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Characterization of a DNA-hydrolyzing DNAzyme for generation of PCR strands of unequal length
Biochimie ( IF 3.9 ) Pub Date : 2020-10-03 , DOI: 10.1016/j.biochi.2020.10.001
Dongling Cao , Wenqian Yu , Jiacui Xu , Fulong Wang , Ying Jiang , Yongjie Sheng , Yanhong Sun , Jin Zhang , Dazhi Jiang

I-R3 DNAzyme is a small, highly active catalytic DNA for DNA hydrolysis. In here, we designed two cis-structure DNAzymes (I-R3N and I-R3S) based on the different locates of the joint linker between I-R3 and its substrate. Data demonstrated that both DNAzymes were highly dependent on Zn2+, and worked at a narrow range around pH 7.0. They exhibited strong anti-interference with Mg2+ and Ca2+, but inhibited by Na+ and K+. Moreover, single and multiple-site mutations were generated within the catalytic core to carry out a comprehensive mutational study of I-R3 motif, in which most nucleotides were highly conserved and the nucleotides A5, T11 and T8 were identified as the mutational hotspots. Furthermore, an efficient variant A5G was obtained and its reaction condition was optimized. Finally, we constructed A5G to the 3’ end of a single-stranded DNA (ssDNA) and applied it for asymmetrical PCR amplification to produce a single and double-stranded DNA mixture, in which A5G within ssDNA can self-cleave to generate a shorter desired ssDNA by denaturing gel separation. This would provide a new non-chemical modification approach for preparation of the expected ssDNA for in vitro selection of DNAzymes.



中文翻译:

表征DNA水解DNA酶以产生长度不等的PCR链

I-R3 DNAzyme是一种小的,高活性的DNA催化DNA。在这里,我们根据I-R3及其底物之间的接头位置不同,设计了两个顺式结构DNA酶(I-R3N和I-R3S)。数据表明,两种DNA酶都高度依赖Zn 2+,并且在pH 7.0左右的狭窄范围内起作用。它们显示出对Mg 2+和Ca 2+的强抗干扰性,但被Na +和K +抑制。此外,在催化核心内产生单和多位点突变以进行I-R3主题的全面突变研究,其中大多数核苷酸高度保守,核苷酸A5,T11和T8被确定为突变热点。此外,获得了有效的变体A5G,并优化了其反应条件。最后,我们在单链DNA(ssDNA)的3'末端构建了A5G,并将其用于不对称PCR扩增以产生单链和双链DNA混合物,其中ssDNA中的A5G可以自我切割以产生较短的通过变性凝胶分离获得所需的ssDNA。这将提供一种新的非化学修饰方法,用于制备预期的ssDNA,用于体外选择DNAzyme。

更新日期:2020-10-12
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