Demethylation of m1A assisted degradation of the signal probe for rapid electrochemical detection of ALKBH3 activity with practical applications

Sensitive Measurement of ALKBH3 Activity using a Dual DNA Amplifier by Coupling DNA Circuits with CRISPR/Cas12a

“…The adding of ALKBH3 proteins generated remarkable fluorescence increase, while pretreating ALKBH3 with 1 μM HUHS015 resulted in significantly decreased fluorescence signal (Figure 4a and b), indicating the inhibition of ALKBH3 activity. We also observed that the dual DNA amplifier delivered gradually decreased fluorescence signals with increasing of the concentrations of HUHS015 from 5 nM to 20 μM, and the halfinhibitory concentration (IC50) value was estimated to be 0.64 μM, which was comparable to the reported values of 0.91 μM using a chemically modified DNA probe [8] (Figure 4c and S3). Together, these results demonstrated that the proposed method hold the potential for effective screening of ALKBH3 inhibitors.…”

“…[6][7] Despite the biological significance of ALKBH3 enzyme, approaches for direct and efficient measurement of ALKBH3 activity are in urgent need. [8] To date, the only fluorescence assay example is reported by Kool and coworkers, [9] which designed a fluorogenic nucleoside analog that the fluorescence was quenched by the neighboring 1MeA lesion. While this work affords a novel method to measure ALKBH3 activity, the lack of signal amplification process limits its sensitivity for monitoring low abundance targets.…”