Synthetic, Structural, and RNA Binding Studies on 2-Aminopyridine-Modified Triplex-Forming Peptide Nucleic Acids

The development of new RNA-binding ligands is attracting increasing interest in fundamental science and the pharmaceutical industry. The goal of this study was to improve the RNA binding properties of triplex-forming peptide nucleic acids (PNAs) by further increasing the pK(a) of 2-aminopyridine (M). Protonation of M was the key for enabling triplex formation at physiological pH in earlier studies. Substitution on M by an electron-donating 4-methoxy substituent resulted in slight destabilization of the PNA-dsRNA triplex, contrary to the expected stabilization due to more favorable protonation. To explain this unexpected result, the first NMR structural studies were performed on an M-modified PNA-dsRNA triplex which, combined with computational modeling identified unfavorable steric and electrostatic repulsion between the 4-methoxy group of M and the oxygen of the carbonyl group connecting the adjacent nucleobase to PNA backbone. The structural studies also provided insights into hydrogen-bonding interactions that might be responsible for the high affinity and unusual RNA-binding preference of PNAs.