Molecular Crowding‐Driven RNA Condensates Enable Förster Resonance Energy Transfer‐Enhanced Small‐Molecule Sensing

ABSTRACT Fluorescent RNA aptamers offer promising opportunities for next‐generation biosensing but are often limited by low signal‐to‐background ratios and unstable folding kinetics. In this work, a label‐free Förster resonance energy transfer (FRET)‐enhanced fluorescent artificial RNA condensate (F‐FARCON) is developed for small‐molecule sensing, leveraging neutral molecular crowders (e.g., polyethylene glycol 8K), and RNA structural motifs to induce multivalent interactions and drive dynamic self‐assembly. As a demonstration, a label‐free FRET system is constructed by integrating a histamine‐responsive RNA aptamer with thioflavin T (ThT) as the fluorescence donor, which increases the signal‐to‐noise ratio while reducing sequence complexity and production costs. Molecular crowders optimize the thermodynamic environment of RNA–ligand and RNA–RNA multivalent interactions, thereby improving folding stability, signal amplitude (dynamic range of up to ∼970‐fold), and target affinity. The platform exhibits fast kinetics (