Towards the Development of Isoenergetic Peptide Nucleic Acid Based Probes Targeting Double-Stranded RNAs Through Enhancing Sequence-Specific Stacking Interactions
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Peptide nucleic acid (PNA), a synthetic nucleic acid analog, exhibits substantial potential in biotechnology and therapeutic applications due to its high binding affinity and nuclease/protease resistance. Chemically modified PNAs capable of forming stable triplex structures with double-stranded RNAs (dsRNAs) under near-physiological conditions further expand their utility by enabling sequence-specific precise targeting and probing of functional RNA structural motifs. However, the presence of inverted Watson-Crick pairs (C-G and U-A) may significantly weaken the triplex formation of the dsRNA-binding PNAs (dbPNAs). Our previous work demonstrated that dbPNA P3 (composed of L, T, and Q monomers for the recognition of G-C, A-U, and C-G base pairs, respectively) can stimulate ribosomal frameshifting by binding to rHP2, a model RNA hairpin structure in an mRNA, albeit with suboptimal efficiency, due to its significantly weakened Q•C-G triple formation. We hypothesize that incorporating s 2 U adjacent to Q may offer unique stacking and hydrogen bonding interactions facilitating the development of isoenergetic dbPNA-based probes binding toward dsRNAs with varied sequences. In this study, we investigate how incorporating s 2 U adjacent to Q residues in P3 influences its binding to rHP2. Bio-layer interferometry (BLI) and non-denaturing polyacrylamide gel electrophoresis (PAGE) analyses demonstrate that substituting T with s 2 U at the N-terminal position adjacent to Q (P3-2QT) enhances binding affinity by ∼10-fold compared to unmodified P3, whereas C-terminal substitution (P3-TQ2) yields only a 2-fold improvement. Consistent with these findings, a cell-free dual-luciferase reporter assay reveals that P3-2QT significantly increases ribosomal frameshifting efficiency compared to P3 and P3-TQ2. Molecular dynamics simulations further indicate that P3-2QT maintains enhanced PNA-PNA stacking stability, particularly between s 2 U3 and Q4, suggesting a structural basis for its superior activity. Intriguingly, analogous s 2 U substitution in the P5 oligomer with the Q replaced by L does not confer a comparable enhancement in binding to the target RNA (rHP1) and frameshifting stimulation, highlighting the context-dependent nature of this modification. To assess the broader applicability of the s 2 U-Q motif, we examined its effect in dbPNAs targeting the RNA panhandle structure of influenza virus A and precursor microRNA-21, respectively. Both PAGE and BLI data confirm that s 2 U incorporation improves binding affinity, reinforcing the generality of this strategy. These findings underscore the potential of sequence-dependent uracil thiolation in optimizing triplex-forming dsRNA-binding PNAs, warranting further exploration of modified nucleobase designs to enhance their binding and functional properties.
Highlights
Thiolation of uracil upstream of Q base in PNA markedly enhances triplex binding affinity and enables isoenergetic and orthogonal targeting/activation.
N-terminal s 2 U modification adjacent to Q residues increases PNA-dsRNA binding by ∼10-fold and significantly boosts ribosomal frameshifting efficiency.
Biophysical assays and molecular dynamics simulations reveal that thiolation improves PNA stacking stability and slows dissociation, thereby enhancing functional activity toward structured RNA targets.
The general applicability of s 2 U modification is demonstrated with influenza A virus RNA panhandle structure and precursor micoRNA-21 targets, underscoring its broad potential for optimizing PNA-based therapeutics and biotechnological tools.
