PROJECT SUMMARY/ABSTRACT We propose herein a methodology to identify synthetic peptide-based binders to non-canonical structural motifs in RNA. Reagents that selectively target these biologically important motifs would be transformative theranostic tools for study and modulation of RNA-governed biology. The long-term goal of our research program is to develop sequence and context-selective reagents for targeting of any such non-canonical structural motif in lncRNAs. The objective of this application is to synthesize a small library of peptide-derived reagents and quantitatively rank their competence in non-canonical hybridization to defined RNA structures, using novel and robust functional screening methods in vitro and in cell culture. In contrast to the striking progress of synthetic biology at the coding interface, non-canonical targeting remains in development. We hypothesize that progress in this area is limited in a number of ways: 1) a prior focus on targeting Watson-Crick (WC) paired bases rather than non-canonical pairs; 2) an over-emphasis on intercalation-driven binding; 3) lack of exploration of secondary structure in RNA targeting reagents; 4) lack of a unified functional assay to rigorously evaluate binding. We further hypothesize that synthetic binding solutions exist for every non-canonical motif; if these solutions could be found, then non-canonical hybridization could be programmed in the same way that duplex hybridization is programmed. Such an advance would enable precise interrogation of nucleic acid biology with novel chemical tools. The rigor of the prior research lies in the known efficiency of synthetic bases in targeting select non-canonical pairs, as well as preliminary data demonstrating tunable and expansive binding selectivity via backbone and base modification. We will test our central hypothesis and accomplish the overall objective of this application via the following three specific aims: 1) Synthesis and evaluation of bPNAs targeting non-canonical sites via base-triple formation; 2) Enzymatic and functional assays for bPNA-RNA targeting efficacy; 3) In vitro and intracellular bPNA targeting and selectivity for structural motifs in native RNAs.