Enhancement of RNA Imaging Platforms by the Use of Peptide Nucleic Acid-Based Linkers

RNA imaging techniques enable researchers to monitor RNA localization, dynamics, and regulation in live or fixed cells. While the MS2-MCP system-comprising the MS2 RNA hairpin and its binding partner, the MS2 coat protein (MCP)-remains the most widely used approach, it relies on a tag containing multiple fluorescent proteins and has several limitations, including the potential to perturb RNA function due to the tag's large mass. Alternative methods using small-molecule binding aptamers have been developed to address these challenges. This protocol describes the synthesis and characterization of RNA-targeting probes incorporating a peptide nucleic acid (PNA)-based linker within the cobalamin (Cbl)-based probe of the Riboglow platform. Characterization in vitro involves a fluorescence turn-on assay to determine binding affinity (K_D) and selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) footprinting analysis to assess RNA-probe interactions at a single nucleotide resolution. To show the advancement of PNA probes in live cells, we present a detailed approach to perform both stress granule (SG) and U-body assays. By combining sequence-specific hybridization with structure-based recognition, our approach enhances probe affinity and specificity while minimizing disruption to native RNA behavior, offering a robust alternative to protein-based RNA imaging systems. Key features • Contains four parts, describing I) cobalamin-PNA probe synthesis, II) fluorescence turn-on assay, III) SHAPE assay, and IV) stress granule (SG) and U-body assays. • Enables high-specificity RNA imaging through avidity, using cobalamin-based probes that incorporate peptide nucleic acid (PNA) linkers for sequence-specific hybridization with the RNA tag. • Increases the dynamic range for the stress granule (SG) assay used in the field to evaluate a fluorescent RNA tag's ability to visualize RNA localization. • Provides insights on how to adapt the described procedures to other RNA-small molecule pairs.