US 5,582,981 AGrant
Method for Identifying an Oligonucleotide Aptamer Specific for a Target
Issue Date:1996-12-10
•12 Claims
•1 Drawing Sheets
Abstract
A method for identifying oligomer sequences which specifically bind target biomolecules is described. The technique involves complexation of the support-bound target molecule with a mixture of oligonucleotides containing random sequences and sequences which serve as primer for PCR under conditions wherein a complex is formed with the specifically binding sequences, but not with the other members of the oligonucleotide mixture. The target-oligonucleotide complexes are then separated from both the support and the uncomplexed oligonucleotides and the complexed members of the oligonucleotide mixture are recovered from the separated complex and subsequently amplified using the polymerase chain reaction. The recovered oligonucleotides may be sequenced, and successive rounds of selection using complexation, separation, amplification and recovery can be employed.
Metadata
Assignee
- Gilead Sciences, Inc.
Inventors
- John J. Toole
- John Latham
- Louis C. Bock
- Linda C. Griffin
Application Information
Application Number:US 2346133
Filing Date:1994-04-28
Priority Date:1991-08-14
Art Unit:187
Classifications
IPC:
C12P 1934C12Q 168
Field of Search:
4359356;91.277
Patent Drawings (1 sheets)
Description
TECHNICAL FIELD
The present invention is directed to a method for identifying oligonucleotide sequences which specifically bind target molecules. More specifically, the invention concerns an improved method to identify specific binding oligonucleotide sequences by incubating a pool of oligonucleotides with support-bound target molecules and detaching the resulting target-oligonucleotide complexes from the support.
BACKGROUND AND RELATED ART
Conventional methods of detection and isolation of proteins and other molecules have employed antibodies and the like which specifically bind such substances. Recently, however, the de novo design of specifically binding oligonucleotides for non-oligonucleotide targets that generally bind nucleic acids has been described. See, e.g., Blackwell, T. K., et al., Science (1990) 250:1104-1110; Blackwell, T. K., et al., Science (1990) 250:1149-1152; Tuerk, C., and Gold, L., Science (1990) 249:505-510; Joyce, G. F., Gene (1989) 82:83-87. Such oligonucleotides have been termed "aptamers" herein. Tuerk and Gold describe the use of a procedure termed "systematic evolution of ligands by exponential enrichment." In this method, a pool of RNAs that are completely randomized at specific positions is subjected to selection for binding by a desired nucleic acid-binding protein which has been fixed on a nitrocellulose filter. The bound RNAs then are recovered and amplified as double-stranded DNA that is competent for subsequent in vitro transcription. The newly transcribed RNA then is recycled through this procedure to enrich for oligonucleotides that have consensus sequences for binding by the cognate protein. The oligonucleotides so obtained then may be sequenced for further study. Tuerk and Gold applied this procedure to identify RNA oligonucleotides which are bound by the RNA binding region of T4 DNA polymerase.
Kinzler, K. W., et al., Nucleic Acids Res. (1989) 17:3645-3653, applied this technique to identify double-stranded DNA sequences that were bound by proteins that bind to DNA and regulate gene expression. In the reported work, total genomic DNA is first converted to a form that is suitable for amplification by PCR by ligation of linker sequences to the genomic DNA fragments and the DNA sequences of interest are selected by binding mediated by the target regulatory protein. The recovered bound sequences are then amplified by PCR. The process of binding by protein and amplification are repeated as needed. The selection and amplification process are repeated as needed. The process as described was applied to identify DNA sequences which bind to the Xenopus laevis transcription factor 3A. The same authors (Kinzler et al.) in a later paper, Mol. Cell Biol. (1990) 10:634-642, applied this same technique to identify the portion of the human genome which is bound by the GLI gene product produced as a recombinant fusion protein. The GLI gene is amplified in a subset of human tumors.
Ellington, A. D., et al., Nature (1990) 346: 818-822, describe the production of a large number of random sequence RNA molecules and identification of those which bind specifically to immobilized target molecules, in the case of this paper, to specific dyes such as Cibacron blue. Randomly synthesized DNA yielding approximately 10.sup.15 individual sequences was amplified by PCR and transcribed into RNA. It was thought that the complexity of the pool was reduced in the amplification/transcription steps to approximately 10.sup.13 different sequences. The pool was then applied to an affinity column containing the dye and the bound sequences subsequently eluted, treated with reverse transcriptase and amplified by PCR. The results showed that about one in 10.sup.10 random sequence RNA molecules folds in such a way as to bind specifically to the ligand.
Thiesen, H.-J., and Bach, C., Nucleic Acids Res. (1990) 18:3203-3208, describe what they call a target detection assay (TDA) to determine double-stranded DNA binding sites for putative DNA binding proteins. In their approach, a purified functionally active DNA binding protein and a pool of random double-stranded oligonucleotides which contain PCR primer sites at each end were incubated with the protein. The resulting DNA complexes with the protein (in their case, the SP-1 regulatory protein) were separated from the unbound oligomers in the random mixture by band-shift electrophoresis and the SP-1 bound oligonucleotides were rescued by PCR and cloned, and then sequenced.
Copending U.S. application Ser. No. 07/586,769, filed Sep. 21, 1990, the entire disclosure of which is hereby incorporated by reference, describes a method utilizing a binding site selection technique which depends on the availability of PCR (polymerase chain reaction). In this approach, selected and amplified oligonucleotides were used to identify consensus DNA recognition sequences used by DNA binding proteins under study.
None of the above references, however, describes a method in which the aptamer DNA-target complexes are isolated by coupling the target to a column support, and then purified by uncoupling the target-aptamer complex.
The invention herein provides such an approach and utilizes a binding site selection method combined with PCR to develop aptamers with selected and amplified binding sites. In a preferred embodiment this process is aided by consensus sequences.
DISCLOSURE OF THE INVENTION
The invention is directed to a method to determine DNA sequences which specifically bind proteins or other targets. The method is especially applicable to DNAs wherein a consensus sequence is identified. In this case, knowledge of the nature of the protein or other target which is bound is not necessarily a requisite. This technique has been applied to describe the nucleotide sequences responsible for binding certain proteins and polypeptides such as bradykinin and thrombin.
Accordingly, in one aspect, the invention is directed to a method to determine an oligonucleotide sequence which binds specifically to a target, which method comprises providing a mixture containing oligomers having portions which form a random set of sequences and portions which permit amplification of the oligomers, incubating the oligomer mixture with the target substance coupled to a support to form complexes between the target and the oligomers bound specifically thereto, removing the unbound members of the oligonucleotide mixture from the support environment, recovering the complexed oligonucleotide(s) by uncoupling the target substance from the support, amplifying the recovered oligonucleotides, and sequencing the recovered and amplified oligonucleotide(s) which had been complexed with the target. In a preferred embodiment, the mixture of oligonucleotides having random sequences also contains a consensus sequence known to bind to the target.
In other aspects, the invention is directed to oligonucleotides identified by the above method, and to oligonucleotide sequences which bind specifically to bradykinin and thrombin.
In still other aspects, the invention is directed to oligomers which contain sequences that bind specifically to target substances, and to the use of these oligomers in therapy, diagnostics, and purification procedures.
BRIEF DESCRIPTION OF THE FIGURE
FIG. 1 is a chart depicting thrombin aptamer consensus-related sequences (SEQ ID NO:18)(SEQ ID NO:19)(SEQ ID NO:20) (SEQ ID NO:21) (SEQ ID NO:22) (SEQ ID NO:23) (SEQ ID NO:24) (SEQ ID NO:25) (SEQ ID NO:26) (SEQ ID NO:27) (SEQ ID NO:28) (SEQ ID NO:29) (SEQ ID NO:30) (SEQ ID NO:31) (SEQ ID NO:32) (SEQ ID NO:33) (SEQ ID NO:34) (SEQ ID NO:35) (SEQ ID NO:36) (SEQ ID NO:37) (SEQ ID NO:38) (SEQ ID NO:39) (SEQ ID NO:40) (SEQ ID NO:41) (SEQ ID NO:42) (SEQ ID NO:43) (SEQ ID NO:44) (SEQ ID NO:45) (SEQ ID NO:46) (SEQ ID NO:47) (SEQ ID NO:48) (SEQ ID NO:49), which correspond to sequences number 1 through 32 in the left column of FIG. 1.
MODES OF CARRYING OUT THE INVENTION
The practice of the present invention encompasses conventional techniques of chemistry, molecular biology, biochemistry, protein chemistry, and recombinant DNA technology, which are within the skill of the art. Such techniques are explained fully in the literature. See, e.g., Oligonucleotide Synthesis (M. J. Gait ed. 1984); Nucleic Acid Hybridization (B. D. Hames & S. J. Higgins, eds., 1984); Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory Manual, Second Edition (1989); PCR Technology (H. A. Erlich ed., Stockton Press); R. K. Scope, Protein Purification Principles and Practice (Springer-Verlag); and the series Methods in Enzymology (S. Colowick and N. Kaplan eds., Academic Press, Inc.).
All patents, patent applications and publications mentioned herein, whether supra or infra, are hereby incorporated by reference in their entirety.
The invention is directed to a method which permits the recovery and deduction or identification of aptamers which bind specifically to desired targets, including polypeptides, short peptides, enzymes, proteins, lipids, glycolipids, phospholipids, leukotrienes, glycoproteins, carbohydrates, or cell surface molecules such as receptors, ion channels or extracellular matrix molecules. More specifically desired targets include thrombin, bradykinin, and PGF2.alpha.. As a result of application of this method, aptamers which contain the specifically binding sequences can be prepared and used in oligonucleotide-based therapy, in the detection and isolation of the target substance, as well as in other applications.
For example, these aptamers can be used as a separation tool for retrieving the targets to which they specifically bind. In these methods, the aptamers function much like monoclonal antibodies in their specificity and usage. By coupling the aptamers containing the specifically binding sequences to a solid support, desired target substances can be recovered in useful quantities. In addition, these aptamers can be used in diagnosis by employing them in specific binding assays for the target substances. When suitably labeled using detectable moieties such as radioisotopes, the specifically binding oligonucleotides can also be used for in vivo imaging or histological analysis.
Furthermore, when the aptamer specifically binds to biologically active sites on a biomolecule, that aptamer can be used therapeutically to affect that biological activity.
"Oligomers" or "oligonucleotides" include RNA or DNA sequences of more than one nucleotide in either single chain or duplex form and specifically includes short sequences such as dimers and trimers, in either single chain or duplex form, which may be intermediates in the production of the specifically binding oligonucleotides. "Nucleic acids", as used herein, refers to RNA or DNA sequences of any length in single-stranded or duplex form.
As used herein, the term "aptamer" or "specifically binding oligonucleotide" refers to an oligonucleotide that is capable of forming a complex with an intended target substance. The complexation is target-specific in the sense that other materials which may accompany the target do not complex to the aptamer. It is recognized that complexation and affinity are a matter of degree; however, in this context, "target-specific" means that the aptamer binds to target with a much higher degree of affinity than it binds to contaminating materials. The meaning of specificity in this context is thus similar to the meaning of specificity as applied to antibodies, for example.
The aptamer may be prepared by any known method, including synthetic, recombinant, and purification methods, and may be used alone or in combination with other aptamers specific for the same target. Further, as described more fully herein, the term "aptamer" specifically includes "secondary aptamers" containing a consensus sequence derived from comparing two or more known aptamers to a given target.
As used herein, the term "binding" refers to an interaction or complexation between a target and an oligonucleotide or aptamer, resulting in a sufficiently stable complex so as to permit separation of oligonucleotide:target complexes from uncomplexed oligonucleotides under given binding complexation or reaction conditions. Binding is mediated through hydrogen bonding or other molecular forces. As used herein, the term "binding" specifically excludes the normal "Watson-Crick"-type binding interactions (i.e., adenine-thymine and guanine-cytosine base-pairing) traditionally associated with the DNA double helix.
In general, a minimum of approximately 3 nucleotides, preferably at least 5 nucleotides, are necessary to effect specific binding. The only apparent limitations on the binding specificity of the target/oligonucleotide complexes of the invention concern sufficient sequence to be distinctive in the binding oligonucleotide and sufficient binding capacity of the target substance to obtain the necessary interaction. Oligonucleotides of sequences shorter than 10 may also be feasible if the appropriate interaction can be obtained in the context of the environment in which the target is placed. Although the oligonucleotides generally described herein are single-stranded or double-stranded, it is contemplated that aptamers may sometimes assume triple-stranded or quadruple-stranded structures.
As further explained below, the specifically binding oligonucleotides need to contain the sequence-conferring specificity, but may be extended with flanking regions and otherwise derivatized.
The aptamers found to bind to the targets may be isolated, sequenced, and then resynthesized as conventional DNA or RNA moieties, or may be "modified" oligomers which are those conventionally recognized in the art. As the resulting aptamers of the invention include intermediates in their synthesis, any of the hydroxyl groups ordinarily present may be replaced by phosphonate groups, phosphate groups, protected by a standard protecting group, or activated to prepare additional linkages to additional nucleotides, or may be conjugated to solid supports. The 5' terminal OH is conventionally free but may be phosphorylated; OH substituents at the 3' terminus may also be phosphorylated. The hydroxyls may also be derivatized to standard protecting groups. One or more phosphodiester linkages may be replaced by alternative linking groups. These alternative linking groups include, but are not limited to embodiments wherein P(O)O is replaced by P(O)S, P(O)NR.sub.2, P(O)R, P(O)OR', CO, or CNR.sub.2, wherein R is H or alkyl (1-20C) and R' is alkyl (1-20C); in addition, this group may be attached to adjacent nucleotide through O or S. Not all linkages in an oligomer need to be identical.
While the randomized portions of the oligonucleotides synthesized by the PCR procedure described below will contain the conventional bases adenine, guanine, cytosine, and thymine or uridine, the isolated aptamers may be sequenced and resynthesized as described above. Included within the invention are synthetic procedures in which the resultant aptamers incorporate analogous forms of purines and pyrimidines.
"Analogous" forms of purines and pyrimidines are those generally known in the art, many of which are used as chemotherapeutic agents. An exemplary but not exhaustive list includes aziridinylcytosine, 4-acetylcytosine, 5-fluorouracil, 5-bromouracil, 5-carboxymethylaminomethyl-2-thiouracil, 5-carboxymethylaminomethyluracil, inosine, N6-isopentenyladenine, 1-methyladenine, 1-methylpseudouracil, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-methyladenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5-methoxyuracil, 2-methylthio-N-6-isopentenyladenine, uracil-5-oxyacetic acid methylester, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid, and 2,6-diaminopurine.
Aptamer oligonucleotides may contain analogous forms of ribose or deoxyribose that are generally known in the art. An exemplary, but not exhaustive list includes 2' substituted sugars such as 2'-O-methyl-, 2'-fluoro- or 2'-azido-ribose, carbocyclic sugar analogs, .alpha.-anomeric sugars, epimeric sugars such as arabinose, xyloses or lyxoses, pyranose sugars, sedoheptuloses, acyclic analogs and abasic nucleoside analogs such as methyl riboside.
In most instances, the conventional sugars and bases will be used in applying the method of the invention; substitution of analogous forms of sugars, purines and pyrimidines may be advantageous in designing the final product. Modified bases are disclosed in the copending U.S. application Ser. No. 07/658,849.
Aptamers containing the specific binding sequences discerned through the method of the invention can also be derivatized in various ways. For example, if the aptamer is to be used for separation of the target substance, conventionally the oligonucleotide will be derivatized to a solid support to permit chromatographic separation. If the oligonucleotide is to be used to label cellular components or otherwise for attaching a detectable moiety to target, the oligonucleotide will be derivatized to include a radionuclide, a fluorescent molecule, a chromophore or the like. If the oligonucleotide is to be used in specific binding assays, coupling to solid support or detectable label is also desirable. If it is to be used therapeutically, the oligonucleotide may be derivatized to include ligands which permit easier transit of cellular barriers, toxic moieties which aid in the therapeutic effect, or enzymatic activities which perform desired functions at the targeted site. The aptamer may also be included in a suitable expression system to provide for in situ generation of the desired sequence.
The oligonucleotides used as starting materials in the process of the invention to determine specific binding sequences may be single-stranded or double-stranded DNA or RNA. In the preferred embodiment of this invention, the sequences are single-stranded DNA. The use of DNA eliminates the need for conversion of RNA aptamers to DNA by reverse transcriptase prior to PCR amplification. Furthermore, DNA is less susceptible to plasma nuclease degradation than RNA. The starting material oligonucleotide will contain a randomized sequence portion, generally including from about 10 to 400 nucleotides, more preferably 20 to 100 nucleotides. The randomized sequence is flanked by primer sequences which permit the application of the polymerase chain reaction to the recovered oligonucleotide from the complex. The flanking sequences may also contain other convenient features, such as restriction sites which permit the cloning of the amplified sequence. These primer hybridization regions generally contain 10 to 30, more preferably 15 to 25, and most preferably 18 to 20, bases of known sequence.
Both the randomized portion and the primer hybridization regions of the initial oligomer population are preferably constructed using conventional solid phase techniques. Such techniques are well known in the art, such methods being described, for example, in Froehler, B., et al., Nucleic Acids Research (1986) 14:5399-5467; Nucleic Acids Research (1988) 16:4831-4839; Nucleosides and Nucleotides (1987) 6:287-291; Froehler, B., Tet Lett (1986) 27:5575-5578. Oligonucleotides may also be synthesized using solution phase methods such as triester synthesis, known in the art. For synthesis of the randomized regions, mixtures of nucleotides at the positions where randomization is desired are added during synthesis.
Any degree of randomization may be employed. Some positions may be randomized by mixtures of only two or three bases rather than the conventional four. Randomized positions may alternate with those which have been specified. Indeed, it is helpful if some portions of the candidate randomized sequence are in fact known.
While the method of the invention is illustrated using thrombin, bradykinin and PGF2.alpha. as the target substances, any biomolecule can be used as the target. Thus, other desired targets, including polypeptides, enzymes, proteins, lipids, glycolipids, phospholipids, leukotrienes, lipoproteins, glycoproteins, carbohydrates, or cell surface molecules, and more specifically thrombin and bradykinin, can be used. Target molecules that are not conventionally considered to be biomolecules are also appropriate for the methods described herein. Examples of "non-biomolecule" targets include intermediates or end-products generated by chemical synthesis of compounds used in therapeutic, manufacturing or cosmetic applications. Aptamer oligonucleotides may be used to specifically bind to most organic compounds and are suitably used for isolation or detection of such compounds.
The Method
Generally, the method for selecting aptamers that bind the desired target substances is as follows. First, a pool of single-stranded random DNA oligomers is generated using conventional synthesis techniques. As described above, each oligomer contains both a randomized sequence as well as flanking regions of known sequence to serve as primer binding sites for PCR amplification. The synthesized single-stranded DNA pool is amplified by PCR. The amplified pool may be left as duplex DNA, or single-stranded DNA may be recovered by subsequent strand separation, and optionally transcribed into RNA.
Next, a column or other support matrix having covalently or noncovalently coupled target molecules is synthesized. Any standard coupling reagent or procedure may be utilized, depending on the nature of the support and the target molecule. For example, covalent binding may include the formation of disulfide, ether, ester or amide linkages. The length of the linkers used may be varied by conventional means. Noncovalent linkages include antibody-antigen interactions, protein-sugar interactions, as between, for example, a lectin column and a naturally-occurring oligosaccharide unit on a peptide.
Lectins are proteins or glycoproteins that can bind to complex carbohydrates or oligosaccharide units on glycoproteins, and are well-described in The Lectins (I. E. Liener et al., eds., Academic Press 1986). Lectins are isolated from a wide variety of natural sources, including peas, beans, lentils, pokeweed and snails. Concanavalin A is a particularly useful lectin.
Other linking chemistries are also available. For example, disulfide-derivatized biotin (Pierce) may be linked to a target molecule by coupling through an amine or other functional group. The resulting target-S-S-biotin complex could then be used in combination with avidin-derivatized support. Oligonucleotide-target complexes could then be recovered by disulfide bond cleavage. Alternatively, target may be coupled via a cis-diol linker, and oligonucleotide-target complexes may be recovered by mild oxidation of the vicinal diol bond using NaIO.sub.4 or other appropriate reagents. Linking chemistries will be selected on the basis of (i) conditions or reagents necessary for maintaining the structure or activity of the target molecule, and/or (ii) chemical groups or moieties on the target molecule available for linking to the support.
Once the oligomer pool and target-coupled support have been synthesized, the oligomers are added to and incubated with the support to permit oligonucleotide-target complexation. Complexes between the oligonucleotide and target molecule are separated from uncomplexed oligonucleotides by removing unbound oligomers from the support environment. For example, if columns are used, nonbinding species are simply washed from the column using an appropriate buffer.
Following removal of unbound oligomers, the target molecules are uncoupled from the support. The uncoupling procedure depends on the nature of the coupling, as described above. Targets bound through disulfide linkages, for example, may be removed by adding a sulfhydryl reagent such as dithiothreitol or .beta.-mercaptoethanol. Targets bound to lectin supports may be removed by adding a complementary monosaccharide (e.g., .alpha.-methyl-mannoside for concanavalin A). Oligonucleotides specifically bound to the target can then be recovered by standard denaturation techniques such as phenol extraction.
The method of elution of target-oligonucleotide complex from a support has superior unexpected properties when compared with standard oligonucleotide elution techniques. This invention is not dependent on the mechanism by which these superior properties occur. However, without wishing to be limited by any one mechanism, the following explanation is offered as to how more efficient elution is obtained. Certain support effects result from the binding of oligonucleotides to the support, or the support in conjunction with oligonucleotide or target. Removing oligonucleotide-target complexes enables the recovery of oligonucleotides specific to target only, while eliminating oligonucleotides binding to the support, or the support in conjunction with oligonucleotide or target. At each cycle of selection, this method may give up to 1,000 fold enrichment for specifically binding species. Selection with targets remaining bound to support gives less enrichment per cycle, making it necessary to go through many more cycles in order to get a good aptamer population.
Once oligonucleotides that bind to the target are separated from the rest of the pool and further separated from the target molecule, the oligonucleotides are amplified by PCR to give a pool of DNA sequences. The PCR method is well known in the art and described in, e.g., U.S. Pat. Nos. 4,683,195 and 4,683,202 and Saiki, R. K., et al., Science (1988) 239:487-491, and European patent applications 86302298.4, 86302299.2 and 87300203.4, as well as Methods in Enzymology (1987) 155:335-350. If RNA is initially used, the amplified DNA sequences are transcribed into RNA. The recovered DNA or RNA, in the original single-stranded or duplex form, is then used in another round of selection and amplification. After three to six rounds of selection/amplification, oligomers that bind with an affinity in the mM to .mu.M range can be obtained for most targets and affinities below the .mu.M range are possible for some targets. PCR may also be performed in the presence of target.
Amplified sequences can be cloned and individual oligonucleotides then sequenced. The entire process can then be repeated using the recovered and amplified oligomers as needed. Once an aptamer that binds specifically to a target has been selected, it may be recovered as DNA or RNA in single-stranded or duplex form using conventional techniques.
When a number of individual, distinct aptamer sequences for a single target molecule have been obtained and sequenced as described above, the sequences may be examined for "consensus sequences." As used herein, "consensus sequence" refers to a nucleotide sequence or region (which may or may not be made up of contiguous nucleotides), which is found in one or more regions of at least two aptamers, the presence of which may be correlated with aptamer-to-target-binding or with aptamer structure. A consensus sequence may be as short as three nucleotides long. It also may be made up of one or more noncontiguous sequences with nucleotide sequences or polymers of hundreds of bases long interspersed between the consensus sequences. Consensus sequences may be identified by sequence comparisons between individual aptamer species, which comparisons may be aided by computer programs and other tools for modeling secondary and tertiary structure from sequence information. Generally, the consensus sequence will contain at least about 3 to 20 nucleotides, more commonly from 6 to 10 nucleotides. When a consensus sequence is identified, oligonucleotides that contain that sequence may be made by conventional synthetic or recombinant means. These aptamers, termed "secondary aptamers," may also function as target-specific aptamers of this invention. A secondary aptamer may conserve the entire nucleotide sequence of an isolated aptamer, or may contain one or more additions, deletions or substitutions in the nucleotide sequence, as long as a consensus sequence is conserved. A mixture of secondary aptamers may also function as target-specific aptamers, wherein the mixture is a set of aptamers with a portion or portions of their nucleotide sequence being random or varying, and a conserved region which contains the consensus sequence.
Aptamers can also be selected using a pool of oligonucleotides that vary in length as the starting material. Thus, several pools of random sequence-containing oligonucleotides that vary in length from, e.g., 10 to 400 bases, and contain the same flanking primer-binding sequences, are synthesized. Equimolar amounts of each pool are mixed and the variable-length pool is then used to select for aptamers that bind to the desired target substance, as described above. This protocol selects for the optimal species for target binding from the starting pool and does not limit aptamers to those of one given length.
Alternatively, several pools of mixed length aptamers can be used in parallel in separate selections and then combined and further selected to obtain the optimal binders from the size range initially used. For example, three pools, A, B and C, can be used. Pool A can consist of oligonucleotides having random sequences that vary in length from 30 to 40 bases; pool B can have sequences varying in length from 40 to 50 bases; and pool C can have sequences varying in length from 50 to 60 bases. It is to be understood that the lengths described above are for illustrative purposes only. After selection to obtain binders from A, B, and C, the selected oligonucleotides are mixed together. A number of rounds of selection are done as described above to obtain the best binders from the initial species selected in the 30- to 60-base range.
It is pointed out that copending, commonly assigned U.S. application Ser. No. 07/659,980 discloses methods for isolating aptamers that have no known flanking sequences. The above-referenced U.S. patent application is incorporated herein by reference for disclosing such methodology. Such amplification methods may be used in combination with the methods disclosed in the present application.
Utility of the Retrieved Sequence
The aptamers of the invention are useful in diagnostic, research and therapeutic contexts. For diagnostic applications, these aptamers are particularly well suited for binding to biomolecules that are identical or similar between different species, where standard antibodies may be difficult to obtain. They are also useful in inhibition assays when the aptamers are chosen to inhibit the biological activity of its target. Antibodies are generally used to bind analytes that are detected or quantitated in various diagnostic assays. Aptamers represent a class of molecules that may be used in place of antibodies for in vitro or in vivo diagnostic and purification purposes.
The aptamers of the invention are therefore particularly useful as diagnostic reagents to detect the presence or absence of the target substances to which they specifically bind. Such diagnostic tests are conducted by contacting a sample with the specifically binding oligonucleotide to obtain a complex which is then detected by conventional means. For example, the aptamers may be labeled using radioactive, fluorescent, or chromogenic labels and the presence of label bound to solid support to which the target substance has been bound through a specific or nonspecific binding means detected. Alternatively, the specifically binding oligonucleotides may be used to effect initial complexation to the support. Means for conducting assays using such oligomers as specific binding partners will track those for standard specific binding partner based assays.
It may be commented that the mechanism by which the specifically binding oligomers of the invention interfere with or inhibit the activity of a target substance is not always established, and is not a part of the invention. The oligomers of the invention are characterized by their ability to bind specific target molecules regardless of the mechanisms of binding or the mechanism of the effect thereof.
For use in research or manufacturing, the specifically binding oligonucleotides of the invention are especially helpful in effecting the isolation and purification of substances to which they bind. For this application, typically, the aptamer containing the specific binding sequences is conjugated to a solid support and used as an affinity ligand in chromatographic separation of the target substance.
In therapeutic applications, the aptamers of the invention can be formulated for a variety of modes of administration, including systemic and topical or localized administration. Techniques and formulations generally may be found in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., latest edition. In general, the dosage required for therapeutic efficacy will range from about 0.1 .mu.g to 20 mg aptamer/kg body weight. Alternatively, dosages within these ranges can be administered by constant infusion over an extended period of time, usually exceeding 24 hours, until the desired therapeutic benefits have been obtained.
For systemic administration, injection is preferred, including intramuscular, intravenous, intraperitoneal, and subcutaneous. For injection, the aptamers of the invention are formulated in liquid solutions, preferably in physiologically compatible buffers such as Hank's solution or Ringer's solution. In addition, the aptamers may be formulated in solid form and redissolved or suspended immediately prior to use. Lyophilized forms are also included.
Systemic administration can also be by transmucosal or transdermal means, or the oligomers can be administered orally. Additional formulations which are suitable for other modes of administration include suppositories, intranasal and other aerosols. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration bile salts and fusidic acid derivatives. In addition, detergents may be used to facilitate permeation. Transmucosal administration may be through nasal sprays, for example, or using suppositories. For oral administration, the oligomers are formulated into conventional oral administration forms such as capsules, tablets, and tonics.
For topical administration, the oligomers of the invention are formulated into ointments, salves, gels, or creams, as is generally known in the art.
The aptamers may also be employed in expression systems, which are administered according to techniques applicable, for instance, in applying gene therapy.
The following examples are meant to illustrate, but not to limit the invention.
EXAMPLE 1
Synthesis of Oligonucleotide Pool
DNA oligonucleotides containing a randomized sequence region were synthesized using standard solid phase techniques and phosphoramidite chemistry (Oligonucleotide Synthesis, Gait, M. J., ed. (IRL Press), 1984; Cocuzza, A., Tetrahedron Letters, (1989) 30:6287-6291). A 1 .mu.M small-scale synthesis yielded 60 nmole of HPLC-purified single-stranded randomized DNA. Each strand consisted of specific 18 mer sequences at both the 5' and 3' ends of the strand and a random 60 mer sequence in the center of the oligomer to generate a pool of 96 mers with the following sequence (N=G, A, T or C):
DNA 18 mers with the following sequences were used as primers for PCR amplification of oligonucleotide sequences recovered from selection columns. The 5' primer sequence was 5' HO-CGTACGGTCGACGCTAGC-OH 3' (SEQ ID NO: 2) and the 3' primer sequence was 5' biotin-O-GGATCCGAGCTCCACGTG-OH 3' (SEQ ID NO: 3). The biotin residue was linked to the 5' end of the 3' primer using commercially available biotin phosphoramidite (New England Nuclear, Cat. No. NEF-707). The biotin phosphoramidite is incorporated into the strand during solid phase DNA synthesis using standard synthesis conditions.
In another, similar experiment, a pool of 98 mers with the following sequence was synthesized:
DNA 19 mers with the following sequences can also be used as primers for PCR amplification of oligonucleotides recovered from selection columns. The 3' primer sequence is
and the 5' primer sequence is
It will be noted that in all cases, the duplex form of the primer binding sites contain restriction enzyme sites.
EXAMPLE 2
Isolation of Thrombin Aptamers Using Thrombin Immobilized on a Lectin Column
A pool of aptamer DNA 96 bases in length was synthesized as described in Example 1, and then PCR-amplified to construct the initial pool. A small amount of the enzymatically-synthesized DNA was further amplified in the presence of .alpha.-.sup.32 P-dNTPs to generate labeled aptamer to permit quantitation from column fractions.
A thrombin column was prepared by washing 1 ml (58 nmole) agarose-bound concanavalin A ("Con-A") (Vector Laboratories, cat. no. AL-1003) with 20 mM Tris-acetate buffer (pH 7.4) containing 1 mM MgCl.sub.2, 1 mM CaCl.sub.2, 5 mM KCl and 140 mM NaCl (the "selection buffer") (4.times.10 ml). 1 ml of settled support was then incubated overnight at 4.degree. C. in 10 ml selection buffer containing 225 .mu.g (6.25 nmole) thrombin (Sigma, Cat. no. T-6759). After shaking overnight to permit thrombin binding to the Con-A beads, the mixture was briefly centrifuged and the supernatant removed. The beads were resuspended in fresh selection buffer and transferred to a column which was then washed with selection buffer (5.times.1 ml). A column containing 1 ml of settled beads had a void volume of approximately 300 .mu.L. A control Con-A column was prepared by adding 1 ml of settled support to a column followed by 5 washes of 1 ml of selection buffer.
Prior to application of the aptamer DNA pool to Con-A columns, the DNA was heated in selection buffer at 95.degree. C. for 3 minutes and then cooled on ice for 10 minutes. The pool, consisting of 100 pmole DNA in 0.5 ml selection buffer, was then pre-run on the control Con-A column at room temperature to remove species that bound to the control support. Three additional 0.5 ml aliquots of selection buffer were added and column fractions 2, 3 and 4 (0.5 ml each) were pooled and then reapplied to the column twice. The DNA in 1.5 ml selection buffer was then recovered. Approximately 1% of total input cpm were retained on the column.
The recovered DNA was then applied to a Con-A-thrombin column as a 0.5 ml aliquot followed by a 1.0 ml aliquot. Flow-through was retained and reapplied to the column twice. DNA added to the column on the final application was left on the column for 1 hour at room temperature. The column was then eluted with 0.5 ml aliquots of selection buffer. 0.5 ml fractions were collected and radioactivity was determined in each fraction. Radioactivity in eluted fractions 7 through 12 were low and relatively constant. After recovery of fraction 12, the column was washed with 0.5 ml aliquots of 0.1M .alpha.-methyl-mannoside (Sigma Cat. no. M-6882) in selection buffer to elute the bound thrombin along with thrombin-bound aptamers. Fractions 14 and 15 showed a significant peak of thrombin enzyme activity, as determined spectrophotometrically by conversion of a chromogenic substrate (Kabi Diagnostica, Cat. no. S-2238). 0.01% of the input DNA eluted in these two fractions.
Aptamer DNA (Round 1 DNA) was recovered from the thrombin by phenol extraction (2.times.0.5 ml). The aqueous phase volume was reduced to about 250 .mu.l by n-butanol extraction. Aptamer DNA was precipitated on dry ice using 3 volumes of ethanol and 20 .mu.g of glycogen as a carrier. The DNA was pelleted, washed once in 70% ethanol and then dried.
EXAMPLE 3
Amplification and Isolation of Single-Stranded DNA
Round 1 DNA from Example 2 was resuspended in 100 .mu.l of H.sub.2 O and amplified by PCR. A 200 .mu.l PCR reaction consisted of the following: 100 .mu.l template 96 mer DNA (approximately 0.01 pmoles); 20 .mu.l 10X buffer (100 mM Tris.Cl (pH 8.3), 500 mM KCl, 20 mM MgCl.sub.2); 32 .mu.l dNTP's (5 mM conc total, 1.25 mM each dATP, dCTP, dGTP, and dTTP); 20 .mu.l primer 1 (biotinylated 18 mer, 50 .mu.M); 20 .mu.l primer 2 (18 mer, 50 .mu.M); 6 .mu.l .alpha.-.sup.32 P-dNTP's (approximately 60 .mu.Ci); and 2 .mu.l Taq I Polymerase (10 units). The reaction was covered with 2 drops NUJOL mineral oil. A control reaction was also performed without template aptamer.
Initial denaturation was at 94.degree. C. for 3 minutes, but subsequent denaturation after each elongation reaction lasted 1 minute. Primer annealing occurred at 60.degree. C. for 1 minute, and elongation of primed DNA strands using the Taq polymerase ran at 72.degree. C. for 2 minutes, with 5-second extensions added at each additional cycle. The final elongation reaction to completely fill in all strands ran for 10 minutes at 72.degree. C., and the reaction was then held at 4.degree. C.
18 rounds of Taq polymerase elongation were carried out in order to amplify the selected aptamer DNA. After the reactions were completed, the aqueous layer was retrieved and any residual NUJOL oil was removed by n-butanol extraction, reducing the volume to 100 .mu.l. A sample may be removed from each of the aptamer and control reaction for quantitation and analytical PAGE. The amplified aptamer pool (100 .mu.l) was run over a Nick column (G-50 Sephadex, washed with 3 ml TE buffer (10 mM Tris.HCl (pH 7.6), 0.1 mM EDTA)) to remove unincorporated NTP's, primers, and salt. 400 .mu.l of TE buffer was then added to the column and the DNA pool was eluted from the column with an additional 400 .mu.l using TE buffer. (A sample may be removed from the eluent for quantitation and analytical PAGE.) The eluent (400 .mu.l) was loaded on an avidin agarose column (Vector Laboratories, Cat. No. A-2010) (500 .mu.l settled support, washed with 3.times.1 ml Tris/NaCl buffer (0.1M Tris, 0.1M NaCl, pH 7.5)). Approximately 90% of the loaded radioactivity remained on the column. The column was washed with Tris/NaCl buffer (4.times.400 .mu.l) and then the nonbiotinylated strand was eluted with 0.15N NaOH (3.times.300 .mu.l fractions). More than 45% of the radioactivity on the column eluted in these three fractions. These fractions (900 .mu.l) were combined and neutralized with approximately 3.5 .mu.l of glacial acetic acid. The neutralized fractions were reduced to 250 .mu.l by speed vacuum or butanol extraction and the nucleic acids were precipitated with EtOH. The resultant pellet was dissolved in 102 .mu.l selection buffer. A 2 .mu.l sample was removed for quantitation and analytical PAGE. The resulting amplified Round 1 Pool was applied to a new Con-A-thrombin column as in Example 2 to obtain Round 2 aptamers.
EXAMPLE 4
Characterization of Round 1 Through Round 5 Aptamers Obtained from Selection on Lectin Columns
Five rounds of thrombin aptamer selection and amplification were carried out using Con-A-thrombin columns as in Examples 2 and 3. As shown in Table 2, the combined fractions 14 and 15 contained a maximum of about 10% of input DNA using the described conditions.
After amplification, round 5 aptamer DNA was analyzed for specificity in a filter binding assay. In this assay, nitrocellulose filters (1 cm diameter) prebound with salmon sperm DNA were used to bind either: (1) An unselected 96 mer oligonucleotide DNA pool, (2) unselected DNA with thrombin (60 pmole), (3) Round 5 aptamer DNA and thrombin (60 pmole), (4) Round 5 aptamer DNA alone, or (5) Round 5 aptamer DNA and ovalbumin (60 pmole). In each case 3.5 pmole of DNA was used and the incubation was in 200 .mu.L selection buffer at room temperature for 1 hour. The filters were then washed 3 times with 3.0 ml of selection buffer and radioactivity was counted to determine the amount of DNA that was retained as a thrombin complex. The results are shown in Table 3.
Unselected DNA did not show significant binding to the thrombin while selected aptamer DNA bound to thrombin. Binding results show specific thrombin binding with no detectable ovalbumin binding.
Round 5 aptamer DNA was then amplified using the following 3' primer sequence:
and the 5' 18 mer primer sequence shown in Example 1. The 36 mer primer was used to generate internal BamH1 restriction sites to aid in cloning. The amplified Round 5 aptamer DNA was then cloned into pGEM 3Z (Promega). 32 of the resulting clones were then amplified directly using the following 5' primer sequence:
and the 3' biotinylated 18 mer primer sequence shown in Example 1, and then sequenced.
Filter binding assays using aptamer DNA from 14 of the clones were used to determine the dissociation constants (K.sub.D) for thrombin as follows: Thrombin concentrations between 10 .mu.M and 1 nM were incubated at room temperature in selection buffer for 5 minutes in the presence of 0.08 pmole of radiolabled 96 mer derived from cloned Round 5 aptamer DNA. After incubation, the thrombin and aptamer mixture was applied to nitrocellulose filters (0.2 micron, 2.4 cm diameter) that were pretreated with salmon sperm DNA (1 mg/ml DNA in selection buffer) and washed twice with 1 ml selection buffer. After application of thrombin mixture, the filters were washed three times with 1 ml selection buffer. The radioactivity retained on the filters was then determined. K.sub.D values for the individual clones ranged from 50 to >2000 nM.
The DNA sequence of the 60-nucleotide randomly-generated region from 32 clones was determined in order to examine both the heterogeneity of the selected population and to identify homologous sequences. Sequence analysis showed each of the 32 clones to be distinct. However, striking sequence conservation was found. The hexamer 5' GGTTGG 3' (SEQ ID NO: 9) was found at a variable location within the random sequence in 31 of 32 clones, and five of the six nucleotides are strictly conserved in all 32. Additionally, in 28 of the 32 clones a second hexamer 5' GGNTGG 3' (SEQ ID NO: 10), where N is usually T and never C, is observed within 2-5 nucleotides from the first hexamer. Thus, 28 clones contain the consensus sequence 5' GGNTGG(N).sub.z GGNTGG 3' (SEQ ID NO:11)(SEQ ID NO:12)(SEQ ID NO:13) (SEQ ID NO:14) where z is an integer from 2 to 5. The remaining 4 clones contain a "close variant sequence" (a sequence differing by only a single base). A compilation of the homologous sequences are shown in FIG. 1. It should be noted that DNA sequencing of several clones from the unselected DNA population or from a population of aptamers selected for binding to a different target revealed no homology to the thrombin-selected aptamers. From these data we conclude that this consensus sequence contains a sequence which is responsible either wholly or in part, for conferring thrombin affinity to the aptamers.
Clotting time for the thrombin-catalyzed conversion of fibrinogen (2.0 mg/ml in selection buffer) to fibrin at 37.degree. C. was measured using a precision coagulation timer apparatus (Becton-Dickinson, Cat. nos. 64015, 64019, 64020). Thrombin (10 nM) incubated with fibrinogen alone clotted in 40 sec, thrombin incubated with fibrinogen and P1 nuclease (Boehringer-Mannheim, Indianapolis, Ind.) clotted in 39 sec, thrombin incubated with fibrinogen and aptamer clone #5 (200 nM) clotted in 115 sec, and thrombin incubated with fibrinogen, clone #5 (200 nM) and P1 nuclease clotted in 40 sec. All incubations were carried out at 37.degree. C. using reagents prewarmed to 37.degree. C. Aptamer DNA or, when present, P1 nuclease, was added to the fibrinogen solution prior to addition of thrombin. These results demonstrated that (i) thrombin activity was inhibited specifically by intact aptamer DNA and (ii) that inhibitory activity by aptamer did not require a period of prebinding with thrombin prior to mixing with the fibrinogen substrate.
Inhibition of thrombin activity was studied using a consensus-related sequence 7 mer, 5' GGTTGGG 3' (SEQ ID NO: 15), and a control 7 mer with the same base composition but different sequence (5' GGGGGTT 3') (SEQ ID NO: 16). Clotting times were measured using the timer apparatus as above. The thrombin clotting time in this experiment was 24 sec using thrombin alone (10 nM), 26 sec with thrombin and the control sequence at 20 .mu.M and 38 sec with thrombin plus the consensus sequence at 20 .mu.M, indicating specificity for thrombin inhibition at the level of the 7 mer.
The inhibitory aptamers were active at physiological temperature under physiologic ion conditions and were able to bind to thrombin in the presence of the fibrinogen substrate, a key requirement for therapeutic efficacy.
EXAMPLE 5
Preparation of Disulfide Linked Bradykinin Affinity Matrix
SPDP (Pharmacia, 60 mg, 192 .mu.mole) was dissolved in EtOH (6 ml) and added in 1 ml aliquots with stirring to bradykinin (Bachem, 213 mg, 157 .mu.mole) in buffer (20 ml, 100 mMNaH.sub.2 PO.sub.4, 100 mM NaCl, pH 7.5). After shaking the reaction for 1 hour at room temperature, dithiothreitol (DTT, 1M, 1.35 ml) was added and the reaction was shaken for an additional hour at room temperature. The thiolated bradykinin was purified by HPLC Dynamax C18 (Rainin, Emeryville, Calif.). Thiopropylbradykinin in 10 ml buffer (100 mM Tris.Cl, pH 7.6, 500 mM NaCl, 1 mM EDTA) was then added to 12 ml of thiol activated Sepharose 6B support (Pharmacia) equilibrated in buffer. The mixture was shaken for 2 hours at room temperature and then washed over a frit with double-distilled water. The amount of bradykinin loading was estimated by determining the concentration of pyridine-2-thione released (A.sub.343 nm =8.08.times.10.sup.3 M.sup.-1, cm.sup.-1). Approximately 12.5 .mu.moles bradykinin per ml support was obtained, giving a 94% overall yield.
The bradykinin column was characterized for its performance with regard to release of bradykinin mediated by 100 mM dithiothreitol (DTT) in selection buffer. It was found that after addition of DTT, disulfide-linked bradykinin (i.e., bradykinin-NH--CO--CH.sub.2 --CH.sub.2 --SH) was immediately released from the column matrix and eluted in the fraction following the void volume. This was true of both a column consisting only of bradykinin and a column consisting of bradykinin with bound round 5 aptamer DNA (prepared in the same procedure of Examples 2 and 3 except using a bradykinin column and eluting with DTT instead of .alpha.-methyl-mannoside). The presence of bound bradykinin aptamer DNA thus did not interfere with release of bradykinin from the column after DTT was added.
Release of bradykinin from the column was monitored by analysis of the eluted fraction after addition of DTT. Fractions (about 300 .mu.L) were extracted in n-butanol and the bradykinin present in the butanol was detected by HPLC. The column fractions were run over a HPLC Dynamax C18 column using an aqueous solvent containing 0.1% trifluoroacetic acid and an acetonitrile gradient (1% to 50% over 30 minutes) at a flow rate of 1 ml/minute (37.degree. C.). Thiol-linkered bradykinin from either column eluted at 22 minutes while native bradykinin eluted at 20 minutes.
Round 5 aptamers for bradykinin prepared by this method had a K.sub.D of 50 .mu.M, as compared to a K.sub.D of >1 mM for unselected DNA. The sequence of one cloned round 5 bradykinin aptamer is as follows:
Claims
We claim:
1. A method for identifying an aptamer which specifically binds to thrombin, wherein the aptamer comprises the sequence 5' GGNTGG 3' wherein N is A, T Or G which method comprises: (a) incubating thrombin reversibly coupled to a support with a randomized mixture of single-stranded DNA oligonucleotide sequences under conditions wherein the member of the mixture to form a support-bound oligonucleotide-thrombin complex; (b) decoupling and recovering support-bound oligonucleotide-thrombin complex to form a free oligonucleotide-thrombin complex; (c) recovering free oligonucleotide from the free oligonucleotide-thrombin complex and recovering and amplifying the free oligonucleotide; and (d) optionally determining the sequence of the recovered oligonucleotide.
2. The method of claim 1 wherein N is T.
3. The method of claim 1 wherein the aptamer comprises the sequence 5' GGNTGG(N).sub.Z GGNTGG 3' wherein Z is an integer from 2 to 5.
4. The method of claim 3 wherein Z is 3.
5. The method of claim 4 wherein the aptamer comprises the sequence 5' GGNTGGTGTGGNTGG 3'.
6. The method of claim 5 wherein N is T.
7. The method of claim 1 wherein the support is a lectin column.
8. The method of claim 1 wherein the support is a solid support.
9. The method of claim 1 wherein the solid support comprises agarose or sepharose.
10. The method of claim 3 wherein the support is a lectin column.
11. The method of claim 3 wherein the support is a solid support.
12. The method of claim 3 wherein the solid support comprises agarose or sepharose.
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