A Small-Molecule Approach to Bypass In Vitro Selection of New Aptamers: Designer Pre-Ligands Turn Baby Spinach into Sensors for Reactive Inorganic Targets

Fluorescent light-up aptamer (FLAP) systems are promising biosensing platforms that can be genetically encoded. Here, we describe how a single FLAP that works with specific organic ligands can detect multiple, structurally unique, non-fluorogenic, and reactive inorganic targets. We developed 4-O-functionalized benzylidene imidazolinones as pre-ligands with suppressed fluorescent binding interactions with the RNA aptamer Baby Spinach. Inorganic targets, hydrogen sulfide (H2S) or hydrogen peroxide (H2O2), can specifically convert these pre-ligands into the native benzylidene imidazolinones, and thus be detected with Baby Spinach. Adaptation of this approach to live cells opened a new opportunity for top-down construction of whole-cell sensors: Escherichia coli transformed with a Baby Spinach-encoding plasmid and incubated with pre-ligands generated fluorescence in response to exogenous H2S or H2O2. Our approach eliminates the requirement of in vitro selection of a new aptamer sequence for molecular target detection, allows for the detection of short-lived targets, thereby advancing FLAP systems beyond their current capabilities. Leveraging the functional group reactivity of small molecules can lead to cell-based sensors for inorganic molecular targets, exploiting a new synergism between synthetic organic chemistry and synthetic biology.