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 pKa 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. RNA binding improved: A 2‐aminopyridine (M) nucleobase enables strong and selective triple helical binding of peptide nucleic acid (PNA) to dsRNA. However, further modification of M with the electron‐donating 4‐methoxy group did not enhance the binding affinity. NMR structural studies showed that this is likely due to steric and electronic repulsion (denoted by the red spheres) between the oxygen of 4‐methoxy group and the oxygen of the carbonyl connecting the adjacent PNA nucleobase to the PNA backbone.