A Small‐Molecule Approach Enables RNA Aptamers to Function as Sensors for Reactive Inorganic Targets

Abstract Fluorescent light‐up aptamer (FLAP) systems are promising (bio)sensing platforms that are genetically encodable. However, FLAP‐mediated detection of each distinct target necessitates either in vitro selection or engineering of nucleic acid sequences. Furthermore, an aptamer that binds an inorganic target or a chemical species with a short lifetime is challenging to realize. Here, we describe a small‐molecule approach that makes it possible for a single FLAP system to detect chemically unique, non‐fluorogenic, and reactive inorganics. We developed functionalized pre‐ligands of RNA aptamers that bind benzylidene imidazolinones (Baby Spinach, Broccolli, Squash). Reactive inorganics, hydrogen sulfide (H 2 S/HS − ) and hydrogen peroxide (H 2 O 2 ), can specifically convert these pre‐ligands into native ligands that fluoresce with FLAPs. Adaptation of this platform to live cells opened an opportunity for constructing whole‐cell sensors: Escherichia coli transformed with a Baby Spinach‐encoding plasmid and incubated with pre‐ligands generated fluorescence in response to exogenous H 2 S/HS − or H 2 O 2 . Leveraging the functional group reactivity of small molecules eliminates the requirement of in vitro selection of a new aptamer sequence or oligonucleotide scaffold engineering for distinct molecular targets. Our method allows for detecting inorganic, short‐lived species, thereby advancing FLAP systems beyond their current capabilities.