TARGETED MRNA CLEAVAGE BY 3 TRNASE AND SMALL GUIDE RNAS

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Antisense and ribozyme methodologies show great promise for inhibiting inappropriate gene expression and ultimately treating various disease states. Unfortunately, these approaches have several drawbacks including difficulty in delivering the nucleic acid to the cell whether by gene therapy or oligonucleotide methods. Oligonucleotides containing as few as seven nucleotides are effective in targeting specific RNAs for cleavage in vitro by a 3' RNase found in all mammalian cells. This cleavage requires that the oligonucleotide (small guide RNA) hybridize at a precise distance from particular secondary structures. This technique has successfully targeted HIV RNAs for cleavage in vitro. The goal of this proposal is to show that mRNAs can be specifically targeted for cleavage in vitro as well as in vivo. The specific aims of the project are to: 1) show that this method can be used to specifically target functional mRNAs including chloramphenicol acetyl transferase (CAT) in vitro, 2) structure map the resulting CAT mRNA/small guide RNA hybrids to investigate whether the proposed computer-generated structures actually form in vitro, and 3) introduce RNA heptamers and/or constructs expressing short RNA molecules which hybridize to reporter mRNAs (e.g CAT), to determine the efficiency of endogenous 3' tRNase to specifically degrade targeted mRNAs in vivo. This approach has many advantages over other oligonucleotide-based methods for modulating inappropriate gene expression in a clinical situation since oligonucleotides containing only seven nucleotides appear to be efficacious in contrast to traditional antisense approaches in which nucleotides containing greater than 15-20 nucleotides are required. Smaller oligonucleotides are much more easily taken up by the cell, may be more resistant to degradation, and are more easily chemically modified to increase their stability and cell membrane permeability.