Chimeric DNA-RNA Catalytic Sequences

This invention pertains to DNA-RNA catalytic molecules. More particularly the invention pertains to chimeric DNA-RNA-DNA-RNA-DNA catalytic molecules effective to cleave HIV-1 RNA sequences, for example.

Ribozymes are structural RNA molecules which mediate a number of RNA self-cleavage reactions. Two distinct trans-acting ribozymes, "hammerhead" and "hairpin," having different secondary structures have been identified. Oncogenes and Aids (1990) [citation] states:

"Another possible synthetic approach is the development of a chimeric molecule containing a ribonucleotide catalytic center and deoxyribonucleotide flanking sequences. It is also conceivable that chimeric catalysts comprised of an RNA catalytic center and DNA flanking sequences will retain biological activity while having greater stability."

Perreault, et al., Nature, 344:565-567 (1990), describes certain mixed deoxyribo and ribooligonucleotides with catalytic activity. No RNA-DNA catalytic molecules of practical therapeutic utility are known.

This invention provides chimeric DNA/RNA catalytic molecules useful to cleave RNA sequences. The invention specifically provides two different chimeric DNA-RNA-DNA-RNA-DNA catalytic molecules which are targeted to cleave HIV-1 RNA sequences. These chimeric molecules include DNA sequences which flank a catalytic RNA center. Interaction with the HIV-1 substrate RNAs is achieved by Watson-Crick base pairing of the DNA flanking sequences with HIV-1 RNA. The catalytic ribonucleotide center cleaves the phosphodiester bond of the substrate HIV-1 RNA at the expected location.

In general the catalytic molecules of the invention function as hammerhead or hairpin ribozymes. The preferred molecular construct consists of two known RNA catalytic sequences each flanked by a DNA sequence at the respective 3' and 5' termini and coupled by a DNA sequence at the corresponding 5' and 3' termini These molecules may accordingly be represented by the formulae I and II::

or

in which X, Y and Z are DNA sequences and AAAG, CAAAG and AGUAGUC are catalytic RNA sequences.

The flanking X and Z components may be any DNA sequences that allow base pairing with the substrate RNA at appropriate positions adjacent to the substrate cleavage site. These flanking sequences may be phosphodiester, phosphorothioate, methyl phosphonate, methyl phosphate or similar moieties.

Y may be any DNA sequence that base pairs inter se in the manner required for catalytic cleavage of the substrate by the RNA sequences preferably as shown in base paired form in Formula III: ##STR1##

The catalytic molecules of this invention can be synthesized in known manner by commercially available DNA synthesizers such as those produced by Applied Biosystems or Milligen. See, e.g., Perreault, et al, supra.

The X and Z sequences may be substituted at the respective 3' and 5' ends with ligands to facilitate cell entry, targeting within the cell and ultimate stability of the catalysts. Such ligands include by way of example but not of limitation: other nuclotides, proteins, carbohydrates, lipids, steroid hormones and cholesterol.

The catalytic molecules of the invention are administered by known and available delivery agents or systems, including, but not limited to, liposomes, defective viral particles, viral capids, and standard DNA/RNA transfective procedures.

FIG. 1 illustrates one catalytic molecule of the invention base paired to an HIV-1 sequence. The RNA portion of the molecule is encircled.

FIG. 2 illustrates a second catalytic molecule of the invention base paired to another HIV-1 sequence. The RNA portion of the molecule is encircled.

FIG. 3A depicts a ribonuclease A digestion of the catalytic molecule of FIG. 1 as compared with an equivalent all DNA molecule. The conditions were 10 units of commercial (Sigma) pancreatic ribonuclease in 2XSSC buffer added to the oligonucleotides which were in 10 microliters of 50 mM Tric-HCl buffer (pH 8.0). The RNAse was incubated with the sample for 10 minutes before the 32-P end labelled DRDRD or DNA molecules were electrophoresed in a 15% polyacrylamide gel containing 8M urea. The gel was autoradiographed for 10 minutes to get the exposure depicted.

FIG. 3B depicts a cleavage reaction involving the catalytic molecule of FIG. 1 under conditions described in Chang, et al., Clinical Biotechnology, 2:23-31 (1990).

The catalytic molecule of FIG. 1 was synthesized in known manner utilizing an automated oligonucleotide synthesizer manufactured by Applied Biosystems, Inc.

The result of ribonuclease A digestion of the catalytic molecule is shown by FIG. 3A.

The catalytic molecule produced, as described, was used to cleave each of a 610 nuleotide long (S-610) and a 170 nucleotide long HIV-1 gag transcript. In brief, the buffer was 50 mM Tris-HCl, pH 7.5, 1mM EDTA, 10mM MgCl.sub.2 at approximately 1 pmole of target, 3 pmole of ribozyme or DNA. The reactions were carried out at 37.degree. C. for 12 hours. The substrate was either a 610 nucleotide long HIV-1 gag containing transcript (S-610) or a 172 nucleotide long HIV-1 gag containing transcript (S-172) . The 5' cleavage product is indicated for both.

In FIG. 3B the 5' cleavage product is shown for both transcripts. The 3' cleavage product for the 610 target is not visible due to poor reproduction of the autoradiograph, but is indicated in its position by a 3' P notation. As a negative control, an all DNA oligonucleotide (D) of the same sequence as the DRDRD molecule was incubated with the same substrates under the same conditions with the result that no cleavage was obtained. Specific cleavage of an HIV-1 5' LTR splice site with a similar catalytic molecule has also been obtained.