Chemical Approaches to Control the Function of Regulatory RNAs

Recent decades have dramatically changed our view of RNA. While RNA was initially believed to be barely a passive messenger in the transfer of genetic information from DNA to proteins, it is now clear that RNA is an exciting and underexplored regulatory molecule that will continue to deliver new discoveries in biology and medicine. Our research program strives to capitalize on these exciting future discoveries by exploring chemical modifications to modulate the structure and function regulatory RNAs. The long-term goals are to 1) develop novel RNA chemical modifications for fundamental studies and biomedical applications, and 2) explore new modes of sequence-specific recognition of double-stranded RNA (dsRNA). Our research program comprises two distinct but interrelated projects: 1) novel backbone modifications to improve specificity of regulatory RNAs, and 2) triplex-forming peptide nucleic acids (PNAs) to modulate structure and biological activity regulatory RNAs. Project 1 replaces internucleotide phosphates with amides and other non-native linkages in short interfering RNAs and RNAs associated with clustered regularly interspaced short palindromic repeats (CRISPR). The goals are to optimize the activity and sequence specificity of these RNAs. The premise is that backbone modifications will remodel and improve RNA-protein interactions and, hence, modulate the activity and specificity. Project 2 explores chemically modified PNA as a ligand for sequence-specific conformational and functional control of biomedically important dsRNA. The goals are to develop PNAs that shift between alternative structures of complex RNAs and to improve the cellular uptake of PNA. The premise is that nucleobase-modified triplex-forming PNAs are uniquely suited for sequence- specific recognition of dsRNA and will enable recognition of biologically important non-coding dsRNA. New future directions will focus on biological activity of PNAs, especially, in phase-separated membrane-less organelles. The projects involve collaborations with structural biochemists (Martin Egli, Janez Plavec), biological chemists (Naoki Sugimoto), and biotech companies (Korro Bio, Inc.). The two projects share a common theme of designing chemical modifications that take advantage of charge complementarity between the RNA target and the ligands and proteins interacting with RNA. The overreaching idea is to develop RNA chemical modifications and RNA binding ligands that avoid unproductive electrostatic repulsion and capitalize on productive electrostatic attraction while concurrently enhancing sequence specificity of molecular interactions. This thrust grows out of our recent discoveries that RNA is unusually receptive to chemical modifications that neutralize the negative charge of phosphate backbone, both in RNA itself and in RNA binding oligonucleotide analogues. If successful, our research will contribute to addressing key gaps in RNA interference, CRISPR, and PNA technologies for recognition of therapeutically relevant RNAs, and will open doors for development of unique research tools and new therapeutic strategies.