In triplex-forming peptide nucleic acid, a novel 2-guanidyl pyridine nucleobase (V) enables recognition of up to two cytosine interruptions in polypurine tracts of dsRNA by engaging the entire Hoogsteen face of C–G base pair.

Triple-helical recognition of any sequence of double-stranded RNA requires high affinity Hoogsteen hydrogen binding to pyrimidine interruptions of polypurine tracts. Because pyrimidines have only one hydrogen bond donor/acceptor on Hoogsteen face, their triple-helical recognition is a formidable problem. The present study explored various five-membered heterocycles and linkers that connect nucleobases to backbone of peptide nucleic acid (PNA) to optimize formation of X * C-G and Y * U-A triplets. Molecular modeling and biophysical (UV melting and isothermal titration calorimetry) results revealed a complex interplay between the heterocyclic nucleobase and linker to PNA backbone. While the five-membered heterocycles did not improve pyrimidine recognition, increasing the linker length by four atoms provided promising gains in binding affinity and selectivity. The results suggest that further optimization of heterocyclic bases with extended linkers to PNA backbone may be a promising approach to triple-helical recognition of RNA.