NNNPERFUME: Detection of 8-oxoguanine DNA glycosylase activity based on primer exchange reaction and functionalized hemin/G-quadruplex DNAzyme

Abstract

Background8-oxoguanine DNA glycosylase can maintain genomic stability and integrity. However, it can interfere with the regular DNA damage repair process, possibly leading to the development of cancer and various other human diseases when its activity becomes abnormal. Current methods for detecting 8-oxoguanine DNA glycosylase activity often suffer from low sensitivity, time-consuming procedures, labor-intensive requirements, and the need for specialized equipment and trained professionals for execution. Consequently, there is an urgent need for a portable, user-friendly 8-oxoguanine DNA glycosylase assay that offers swift results and supports real-time testing. ResultsWe've developed a PERFUME method that combines primer exchange reaction and functionalized G-quadruplex/hemin DNAzyme for sensitive detection of Fpg, a typical 8-oxoguanine DNA glycosylase. Utilizing a single probe and T4 Polynucleotide Kinase (PNK) simplifies the experiment to a one-step reaction at 37 °C in 3 h, reducing sample consumption and improving sensitivity. We chose functionalized hemin cofactors, significantly improving catalytic efficiency and enhancing detection capability. This biosensor detects Fpg activity with a sensitivity as low as 0.11 U mL−1, displaying exceptional sensitivity, selectivity, and interference resistance in human serum and bacterial cell extracts under isothermal conditions. The biosensor demonstrates remarkable selectivity and ability for Fpg inhibitors screening. In addition, this biosensor enables reading the sample's RGB values using a smartphone, facilitating accurate quantification of Fpg activity without the necessity for specialized equipment. SignificancePERFUME simplifies Fpg detection by using a single hairpin and PNK in a one-step process. We utilize FUME to enhance catalytic efficiency, it surpassing the performance of traditional G-quadruplex/hemin DNAzyme methods. This approach excels in analyzing Fpg in human serum and bacterial extracts. It allows quantitative Fpg detection using UV–Vis and smartphones under isothermal conditions, making it valuable for clinical diagnosis.