通过紫外光谱、圆二色光谱和量热技术监测G-四链体的温度解折叠。

Monitoring the temperature unfolding of G-quadruplexes by UV and circular dichroism spectroscopies and calorimetry techniques.

作者信息

Olsen Chris M, Marky Luis A

机构信息

Department of Pharmaceutical Sciences, University of Nebraska Medical Center, Omaha, NE, USA.

出版信息

Methods Mol Biol. 2010;608:147-58. doi: 10.1007/978-1-59745-363-9_10.

DOI:10.1007/978-1-59745-363-9_10

PMID:20012421

Abstract

DNA oligonucleotides containing guanine repeat sequences can adopt G-quadruplex (GQ) structures in the presence of specific metal ions. We report on how to use a combination of spectroscopic and calorimetric techniques to determine the spectral characteristics and thermodynamic parameters for the temperature-unfolding of GQs. Specifically, we investigated the unfolding of d(G(2)T(2)G(2)TGTG(2)T(2)G(2)), G2, and d(G(3)T(2)G(3)TGTG(3)T(2)G(3)), G3 by a combination of UV and circular dichroism (CD) spectroscopies, and differential scanning calorimetry (DSC).Analysis of the UV and CD spectra of these GQs at low (100% helix) and high (100% random coil) temperatures yielded the optimal wavelengths to determine the melting curves. In addition, the CD spectra yielded the particular conformation(s) that each GQ adopted at low temperature. DSC curves yielded complete thermodynamic profiles for the unfolding of each GQ. We use these profiles to determine the thermodynamic contributions for the formation of a G-quartet stack.

摘要

含有鸟嘌呤重复序列的DNA寡核苷酸在特定金属离子存在的情况下可以形成G-四链体（GQ）结构。我们报告了如何结合使用光谱和量热技术来确定GQ温度解折叠的光谱特征和热力学参数。具体而言，我们通过紫外光谱和圆二色光谱（CD）以及差示扫描量热法（DSC）相结合的方法，研究了d(G(2)T(2)G(2)TGTG(2)T(2)G(2))（G2）和d(G(3)T(2)G(3)TGTG(3)T(2)G(3))（G3）的解折叠情况。对这些GQ在低温（100%螺旋）和高温（100%无规卷曲）下的紫外光谱和CD光谱进行分析，得出了用于确定熔解曲线的最佳波长。此外，CD光谱还给出了每个GQ在低温下所采用的特定构象。DSC曲线给出了每个GQ解折叠的完整热力学曲线。我们利用这些曲线来确定形成G-四重体堆积的热力学贡献。