Homogeneously Sensitive Detection of Multiple DNA Glycosylases with Intrinsically Fluorescent Nucleotides

DNA glycosylases are responsible for recognition and excision of the damaged bases in the base excision repair pathway, and all mammals express multiple DNA glycosylases to maintain genome stability. However, simultaneous detection of multiple DNA glycosylase still remains a great challenge. Here, we develop a rapid and sensitive fluorescent method for simultaneous detection of human 8-oxoG DNA glycosylase 1 (hOGG1) and uracil DNA glycolase (UDG) using exonuclease-assisted recycling signal amplification in combination with fluorescent bases 2-aminopurine (2-AP) and pyrrolo-dC (P-dC) as the fluorophores. We design a bifunctional DNA probe modified with one 8-oxoG and five uracil bases, which can hybridize with the trigger probes to form a sandwiched DNA substrate for hOGG1 and UDG. In addition, we design 2-AP and P-dC signal probes as the hairpin structures with 2-AP and P-dC in the stems. The presence of hOGG1 and UDG may initiate the signal amplification process by the recycling lambda exonuclease digestion and generates distinct fluorescence signals, with 2-AP indicating the presence of hOGG1 and P-dC indicating the presence of UDG. This method can simultaneously detect multiple DNA glycosylases with the detection limits of 0.0035 U/mL for hOGG1 and 0.0025 U/mL for UDG, and it can even measure DNA glycosylases at the single-cell level. Moreover, this method can be applied for the measurement of enzyme kinetic parameters and the screening of DNA glycosylase inhibitors, holding great potential for further applications in biomedical research and clinical diagnosis.