Name: single-molecule manipulation of g-quadruplexes by magnetic tweezers
Uploaded: 2017-09-19T14:00:00.000+00:00
Duration: 8 min 28 s
Description: Watch this Scientific Journal Video about Single-molecule Manipulation of G-quadruplexes by Magnetic Tweezers at JoVE.com

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。

1. 单分子拉伸的 G4 DNA 的制备 <ol> <li> 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 . </li>
	<li> 根据制造商和 #39 的协议, 使用商业纯化试剂盒纯化 PCR 产品, 并用 BstXI 限制酶进行消化. </li>
	<li> 结扎 G4 形成 ssDNA, 侧面 ssDNA 和 dsDNA 处理使用 T4 DNA 连接根据制造商和 #39; s 协议。根据制造商和 #39 的协议, 使用商业纯化试剂盒, 用凝胶萃取法净化结扎后的产品. </li>
</ol> 2。流道的制备 <ol> <li> 片清洗和表面功能化 <ol> <li> 将底部片 (#1 5、22毫米和 #215; 32 mm) 和顶部片 (#1 5、20 mm x 20 mm) 放入覆盖玻璃染色罐 (每个罐子可容纳7件片,音量是 ~ 20 毫升)。用蒸馏水冲洗罐子里的片2-5 次. </li>
		<li> 在每个罐子中加入〜20毫升的5-40% 洗涤剂溶液, 然后放置在一个超声波清洗浴30分钟, 用蒸馏水冲洗 #62; 10 次清除洗涤剂. </li>
		<li> 烘干罐中的片 (〜150和 #176; 警告 、热) 或 N 2 气体。将干的顶片在干燥的柜子里. </li>
		<li> 使用等离子 (O 2 气体) 清洗罐中的片10分钟。在10分钟, 准备20毫升的 1% (3-氨基丙基) 烷 (APTES) 溶液在甲醇 ( 小心 , 有毒/易燃)。使用化学油烟机来溶解甲醇中的 APTES. 
		注意: 在存储 APTES 解决方案时避免湿度。老 APTES 常引起片表面纳米的问题. </li>
		<li> 在等离子清洗后立即将所有 1% APTES 溶液加入罐中, 并孵育1小时. 将废物倒入专用于 1% APTES 甲醇的废弃瓶中。执行与易燃化学品有关的烟罩中的甲醇相关步骤. </li>
		<li> 用甲醇和 #62 冲洗罐一次; 蒸馏水10次, 然后用烤箱烘干 (〜150和 #176; 警告 , 热)。将 APTES 涂层的底部片在干燥的柜子里, 如果不使用长达两周. 
		注意: 在每个步骤从2.1.1 到2.1.4 之后, 清洗过程可以在下一个步骤之前暂停. </li>
	</ol> </li> <li> 组装流通道 <ol> <li> 准备两个和 #34; 间隔和 #34; , 即 、膜或双面胶带 (〜 4 mm 和 #215; 20 mm), 用于每个通道。将两块垫片放置在 long-edge 的底部片上。在间隔处放置一个顶部片, 形成一个介于 (~ 10 毫米和 #215 之间的流动单元; 20 mm 区域, 图 2A , B ). </li>
		<li> 如果膜用作间隔, 则将流通道放置在加热器 (60-120 和 #176; C; 警告 , 热) 为5-10 秒, 而轻轻地按下顶部片的两侧, 以坚持两个片一起由膜。所产生的流量通道的高度为 ~ 100 和 #181; m, 从而该通道的音量是 ~ 20 和 #181; L. </li>
		<li> 用硅胶密封通道的长边以避免渗漏。使用硅胶胶水, 使一个小的水槽状结构在每个开放边缘的流通道, 作为一个入口和解决方案的出口. 
		注: 输入和退出也可以通过其他方式, 例如 , 通过粘附小塑料环使用蜡. </li>
		<li> 将通道存储在干燥的机箱中长达4周. </li>
	</ol> </li> <li> 将链式 DNA 放到流通道的底部表面 <ol> <li> 将氨基包覆的聚苯乙烯微珠 (直径: 3 和 #181; m) 在蒸馏1X 磷酸缓冲盐 (PBS) 缓冲液中稀释200X。涡珠溶液, 然后流入渠道。在通道中孵育30分钟的珠液, 用200和 #181 冲洗掉任何不通珠; 1X PBS 缓冲器. </li>
		<li> 调整 (增加/减少) 孵化时间, 以达到每 50 mm x 50 mm 面积1-5 珠的表面密度。存储与参考珠子沉积的通道长达3天. </li>
		<li> 稀释 sulfosuccinimidyl 4-(n-基) 环己烷-1-羧酸 (磺会) 粉末成 1X PBS 溶液 (~ 0.5 毫克/毫升)。漩涡的解决方案, 然后流入通道. 
		注意: 在使用前准备新鲜的磺-会溶液, 并立即将其添加到通道中, 以避免会的水解. </li>
		<li> 在通道中孵育会解决方案30分钟. 通过使用 1X PBS 解决方案中的大量 (1 毫升、50X 的通道容量) 清洗, 删除会解决方案. 
		注意: 仔细清洗多余的会。这一步对于 DNA 在片上的结合非常重要. </li>
		<li> dna 中继 <ol> <li> 将硫醇-生物素标记为 1X PBS, 其 dna 浓度为 0.3 nM。轻轻吸管混合溶液, 将 DNA 溶液流进会涂层通道, 室温孵育30分钟 (约23和 #176; C). </li>
		</ol> </li> <li> 用200和 #181 轻轻冲洗游离的 DNA; L 的堵塞解决方案, 含有 1X PBS 与10毫克/毫升牛血清白蛋白 (BSA) 和 0.01% 2-基. </li>
		<li> 通过在阻塞解决方案中孵化通道 (10 毫克/毫升 BSA, 0.01% 2-基) #62; 2 h; 在这一步之后, 通道就可以进行实验了。所准备的通道可保持在4和 #176; C 为 ~ 1 天. 
		注意: 阻断步骤对于减少 DNA 和磁珠对片的非特异性结合是非常重要的. </li>
	</ol> </li> </ol> 3。磁性镊子单 dsDNA 绳的设置和识别 <ol> <li> 磁性镊子设置 <ol> <li> 启动磁镊控制程序。在这里, 磁性镊子是由一个 in-house 编写的 LabVIEW 程序控制的. </li>
		<li> 在安装通道前对齐磁铁中心。使用4X 物镜来调整显微镜光轴中磁体的 x 和 y-axis. </li>
		<li> 使用计算机控制的机动机械手将磁体沿 z 方向移动 ( 图 2A ), 并将距离设置为 d = 0 ( d : 磁铁和片之间的距离) 当磁铁附着到显微镜上的片. </li>
		<li> 程序通过机械手的运动来实现对力的控制, 包括恒定力 ( 即 、常数 d ) 和时变力 F ( t ) ( 即 , 时间变化 d ( t)).</li>
		<li> 使用明亮的发光二极管 (LED) 光源, 用于后分散照明的珠子通过目标。用电荷耦合器件 (CCD) 摄像机采集100赫兹采样率的磁珠图像。i >> </ol> </li> <li> 示例设置和标识单 dsDNA 绳 <ol> <li> 系缆编队 <ol> 在200X 稀释 M-280 中轻轻流动, 顺磁性珠在化验溶液中 (100 毫米氯化钾, 2 毫米氯化镁 <li> 2 , 10 mm 三-pH 7。4) 变成一个通道。孵育10分钟, 使珠子绑定到生物素标记的 DNA 分子固定在会涂层表面通过硫醇标记结束。用200和 #181 的标准反应溶液, 轻轻地洗去 un-tethered 珠. 
			注: 与其它商业磁珠 (如 M-270) 相比, M-280 珠对表面具有较低的非特异性约束力. </li>
		</ol> </li> <li> 将通道安装到显微镜阶段。搜索底部表面的珠子使用100X 油浸泡目标. </li>
		<li> 选取曲面上的参照珠和移动的栓珠。在不同的离焦平面上建立参考珠和栓珠的初始图像库. </li>
		<li> 对珠子图像的校准 <ol> <li> 在实验之前, 使用一个客观的压电驱动器, 以获得在不同的离焦平面上的参考和拴珠图像 20-50 nm, 这是存储为两个独立的珠图像库并分别用散焦距离 ( 图 2D ) 对其进行索引. </li>
		在实验过程中, </ol> </li> <li> x、y、z 位置确定 <ol> <li> 在 x-y 平面上通过珠形质心确定磁珠的位置。通过比较当前的磁珠图像和存储在库中的图象, 确定栓珠的高度变化。用自函数分析了磁珠图像的傅里叶变换的功率谱 16 . </li>
		</ol> </li> <li> 在实验过程中使用参照珠 <ol> 的反漂移反馈, 使用压电 #34; 锁定和 #34; 通过低频存储在库中的目标与特定参考磁珠图像之间的距离反馈控制, 使卡住的磁珠图像与存储在库中的特定图像 ( 图 2D ) 具有最佳的相关性. </li>
		</ol> </li> <li> 通过应用 ~ 65 pN 力确定一个单一的 dsDNA 分子是否是一个单一的 dsDNA 分子, 如果系绳经历特征 DNA 过度过渡 17 , 18 , 19 . 重复该过程, 直到找到一个 dsDNA 的绳索。(请参阅下一节的力量校准和 DNA 过度过渡). </li>
	</ol> </li> </ol> 4。磁力钳力校准 <ol> <li> 直接确定长 dna (48502 bp 和 #955;-脱氧核糖核酸) 分子的力 (最多 100 pN), 使用磁珠起伏: <img alt="Equation 1" src="/files/ftp_upload/56328/56328eq1.jpg"/> ,其中 k B 是玻尔兹曼常量, T 是开尔文刻度中的温度, 和 #948; 2 y 是垂直于磁场和力的方向上的磁珠波动的方差方向.在这个方向上, 磁珠的运动可以被描述为一个钟摆的波动, 长度为 l + r 0 , 其中 l 是沿 DNA 的力方向 (扩展) 的端对边距离, r 0 是珠子的半径 10 ( 图 3A )。
	<li> 确定力的校准曲线 F 作为磁体到磁珠距离的函数 d , 以及 f ( d ), 用于使用长和 #955 的不同的珠子;-脱氧核糖核酸。通常, 磁铁和片之间的距离被用来作为 DNA 系绳长度可以被忽略。使 F - d 曲线符合 double-exponential 衰减函数: <img alt="Equation 2" src="/files/ftp_upload/56328/56328eq2.jpg"/> , 其中的管接头参数和 #945; 1、#945; 2、#947; 1, #947; 2 是由磁性镊子和参数 C 由磁珠的异质性决定。通过移动 C , 从不同的珠子获得的 F - d 曲线可以重叠 10 ( 图 3B )。
	<li> 对单个磁珠进行短 DNA 实验的参数 C 的校准。
	<ol> <li> 确定基于波动的力需要在比拐角频率高的频率上记录磁珠位置: <img alt="Equation 3" src="/files/ftp_upload/56328/56328eq3.jpg"/> , 位置和 #947; 拖动珠的系数。对于高力的短 DNA, 它需要记录频率比100赫兹快, 因此, 不能直接测量的力量, 根据波动与相机。但是, 参数 C 可以通过使用涨落来测量低力范围 (和 #60; 15 pN) 中的力, 并用校准的 F - d 曲线来拟合数据, 以获取参数 C 20 . </li>
		<li> 或者, 对于 dsDNA 的实验, 确定参数 C 使用 65 pN ( 图 3C ) 中的 DNA 过度转换, 21 , 22 , 23 通过记录 d 位置, dsDNA 显示了扩展跃迁 (0.6X 长度增加的轮廓长度)。在确定参数 C 后, 计算栓珠的力. </li>
	</ol> </li> <li> 通过将磁体的运动通过编程来控制加载速率 F ( d ) 的反函数。磁铁应该接近样品双指数. 
	注: 这种外推法确定的相对力误差为 10%, 主要由珠半径非均质性 20 引起. </li>
</ol> 5。G4 在结合蛋白存在和缺失时的单分子操作 <ol> <li> 强制斜坡实验 <ol> 在识别 dsDNA 绳后, 执行强制增加扫描, 其加载速率为 0.2 pN/秒, 后跟-0.2 pN/秒的强迫死亡扫描。在每一个拉伸周期之后, 在三十年代的 1 pN 中持有 DNA 分子, 让 ssDNA 再到 G4. </li>
	</ol> </li> <li> 强制跳转实验 <ol> <li> 在三十年代的 54 pn 之间循环, 折叠的 G4 可以展开, 1 pn 为 60s, 折叠的 G4-15T 可以再. </li>
	</ol> </li> <li> 流动蛋白溶液 <ol> 在 20 pN 力时流动蛋白质溶液, 以避免将珠子附着在片上。DEAH 盒 RHAU 旋, 它显示了高特异性 G4 结构, 使用了 24 。重组的 果蝇 RHAU (DmRHAU) 旋是在 大肠杆菌 中表达的, 如前所述 25 所描述的那样纯化。在 tetramolecular G4 DNA 基质上测定了 DmRHAU 的 G4 展开活性 13 . </li>
	</ol> </li> <li> 使用 in-house 编写的 Matlab 程序分析展开的力 14 . </li> </ol>

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J. 82 (6), 3314-3329 (2002).</li><li>Fu, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transition+dynamics+and+selection+of+the+distinct+S-DNA+and+strand+unpeeling+modes+of+double+helix+overstretching.">Transition dynamics and selection of the distinct S-DNA and strand unpeeling modes of double helix overstretching.</a> Nucleic Acids Res. 39 (8), 3473-3481 (2011).</li><li>Zhang, X., Chen, H., Fu, H., Doyle, P. S., Yan, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two+distinct+overstretched+DNA+structures+revealed+by+single-molecule+thermodynamics+measurements.">Two distinct overstretched DNA structures revealed by single-molecule thermodynamics measurements.</a> Proc Natl Acad Sci U S A. 109 (21), 8103-8108 (2012).</li><li>Zhang, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Revealing+the+competition+between+peeled+ssDNA,+melting+bubbles,+and+S-DNA+during+DNA+overstretching+by+single-molecule+calorimetry.">Revealing the competition between peeled ssDNA, melting bubbles, and S-DNA during DNA overstretching by single-molecule calorimetry.</a> Proc Natl Acad Sci U S A. 110 (10), 3865-3870 (2013).</li><li>Chen, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+High-Force+Magnetic+Tweezers+for+Stretching+and+Refolding+of+Proteins+and+Short+DNA.">Improved High-Force Magnetic Tweezers for Stretching and Refolding of Proteins and Short DNA.</a> Biophys. J. 100 (2), 517-523 (2011).</li><li>Fu, H. X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transition+dynamics+and+selection+of+the+distinct+S-DNA+and+strand+unpeeling+modes+of+double+helix+overstretching.">Transition dynamics and selection of the distinct S-DNA and strand unpeeling modes of double helix overstretching.</a> Nucleic Acids Res. 39 (8), 3473-3481 (2011).</li><li>Zhang, X., Chen, H., Fu, H., Doyle, P. S., Yan, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two+distinct+overstretched+DNA+structures+revealed+by+single-molecule+thermodynamics+measurements.">Two distinct overstretched DNA structures revealed by single-molecule thermodynamics measurements.</a> Proc. Natl. Acad. Sci. U.S.A. 109 (21), 8103-8108 (2012).</li><li>Zhang, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Revealing+the+competition+between+peeled+ssDNA,+melting+bubbles,+and+S-DNA+during+DNA+overstretching+by+single-molecule+calorimetry.">Revealing the competition between peeled ssDNA, melting bubbles, and S-DNA during DNA overstretching by single-molecule calorimetry.</a> Proc. Natl. Acad. Sci. U.S.A. 110 (10), 3865-3870 (2013).</li><li>Vaughn, J. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+DEXH+protein+product+of+the+DHX36+gene+is+the+major+source+of+tetramolecular+quadruplex+G4-DNA+resolving+activity+in+HeLa+cell+lysates.">The DEXH protein product of the DHX36 gene is the major source of tetramolecular quadruplex G4-DNA resolving activity in HeLa cell lysates.</a> J Biol Chem. 280 (46), 38117-38120 (2005).</li><li>Giri, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=G4+resolvase+1+tightly+binds+and+unwinds+unimolecular+G4-DNA.">G4 resolvase 1 tightly binds and unwinds unimolecular G4-DNA.</a> Nucleic Acids Res. 39 (16), 7161-7178 (2011).</li><li>De Vlaminck, I., Dekker, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+advances+in+magnetic+tweezers.">Recent advances in magnetic tweezers.</a> Annu Rev Biophys. 41, 453-472 (2012).</li><li>Yan, J., Skoko, D., Marko, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Near-field-magnetic-tweezer+manipulation+of+single+DNA+molecules.">Near-field-magnetic-tweezer manipulation of single DNA molecules.</a> Phys Rev E Stat Nonlin Soft Matter Phys. 70 (1 Pt 1), 011905 (2004).</li><li>Le, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disturbance-free+rapid+solution+exchange+for+magnetic+tweezers+single-molecule+studies.">Disturbance-free rapid solution exchange for magnetic tweezers single-molecule studies.</a> Nucleic Acids Res. 43 (17), e113 (2015).</li><li>Neidle, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quadruplex+Nucleic+Acids+as+Novel+Therapeutic+Targets.">Quadruplex Nucleic Acids as Novel Therapeutic Targets.</a> J Med Chem. 59 (13), 5987-6011 (2016).</li><li>Simone, R., Fratta, P., Neidle, S., Parkinson, G. N., Isaacs, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=G-quadruplexes:+Emerging+roles+in+neurodegenerative+diseases+and+the+non-coding+transcriptome.">G-quadruplexes: Emerging roles in neurodegenerative diseases and the non-coding transcriptome.</a> FEBS Lett. 589 (14), 1653-1668 (2015).</li><li>Balasubramanian, S., Hurley, L. H., Neidle, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+G-quadruplexes+in+gene+promoters:+a+novel+anticancer+strategy?.">Targeting G-quadruplexes in gene promoters: a novel anticancer strategy?.</a> Nat Rev Drug Discov. 10 (4), 261-275 (2011).</li><li>Amato, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toward+the+Development+of+Specific+G-Quadruplex+Binders:+Synthesis,+Biophysical,+and+Biological+Studies+of+New+Hydrazone+Derivatives.">Toward the Development of Specific G-Quadruplex Binders: Synthesis, Biophysical, and Biological Studies of New Hydrazone Derivatives.</a> J Med Chem. 59 (12), 5706-5720 (2016).</li><li>Wells, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-B+DNA+conformations,+mutagenesis+and+disease.">Non-B DNA conformations, mutagenesis and disease.</a> Trends Biochem Sci. 32 (6), 271-278 (2007).</li></ol>

作者没有什么可透露的。

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?...

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。

<table><tbody><tr><td>DNA PCR primers</td><td>IDT</td><td></td><td>DNA preparations</td></tr><tr><td>DNA PCR chemicals</td><td>NEB</td><td></td><td>DNA preparations</td></tr><tr><td>restriction enzyme BstXI</td><td>NEB</td><td>R0113S</td><td>DNA preparations</td></tr><tr><td>coverslips (#1.5, 22*32 mm, and 20*20 mm)</td><td>BMH.BIOMEDIA</td><td>72204</td><td>flow channel preparation</td></tr><tr><td>Decon90</td><td>Decon Laboratories Limited</td><td></td><td>flow channel preparation</td></tr><tr><td>APTES</td><td>Sigma</td><td>440140-500ML</td><td>flow channel preparation</td></tr><tr><td>Sulfo-SMCC</td><td>ThermoFisher Scientific</td><td>22322</td><td>flow channel preparation</td></tr><tr><td>M-280, paramganetic beads,streptavidin</td><td>ThermoFisher Scientific</td><td>11205D</td><td>flow channel preparation</td></tr><tr><td>Polybead Amino Microspheres 3.00 &amp;mu;m</td><td>Polysciences, Inc</td><td>17145-5</td><td>flow channel preparation</td></tr><tr><td>2-Mercaptoethanol</td><td>Sigma</td><td>M6250-250ML</td><td>flow channel preparation</td></tr><tr><td>Olympus Microscopes IX71</td><td>Olympus</td><td>IX71</td><td>Magnetic tweezers setup</td></tr><tr><td>Piezo-Z Stages P-721</td><td>Physik Instrumente</td><td>P-721</td><td>Magnetic tweezers setup</td></tr><tr><td>Olympus Objective lense MPLAPON-Oil 100X</td><td>Olympus</td><td>MPLAPON-Oil 100X</td><td>Magnetic tweezers setup</td></tr><tr><td>CCD/CMOS camera</td><td>AVT</td><td>Pike F-032B</td><td>Magnetic tweezers setup</td></tr><tr><td>Translation linear stage</td><td>Physik Instrumente</td><td>MoCo DC</td><td>Magnetic tweezers setup</td></tr><tr><td>LED</td><td>Thorlabs</td><td>MCWHL</td><td>Magnetic tweezers setup</td></tr><tr><td>Cubic Magnets</td><td>Supermagnete</td><td></td><td>Magnetic tweezers setup</td></tr><tr><td>Labview</td><td>National Instruments</td><td></td><td>Magnetic tweezers setup</td></tr><tr><td>OriginPro/Matlab</td><td>OriginLab/MathWorks</td><td></td><td>Data analysis</td></tr></tbody></table>

single-molecule manipulation of g-quadruplexes by magnetic tweezers

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。

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...

Watch this Scientific Journal Video about Single-molecule Manipulation of G-quadruplexes by Magnetic Tweezers at JoVE.com

Single-molecule Manipulation of G-quadruplexes by Magnetic Tweezers

磁性镊子 quadruplexes 单分子操作

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。

Non-canonical nucleic acid secondary structure G-quadruplexes (G4) are involved in diverse cellular processes, such as DNA replication, transcription, RNA ...

single-molecule-manipulation-of-g-quadruplexes-by-magnetic-tweezers

Research

JoVE Journal

Biochemistry

7.9K 次观看.  Huazhong University of Science and Technology. 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。

视频: Single-molecule Manipulation of G-quadruplexes by Magnetic Tweezers

A G-quadruplex DNA-affinity Approach for Purification of Enzymatically Active G4 Resolvase1

Higher-order nucleic acid structures called G-quadruplexes (G4s, G4 structures) can form in guanine-rich regions of both DNA and RNA and are highly thermally stable. There are &#62;375,000 putative G4-forming sequences in the human genome, and they are enriched in promoter regions, untranslated regions (UTRs), and within the telomeric repeat. Due to the potential for these structures to affect cellular processes, such as replication and transcription, the cell has evolved enzymes to manage them. One such enzyme is G4 Resolvase 1 (G4R1), which was biochemically co-characterized by our laboratory and Nagamine et al. and found to bind extremely tightly to both G4-DNA and G4-RNA (Kd in the low-pM range). G4R1 is the source of the majority of G4-resolving activity in HeLa cell lysates and has since been implicated to play a role in telomere metabolism, lymph development, gene transcription, hematopoiesis, and immune surveillance. The ability to efficiently express and purify catalytically active G4R1 is of importance for laboratories interested in gaining further insight into the kinetic interaction of G4 structures and G4-resolving enzymes. Here, we describe a detailed method for the purification of recombinant G4R1 (rG4R1). The described procedure incorporates the traditional affinity-based purification of a C-terminal histidine-tagged enzyme expressed in human codon-optimized bacteria with the utilization of the ability of rG4R1 to bind and unwind G4-DNA to purify highly active enzyme in an ATP-dependent elution step. The protocol also includes a quality-control step where the enzymatic activity of rG4R1 is measured by examining the ability of the purified enzyme to unwind G4-DNA. A method is also described that allows for the quantification of purified rG4R1. Alternative adaptations of this protocol are discussed.

G4 Resolvase1 binds to G-quadruplex (G4) structures with the tightest reported affinity for a G4-binding protein and represents the majority of the G4-DNA unwinding activity in HeLa cells. We describe a novel protocol that harnesses the affinity and ATP-dependent unwinding activity of G4-Resolvase1 to specifically purify catalytically active recombinant G4R1.

对酶活性G4 Resolvase1净化用G-四DNA亲和方法

Higher-order nucleic acid structures called G-quadruplexes (G4s, G4 structures) can form in guanine-rich regions of both DNA and RNA and are highly ...

科研

生物化学

High Precision FRET at Single-molecule Level for Biomolecule Structure Determination

A protocol on how to perform high-precision interdye distance measurements using F&#246;rster resonance energy transfer (FRET) at the single-molecule level in multiparameter fluorescence detection (MFD) mode is presented here. MFD maximizes the usage of all &#34;dimensions&#34; of fluorescence to reduce photophysical and experimental artifacts and allows for the measurement of interdye distance with an accuracy up to ~1 &#197; in rigid biomolecules. This method was used to identify three conformational states of the ligand-binding domain of the N-methyl-D-aspartate (NMDA) receptor to explain the activation of the receptor upon ligand binding. When comparing the known crystallographic structures with experimental measurements, they agreed within less than 3 &#197; for more dynamic biomolecules. Gathering a set of distance restraints that covers the entire dimensionality of the biomolecules would make it possible to provide a structural model of dynamic biomolecules.

A protocol for high-precision FRET experiments at the single molecule level is presented here. Additionally, this methodology can be used to identify three conformational states in the ligand-binding domain of the N-methyl-D-aspartate (NMDA) receptor. Determining precise distances is the first step towards building structural models based on FRET experiments.

单分子水平的高精度FRET用于生物分子结构测定

A protocol on how to perform high-precision interdye distance measurements using F&#246;rster resonance energy transfer (FRET) at the single-molecule ...

Multiplexed Single-molecule Force Proteolysis Measurements Using Magnetic Tweezers

The generation and detection of mechanical forces is a ubiquitous aspect of cell physiology, with direct relevance to cancer metastasis1, atherogenesis2 and wound healing3. In each of these examples, cells both exert force on their surroundings and simultaneously enzymatically remodel the extracellular matrix (ECM). The effect of forces on ECM has thus become an area of considerable interest due to its likely biological and medical importance4-7.
	Single molecule techniques such as optical trapping8, atomic force microscopy9, and magnetic tweezers10,11 allow researchers to probe the function of enzymes at a molecular level by exerting forces on individual proteins. Of these techniques, magnetic tweezers (MT) are notable for their low cost and high throughput. MT exert forces in the range of ~1-100 pN and can provide millisecond temporal resolution, qualities that are well matched to the study of enzyme mechanism at the single-molecule level12. Here we report a highly parallelizable MT assay to study the effect of force on the proteolysis of single protein molecules. We present the specific example of the proteolysis of a trimeric collagen peptide by matrix metalloproteinase 1 (MMP-1); however, this assay can be easily adapted to study other substrates and proteases.

In this article we describe the use of magnetic tweezers to study the effect of force on enzymatic proteolysis at the single molecule level in a highly parallelizable manner.

警队复用单分子蛋白水解测量磁性镊子

The  generation and detection of mechanical forces is a ubiquitous aspect of cell physiology,  with direct relevance to cancer metastasis1, atherogenesis2 ...

生物工程

Magnetic Tweezers for the Measurement of Twist and Torque

Single-molecule techniques make it possible to investigate the behavior of individual biological molecules in solution in real time. These techniques include so-called force spectroscopy approaches such as atomic force microscopy, optical tweezers, flow stretching, and magnetic tweezers. Amongst these approaches, magnetic tweezers have distinguished themselves by their ability to apply torque while maintaining a constant stretching force. Here, it is illustrated how such a &#8220;conventional&#8221; magnetic tweezers experimental configuration can, through a straightforward modification of its field configuration to minimize the magnitude of the transverse field, be adapted to measure the degree of twist in a biological molecule. The resulting configuration is termed the freely-orbiting magnetic tweezers. Additionally, it is shown how further modification of the field configuration can yield a transverse field with a magnitude intermediate between that of the &#8220;conventional&#8221; magnetic tweezers and the freely-orbiting magnetic tweezers, which makes it possible to directly measure the torque stored in a biological molecule. This configuration is termed the magnetic torque tweezers. The accompanying video explains in detail how the conversion of conventional magnetic tweezers into freely-orbiting magnetic tweezers and magnetic torque tweezers can be accomplished, and demonstrates the use of these techniques. These adaptations maintain all the strengths of conventional magnetic tweezers while greatly expanding the versatility of this powerful instrument.

Magnetic tweezers, a powerful single-molecule manipulation technique, can be adapted for the direct measurements of the twist (using a configuration called freely-orbiting magnetic tweezers) and torque (using a configuration termed magnetic torque tweezers) in biological macromolecules. Guidelines for performing such measurements are given, including applications to the study of DNA and associated nucleo-protein filaments.

磁性镊子的扭曲和扭矩的测量

Single-molecule techniques make it possible to investigate the behavior of individual biological molecules in solution in real time. These techniques ...

Nanomanipulation of Single RNA Molecules by Optical Tweezers

A large portion of the human genome is transcribed but not translated. In this post genomic era, regulatory functions of RNA have been shown to be increasingly important. As RNA function often depends on its ability to adopt alternative structures, it is difficult to predict RNA three-dimensional structures directly from sequence. Single-molecule approaches show potentials to solve the problem of RNA structural polymorphism by monitoring molecular structures one molecule at a time. This work presents a method to precisely manipulate the folding and structure of single RNA molecules using optical tweezers. First, methods to synthesize molecules suitable for single-molecule mechanical work are described. Next, various calibration procedures to ensure the proper operations of the optical tweezers are discussed. Next, various experiments are explained. To demonstrate the utility of the technique, results of mechanically unfolding RNA hairpins and a single RNA kissing complex are used as evidence. In these examples, the nanomanipulation technique was used to study folding of each structural domain, including secondary and tertiary, independently. Lastly, the limitations and future applications of the method are discussed.

Optical tweezers have been used to study RNA folding by stretching individual molecules from their 5&#8217; and 3&#8217; ends. Here common procedures are described to synthesize RNA molecules for tweezing, calibration of the instrument, and methods to manipulate single molecules.

单个RNA分子的光镊纳米操作

A large portion of the human genome is transcribed but not translated. In this post genomic era, regulatory functions of RNA have been shown to be ...

Magnetic Tweezers in a Microplate Format

Mechanobiology describes how the physical forces and mechanical properties of biological material contribute to physiology and disease. Typically, these approaches are limited single-molecule methods, which restricts their availability. To address this need, a microplate assay was developed that enables mechanical manipulation while performing standard biochemical assays. This is achieved using magnets incorporated into a microplate lid to create multiple magnetic tweezers. In this format, force is exerted across biomolecules connected to paramagnetic beads, equivalent to a typical magnetic tweezer. The study demonstrates the application of this tool with FRET-based assays to monitor protein conformations. However, this approach is widely applicable to different biological systems ranging from measuring enzymatic activity through to the activation of signaling pathways in live cells.

Here, we describe the use of a novel microplate assay to enable mechanical manipulation of biomolecules while performing ensemble biochemical assays. This is achieved using a microplate lid modified with magnets to create multiple static magnetic tweezers across the microplate.

Mechanobiology describes how the physical forces and mechanical properties of biological material contribute to physiology and disease. Typically, these ...

使用化学作图鉴定 DNA 模板中的 G-四链体

In Vitro Chemical Mapping of G-Quadruplex DNA Structures by Bis-3-Chloropiperidines

G-quadruplexes (G4s) are biologically relevant, non-canonical DNA structures that play an important role in gene expression and diseases, representing significant therapeutic targets. Accessible methods are required for the in vitro characterization of DNA within potential G-quadruplex-forming sequences (PQSs). B-CePs are a class of alkylating agents that have proven to be useful chemical probes for investigation of the higher-order structure of nucleic acids. This paper describes a new chemical mapping assay exploiting the specific reactivity of B-CePs with the N7 of guanines, followed by direct strand cleavage at the alkylated Gs. 
Namely, to distinguish G4 folds from unfolded DNA forms, we use B-CeP 1 to probe the thrombin-binding aptamer (TBA), a 15-mer DNA able to assume the G4 arrangement. Reaction of B-CeP-responding guanines with B-CeP 1 yields products that can be resolved by high-resolution polyacrylamide gel electrophoresis (PAGE) at a single-nucleotide level by locating individual alkylation adducts and DNA strand cleavage at the alkylated guanines. Mapping using B-CePs is a simple and powerful tool for the in vitro characterization of G-quadruplex-forming DNA sequences, enabling the precise location of guanines involved in the formation of G-tetrads.

作者聚焦：通过基于 Bis-3-氯哌啶的化学定位表征 DNA G-四链体 

Bis-3-chloropiperidines (B-CePs) are useful chemical probes to identify and characterize G-quadruplex structures in DNA templates in vitro. This protocol details the procedure to perform probing reactions with B-CePs and to resolve reaction products by high-resolution polyacrylamide gel electrophoresis.

体外研究双-3-氯哌啶对G-四链体DNA结构的化学定位

G-quadruplexes (G4s) are biologically relevant, non-canonical DNA structures that play an important role in gene expression and diseases, representing ...

High-Speed Magnetic Tweezers for Nanomechanical Measurements on Force-Sensitive Elements

Single-molecule magnetic tweezers (MTs) have served as powerful tools to forcefully interrogate biomolecules, such as nucleic acids and proteins, and are therefore poised to be useful in the field of mechanobiology. Since the method commonly relies on image-based tracking of magnetic beads, the speed limit in recording and analyzing images, as well as the thermal fluctuations of the beads, has long hampered its application in observing small and fast structural changes in target molecules. This article describes detailed methods for the construction and operation of a high-resolution MT setup that can resolve nanoscale, millisecond dynamics of biomolecules and their complexes. As application examples, experiments with DNA hairpins and SNARE complexes (membrane-fusion machinery) are demonstrated, focusing on how their transient states and transitions can be detected in the presence of piconewton-scale forces. We expect that high-speed MTs will continue to enable high-precision nanomechanical measurements on molecules that sense, transmit, and generate forces in cells, and thereby deepen our molecular-level understanding of mechanobiology.

Here, we describe a high-speed magnetic tweezer setup that performs nanomechanical measurements on force-sensitive biomolecules at the maximum rate of 1.2 kHz. We introduce its application to DNA hairpins and SNARE complexes as model systems, but it will be also applicable to other molecules involved in mechanobiological events.

高速磁镊，用于力敏元件的纳米力学测量

Single-molecule magnetic tweezers (MTs) have served as powerful tools to forcefully interrogate biomolecules, such as nucleic acids and proteins, and are ...

磁性镊子 quadruplexes 单分子操作

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对酶活性G4 Resolvase1净化用G-四DNA亲和方法

单分子水平的高精度FRET用于生物分子结构测定

警队复用单分子蛋白水解测量磁性镊子

磁性镊子的扭曲和扭矩的测量

单个RNA分子的光镊纳米操作

Magnetic Tweezers in a Microplate Format

体外研究双-3-氯哌啶对G-四链体DNA结构的化学定位

体外研究双-3-氯哌啶对G-四链体DNA结构的化学定位

体外研究双-3-氯哌啶对G-四链体DNA结构的化学定位

高速磁镊，用于力敏元件的纳米力学测量