Improved Triplex‐Forming Isoorotamide PNA Nucleobases for A‐U Recognition of RNA Duplexes

Four new isoorotamide (Io) containing PNA nucleobases were designed for A‐U recognition of double helical RNA. New PNA monomers were prepared efficiently and incorporated into PNA 9‐mers for binding A‐U in a PNA:RNA2 triplex. Isothermal titration calorimetry and UV thermal melting experiments revealed slightly improved binding affinity for singly‐modified PNA compared to known A‐binding nucleobases. Molecular dynamics simulations provided further insights into binding of Io bases in the triple helix. Together, data revealed interesting insights into binding modes including the notion that three Hoogsteen‐hydrogen bonds are unnecessary for strong selective binding of an extended nucleobase. Cationic monomer Io8 additionally gave the highest affinity observed for an A‐binding nucleobase to date. These results will help inform future nucleobase design toward the goal of recognizing any sequence of double helical RNA.