连接环、 末端碱基及一价阳离子对G-四链体结构的影响

doi:10.7503/cjcu20140408

高等学校化学学报 ›› 2014, Vol. 35 ›› Issue (12): 2556.doi: 10.7503/cjcu20140408

连接环、末端碱基及一价阳离子对G-四链体结构的影响

常天俊¹(), 李刚¹, 赵可旭¹, 班琳¹, 卞文秀¹, 邴涛², 上官棣华²()

1. 河南理工大学资源环境学院, 焦作 454000
2.中国科学院化学研究所, 北京分子科学国家实验室, 活体分析化学院重点实验室, 北京100190

收稿日期:2014-04-29 出版日期:2014-12-10 发布日期:2014-11-29
作者简介:联系人简介: 常天俊, 男, 博士, 副教授, 主要从事功能核酸的分子识别与应用研究. E-mail:tjchang@iccas.ac.cn;上官棣华, 男, 博士, 研究员, 博士生导师, 主要从事生化分析与分子识别研究. E-mail:sgdh@iccas.ac.cn
基金资助:
北京分子科学国家实验室开放课题基金(2012)、河南省教育厅科学技术研究重点项目(批准号: 13A180328)和河南理工大学博士基金(批准号: B2013-068)资助

Effects of Loops, Flanking Bases and Monovalent Cations on the Structures of G-Quadruplexes^†

CHANG Tianjun^1,^*(), LI Gang¹, ZHAO Kexu¹, BAN Lin¹, BIAN Wenxiu¹, BING Tao², SHANGGUAN Dihua^2,^*()

1. Institute of Resources and Environment, Henan Polytechnic University, Jiaozuo 454000, China
2. Beijing National Laboratory for Molecular Sciences, Key Laboratory of Analytical Chemistry for Living Biosystems, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190, China

Received:2014-04-29 Online:2014-12-10 Published:2014-11-29
Contact: CHANG Tianjun,SHANGGUAN Dihua E-mail:tjchang@iccas.ac.cn;sgdh@iccas.ac.cn
Supported by:
† Supported by the Beijing National Laboratory for Molecular Sciences, China(2012), the Science and Technology Research Key Projects of Education Department of Henan Province, China(No.13A180328) and the Doctoral Program of Henan Polytechnic University, China(No.B2013-068).

摘要/Abstract

摘要：

研究了G-四链体中的连接环(Loop)、末端碱基和一价阳离子对其结构的影响, 发现在K⁺溶液中Loop短的序列易形成平行结构, 无末端碱基时容易形成多聚体, 而反平行或混合平行/反平行的G-四链体则难以形成多聚体; 一价阳离子K⁺, N $H 4 +$ 和Na⁺促进形成平行结构及多聚体的能力依次减弱. 在平行G-四链体的3'或5'端增加非G碱基, 或改变阳离子使其形成非平行结构, 均可抑制多聚体的形成. Loop长度影响G-四链体的热稳定性, Loop短的序列可形成很稳定的分子内结构; 无末端碱基的G-四链体多聚体的稳定性低于单个G-四链体, 且多聚体随着温度升高而变小. 结果表明, 在K⁺溶液中, 无末端碱基的平行G-四链体序列首先形成分子内结构, 然后通过π-π堆积形成多聚体; 末端碱基及反平行或混合平行/反平行G-四链体中的Loop可阻碍末端堆积作用, 抑制多聚体的形成. 本研究为G-四链体的结构与功能研究提供了有用信息.

关键词: G-四链体, 拓扑结构, DNA, 自组装, 多聚体

Abstract:

G-rich tandem repeats sequences are able to fold into structures called G-quadruplexes(G4s). G4s have complicated topological structures, which closely depend on the sequences and the solution conditions. In this work, the effects of loops, flanking bases and monovalent cations on the structure of G4s were investigated by gel electrophoresis, circular dichroism spectra and UV-Melting. The results showed that G4 forming sequences with short loops were favorable to form parallel structure in K⁺ solution, and the parallel G4s without flanking bases were able to further form G4 multimers, but the anti-parallel or mixed parallel/anti-parallel G4s were not. The monovalent cations had an influence on the adopted structures; K⁺ was more favorable for the formation of parallel G4 and G4 multimers than N $H 4 +$ and Na⁺. The addition of non-guanine bases to the terminals of parallel G4s, or the transformation of the parallel G4s to non-parallel structures by changing the cations, could inhibit the formation of G4 multimers. Furthermore, loop length could affect the thermo-stability of G4s; G4s with short loops had higher thermo-stability. The G4 multimers showed lower thermo-stability than G4 monomer, and their size and amount became smaller with the temperature increase. These results suggest that G4s with short loops and no flanking bases fold into intramolecular parallel structures firstly, and then form multimers through end stacking. The flanking bases and loops located on the G-quartets at 3' or 5' terminal hinder the stacking interactions, and inhibit the multimer assembly. Our results may offer useful clues for the structure and function studies of G4s.

Key words: G-Quadruplex, Topological structure, DNA, Self-assembly, Multimer

中图分类号:

O629.74

TrendMD:

常天俊, 李刚, 赵可旭, 班琳, 卞文秀, 邴涛, 上官棣华. 连接环、末端碱基及一价阳离子对G-四链体结构的影响. 高等学校化学学报, 2014, 35(12): 2556.

CHANG Tianjun, LI Gang, ZHAO Kexu, BAN Lin, BIAN Wenxiu, BING Tao, SHANGGUAN Dihua. Effects of Loops, Flanking Bases and Monovalent Cations on the Structures of G-Quadruplexes^†. Chem. J. Chinese Universities, 2014, 35(12): 2556.

图/表 5

Fig.1 Schematic representation of G-quartet and G-quadruplex structures (A) Chemical structure of G-quartet; schematic of intramolecular parallel G-quadruplex(B), intramolecular antiparallel G-quadruplex(C), mixed intramolecular parallel/antiparallel G-quadruplex(D), bimolecular antiparallel G-quadruplex(E), tetramolecular parallel G-quadruplex(F), dimeric parallel G-quadruplex of c-kit2[18](G) and dimeric parallel G-quadruplexes with 5'-5' stacking motif[19](H).

Table 1 DNA sequences and their Tm values in 20 mmol/L KCl*

Fig.2 CD spectra of DNAs without flanking base in K+ solution Sequences from a to i: VEGF18, EAD15, c-Myc16,c-Myc(b)20, c-kit19, CEB23, Bcl-2, HT, ILPR.

Fig.3 DNA analysis by denaturing-PAGE(A) and native-PAGE(B) (A) The gels for denaturing-PAGE contains 7 mol/L urea; (B) both the gels and the running buffers contain 20 mmol/L KCl for native-PAGE. The sequences were indicated on top of the gels. The horizontal dashed lines indicate the position of the molecular weight markers(Mw). The Mw of the sequences are: 15-mer,(AAGT)3AAG; 20-mer,(AAGT)5; 30-mer, (AAGT)7AA; 40-mer,(AAGT)10.

Fig.4 Native-PAGE of G-quadruplex forming sequences without flanking bases folded in buffers containing various concentrations of K+(A), Na+(B) and NH4+(C) Both the gels and running buffers contain 20 mmol/L K+, Na+, and N H 4 + ,respectively. (A1—C1) EAD15; (A2—C2) c-Myc16; (A3—C3) VEGF18; (A4—C4) c-kit19; (A5—C5) c-Myc(b)20; (A6—C6) CEB23. Concentration/(mmol·L-1): a. 20; b. 50; c. 80; d. 100.

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连接环、末端碱基及一价阳离子对G-四链体结构的影响

Effects of Loops, Flanking Bases and Monovalent Cations on the Structures of G-Quadruplexes^†

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连接环、 末端碱基及一价阳离子对G-四链体结构的影响

Effects of Loops, Flanking Bases and Monovalent Cations on the Structures of G-Quadruplexes†

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连接环、末端碱基及一价阳离子对G-四链体结构的影响

Effects of Loops, Flanking Bases and Monovalent Cations on the Structures of G-Quadruplexes^†