Single-molecule Manipulation of G-quadruplexes by Magnetic Tweezers

Huijuan You; Shimin Le; Hu Chen; Linyan Qin; Jie Yan

doi:10.3791/56328

JoVE Journal > Biochemistry

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Biyokimya

磁性镊子 quadruplexes 单分子操作

Published: September 19, 2017

doi:

10.3791/56328

Huijuan You*¹, Shimin Le*^2,3, Hu Chen^2,4, Linyan Qin¹, Jie Yan^2,4,5

¹School of Pharmacy, Tongji Medical College,Huazhong University of Science and Technology, ²Mechanobiology Institute,National University of Singapore, ³Fizik Bölümü,National University of Singapore, ⁴Research Institute for Biomimetics and Soft Matter, Fujian Provincial Key Lab for Soft Functional Materials Research, Department of Physics,Xiamen University, ⁵Center for Bioimaging Sciences,National University of Singapore

Özet

报道了一种分子磁镊 quadruplexes 的操作平台, 可以对各种蛋白质的 G4 稳定性和调节进行研究。

Abstract

非标准核酸二级结构 G quadruplexes (G4) 涉及不同的细胞过程, 如 DNA 复制, 转录, RNA 处理, 和端粒伸长。在这些过程中, 各种蛋白质结合, 并解决 G4 结构, 以履行其功能。由于 G4 的功能往往取决于其折叠结构的稳定性, 所以研究 G4 结合蛋白如何调节 G4 的稳定性是很重要的。这项工作提出了一种使用磁性镊子操作单 G4 分子的方法, 它能够研究 G4 结合蛋白在单个 G4 分子上的实时调节。一般而言, 这种方法适用于研究蛋白质/配体相互作用和各种 DNA 或 RNA 二级结构的规则的广泛应用。

Introduction

四-搁浅的 DNA 或 RNA G4 结构在许多重要的生物过程中扮演重要角色¹。许多蛋白质涉及 G4 结合和调节, 包括端粒结合蛋白 (端粒酶, POT1, RPA, TEBPs, TRF2)¹^,², 转录因子 (核仁, PARP1)³, RNA 处理蛋白 (hnRNP A1,hnRNP A2)⁴, 旋 (土地管理局, FANCJ, RHAU, 警告, Dna2, Pif1)⁵, 和 DNA 复制相关的蛋白质 (Rif1, REV1, PrimPolymerase)⁶。蛋白质结合能稳定或破坏 G4 结构;从而调节后续的生物功能。采用紫外 (UV) 或循环二 (CD) 方法测定了 G4 的稳定性.⁷。然而, 这些条件是不生理相关的, 很难适用于研究结合蛋白的影响⁷。

在分子操作技术的快速发展, 使研究的折叠和展开的生物, 如 DNA 或蛋白质, 在分子水平与纳米分辨率实时⁸。原子力显微镜 (AFM)、光镊和磁性镊子是最常用的分子操作方法。与 AFM 和光学镊子⁹相比, 磁性镊子可以使用抗技术¹⁰^、¹¹, 在几天内稳定地测量单个分子的折叠展开动力学。

在这里, 一个分子操作平台使用磁性镊子研究 G4 稳定性的调节结合蛋白报告¹²^,¹³。这项工作概述了基本的方法, 包括样品和流动通道的准备, 磁性镊子的设置, 和力量校准。3步所述的力控制和抗协议允许在各种力控制下进行长时间测量, 如恒定力 (力钳) 和恒定加载速率 (力-斜坡) 和力跃测量。4步中描述的力校准协议使 #60 的力标定; 1 µm 短系超过 100 pN, 相对误差在10% 以内。在解析 rna G4R1 中扮演重要角色的 rna 旋与富金元素 (RHAU) 旋 (别名 DHX36, G4) 的稳定性的调节示例用于演示此平台¹³的应用程序。

Protocol

1. 单分子拉伸的 G4 DNA 的制备准备5和 #39;-巯基标记和5和 #39;-生物素标记 dsDNA 处理, 使用 DDNA 聚合酶在 lambda 噬菌体 DNA 模板上使用5和 #39;-巯基和5和 #39;-生物素引物 14 ( 图 1 )。两个 dsDNA 处理都具有高 GC 含量 (和 #62; 60%), 以防止 dna 在高力量或 dna 过度过渡过程中的 dna 熔化 15 . 根据制造商和 #39 的协议, 使用商业纯化试…

Representative Results

在图 4中显示了拉伸单个 G4 分子的实验装置。在两个 dsDNA 手柄之间的单 G4 形成序列被拴在片和顺磁珠之间。为了找到一个单一的 dsDNA 栓珠, 通过在恒定加载速率下增加力来进行过度试验。三种测量方法通常用于研究生物分子的折叠和展开: (i) 恒定力测量, (ii) 力-斜坡测量, 和 (iii) 力跳跃测量。由于这个 G4 结构的非常缓慢的展开速率, 平衡折叠展开的…

Discussion

如上所述, 一个研究 G4 DNA 的机械稳定性的平台和 G4 使用分子磁性镊子的蛋白质相互作用的报告。在该平台的基础上, 开发了高效的 G4 DNA 链, 并对 G4 结构的折叠展开动力学和稳定性进行了测量。焦平面锁定使高度稳定的抗控制, 这是重要的检测小结构转变, 如 G4 (步长〜 7 nm) 和与蛋白质的相互作用在缓冲交换实验。该平台最近被用于研究端 G4¹⁴和 c-myc 启动子 G4¹²?…

Açıklamalar

The authors have nothing to disclose.

Acknowledgements

作者感谢孟潘校对手稿。这项工作得到新加坡教育部学术研究基金3层 (MOE2012-T3-1-001) 对彼莱的支持;国家研究基金会通过 Mechanobiology 研究所新加坡彼莱;国家研究基金会, 新加坡总理办公室, 根据其 NRF Investigatorship 方案 (NRF Investigatorship 奖 No。NRF-NRFI2016-03 到彼莱;中央大学基础研究基金 (2017KFYXJJ153) h.y。

Materials

DNA PCR primers	IDT		DNA preparations
DNA PCR chemicals	NEB		DNA preparations
restriction enzyme BstXI	NEB	R0113S	DNA preparations
coverslips (#1.5, 2232 mm, and 2020 mm)	BMH.BIOMEDIA	72204	flow channel preparation
Decon90	Decon Laboratories Limited		flow channel preparation
APTES	Sigma	440140-500ML	flow channel preparation
Sulfo-SMCC	ThermoFisher Scientific	22322	flow channel preparation
M-280, paramganetic beads,streptavidin	ThermoFisher Scientific	11205D	flow channel preparation
Polybead Amino Microspheres 3.00 μm	Polysciences, Inc	17145-5	flow channel preparation
2-Mercaptoethanol	Sigma	M6250-250ML	flow channel preparation
Olympus Microscopes IX71	Olympus	IX71	Magnetic tweezers setup
Piezo-Z Stages P-721	Physik Instrumente	P-721	Magnetic tweezers setup
Olympus Objective lense MPLAPON-Oil 100X	Olympus	MPLAPON-Oil 100X	Magnetic tweezers setup
CCD/CMOS camera	AVT	Pike F-032B	Magnetic tweezers setup
Translation linear stage	Physik Instrumente	MoCo DC	Magnetic tweezers setup
LED	Thorlabs	MCWHL	Magnetic tweezers setup
Cubic Magnets	Supermagnete		Magnetic tweezers setup
Labview	National Instruments		Magnetic tweezers setup
OriginPro/Matlab	OriginLab/MathWorks		Data analysis

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Etiketler

G-quadruplex Magnetic Tweezers Single-molecule Manipulation Folding And Unfolding Binding Proteins DNA-protein Interactions Helicase Activity DNA Tether Formation Paramagnetic Beads Nanometer-scale Dynamics

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You, H., Le, S., Chen, H., Qin, L., Yan, J. Single-molecule Manipulation of G-quadruplexes by Magnetic Tweezers. J. Vis. Exp. (127), e56328, doi:10.3791/56328 (2017).