Isoenergetic peptide nucleic acid probes for targeting double-stranded RNAs via enhanced sequence-specific stacking interactions

RNA secondary structures include double-stranded RNA (dsRNA) and single-stranded RNA (ssRNA) regions. dsRNAs can be targeted by chemically modified dsRNA-binding peptide nucleic acids (dbPNAs) via triplex formation, but inverted C-G pairs weaken affinity due to a relatively weakened Q⋅C-G triple. We show, using bio-layer interferometry (BLI) and non-denaturing polyacrylamide gel electrophoresis (PAGE), that introducing 2-thiouracil (s 2 U) upstream of Q significantly enhances affinity by reducing dissociation and/or increasing association rates. This modification also increases ribosomal frameshifting efficiency in a cell-free dual-luciferase reporter assay. Molecular dynamics simulations reveal improved stacking of s 2 U upstream of Q, which, however, is highly dependent on the local RNA structure, due to an energy coupling between the structure of dsRNA and dbPNA binding. The enhancement generalizes to RNAs such as influenza A viral RNA promoter, precursor microRNA 21 (pre-miR-21), and a model RNA containing a dsRNA-ssRNA junction. This strategy enables the design of isoenergetic, high-affinity PNAs to target diverse dsRNA sequences, thereby advancing RNA-targeted therapeutics and biotechnology.