Abasic Site-Assisted Inhibition of Nicking Endonuclease Activity for the Sensitive Determination of Uracil DNA Glycosylase,Biotechnology Journal

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Abasic Site-Assisted Inhibition of Nicking Endonuclease Activity for the Sensitive Determination of Uracil DNA Glycosylase
Biotechnology Journal ( IF 3.2 ) Pub Date : 2017-12-07 , DOI: 10.1002/biot.201700603
Jun Ki Ahn ₁ , Chang Yeol Lee ₁ , Ki Soo Park ₂ , Hyun Gyu Park ₁

Affiliation

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).

中文翻译：

碱基定位辅助抑制核酸内切酶活性的敏感性测定尿嘧啶脱氧核糖核酸糖基化酶

我们在此描述一种新的策略，其基于以下发现的结果描述了一种精确确定尿嘧啶DNA糖基化酶（UDG）活性的新策略：通过内切酶位点的存在可以调节切口内切核酸酶辅助的裂解反应。该策略利用合理设计的DNA探针，在切割位点包含尿嘧啶碱基以形成切口内切核酸酶，该探针与等温切口内切核酸酶扩增反应（NEAR）方法耦合。在没有UDG的情况下，完整的DNA探针会通过NEAR产生大量的双链（ds）DNA产物，但是将尿嘧啶碱基转化为无碱基位点的UDG的存在会抑制切口内切核酸酶活性和随后的NEAR。结果，不会产生dsDNA产物，只需使用dsDNA特异性荧光染料SYBR green I即可对其进行监控。通过采用这种策略，^-1具有比其他碱基切除酶高的特异性。此外，通过可靠地测定人血清样品中存在的UDG，已成功验证了该方法的诊断能力。基于以下发现，发明了一种确定尿嘧啶DNA糖基化酶活性的新策略：发现缺碱基内切酶可以调节切口内切核酸酶辅助的裂解反应，该碱基与随后的等温切口内切核酸酶扩增反应（NEAR）偶联。

更新日期：2017-12-14

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