Rational Design Of A Novel Aptamer-Based Biosensor For A Target Enzyme Via Modification Of Gfp-Like Fluorogens: Carboxylesterase 2a As A Case Study

Herein, a rational design strategy was used to construct a novel and practical aptamer-based biosensor for sensing and imaging human carboxylesterase 2A (CES2A), via modification of a green fluorescent protein (GFP)-like fluorogen as a key recognition element. For this purpose, series of 3, 5-difluoro-4-hydroxybenzylidene imidazolinone (DFHBI) esters were designed based on the substrate preference of the target enzyme and the mechanism of RNA aptamer-fluorogen complex for fluorescence detection, while their potentials as CES2A substrates were evaluated by docking simulations and experimental assays. The results demonstrated that at least four DFHBI esters could be readily hydrolyzed by CES2A, while T-DFHBI (DFHBI trimethylacetyl ester) showed the best combination of chemical stability, fast response and high specificity towards CES2A. Aided by the signal enhancer (Broccoli, a RNA aptamer), T-DFHBI measured CES2A at the level as low as 8.8 ng/mL. The newly developed aptamer-based biosensor displayed good performance for sensing and imaging of CES2A activities in real samples, including tissue preparations and living cells. Collectively, this study demonstrates a new strategy for rational design and development of aptamer-based biosensors for target enzyme(s), while such strategy can be expanded to develop other light-up aptamer-based biosensors for a wide range of enzymes, particularly for the hydrolases.