RNA recognition by helix-threading peptides and their utility as inhibitors of protein-RNA interactions

Many important biological processes, from the interferon antiviral response to the generation of microRNA regulators of translation, involve duplex RNA. Small-molecules capable of binding duplex RNA structures with high affinity and selectivity will be useful in regulating these processes and, as such, are valuable research tools and potential therapeutics. To this end, we have endeavored to design compounds of the threading intercalator type, which we have called helix-threading peptides (HTPs).; HTPs constitute a new class of small-molecules that bind selectively to duplex RNA structures adjacent to helix defects and project peptide functionality into the helix grooves. An understanding of their RNA recognition properties originated from in vitro selection of RNA aptamers that were evolved to recognize HTPs. Strong sequence and structural consensus among the aptamers revealed important information regarding preferred RNA architecture for threading intercalation. Directed hydroxyl radical affinity cleavage experiments with EDTA•Fe-modified HTPs allowed us to further examine the effects of manipulating the their high-affinity binding sites. Additionally, the affinity cleaving reagents enabled us to define the binding orientation of an HTP bound to RNA. Armed with these RNA recognition principles, we identified naturally occurring RNA molecules with secondary structure meeting the criteria for HTP binding. One of these RNAs was helix 22 of the prokaryotic ribosomal 16S RNA. This helix is a component of the binding site for the ribosomal protein S15. In addition, the S15--16S RNA interaction is important for the ordered assembly of the bacterial ribosome. In pursuit of our goals, we have characterized HTPs that bind selectively to this RNA. These compounds bind helix 22 by threading intercalation placing their N-termini in the minor groove and the C-termini in the major groove. Binding is dependent on the presence of a highly conserved purine-rich internal loop in the RNA, whereas removal of the loop minimally affects binding of the classical intercalators ethidium bromide and methidiumpropyl-EDTA•Fe (MPE•Fe). Moreover, binding selectivity translates into selective inhibition of formation of the S15--16S complex. The final chapter of this dissertation elaborates upon methods we are currently utilizing to build upon the inherent selectivity HTPs possess.