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一种关于内切酶 BspD6I 及其通过修饰寡核苷酸的刺激响应切换的研究。

A study on endonuclease BspD6I and its stimulus-responsive switching by modified oligonucleotides.

机构信息

Department of Chemistry and A.N. Belozersky Institute of Physico-Chemical Biology, M.V. Lomonosov Moscow State University, Moscow, Russia.

Skolkovo Institute of Science and Technology, Skolkovo, Moscow region, Russia.

出版信息

PLoS One. 2018 Nov 26;13(11):e0207302. doi: 10.1371/journal.pone.0207302. eCollection 2018.

DOI:10.1371/journal.pone.0207302
PMID:30475809
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC6261011/
Abstract

Nicking endonucleases (NEases) selectively cleave single DNA strands in double-stranded DNAs at a specific site. They are widely used in bioanalytical applications and in genome editing; however, the peculiarities of DNA-protein interactions for most of them are still poorly studied. Previously, it has been shown that the large subunit of heterodimeric restriction endonuclease BspD6I (Nt.BstD6I) acts as a NEase. Here we present a study of interaction of restriction endonuclease BspD6I with modified DNA containing single non-nucleotide insertion with an azobenzene moiety in the enzyme cleavage sites or in positions of sugar-phosphate backbone nearby. According to these data, we designed a number of effective stimulus-responsive oligonucleotide inhibitors bearing azobenzene or triethylene glycol residues. These modified oligonucleotides modulated the functional activity of Nt.BspD6I after cooling or heating. We were able to block the cleavage of T7 phage DNA by this enzyme in the presence of such inhibitors at 20-25°C, whereas the Nt.BspD6I ability to hydrolyze DNA was completely restored after heating to 45°C. The observed effects can serve as a basis for the development of a platform for regulation of NEase activity in vitro or in vivo by external signals.

摘要

尼克内切核酸酶 (NEases) 能够在双链 DNA 中特定位置选择性地切割单链 DNA。它们被广泛应用于生物分析和基因组编辑领域；然而，大多数尼克内切核酸酶的 DNA-蛋白质相互作用的特点仍研究甚少。先前已经表明，异二聚体限制性内切核酸酶 BspD6I 的大亚基（Nt.BstD6I）充当尼克内切核酸酶。在这里，我们研究了限制内切核酸酶 BspD6I 与含有单个非核苷酸插入物的修饰 DNA 的相互作用，该插入物在酶切割位点或附近糖-磷酸主链的位置处带有偶氮苯部分。根据这些数据，我们设计了一系列带有偶氮苯或三乙二醇残基的有效刺激响应性寡核苷酸抑制剂。这些修饰的寡核苷酸在冷却或加热后调节了 Nt.BspD6I 的功能活性。我们能够在存在这种抑制剂的情况下在 20-25°C 下阻止该酶对 T7 噬菌体 DNA 的切割，而在加热至 45°C 后，Nt.BspD6I 水解 DNA 的能力完全恢复。观察到的效应可以作为体外或体内通过外部信号调节尼克内切核酸酶活性的平台的开发基础。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/97b5/6261011/68b1d8656187/pone.0207302.g007.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/97b5/6261011/fb3c10ed21f4/pone.0207302.g001.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/97b5/6261011/f8430abbb6fa/pone.0207302.g002.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/97b5/6261011/140bfc29a30b/pone.0207302.g003.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/97b5/6261011/4044f174f3a5/pone.0207302.g004.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/97b5/6261011/6b266729a372/pone.0207302.g005.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/97b5/6261011/f400d9061346/pone.0207302.g006.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/97b5/6261011/68b1d8656187/pone.0207302.g007.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/97b5/6261011/fb3c10ed21f4/pone.0207302.g001.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/97b5/6261011/f8430abbb6fa/pone.0207302.g002.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/97b5/6261011/140bfc29a30b/pone.0207302.g003.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/97b5/6261011/4044f174f3a5/pone.0207302.g004.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/97b5/6261011/6b266729a372/pone.0207302.g005.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/97b5/6261011/f400d9061346/pone.0207302.g006.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/97b5/6261011/68b1d8656187/pone.0207302.g007.jpg

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