Isolation of novel fluorogenic RNA aptamers via in vitro compartmentalization using microbead-display libraries

Fluorogenic RNA aptamers, which specifically bind to fluorogens and dramatically enhance their fluorescence, are valuable for imaging and detecting RNAs and metabolites in living cells. Most fluorogenic RNA aptamers have been identified and engineered through iterative rounds of in vitro selection based on their binding to target fluorogens. While such selection is an efficient approach for generating RNA aptamers, it is less efficient for isolating fluorogenic aptamers because it does not directly screen for fluorogenic properties. In this study, we combined a fluorescence-based in vitro selection technique using water-in-oil microdroplets with an affinity-based selection technique to obtain fluorogenic RNA aptamers. This approach allowed us to identify novel fluorogenic aptamers for a biotin-modified thiazole orange derivative. Our results demonstrate that our approach can expand the diversity of fluorogenic RNA aptamers, thus leading to new applications for the imaging and detection of biomolecules.