQLKEAKHIAEDADRKYEEV
			ARKLVIIESDLERAEERAELSEGKCAELEEELK
			TVTNNLKSLEAQAEKYSQKEDKYEEEIKVLSDK
			LKEAETRAEFAERSVTKLEKSIDDLEEKVAHAK
			EENLSMHQMLHQTLLELNNM
			(SEQ ID NO: 62)
SWISS_PR	P18342	Tropomyosin	MDAIKKKMQMLKLDKENALDRAEQAEADKKAAE
OT:		alpha chain,	DRSKQLEDELVSLQKKLKATEDELDKYSEALKD
TPMX_RAT		brain-1	AQEKLELAEKKATDAEADVASLNRRIQLVEEEL
		(TMBR-1)	DRAQERLATALQKLEEAEKAADESERGMKVIES
			RAQKDEEKMEIQETQLKEAKHIAEDADRKYEEV
			ARKLVIIESDLERAEERAELSEGKCAELEEELK
			TVTNNLKSLEAQAEKYSQKEDKYEEEIKVLSDK
			LKEAETRAEFAERSVTKLEKSIDDLEDQLYHQL
			EQNRRLTNELKLALNED
			(SEQ ID NO: 63)
SWISS_PR	P18343	Tropomyosin	MAGSSSLEAVRRKIRSLQEQADAAEERAGSLQR
OT:		alpha chain,	ELDQERKLRETAEADVASLNRRIQLVEEELDRA
TPMY_RAT		brain-2	QERLATALQKLEEAEKAADESERGMKVIESRAQ
		(TMBR-2)	KDEEKMEIQEIQLKEAKHIAEDADRKYEEVARK
			LVIIESDLERAEERAELSEGKCAELEEELKTVT
			NNLKSLEAQAEKYSQKEDKYEEEIKVLSDKLKE
			AETRAEFAERSVTKLEKSIDDLEDKFLCFSPPK
			TPSSSRMSHLSELCICLLSS
			(SEQ ID NO: 64)
SWISS_PR	P18344	Tropomyosin	MAGSSSLEAVRRKIRSLQEQADAAEERAGSLQR
OT:		alpha chain,	ELDQERKLRETAEADVASLNRRIQLVEEELDRA
TPMZ_RAT		brain-3 (TMBR-	QERLATALQKLEEAEKAADESERGMKVIESRAQ
		3)	KDEEKMEIQEIQLKEAKHIAEDADRKYEEVARK
			LVIIESDLERAEERAELSEGKCAELEEELKTVT
			NNLKSLEAQAEKYSQKEDKYEEEIKVLSDKLKE
			AETRAEFAERSVTKLEKSIDDLEDQLYHQLEQN
			RRLTNELKLALNED
			(SEQ ID NO: 65)
SWISS_PR	P41541	General	MNFLRGVMGGQSAGPQHTEAETIQKLCDRVASS
OT:		vesicular	TLLDDRRNAVRALKSLSKKYRLEVGIQAMEHLI
VDP_BOVI		transport	HVLQTDRSDSEIIGYALDTLYNIISNDEEEEVE
N		factor p115	ENSTRQSEDLGSQFTEIFIKQQENVTLLLSLLE
		(Transcytosis	EFDFHVRWPGVKLLTSLLKQLGPQVQQIILVSP
		associated	MGVSRLMDLLADSREVTRNDGVLLLQALTRSNG
		protein)(TAP)	AIQKIVAFENAEERLLDIITEEGNSDGGIVVED
		(Vesicle	CLILLQNLLKNNNSNQNFFKEGSYIQRMKPWEE
		docking	VGDENSGWSAQKVTNLHLMLQLVRVLVSPNNPP
		protein)	GATSSCQKAMFQCGLLQQLCTTLMATGVPADIL
			TETINTVSEVIRGCQVNQDYEASVNAPSNPPRP
			AIVVLLMSMVNERQPFVLRCAVLYCFQCFLYKN
			QKGQGEIVSTLLPSTIDATGNTVSAGQLLCGGL
			FSTDSLSNWCAAVALAHALQENATQKEQLLRVQ
			LATSIGNPPVSLLQQCTNILSQGSKIQTRVGLL
			MLLCTWLSNCPIAVTHFLHNSANVPFLTGQIAE
			NLGEEEQLVQGLCALLLGISIYFNDNSLETYMK
			EKLKQLIEKRIGKENFIEKLGFISKHELYSRAS
			QKPQPNFPSPEYMIFDHEFTKLVKELEGVITKA
			IYKSSEEDKKEEEVKKTLEQHDSIVTHYKNMIR
			EQDLQLEELKQQISTLKCQNEQLQTAVTQQVSQ
			IQQHKDQYNLLKVQLGKDSQHQGPYTDGAQMNG
			VQPEBISRLREEIEELKSNRELLQSQLAEKDSL
			IENLKSSQLSPGTNEQSSATAGDSEQIAELKQE
			LATLKSQLNSQSVEITKLQTEKQELLQKTEAPA
			KSAPVPGESETVIATKTTDVEGRLSALLQETKE
			LKNEIKALSEERTAIKEQLDSSNSTIAILQNEK
			NKLEVDITDSKKEQDDLLVLLADQDQKIFSLKN
			KLKELGHPVEEEDELESGDQDDEDDEDEDDGKE
			QGHI
			(SEQ ID NO: 66)
SWISS_PR	P41542	General	MNFLRGVMGGQSAGPQHTEAETIQKLCDRVASS
OT:		vesicular	TLLDDRRNAVRALKSLSKKYRLEVGIQAMEHLI
VDP_RAT		transport	HVLQTDRSDSEIIAYALDTLYNIISNDEEEEVE
		factor p115	ENSTRQSEDLGSQFTEIFIKQPENVTLLLSLLE
		(Transcytosis	EFDFHVRWPGVRLLTSLLKQLGPPVQQIILVSP
		associatedpro-	MGVSKLMDLLADSREIIRNDGVLLLQALTRSNG
		tein) (TAP)	AIQKIVAFENAFERLLDIITEEGNSDGGIVVED
		(Vesicle	CLILLQNLLKNNNSNQNFFKEGSYIQRMKAWFE
		docking	VGDENPGWSAQKVTNLHLMLQLVRVLVSPTNPP
		protein)	GATSSCQKAMFQCGLLQQLCTILMATGIPADIL
			TETINTVSEVIRGCQVNQDYFASVNAPSNPPRP
			AIVVLLMSMVNERQPFVLRCAVLYCFQCFLYKN
			EKGQGEIVATLLPSTIDATGNSVSAGQLLCGGL
			ESTDSLSNWCAAVALAHALQGNATQKEQLLRVQ
			LATSIGNPPVSLLQQCTNILSQGSKIQTRVGLL
			MLLCTWLSNCPIAVTHFLHNSANVPPLTGQIAE
			NLGEEEQLVQGLCALLLGISIYFNDNSLENYTK
			EKLKQLIEKRIGKENYIEKLGFISKHELYSRAS
			QKPQPNFPSPEYMIFDHEFTKLVKELEGVITKA
			IYKSSEEDKKEEEVKKTLEQFIDNIVTHYKNMIR
			EQDLQLEELKQQVSTLKCQNEQLQTAVTQQASQ
			IQQHKDQYNLLKVQLGKDNHHQGSHSDGAQVNG
			IQPEEISRLREEIEELRSHQVLLQSQLAEKDTV
			IENLRSSQVSGMSEQALATCSPRDAEQVAELKQ
			ELSALKSQLCSQSLEITRLQTENSELQQRAETL
			AKSVPVEGESELVTAAKTTDVEGRLSALLQETK
			ELKNEIKALSEERTAIQKQLDSSNSTIAILQTE
			KDKLYLEVTDSKKEQDDLLVLLADQDQKILSLK
			SKLKDLGHPVEEEDESGDQEDDDDELDDGDRDQ
			DI
			(SEQ ID NO: 67)
TREMBL:	Q21049	PUTATIVE	LIPRINMSYSNGNINCDIMPTISEDGVDNGGPIDEPSDR
Q21049		ALPHA (LAR-	DNIEQLMMNMLEDRDKLQEQLENYKVQLENAGL
		INTERACTING	RTKEVEKERDMMKRQEEVHTQNLPQELQTMTRE
		PROTEIN ALPHA)	LCLLKEQLLEKDEEIVELKAERNNTRLLLEHLE
			CLVSRHERSLRMTVMKRQAQNHAGVSSEVEVLK
			ALKSLFEHHKALDEKVRERLRVAMERVATLEEE
			LSTKGDENSSLKARIATYAAEAEEAMASNAPIN
			GSISSESANRLIEMQEALERMKTELANSLKQST
			EITTRNAELEDQLTEDAREKHAAQESIVRLKNQ
			ICELDAQRTDQETRITTFESRFLTAQRESTCIR
			DLNDKLEHQLANKDAAVRLNEEKVHSLQERLEL
			AEKQLAQSLKKAESLPSVEAELQQRMEALTAAE
			QKSVSAEERIQRLDRNIQELSAELERAVQRERM
			NEEHSQRLSSTVDKLLSESNDRLQLHLKERMQA
			LDDKNRLTQQLDGTKKIYDQAERIKDRLQRDNE
			SLRQEIEALRQQLYNARTAQFQSRMHAIPFTHA
			QNIVQQQPQASIAQQSAYQMYKQQPAQQYQTVG
			MRRPNKGRISALQDDPNKVQTLNEQEWDRLQQA
			HVLANVQQAFSSSPSLADVGQSTLPRPNTAVQH
			QQDDMMNSGMGMPSGMQGGMQGGMGGGQDAQML
			ASMLQDRLDAINTEIRLIQQEKHHAERVAEQLE
			RSSREFYDDQGISTRSSPRASPQLDNMRQHKYN
			TLPANVSGDRRYDIYGNPQFVDDRMVRDLDYEP
			RRGYNQFDEMQYERDRMSPASSVASSTDGVLGG
			KKKRSNSSSGLKTLGRFFNKKKNSSSDLFKRNG
			DYSDGEQSGTEGNQKADYDRRKKKKHELLEEAM
			KARTPFALWNGPTVVAWLELWVGMPAWYVAACR
			ANVKSGAIMSALSDQEIQKEIGISNPLHRLKLR
			LAIQEMVSLTSPSAPRTARLTLAFGDMNHEYIG
			NDWLPCLGLAQYRSAFMECLLDARMLEHLSKRD
			LRTHLRMVDTFHRTSLQYGIMCLKKVNYDKKVL
			ADRRKACDNINTDLLVWSNERVQRWVEEIGLGV
			FSRNLVDSGIHGALIALDETFDASAFAYALQIG
			SQDVPNRQLLEKKFIGLVNDHRQQSDPHPRSGS
			SRKNDSIAKSYEFHLYT
			(SEQ ID NO: 68)
TREMBL:	Q94071	PUTATIVE LIPRIN	MYSRHSISDAYGAVCILPEDTLTVSSSQNSHID
Q94071		BETA (LAR-	AFAALVDRERDSSRSSGSGNIFKDNGSIKRRQA
		INTERACTING	LPYVTHYSDSGFGSAPSAGSSCSYLPPPPPYRM
		PROTEIN BETA)	RGSGGLSSKPQHKIHRSLSDSKYTASLMTTGVP
			TLPLLSMTPFNQLQSRDARGASWISLVRAPNFH
			LYCFFVFFFSFNIDETFRNSNISSPSPSMSTVS
			CPEYPELQDKLHRLAMARDSLQLQVSVLSEQVG
			AQKEKIKDLETVIALKRNNLTSTEELLQDKYHR
			IDECQELESKKMDLLAEVSSLKLRYATLEREKN
			ETEKKLRLSQNEMDRVNQSMHCMVVQQCLARHT
			NGHSSGGYMSPLREHRSEKNDDEMSQLRTAVQR
			LMADNEHKSLQINTLRNALDEQMRSRSQQEDFY
			ASQRNYTDNFDVNAQIRRILMDEPSDSMSHSTS
			FPVSLSSTTSNGKGPRSTVQSSSSYNSSLSAVS
			PQHNWSSAGAGTPRQLHPIGGNQRVNNITSAQY
			CSPSPPAARQLAAELDELRRNGNEGANHNYSSA
			SLPRGVGKASSTLTLPSKKLSVASGTSVVESDD
			EIARGRNLNNATSQSNLKNFSRERTRSSLRNIF
			SKLTRSTSQDQSNSFRRGSAARSTSTARLGSTN
			HLGTVSKRPPLSQFVDWRSEQLADWIAEIGYPQ
			YMNEVSRHVRSGRHFLNMSMNEYEGVLNIKNPV
			HRKRVAILLRRIEEDIMEPANKWDVHQTLRWLD
			DIGLPQYKDVFAENVVDGPLLLSLTANDAVEMK
			VVNAHHYATLARSIQFLKKADFRFNAMEKLIDQ
			NIVEKYPCPDVVVRWTHSATCEWLRKIDLAEFT
			QNLLFAGVPGALMIYEPSFTAESLAEILQMPPH
			KTLLRRHLTSHFNQLLGPKIIADKRDFLAAGNY
			PQISPTGRVKVVKKGFSLTRKKAKNEICLEPEE
			LLCPQVLVHKYPTGAGDNSSFESSNV
			(SEQ ID NO: 69)