通过合成含 8-三氟甲基-2-脱氧鸟苷的寡核苷酸 DNA 观察 Z-DNA 结构。

Observation of Z-DNA Structure via the Synthesis of Oligonucleotide DNA Containing 8-Trifluoromethyl-2-Deoxyguanosine.

机构信息

Division of Chemistry, Department of Medical Sciences, Faculty of Medicine, University of Miyazaki, 5200 Kihara, Kiyotake, Miyazaki, Japan.

出版信息

Curr Protoc. 2021 Jan;1(1):e28. doi: 10.1002/cpz1.28.

DOI:10.1002/cpz1.28

PMID:33484490

Abstract

This article contains detailed synthetic protocols for the preparation of DNA oligonucleotides containing 8-trifluoromethyl-2'-deoxyguanosine ( dG) and their application to observe Z-DNA structure in vitro and in living HeLa cells. First, using a catalytic system consisting of FeSO , H SO , and H O in DMSO, we achieved a one-step synthesis of dG through a radical reaction between deoxyguanosine (dG) and CF I, with a yield of 45%. We then obtained the 3'-phosphoramidite of dG through a routine three-step procedure. Next, we employed the dG phosphoramidite monomer in the synthesis of oligonucleotides on a solid-phase DNA synthesizer. Finally, we used the dG-modified DNA oligonucleotides to observe Z-DNA structure in vitro and in living HeLa cells through F NMR spectroscopy. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Synthesis of dG phosphoramidites Basic Protocol 2: Preparation of dG-modified DNA oligonucleotides Basic Protocol 3: Evaluation of dG stabilization of Z-DNA structure by CD spectroscopy Basic Protocol 4: Investigation of Z-DNA structure in vitro and in HeLa cells with dG-modified DNA oligonucleotides and F NMR spectroscopy.

摘要

这篇文章包含详细的合成方案，用于制备含有 8-三氟甲基-2'-脱氧鸟苷（dG）的 DNA 寡核苷酸，并将其应用于体外和活 HeLa 细胞中观察 Z-DNA 结构。首先，我们使用由 FeSO4、H2SO4 和 H2O2 在 DMSO 中的催化体系，通过脱氧鸟苷（dG）与 CF3I 之间的自由基反应，一步法合成 dG，产率为 45%。然后，我们通过常规的三步法获得了 dG 的 3'-磷酰胺。接下来，我们在固相 DNA 合成仪上使用 dG 磷酰胺单体合成寡核苷酸。最后，我们使用 dG 修饰的 DNA 寡核苷酸通过 F NMR 光谱法在体外和活 HeLa 细胞中观察 Z-DNA 结构。©2021Wiley Periodicals LLC. 基础方案 1：dG 磷酰胺的合成基础方案 2：dG 修饰的 DNA 寡核苷酸的制备基础方案 3：通过 CD 光谱法评估 dG 稳定 Z-DNA 结构的能力基础方案 4：使用 dG 修饰的 DNA 寡核苷酸和 F NMR 光谱法在体外和 HeLa 细胞中研究 Z-DNA 结构。