Ultrasensitive evaluation of Ribonuclease H activity using a DNAzyme-powered on-particle DNA walker
Ultrasensitive evaluation of Ribonuclease H activity using a DNAzyme-powered on-particle DNA walker
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使用 DNAzyme 驱动的粒子 DNA 步行器对核糖核酸酶 H 活性进行超灵敏评估
DOI：
10.1016/j.snb.2019.127380
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发表时间：
2020-02
期刊：
影响因子：
--
通讯作者：
Han Heyou
中科院分区：
文献类型：
--
作者：
Wang Wenjing;Shu Mingbo;Nie Axiu;Han Heyou
核糖核酸酶H（Ribonuclease H，RNase H）活性的测定对于抗HIV药物的开发以及DNA复制、DNA修复和转录等细胞过程的研究具有重要意义。然而，用简单的策略高度灵敏地检测RNase H活性仍然具有挑战性。在这里，提出了一种DNA酶驱动的粒子DNA步行者作为信号放大器，以评估低至0.00847 U mL−1水平的RNA酶H活性。在金纳米颗粒的界面处修饰Mn 2+特异性DNA酶的预笼化酶链和底物链，形成DNA步行者探针。在不存在RNase H的情况下，由于金纳米颗粒淬灭标记底物链的荧光团的优异能力，观察到最小的荧光信号。在RNase H存在下，DNA/RNA杂合复合物被水解以释放单链DNA，其能够与笼住酶链的锁链杂交。加入Mn 2+后，活性形式的DNA酶在底物链的RNA位点被切割，释放增强的荧光分子。最后，步行者链像机器一样在AuNP上移动以放大信号，从而间接测量RNase H的活性。这种方法被证明是一种超灵敏和简单的方法，用于评估细胞提取物中的RNase H活性，也已成功地用于评估抑制剂的作用。
The evaluation of Ribonuclease H (RNase H) activity is very important for anti-HIV drug development and the study of cellular processes, including DNA replication, DNA repair and transcription. However, the highly sensitive detection of RNase H activity with a simple strategy remains challenging. Here, a DNAzyme-powered on-particle DNA walker was proposed as a signal amplifier to evaluate the RNase H activity at levels as low as 0.00847 U mL−1. Pre-caged enzyme strands and substrate strands of Mn2+-specific DNAzyme were modified at the interface of gold nanoparticles, forming the DNA walker probes. In the absence of RNase H, a minimum fluorescent signal was observed due to excellent ability of gold nanoparticles to quench the fluorophore that labeled the substrate strand. In the presence of RNase H, the DNA/RNA hybrid complex was hydrolyzed to release single stranded DNA, which was able to hybridize to the locker strand that caged the enzyme strand. Following the addition of Mn2+, the active forms of DNAzymes were cleaved at the RNA site of substrate strands, releasing enhanced fluorescent molecules. Finally, the walker strands traveled over the AuNPs like a machine to amplify the signal, thus indirectly measuring the activity of RNase H. This approach proved to be an ultrasensitive and simple method for the evaluation of RNase H activity in cell extracts and has also been successfully used to evaluate the effects of inhibitors.
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影响因子：
7.4
作者：
Chen, Junbo;Zuehlke, Albert;Zhang, Hongquan
通讯作者：
Zhang, Hongquan
DOI：
10.1111/j.1432-1033.1995.0557d.x
发表时间：
1995-08
期刊：
European journal of biochemistry
影响因子：
--
作者：
E. Kanaya;S. Kanaya
通讯作者：
E. Kanaya;S. Kanaya
影响因子：
5.6
作者：
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通讯作者：
Arnold E
影响因子：
16.6
作者：
Peng H;Li XF;Zhang H;Le XC
通讯作者：
Le XC
DOI：
10.1074/jbc.272.35.22023
发表时间：
1997-08
期刊：
The Journal of Biological Chemistry
影响因子：
--
作者：
X. Zhan;R. Crouch
通讯作者：
X. Zhan;R. Crouch