Simple label-free and sensitive fluorescence determination of human 8-oxoG DNA glycosylase 1 activity and inhibition via TdT-assisted sequence extension amplification

Abstract

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.