Unveiling the promise of peptide nucleic acids as functional linkers for an RNA imaging platform

Linkers in chemical biology provide more than just connectivity between molecules; their intrinsic properties can be harnessed to enhance the stability and functionality of chemical probes. In this study, we explored the incorporation of a peptide nucleic acid (PNA)-based linker into RNA-targeting probes to improve their affinity and specificity. By integrating a PNA linker into a small molecule probe of the Riboglow platform, we enabled dual binding events: cobalamin (Cbl)-RNA structure-based recognition and sequence-specific PNA-RNA interaction. We show that incorporating a six-nucleotide PNA sequence complementary to a region of wild type RNA aptamer (<i>env</i>8) results in a 30-fold improvement in binding affinity compared to the probe with a nonfunctional PEG linker. Even greater improvements are observed when the PNA probe was tested against truncated versions of the RNA aptamer, with affinity increasing by up to 280-fold. Additionally, the PNA linker is able to rescue the Cbl-RNA interaction even when the cobalamin binding pocket is compromised. We demonstrate that PNA probes effectively bind RNA both <i>in vitro</i> and in live cells, enhancing visualization of RNA in stress granules and U-bodies at low concentrations. The modular nature of the Riboglow platform allows for flexible modifications of the PNA linker, fluorophore, and RNA tag, while maintaining high specificity and affinity. This work establishes a new approach for enhancing RNA imaging platforms through the use of PNA linkers, highlighting the potential of combining short oligonucleotides with small molecules to improve the affinity and specificity of RNA-targeting probes. Furthermore, this dual-binding approach presents a promising strategy for driving advancements in RNA-targeted drug development.