Engineering Fluorophore Recycling in a Fluorogenic RNA Aptamer

Fluorogenic aptamers can potentially show minimal photobleaching during continuous irradiation since any photobleached fluorophore can exchange with fluorescent dyes in the media. However, fluorophores have not been designed to maximize “fluorophore recycling.” Here we describe TBI, a novel fluorophore for the Broccoli fluorogenic aptamer. Previous fluorophores either fail to rapidly dissociate when they undergo photobleaching via cis–trans isomerization, or bind slowly, resulting in extended periods after dissociation of the photobleached fluorophore when no fluorophore is bound. By contrast, photobleached TBI dissociates rapidly from Broccoli, and TBI from the media rapidly replaces dissociated photobleached fluorophore. Using TBI, Broccoli exhibits markedly enhanced fluorescence in cells during continuous imaging. These data show that designing fluorophores to optimize fluorophore recycling can lead to enhanced fluorescence of fluorogenic aptamers. Fluorogenic RNA aptamers bind to and activate the fluorescence of otherwise nonfluorescent dyes. Here we show an approach to maximize their fluorescence by reducing the impact of photobleaching. We engineered TBI, a fluorophore that rapidly dissociates upon photobleaching and can be rapidly replaced by TBI in the media. By maximizing “fluorophore recycling”, we achieve higher fluorescence and enable long‐term fluorescence imaging.