Self-primer and self-template recycle rolling circle amplification strategy for sensitive detection of uracil-DNA glycosylase activity
Self-primer and self-template recycle rolling circle amplification strategy for sensitive detection of uracil-DNA glycosylase activity
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自引物和自模板循环滚环扩增策略用于灵敏检测尿嘧啶-DNA糖基化酶活性
DOI：
10.1016/j.aca.2017.11.036
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发表时间：
2018
影响因子：
6.2
通讯作者：
Jiang Wei
中科院分区：
文献类型：
--
作者：
Zhang Pingping;Wang Lei;Zhao Haiyan;Xu Xiaowen;Jiang Wei
灵敏、准确地检测尿嘧啶- dna糖基酶（UDG）活性，可用于评估和验证其在尿嘧啶碱基切除修复（UBER）通路和临床诊断中的功能。本研究基于自引物和自模板循环滚圈扩增（Self-RRCA）策略，建立了一种灵敏、准确的UDG活性检测方法。首先，设计了一个含有尿嘧啶碱基和Nt.BbvCI缺口位点的未成熟模板（IT），该模板可与设计的引物杂交形成前置扩增子探针（PA探针）。在UDG的作用下，PA探针中的尿嘧啶碱基可以被去除，生成ap嘧啶（ap嘧啶）位点。然后用核酸内切酶IV （endo IV）切除生成的AP位点，使PA探针通过重组形成RCA扩增子。随后使用RCA扩增子触发RCA，在Nt.BbvCI nick反应后，释放新的扩增子启动下一个RCA，构成Self-RRCA。在该方法中，设计的IT在连接部分与引物不完全互补，可以有效避免非特异性连接反应，最终有效避免非特异性扩增。与线性RCA相比，Self-RRCA具有更高的扩增效率。该方法灵敏度高，检测限为4.68 × 10−5U mL−1。采用该方法筛选UDG抑制剂，测定UDG在HeLa细胞裂解液中的活性。该方法将为进一步开展UDG的生物医学研究和临床诊断提供一种有前景的分析工具。
Sensitive and accurate detection of uracil-DNA glycosylase (UDG) activity is available for evaluating and validating their function in uracil base-excision repair (UBER) pathway and clinical diagnosis. Here, a sensitive and accurate method for UDG activity detection was developed on the basis of self-primer and self-template recycle rolling circle amplification (Self-RRCA) strategy. First, an immature template (IT) with a uracil base and an Nt.BbvCI nicking site was designed, which could hybridize with a designed primer to form a pre-amplicon probe (PA probe). Under the action of UDG, the uracil base in the PA probe could be removed to generate an apyrimidinic (AP) site. Then the generated AP site was excised by endonuclease IV (endo IV), making the PA probe form a RCA amplicon through reconformation. The RCA amplicon subsequently was used to trigger the RCA, and after Nt.BbvCI nicking reaction, new amplicons were released to initiate next RCA, constituting a Self-RRCA. In this method, the designed IT was not fully complementary with the primer in the ligation part, which could effectively avoid nonspecific ligation reaction and eventually effectively avoid nonspecific amplification. Compared with the linear RCA, the Self-RRCA exhibited higher amplification efficiency. Due to above advantages, a sensitive and accurate detection method was achieved with a limit of 4.68 × 10−5U mL−1. Furthermore, the method was adopted to screen the inhibitor of UDG and assay the activity of UDG in HeLa cell lysate. This method will offer a promising analysis tool for further biomedical research of UDG and clinical diagnosis.