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

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 (H₂S/HS^-) and hydrogen peroxide (H₂O₂), 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 H₂S/HS^- or H₂O₂. 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.