Structure‐Guided Design of Pepper‐Binding Fluorophores Through Hydrogen‐Bonding Engineering

ABSTRACT Pepper fluorescent RNAs (FRs) enable spatiotemporal imaging of intracellular RNA with high resolution. Although spectral optimization of the HBC scaffold has extended coverage from cyan to red, systematic refinement of binding affinity, photostability, and brightness remains largely unexplored. We describe a hydrogen‐bonding strategy that strengthens fluorophore‐aptamer interactions by incorporating hydroxyl groups or polyethylene glycol (PEG) moieties into HBC derivatives. The resulting compounds, MBC and PBC620, bind Pepper over 6‐fold and 10‐fold more tightly than their parent dyes HBC and HBC620, respectively, and exhibit correspondingly higher signal‐to‐noise ratios and enhanced photostability, with 2.1‐fold and 1.5‐fold increases in cellular brightness. Molecular models reveal that improved affinity stems from optimally positioned hydrogen bonds with RNA nucleotides. MBC enables real‐time tracking of genomic loci with enhanced contrast compared to standard HBC. This work establishes structure‐guided interface engineering as a general strategy for improving fluorescent RNA systems.