Fluorogenic Aptamer Optimization on a Massively Parallel Sequencing Platform

chip, we identify the C14T mutant which exhibits an improved dissociation constant, increased quantum yield, extended fluorescence lifetime, and enhanced emission intensity. Co-crystal structures of the aptamer/fluorogen complexes reveal that π-π stacking interactions are critical for the stable coordination of TO1-biotin within Lettuce. When tested in a cellular environment, both the screening-identified C14T mutant and a structure-guided rationally designed variant, C14dU, exhibit 15% and 18% stronger fluorescence intensities, respectively, compared with canonical Lettuce. Our massive screening and molecular dynamics simulation pipeline enable efficient FAP optimization without prior structural knowledge, yielding not only improved probes for fluorescence sensing but also deeper insights into aptamer-fluorogen interactions.