Simple label-free and sensitive fluorescence determination of human 8-oxoG DNA glycosylase 1 activity and inhibition via TdT-assisted sequence extension amplification
Human 8-oxoG DNA glycosylase 1 (hOGG1) represents one of the important base excision repair enzymes in the human genome, and its variation in expression levels is closely related to cancers. Here, we show the establishment of a simple label-free fluorescence approach for the determination of hOGG1 activity and inhibition with high sensitivity by using the terminal deoxynucleotidyl transferase (TdT)-aided sequence extension amplification. The target hOGG1 specifically recognizes and excises the 8-oxoG site on one of the sequences in a dsDNA probe with recessed 3′-termini. Such an enzymatic reaction leads to the transformation of dsDNA into a 3′-terminus protruding duplex, which is a favorable template for TdT-assisted extension formation of a long sequence with repeated G-quadruplex sequences with a well-tuned ratio of dGTP to dATP. These G-quadruplex sequences further bind the thioflavin dye to exhibit drastically enhanced fluorescence for detecting hOGG1 with high sensitivity in the dynamic range between 0.005 and 2 U mL−1. The developed method shows a calculated detection limit of 0.002 U mL−1, and a high selectivity toward hOGG1 against other proteins has been demonstrated. Besides, this approach can also be used to assess hOGG1 activity inhibition by the CdCl2 inhibitor, offering great potential for disease diagnosis and clinical research.