Abasic Site‐Assisted Inhibition of Nicking Endonuclease Activity for the Sensitive Determination of Uracil DNA Glycosylase

• Published in: Biotechnology journal 2018-04, Vol.13 (4), p.e1700603-n/a
• Main Authors: Ahn, Jun Ki, Lee, Chang Yeol, Park, Ki Soo, Park, Hyun Gyu
• Format: Article
• Language: English
• Subjects: abasic site; biosensors; DNA amplification; nicking endonuclease; uracil DNA glycosylase
• ISSN: 1860-6768; 1860-7314
• DOI: 10.1002/biot.201700603

We herein describe A novel strategy to accurately determine uracil DNA glycosylase (UDG) activity is described based on the finding that nicking endonuclease‐assisted cleavage reaction can be regulated by the presence of abasic site. This strategy utilizes DNA probes rationally designed to contain uracil base at the cleavage site for nicking endonuclease, which is coupled to the isothermal nicking endonuclease amplification reaction (NEAR) method. In the absence of UDG, intact DNA probes generate a large number of double‐stranded (ds) DNA products through the NEAR, but the presence of UDG that converts uracil base into abasic site suppresses nicking endonuclease activity and the subsequent NEAR. As a result, dsDNA products are not produced, which is simply monitored by the dsDNA specific fluorescence dye, SYBR green I. By employing this strategy, we sensitively determined the UDG activity down to 0.003 U mL−1 with high specificity over other base excision enzymes. In addition, the diagnostic capability of this method was successfully verified by reliably assaying UDG present in a human serum sample. A new strategy to determine uracil DNA glycosylase activity is devised based on the finding that nicking endonuclease‐assisted cleavage reaction can be regulated by the presence of abasic site, which is coupled to the subsequent isothermal nicking endonuclease amplification reaction (NEAR).