Branched Sidechain bPNA + Reports on RNA-Loop Tertiary Interactions via Triplex Hybridization Stem Replacement at Internal Domains

We report herein the synthesis and characterization of bPNA+, a new variant of bifacial peptide nucleic acid (bPNA) that binds oligo T/U nucleic acids to form triplex hybrids with good affinity but half the molecular footprint of bPNA. This is accomplished via display of two melamine (M) bases melamine per lysine sidechain on bPNA+ rather than one as previously reported. Lysine derivatives bearing two bases were prepared by double reductive alkylation with melamine acetaldehyde, resulting in a tertiary amine branch point. Importantly, this amino sidechain fosters oligo T/U binding through both base-triple formation and electrostatic interactions, while maintaining selectivity and peptide solubility. The bPNA+ binding site roughly the size of a 6 base-pair stem; this relatively compact perturbation can be genetically encoded at virtually any position within an RNA transcript, and may replace existing stem elements. Subsequent triplex hybridization with fluorophore-labeled bPNA+ triplex hybrids thus accomplishes site-specific labeling of internal RNA locations without the need for chemical modification. We demonstrate herein the use of this strategy for reporting on intermolecular RNA-RNA kissing loop interactions, RNA-protein binding as well as intramolecular RNA tetraloop-tetraloop receptor binding. We anticipate that bPNA+ will have utility as structural probes for dynamic tertiary interactions in long noncoding RNAs.