5-Triazolyl uracil improves nucleobase stacking, pyrimidine recognition, biological activity, and specificity of RNA-binding triplex-forming peptide nucleic acids

Cellular RNA folds into complex helical structures that are attractive targets for controlling biological activity and treating diseases using RNA-binding ligands. Peptide nucleic acid (PNA) is a neutral DNA analog uniquely suited for triple-helical recognition of RNA. However, the applications of triplex-forming PNAs have been limited by sequence restrictions, as stable base triples are formed only with purines. Hoogsteen recognition of pyrimidines has been a long-standing challenge. The present study explores a new approach to improve pyrimidine recognition by enhancing nucleobase stacking in the PNA strand. Placing 5-triazolyl uracil adjacent to the modified nucleobases designed for pyrimidine recognition increased the binding affinity while maintaining sequence specificity. Molecular dynamics simulations confirmed the beneficial effect of synergistic stacking interactions between the triazole and neighboring nucleobases. The increased binding affinity improved the biological activity of triplex-forming PNAs targeting microRNA precursors in a cell-based dual fluorescent protein assay. RNA-seq analysis showed that a PNA recognizing a mixed sequence of three nucleobases (G, A, and U) had higher specificity than a similar PNA featuring a uniform purine recognition tract. These results demonstrate a novel strategy for addressing the issue of pyrimidine recognition, a critical bottleneck in the triple helical targeting of nucleic acids.