Ribonucleic acid (RNA) function is central to all life, including that of viruses and bacteria. Antibacterial agents such as neomycin and erythromycin are examples of existing drugs that target sites in bacterial ribosomal RNAs. Unfortunately, bacteria are becoming increasingly resistant to these compounds via adaptation that allows for the modification of the RNA target or modification of the antibiotic. Human immunodeficiency virus (HIV), adenovirus and Hepatitis C virus (HCV) are examples of human pathogens that have unique RNA structures that appear necessary for replication. These RNAs are potential targets for drug intervention. Unfortunately, our lack of understanding of the recognition of RNA by small molecules limits our ability to design high affinity ligands. The overall goal of this research plan is to develop small molecules capable of controlling the function of an RNA target, such as a regulatory element from a viral genome, inside eukaryotic cells. To achieve this goal the following criteria must be met: a) the ligand must bind the target RNA with high affinity and selectivity and b) the ligand must be cell permeable with favorable subcellular localization and stability in the intracellular environment.
One specific aim of this project is to define the physical basis for binding observed with peptide-intercalator conjugates (PICs) and RNA aptamers isolated through in vitro evolution. This will facilitate the rational design of new PICs to selectively target viral RNAs that have similar structures. Another specific aim is to discover new PICs capable of binding selectively to viral RNA targets using library synthesis and screening approaches. Also, RNA structures predisposed to bind PICs will be identified by a) cleaving viral RNAs with EDTA-Fe-modifed PICs and by b) using in vitro evolution from sequence random RNA libraries. Finally, the extent to which PICs are capable of entering eukaryotic cells and binding intracellular RNA targets will be assessed. These experiments will ultimately lead to an understanding of how to prepare a low molecular weight ligand that binds selectively to certain RNA motifs. In addition, these new compounds would have the potential to be developed into antiviral or antibacterial therapeutics. ? ?
