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用于研究蛋白质 - DNA 相互作用的新型环境敏感多通道 DNA 荧光标记物。

New environment-sensitive multichannel DNA fluorescent label for investigation of the protein-DNA interactions.

作者信息

Kuznetsova Alexandra A, Kuznetsov Nikita A, Vorobjev Yuri N, Barthes Nicolas P F, Michel Benoît Y, Burger Alain, Fedorova Olga S

机构信息

Siberian Branch of the Russian Academy of Sciences, Institute of Chemical Biology and Fundamental Medicine, Novosibirsk, Russia and Department of Natural Sciences, Novosibirsk State University, Novosibirsk, Russia.

Institut de Chimie de Nice, UMR 7272, Université de Nice Sophia Antipolis, CNRS, Nice, France.

出版信息

PLoS One. 2014 Jun 12;9(6):e100007. doi: 10.1371/journal.pone.0100007. eCollection 2014.

DOI:10.1371/journal.pone.0100007
PMID:24925085
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC4055743/
Abstract

Here, we report the study of a new multichannel DNA fluorescent base analogue 3-hydroxychromone (3HC) to evaluate its suitability as a fluorescent reporter probe of structural transitions during protein-DNA interactions and its comparison with the current commercially available 2-aminopurine (aPu), pyrrolocytosine (Cpy) and 1,3-diaza-2-oxophenoxazine (tCO). For this purpose, fluorescent base analogues were incorporated into DNA helix on the opposite or on the 5'-side of the damaged nucleoside 5,6-dihydrouridine (DHU), which is specifically recognized and removed by Endonuclease VIII. These fluorophores demonstrated different sensitivities to the DNA helix conformational changes. The highest sensitivity and the most detailed information about the conformational changes of DNA induced by protein binding and processing were obtained using the 3HC probe. The application of this new artificial fluorescent DNA base is a very useful tool for the studies of complex mechanisms of protein-DNA interactions. Using 3HC biosensor, the kinetic mechanism of Endonuclease VIII action was specified.

摘要

在此，我们报告了对一种新型多通道DNA荧光碱基类似物3-羟基色酮（3HC）的研究，以评估其作为蛋白质-DNA相互作用过程中结构转变的荧光报告探针的适用性，并将其与目前市售的2-氨基嘌呤（aPu）、吡咯胞嘧啶（Cpy）和1,3-二氮杂-2-氧代吩恶嗪（tCO）进行比较。为此，将荧光碱基类似物掺入受损核苷5,6-二氢尿苷（DHU）相对侧或5'侧的DNA螺旋中，DHU可被核酸内切酶VIII特异性识别并切除。这些荧光团对DNA螺旋构象变化表现出不同的敏感性。使用3HC探针可获得关于蛋白质结合和加工诱导的DNA构象变化的最高灵敏度和最详细信息。这种新型人工荧光DNA碱基的应用是研究蛋白质-DNA相互作用复杂机制的非常有用的工具。使用3HC生物传感器，确定了核酸内切酶VIII的作用动力学机制。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/1a2a/4055743/e1f009c5cef3/pone.0100007.g015.jpg
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