Name: studying dna looping by single-molecule fret
Uploaded: 2014-06-28T15:00:00.000+00:00
Duration: 11 min 27 s
Description: Watch this Scientific Journal Video about Studying DNA Looping by Single-Molecule FRET at JoVE.com

我们感谢詹姆斯·沃特斯，山墙沃兹沃斯和博Broadwater的紧要阅读手稿。我们也感谢4匿名审稿人提供有用的意见。我们承认从乔治亚理工学院，宝来惠康基金事业奖在科学界面，并从生命系统的NSF物理学学生研究网络赠款资金支持。

1，双链DNA样品制备<ol><li>通过重复10个碱基序列设计全局弯曲的DNA。例如，5&#39;-GTGCCAGCAACAGATAGC - （TTTATCATCCTTTATCATCC 倍 ）7 - TTTCATTCGAGCTCGTTGTTG-3&#39;是一个186-bp的DNA弯曲，其中X是一个随机额外的碱和序列侧翼的重复10 -聚体序列的接头序列。 注意:在这个例子中两个10聚体与基于由Kaplan 等大规模的核小体占据的研究相反的喜好来核小体形成15被选中。由于双链DNA的螺旋重复接近10基点的10 -聚体的螺旋轴的任何净挠度会积累，产生类似的圆弧（ 图3A）的形状。由于螺旋周期为越接近10.5沸点，一个额外的基底被插入每两个重复序列后，以保持弯曲的结构的平面成为可能。这些序列大于200 bp的短可以从companie订购s表示提供基因合成服务。是很方便的侧翼这些序列与用于后续步骤中共同接头序列。该程序是图式在图3B中。 </li><li>用引物1（GTGCCAGCAACAGATAGC）和引物2（/ 5Cy3/TAAATTCCTACAACAACGAGCTCGAATG）进行PCR。注:引物2被标记的FRET供体Cy3标记的5&#39;末端。一个典型的PCR配方和骑自行车的协议列于表1和表2。 </li><li>用引物3（/ 5BioTEG/GAAACATAG/iCy5/GAATTTACCGTGCCAGCAACAGATAGC）和引物4（CAACAACGAGCTCGAATG）进行PCR。注:引物3被标记通过骨干和生物素 - 接头在5&#39;端的FRET受体的Cy5。 PCR技术配方和骑自行车的协议如上。 </li><li>使用PCR净化试剂盒纯化PCR产物。 </li><li>以最终浓度为0.4和混合在一个缓冲区链交换（100mM的氯化钠，10mM的Tris-盐酸的pH 7.0,1 mM EDTA）中的Cy3标记产物和Cy5标记产物＃181，M和0.1微米。注意:过量的Cy3-DNA增加了双面同时携带Cy3和Cy5的浓度为链交换的结果。 </li><li>通过在98.5℃下培养2分钟交换链，逐渐冷却到5 ℃，0.1℃/秒的升温速率，并温育5 &#160; °下加热2小时。 </li></ol> 2，凝胶电泳来检测双链DNA曲率<ol><li>通过将丙烯酰胺和双丙烯酰胺溶液在29.2:0.8比例倒入聚丙烯酰胺凝胶16,17，5％（重量/体积）在1X的Tris /硼酸/ EDTA（TBE）缓冲液，pH 8.0。注:10的凝胶溶液包含:1.217毫升40％的丙烯酰胺，0.667毫升2％双丙烯酰胺，加入1ml TBE 10X，100升的过硫酸铵（APS），10％，10升TEMED，其余的是DH 2 O。凝胶的充分固化需要大约30分钟。 </li><li>加载聚丙烯酰胺凝胶用DNA样品和在1X上样缓冲液的DNA梯状条带（5％甘油，0.03％（重量/体积）溴酚BLUE），并在5-8运行凝胶V / cm时，在4℃下进行45分钟，或直至染料前沿接近凝胶的末端。 </li><li>使用1×TBE缓冲液包含0.5克/毫升的溴化乙锭进行30分钟染色的凝胶。确定在紫外光照射下的DNA条带。比较频带位置与大小标记（100 bp的DNA梯状条带）来计算DNA分子的表观体积。注:弯的DNA通常比移动笔直的DNA慢。 </li></ol> 3，流通池准备<ol><li>钻6-7对孔沿载玻片用钻床和金刚石钻头的两个相对的边缘（3&#39;×1&#39;）。钻孔后，擦在流水，除去可见的玻璃粉末的幻灯片。注:孔作为灌注入口和出口。在钻井过程中，冷却水的滑动是非常重要的，以防止开裂。 </li><li>将玻片直立在一个玻璃罐子，装满水。超声处理15分钟，然后将其转移到另一个玻璃瓶德dicated丙酮清洗。它用丙酮进行超声波处理15分钟，加满。用一个喷雾瓶，然后用清水冲洗，用乙醇的幻灯片。将它们放置在聚丙烯罐，用5个M氢氧化钾填写，超声15分钟。最后，超声处理在水中载玻片15分钟。使用相同的协议清洁的盖玻片（第1号，24×40毫米）。注意:清洗载玻片和盖玻片可以存储在DH 2 O长期使用。 </li><li>拌1毫克生物素-PEG-硅烷（分子量3400）与80毫克的mPEG-硅烷（分子量2000）在340升0.1M碳酸氢钠溶液中。搅拌均匀，离心混合物短暂地摆脱泡沫。注意:用聚乙二醇（PEG）表面的官能化有助于减少DNA与表面的非特异性结合。 </li><li>把80升的PEG溶液的每张幻灯片上，并轻轻地放下盖玻片了它。等待45分钟。从幻灯片用镊子分开盖玻片，用DH 2 O的丰富量冲洗，并令t下摆干在露天。 </li><li>把细条双面胶带穿过滑动，以形成通道。对齐盖玻片过它，并用力按下盖玻片对滑动，形成液体密封通道。使用5分钟环氧树脂密封的通道的边缘。 </li></ol> 4制备水溶性维生素E的解决方案<ol><li>放〜30毫克Trolox的和10ml DH 2 O中的烧瓶中，并使用一个磁铁搅拌棒搅拌该溶液在开放的空气中18小时。 </li><li>使用0.2微米的过滤器过滤该溶液，并通过添加〜6微升的1M Tris碱（pH为11），将pH调节至7。注意:Trolox的是抗闪烁试剂也就是通常在单分子用于研究18。 Trolox的的抗褪色作用来源于其氧化衍生物，其存在于部分降解的Trolox的溶液19。由于低效的氧化，在甲醇或高pH的Tris溶液迅速溶解Trolox的，应该避免。 </li></ol> 5，单摩尔ecule成像<ol><li>注射15微升的NeutrAvidin溶液（0.5毫克/毫升）进入通道，并等待2分钟，加入100μlT50缓冲液（10mM的Tris-HCl，50 mM氯化钠，pH 7.0）中漂洗之前。 </li><li>注入50μl的DNA样品（50-PM）的进入通道。等待5分钟，用100升的T50缓冲冲洗未结合DNA的路程。注意:DNA分子将具体通过的NeutrAvidin - 生物素相互作用结合到表面上。 </li><li>填补了通道与包含氧清除系统20（100毫米PCD，5毫米的PCA，1 mM的水溶性维生素E和500 mM氯化钠）的成像缓冲区。 </li><li>设置在帧传输模式的EMCCD流2×2的离散化的图像（256×256）到计算机每秒25帧。 </li><li>放置在显微镜物镜浸油，并用试样夹固定在显微镜载物台上的流动池。通过观察在墙壁上的激光反射图案粗调整焦点。用荧光IM的即时画面精细调焦年龄在监视器上。 </li><li>开始数据采集与532 nm激光。检查现场查看，并在必要时调整对焦。停止数据采集，当大多数分子的光漂白。 注意:在这个设施，实验室编写C程序来控制显微镜和显示器上显示实时图像，因为它们被保存到硬盘上使用。 </li></ol> 6，图像处理和数据分析注:时间序列的256×256的图像是由一个MATLAB代码处理，生成的Cy3和Cy5强度单分子时间的痕迹。配对的供体通道和分割后的视点图像的受体通道之间的像素，6-7对Cy3和Cy5的斑点，每对来自同一分子穿过视场均匀地分散，是手工采摘，并且仿射变换使用这些点的坐标作为支撑点的计算方法。 <ol><li>使用MATLAB脚本，通过所有的单分子时间看痕迹吨低和高的FRET信号之间的帽子秀多个过渡。确定循环和不成圈的状态。 注:FRET信号被定义为Cy5 ​​的强度（I 一 ）除以的Cy3和Cy5的强度之和（I A + I D）。循环状态，具有较高的荧光共振能量转移的价值，同时不成圈状态，具有低FRET值。从一个单分子FRET信号的直方图是双峰分布，因为可逆循环和unlooping。 </li><li>发现，通过确定两个拟合高斯曲线之间的交叉点隔开的两个分布的阈值。 </li><li>计算FRET效率<img fo:content-width="0.6in" src="/files/ftp_upload/51667/eq7.jpg" />并指派具有高FRET值与不成圈的状态与低FRET值的循环状态。 </li><li>使用MATLAB脚本，分析分子（N（t））的累计数环（或由于在数据采集的开始时间不同的失误达到了高FRET状态）。注:由于双链DNA分子可以在数据采集开始在的不成圈状态的任何构象开始，增加了环形人口的比率，反映的平均循环率平均超过初始构象。提取循环率 k 循环用指数函数拟合N（T）: <img fo:content-width="1.8in" src="/files/ftp_upload/51667/eq1.jpg" />如果增加biphasically，它可以被装配有双指数函数: <img fo:content-width="2.2in" src="/files/ftp_upload/51667/eq2.jpg" />在这种情况下，K 循环从获得: <img fo:content-width="1.7in" src="/files/ftp_upload/51667/eq3.jpg" />注意:从理论上讲，生存概率的聚合物并没有循环在时间t不是单一的或双指数函数12。指数FITTing是用来作为一种实用的装置，提取该平均循环时间。 </li></ol> 7，确定第j因子注:歼因子代表如何集中一个双链DNA的一端周围的另一端。它可以通过内插的DNA中的一个端部段，将产生相同的反应速率与另一端部分作为测量循环速率的浓度来确定。在实验上，其一端部分被固定在表面上，而另一端段是在一定的浓度c引入。如果两端之间测量的退火速率为k 退火 ，然后歼因子21由下式给出<img fo:content-width="0.6in" src="/files/ftp_upload/51667/eq4.jpg" /> 。退火速率常数（k 退火 = K 退火 / C）是独立于探头的浓度。 <ol><li>流20μl的30-50 PM生物素Cy5的寡核苷酸（引物3）我成一的NeutrAvidin涂层通道。冲洗通道用100微升T50洗去未结合的寡核苷酸。 </li><li>准备成像缓冲器中的部分5与在50nM的终浓度添加Cy3标记的寡核苷酸（引物2）的说明。流入该成像缓冲器进入通道。 </li><li>在保持532 nm激光上，简要地打开640 nm激光，以确定表面结合Cy5标记的寡核苷酸的位置。关闭640 nm激光，并开始监测FRET信号。 注意:当一个Cy3标记的寡到一个表面结合Cy5标记的寡核苷酸的杂交，Cy5的荧光将产生FRET。 </li><li>使用MATLAB脚本，分析分子开始在绑定状态（低Cy5的强度），但后来变成了退火状态（高Cy5的强度）作为时间从Cy5的强度痕迹函数的数量。 </li><li>剧情退火分子随时间的这个数字。适合这个曲线用单指数函数（ <img fo:content-width="1.8in" src="/files/ftp_upload/51667/eq5.jpg"/&gt;）获得退火速率（K 退火 ）。 </li><li>重复此实验在不同的Cy3标记的寡核苷酸的浓度（60，100，和180纳米），以确认退火速率和反应物浓度之间的线性关系。解二阶退火速率常数（k 退火 ）从斜率。 </li><li>计算从歼因素<img fo:content-width="0.6in" src="/files/ftp_upload/51667/eq6.jpg" /> ，其中k是循环在相同缓冲液条件下测得的循环速率。 </li></ol>

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Acids Res. 37, 6984-6990 (2009).</li><li>Vologodskii, A., 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=Bending+of+short+DNA+helices.">Bending of short DNA helices.</a> Artif DNA PNA XNA. 4, (2013).</li><li>Hoover, D. M., Lubkowski, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNAWorks:+an+automated+method+for+designing+oligonucleotides+for+PCR-based+gene+synthesis.">DNAWorks: an automated method for designing oligonucleotides for PCR-based gene synthesis.</a> Nucl. Acids Res. 30, (2002).</li><li>Waters, J. T., Kim, H. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Equilibrium+Statistics+of+a+Surface-Pinned+Semiflexible+Polymer.">Equilibrium Statistics of a Surface-Pinned Semiflexible Polymer.</a> Macromolecules. 46, 6659-6666 (2013).</li><li>Mills, J. 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=Electrophoretic+evidence+that+single-stranded+regions+of+1+or+more+nucleotides+dramatically+increase+the+flexibility+of+DNA.">Electrophoretic evidence that single-stranded regions of 1 or more nucleotides dramatically increase the flexibility of DNA.</a> Biochemistry. 33, 1797-1803 (1994).</li></ol>

作者宣称没有利益冲突。

基于FRET一个简单的单分子实验来研究不同的内在形状DNA的循环动力学。弯曲的DNA可以通过重复一个10 - 聚体序列中的相位与10.5沸点螺旋周期来制备，并且其曲率可以用PAGE进行估计。这些dsDNAs都设计有粘性末端，让短暂的环路稳定性。我们从指数上升的环的分子数随时间提取的循环速率。断开连接的粘性末端段之间的退火速率常数是用来确定该浓度相当于该环形概率密度，这是被称为歼因子。 在连接酶为基础的DNA环化法，DNA闭合概率的测量是将连接酶浓度敏感，歼因子可以如连接酶是过量9被高估。非特异性DNA连接酶与DNA的结合也可影响循环10。此外，ligasE活动依赖于盐和随着时间衰减。因此它是难以研究的DNA循环中的条件不理想的用于连接酶活性。与此相反，一个基于FRET的循环测定法是从所有的这些问题。 我们的实验方案是类似Vafabakhsh和哈13中，除了两个重要的方面这是值得讨论的。 Vafabakhsh和13哈结扎几种合成寡核苷酸构成的双链DNA分子。虽然每个核苷酸的错误（插入，缺失或替换）速率在 ​​寡核苷酸合成是可以忽略的（F 0.181％）23，非预期的DNA序列的部分线性寡核苷酸的长度增加而增加。因此，制备本协议的100-bp的双链样品可以包含多达〜33％的不完全双链这可能导致更高的循环速率24。相比之下，我们的协议采用聚合酶链反应（PCR），合成的双链DNA分子，其具有更高的保真度比DNA枝mosynthesis 25。根据制造商的数据，我们使用高保真聚合酶将后PCR扩增30个循环产生一个正确的100-bp的DNA分子，在99.8％的概率。 这两项研究之间的另一个主要区别是表面固定方案。在我们的研究中，DNA分子通过它们的端部附连到表面，而在协议由Vafabakhsh和哈13，它们通过一个内部基极连接。据预测，仅基于约束效应，内部固定的双链DNA可循环比一个免费的dsDNA更频繁，多达五个因素取决于内部牵制26的位置。因此，测量歼因素，因为DNA的轮廓长度的函数时，内部牵制可以引入意想不到的变化。它也有可能是与连接子修饰碱基具有弱碱基配对的能力，这可以导致更高的循环速率。然而，DESP伊特这些明显的差异，这两个协议产生类似Ĵ因素在100个基点（〜3纳米与〜2纳米）。 这FRET为基础的循环试验有希望的研究范围广泛的涉及到双链DNA循环，包括不匹配，刻痕或缺口的双链DNA上的作用循环27的DNA结合蛋白的双链DNA循环的影响，以及温度的双链DNA的灵活性依赖现象。这些课题将难以解决与常规的连接酶为基础的环化反应。在基于FRET的循环测定法，也可用于测量歼因子与轮廓长度，这是标准的关系来测试聚合物的模型。但是，我们这里提出的协议并不适合来研究双链DNA循环大于100 bp的短。低于这个长度，双链DNA的循环变得非常慢，因此，所测量的速度会受到其他非预期过程，如漂白。人们可以通过我们加快明显的循环动力学ing高盐浓度（[Na +]介绍&gt; 500毫米），但这种生理测量的相关性变得可疑。因此，双链DNA的调查，在低于100 bp的长度尺度弯曲将从做法正交利于​​环为基础的检测。

了解双链DNA的力学性能是极为重要的基础科学和工程应用。双链DNA的结构比直螺旋阶梯更加复杂，因为连续的碱基对之间滚动，倾斜和扭转角可以与序列有所不同。热波动可能会导致双链DNA，以进行构象波动的不同模式，如弯曲，扭转和伸展。过渡，例如熔化和扭结，也可能发生在极端条件下。 在这些议案，双链DNA弯曲有最明显的生物学影响1。双链DNA弯曲与基因抑制或激活通过将两个遥远接近彼此关联。它也起着在细胞核内的DNA包装或病毒衣壳中起重要作用。双链DNA的弯曲变形可以通过实验由高分辨率显微镜（AFM 2和TEM 3），以及thermodyn进行可视化AMICS和动力学可以通过循环检测，其化学连接的双链DNA并列的网站进行研究。 一个这样的实验是连接酶依赖的环化4。在该试验中，双链DNA分子的"粘性"（聚）端部发送或通过DNA连接酶二聚化。通过比较圆和二聚物的形成速率，可以得到在其另一端，这是已知的歼因子附近的DNA的一端的有效摩尔浓度。这j数目是二维等价​​于求该DNA的一端从另一端，在短距离的概率密度，从而反映了DNA的灵活性。测量歼因子的DNA长度的函数揭示了DNA的机制包括持续长度4,5诸多特点。 该蠕虫状链（WLC）模型已被广泛认为是基于其在expla成功双链DNA力学的规范聚合物模型伊宁DNA的拉动实验6，正确预测dsDNAs的歼因素大于200 bp的7所得到的力-延伸曲线。但是，使用双链DNA上的分子越短100 bp的环化法，克卢捷和Widom测量歼因素是几个数量级比WLC模型预测高8。一年后，杜等人 。生产Ĵ因素与使用环化检测与低连接酶的浓度在WLC模型协议，并归因于异常结果从Widom组用9高连接酶的浓度。使用传统的分析9时，这场争论体现了DNA连接酶环化上动力学不可避免的影响。此外，DNA连接酶还可以通过非特异性结合10,11影响DNA结构和刚度。 为了消除蛋白依赖的循环试验的技术问题，我们最近展示了普罗特基于荧光共振能量转移（FRET）12 EIN自由循环试验。在该方法中，环的构象是通过FRET附近的DNA分子的粘性末端的供体和受体之间进行检测。客观型全内反射荧光显微镜（TIRFM）是用来记录可逆循环，并从表面固定的单个DNA分子为长时间unlooping事件的轨迹。此方法具有的DNA分子的PCR为基础的组件以产生错配的游离DNA分子，这是通过由Vafabakhsh和哈13类似的方法的一个关键的改进。此协议的单分子方面允许除了分布测量到总体均值而FRET方面允许一个反复从同一分子测定DNA循环动力学，甚至在可能损害连接酶活性的条件。 该TIRFM设置如图1。自定义设计的样品载物台放置在奥林巴斯IX61显微镜体。 532纳米和640纳米激光器从侧面引入并通过微小的椭圆镜14进入高NA物镜被反射来实现入射临界角在盖玻片-水界面。我们注意到，更广泛地通过目标使用二向色反射镜或棱镜型TIR TIR设置也可用于本申请FRET。由显微镜所形成的荧光图像通过分色镜分成供体和受体的图像。然后将它们重新成像到一个EMCCD的两半。附加的长通发射滤波器被用于减少背景信号。 温度控制是获得可重复的动力学数据是必不可少的。对于温度控制，目标是从镜身，以减少热传递，并从温度控制冷却器/加热器水的分离物镜转换器通过一个黄铜项圈紧密配合循环围绕目标外套下的内部金属。这种设置能够实现强大的温度控制在（ 图2），15至50℃的盖玻片表面。在这项工作中，将样品的温度保持在24℃。 以下协议给出了一步一步的过程进行DNA施工，估计DNA的形状，单分子实验，和J的因素决定。

<table><tbody><tr><td>Small DNA FRAG Extract Kit-100PR</td><td>VWR</td><td>97060-558</td><td></td></tr><tr><td>Acrylamide 40% solution 500 ml</td><td>VWR</td><td>97064-522</td><td></td></tr><tr><td>Bis-acrylamide 2% (w/v) solution 500 ml</td><td>VWR</td><td>97063-948</td><td></td></tr><tr><td>GeneRuler 100 bp DNA Ladder, 100-1,000 bp</td><td>Fermentas</td><td>SM0241</td><td></td></tr><tr><td>Mini Vertical PAGE System</td><td>VWR</td><td>89032-300</td><td></td></tr><tr><td>Syringe filter 0.2 &amp;mu;m CS50</td><td>VWR</td><td>A2666</td><td></td></tr><tr><td>Trolox</td><td>Sigma-Aldrich</td><td>238813-1G</td><td>triplet state quencher</td></tr><tr><td>Protocatechuic acid (PCA)</td><td>Sigma-Aldrich</td><td>08992-50MG</td><td>oxygen scavenging system</td></tr><tr><td>Protocatechuate 3,4-Dioxygenase (PCD)</td><td>Sigma-Aldrich</td><td>P8279-25UN</td><td>oxygen scavenging system</td></tr><tr><td>mPEG-silane, MW 2,000 1 g</td><td>Laysan Bio</td><td>MPEG-SIL-2000-1g</td><td></td></tr><tr><td>Biotin-PEG-Silane, MW 3,400</td><td>Laysan Bio</td><td>Biotin-PEG-SIL-3400-1g</td><td></td></tr><tr><td>Avidin, NeutrAvidin Biotin-binding Protein</td><td>Invitrogen</td><td>A2666</td><td></td></tr><tr><td>Phusion Hot Start High-Fidelity DNA Polymerase</td><td>New England Biolabs</td><td>F-540L</td><td></td></tr><tr><td>Gel/PCR DNA Fragments Extraction Kit</td><td>IBI Scientific</td><td>IB47020</td><td></td></tr><tr><td>Premium plain glass microscope slides</td><td>Fisher Scientific</td><td>12-544-1</td><td></td></tr><tr><td>VWR micro cover glass, rectangular, no. 1</td><td>VWR</td><td>48404-456</td><td></td></tr><tr><td>Fisher Scientific Isotemp 1006S Recirculating Chiller/Heater</td><td>Fisher Scientific</td><td></td><td>temperature control</td></tr><tr><td>Objective Cooling Collar</td><td>Bioptechs</td><td>150303</td><td>temperature control</td></tr><tr><td>KMI53 Biological Micrometer Measuring Stage</td><td>Semprex</td><td>KMI53</td><td></td></tr><tr><td>High Performance DPSS Laser 532 nm 50 mW</td><td>Edmund optics</td><td>NT66-968</td><td>Cy3 excitation</td></tr><tr><td>CUBE Fiber Pigtailed 640 nm, 30 mW, Fiber, FC/APC Connector</td><td>Coherent</td><td>1139604</td><td>Cy5 excitation</td></tr><tr><td>650 nm BrightLine Dichroic Beamsplitter</td><td>Semrock</td><td>FF650-Di01-25x36</td><td>splitting dichroic</td></tr><tr><td>LaserMUX Beam Combiner, reflects 514.5, 532, &amp;amp; 543.5 nm lasers, 25 mm</td><td>Semrock</td><td>LM01-552-25</td><td>combining dichroic</td></tr><tr><td>Brightline Fluorescence Filter 593/40</td><td>Semrock</td><td>FF01-593/40-25</td><td>Cy3 emission filter</td></tr><tr><td>635 nm EdgeBasic LWP longpass Filter, 25 mm</td><td>Semrock</td><td>BLP01-635R-25</td><td>Cy5 emission filter</td></tr><tr><td>EMCCD iXon+</td><td>Andor Technology</td><td>DU-897E-CS0-#BV</td><td></td></tr><tr><td>IX51 inverted microscope frame</td><td>Olympus</td><td></td><td></td></tr><tr><td>Objective UApo N 100X/1.49 Oil TIRF</td><td>Olympus</td><td></td><td></td></tr><tr><td>Immersion oil type-F for fluorescence microscopy</td><td>Olympus</td><td>IMMOIL-F30CC</td><td></td></tr><tr><td>2 mm Diameter 45&amp;deg; Rod Lens Aluminum Coated&amp;nbsp;</td><td>Edmund optics</td><td>54-092</td><td>miniature mirror</td></tr><tr><td>1/4&quot; Travel Single-Axis Translation Stage</td><td>Thorlabs</td><td>MS-1</td><td>translation of miniature mirror</td></tr><tr><td>&amp;Oslash;1&quot; Achromatic Doublet, ARC: 400-700 nm, f=200 mm</td><td>Thorlabs</td><td>AC254-200-A</td><td>focusing lens</td></tr><tr><td>Adjustable Mechanical Slit</td><td>Thorlabs</td><td>VA100</td><td></td></tr><tr><td>Dielectric Mirror</td><td>Thorlabs</td><td>BB1-E02</td><td></td></tr><tr><td>&amp;Oslash;1&quot; Achromatic Doublet, f = 100 mm</td><td>Thorlabs</td><td>AC254-100-A</td><td>relay lens</td></tr><tr><td>Lens Mount for &amp;Oslash;1&quot; Optics</td><td>Thorlabs</td><td>LMR1</td><td></td></tr><tr><td>Dichroic Filter Mount</td><td>Thorlabs</td><td>FFM1</td><td></td></tr><tr><td>Fixed Cage Cube Platform</td><td>Thorlabs</td><td>B3C</td><td></td></tr><tr><td>Kinematic Mount for &amp;Oslash;1&quot; Optics</td><td>Thorlabs</td><td>KM100</td><td></td></tr><tr><td>N-BK7 Plano-Convex Lens, &amp;Oslash;1&quot;, f = 40 mm</td><td>Thorlabs</td><td>LA1422-A</td><td>collimating lens</td></tr><tr><td>N-BK7 Plano-Convex Lense, &amp;Oslash;6.0 mm, f = 15 mm</td><td>Thorlabs</td><td>LA1222-A</td><td>telescope lens</td></tr><tr><td>N-BK7 Plano-Convex Lense, &amp;Oslash;6.0 mm, f = 150 mm</td><td>Thorlabs</td><td>LA1433-A</td><td>telescope lens</td></tr></tbody></table>

studying dna looping by single-molecule fret

弯曲的双链DNA（dsDNA）的是与许多重要的生物学过程，如DNA-蛋白质识别和DNA包装成核小体相关联。双链DNA弯曲的热力学研究了它依赖于DNA连接酶共价连接一个双链DNA的短粘性末端的方法称为环化。然而，连接效率可受许多不相关的双链DNA循环如围绕接合的粘性末端的DNA结构，和连接酶的因素也可以通过诸如非特异性结合的影响明显的循环速率。在这里，我们将展示如何通过检测瞬态DNA环的形成由FRET（荧光共振能量转移）来衡量双链DNA循环动力学无连接酶。双链DNA分子使用的是简单的基于PCR的协议与FRET对和生物素连接器构成。称为歼因子的循环概率密度是从两个断路之间的循环速率和退火速率提取特德粘性末端。通过测试2 dsDNAs具有不同本征曲率，我们表明，歼因子是对双链DNA的固有形状敏感。

用于循环研究DNA分子组成的可变序列和长度和单链突出端是彼此互补的双链区。的突出端，其中有7个碱基长，可彼此退火来捕获绕环状态。每个悬包含任何的Cy3和Cy5是通过亚酰胺化学联在DNA骨架。 Cy5的突出端还与生物素-TEG（15-原子四甘醇间隔），用于表面固定（参见图4A）相连。所有这些修改可以被掺入到双链以下以PCR为基础的协议（协议1和图3B）。所选择的长度出挑和顺序运行良好，生产正常实验条件下检测到的和可逆的荧光共振能量转移的事件。的循环和DNA环的unlooping动力学是对周围温度敏感的，因此，温度调节装置是用于再现性是必不可少的。这种控器缪勒可以容易地掺入到显微镜（ 图2）。 使用TIRFM，反相关的Cy3（供体）和Cy5（受体）从表面固定化的双链DNA分子的信号进行观察（ 图4B）的波动。在环形状态下，两种染料之间的距离是小于5nm，这将导致高的Cy5信号和低Cy3的信号，由于高的FRET效率（ 图4A和4C）。几个控制实验，以证明高FRET状态表示环形状态。高FRET状态的寿命的增加而增加悬垂长度，这表明高的FRET状态是由两个突出端之间的碱基配对稳定。高FRET态的寿命也降低与减少DNA的长度，这是预料之中，因为环自由能随循环变小12。根据这一证据，高，低FRET站TES，可以分别分配给环和不成圈的状态。 要测试的dsDNA固有曲率对循环的效果，2 186 bp的dsDNAs用不同的曲率被构造，其被命名为"直"（S-DNA）和"弯曲"（C-DNA），基于它们的曲率。重复10 - 聚体序列是TTTATCATCC对于C-DNA和TGACAGCAAC为S-DNA。每个这些dsDNAs的总体曲率是由PAGE（聚丙烯酰胺凝胶电泳），这是最广泛使用的方法来估计的dsDNA曲率16,22格子图5示出了S-DNA同样运行于相同大小的DNA在DNA梯状条带（比较泳道1和泳道2在图5）。另一方面，在C-DNA显示了一个更大的表观大小相比，其在DNA梯状条带对应物（〜1.2比其实际尺寸，见泳道1和泳道3）。这一观察结果表明，C-DNA具有更大的内在立方米rvature比S-DNA。 图6示出了从C-DNA（ 图6A）和相应的荧光共振能量转移曲线（ 图6B）有代表性的荧光时间跟踪。相比于S-DNA（ 图4B和4C），该C-DNA的过程中在相同的时间期间内产生更多的优质FRET事件;在C-DNA循环4次的频率比在同一取样时间的S-DNA（ 图7）。该结果表明，DNA的固有曲率显著影响循环动力学。 提取的循环率歼因素，需要测量两个断开出挑的浓度无关的二级速率常数杂交。一个实现这种测量如图8A。 Cy3标记的寡核苷酸与固定Cy5的寡核苷酸杂交产生Cy5的强度阵阵由于FRET。从多个MEAsurements不同Cy3标记的寡核苷酸的浓度（ 见图8B），可以在统计上可靠的方法提取。在我们的实验中，500 mM氯化钠两个寡核苷酸之间的退火速率常数被确定为0.45±0.04×10 6 M -1秒-1。歼因子进行了计算由第二阶退火速率常数（k&#39; 退火 ）除以循环速率（K 循环 ）。这是61±3 nm处的S-DNA和265±48纳米的C-DNA。将S-DNA（ 图9）的第j因素是与以前的测量在类似的长度为7公平的协议。 <img alt="图1" src="/files/ftp_upload/51667/51667fig1highres.jpg" /> 图1:目标型TIRFM A）激励光学。在532 nm和640 nm的激光器分别准直到SIMIL芳直径，并入相同的路径通过一个分色镜，并聚焦到一个小椭圆镜放置在目标背孔的下方。使用消色差透镜，用于聚焦，以减少色差。 乙）显微镜光学器件是很重要的。微型反射镜的横向位置，必须精确地调整，以便它可以通过目标反射激光束，同时最低限度地阻断荧光发射。因此，它必须被装在一个小的平移台上。在相反侧的另一个镜子反射的输出激光，以防止它进入检测光学系统。在显微镜主体以外的图像平面中，可调节的狭缝被放置到图像裁剪到CCD的检测区域的尺寸的一半。荧光发射被分离的分色镜分成两个通道，而中继透镜形成在CCD上的第二图像，用1:1的放大倍率。一个反射镜的稍微转动，以抵消供体和受体图像。带通和长通滤波器用于降低背景的供体和受体通道，分别为。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/51667/51667fig1highres.jpg" target="_blank">点击这里查看大图。</a> <img alt="图2" src="/files/ftp_upload/51667/51667fig2highres.jpg" /> 图2。温度控制单分子实验的设定温度（T s）是对水冷却器/加热器读数。实际温度（T A）是由一热电偶的接触显微镜盖玻片的上表面测定。的实际温度与六种不同的设定温度（黑方块）的图显示了出色的线性度（绿色虚线）。控制器的鲁棒性表现为在以后的时间（红钻）做随机测量。插图是温度共同的图片ntrolling单元和物镜在其最后的组装。红线代表团结的斜率。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/51667/51667fig2highres.jpg" target="_blank">点击这里查看大图。</a> <img alt="图3" src="/files/ftp_upload/51667/51667fig3highres.jpg" /> 图3的DNA设计A）曲线双链DNA。有10个碱基的双链DNA被表示为一个弯曲的管段，并且两个单链为虚线。任何10 - 聚体DNA螺旋轴不是完全直的，并且因此这样的10 - 聚体的每个级联会导致螺旋轴在同一方向上的增量倾斜。这种效果可被利用以产生一个平面的，超螺旋分子B）的基于PCR的制备双链DNA。单链DNA被示为一水平线。它们的实际曲率此处未示出。在黑色的中央部分代表<html>的DNA片段的独特部分。以浅灰色显示是共同的，在本研究中使用的所有的DNA的接头序列。引物1和4退火的正面和背面的适配器，分别。引物2是用Cy3标记在5&#39;端。引物3具有生物素标记的5&#39;-末端和一个内部连接的Cy5。引物2和3的光的蓝色区域包含充当粘性末端互补的序列。无论是质粒或基因组DNA，包含与靶序列被用作在两个独立的PCR反应的模板。 Cy3标记的双链DNA使用引物1和2中生产的，和生物素标记的Cy5标记的DNA用引物3和4产生的，这两个PCR产物进行加热和冷却，一起为链交换。四个不同的dsDNAs形成，其中只有一个包含的Cy3，Cy5标记，生物素等。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/51667/51667fig3highres.jpg" target="_blank">点击这里查看大图。</a> 总是"&gt; <img alt="图4" src="/files/ftp_upload/51667/51667fig4highres.jpg" /> 图4。实验方案。A）单分子荧光共振能量转移实验。在一侧和Cy5（红色）对其他用Cy3标记（绿色）的双链DNA分子被固定在PEG（聚乙二醇）涂覆的表面。 dsDNAs可逆循环和unlooping导致高FRET（环形）和低荧光共振能量转移（不成圈）状态，分别为B）的供体（绿色典型的单分子实时跟踪）和受体（红色）荧光强度。高和低的FRET状态被确定为环和不成圈状态，分别。还指出的是，用于计算的循环速率所述第一循环时间。 （插图）的放大图显示了Cy3和Cy5强度的反相关。 三）从供体和受体的强度计算荧光共振能量转移效率的时间一丝痕迹B 中 。 图4B和<s<html> TRONG&gt; 4C分别取自直DNA（S-DNA），在正文中描述。 0.2低FRET是由于Cy5的由532 nm激光直接激发，并证实了荧光激活Cy5的存在。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/51667/51667fig4highres.jpg" target="_blank">点击这里查看大图。</a> <img alt="图5" src="/files/ftp_upload/51667/51667fig5highres.jpg" /> 图5。聚丙烯酰胺凝胶电泳 （29.2:0.8丙烯酰胺/双丙烯酰胺，5％的TBE缓冲液，pH 8.0）中合成DNA，从左至右，车道含有1 kb的标记，186 bp的直DNA和186个基点弯曲的DNA。这个数字是部分改编自以前的出版物12的许可。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/51667/51667fig5highres.jpg" target="_blank">点击这里查看大图。</a> _content"FO:保持together.within页="总是"&gt; <img alt="图6" src="/files/ftp_upload/51667/51667fig6highres.jpg" /> 强度的图6。从弯曲脱氧核糖核酸（C-DNA）的（A）和与其对应的FRET的迹线（B）代表的痕迹。比较与FRET S-DNA（ 图4B和4C）和C-DNA（ 图6A之间的痕迹和图6B）清楚地表明，在弯曲的DNA循环比在相同缓冲液条件下的直链DNA的更加频繁。 （插图）的放大图显示了Cy3和Cy5强度的反相关。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/51667/51667fig6highres.jpg" target="_blank">点击这里查看大图。</a> <img alt="图7" src="/files/ftp_upload/51667/51667fig7highres.jpg" /> 图7的直线和弯曲的DNA的平均首循环时间。各个tIME是从〜500分子中提取5个不同的领域。误差棒代表3次独立实验计算出的平均值（SEM）的标准误差。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/51667/51667fig7highres.jpg" target="_blank">点击这里查看大图。</a> <img alt="图8" src="/files/ftp_upload/51667/51667fig8highres.jpg" /> 图8。确定从杂交的寡核苷酸的退火速率常数。退火速率测量的）示意图。 Cy5标记引物被固定在表面上，并且Cy3标记的引物是存在于50-180纳米。绑定和解除绑定的Cy3标记引物为50纳米可逆地发生，并导致检测受阵阵，这是显示的插图B）中的退火速率线性依赖于自由寡核苷酸的浓度。的直线的斜率表示了二阶退火速率常数（k&#39;annea<html> L）。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/51667/51667fig8highres.jpg" target="_blank">点击这里查看大图。</a> <img alt="图9" src="/files/ftp_upload/51667/51667fig9highres.jpg" /> 图9纳米计算的直线和曲线的DNA的第j因素基于两种衡量标准歼因素确定:。的双链DNA环和自由互补突出的退火速率常数的循环率<a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/51667/51667fig9highres.jpg" target="_blank">点此查看大图。</a> <table border="0" cellpadding="0" cellspacing="0" width="594"><tbody><tr height="50"><td height="50" style="height:50px;width:351px;">元件</td><td style="width:123px;"> Volume/50微升反应</td><td style="width:121px;">终浓度</td></tr><tr height="25">八:25PX;宽度:351px;"&gt;水</td><td style="width:123px;"> 36微升</td><td style="width:121px;"></td></tr><tr height="25"><td height="25" style="height:25px;width:351px;"> 5倍的Phusion缓冲</td><td style="width:123px;"> 10微升</td><td style="width:121px;"> 1X </td></tr><tr height="25"><td height="25" style="height:25px;width:351px;"> 10毫米的dNTP </td><td style="width:123px;"> 1微升</td><td style="width:121px;">每200微米</td></tr><tr height="25"><td height="25" style="height:25px;width:351px;">正向引物（25微米） </td><td style="width:123px;"> 1微升</td><td style="width:121px;"> 0.5微米</td></tr><tr height="25"><td height="25" style="height:25px;width:351px;">反向引物（25微米） </td><td style="width:123px;"> 1微升</td><td style="width:121px;"> 0.5微米</td></tr><tr height="25"><td height="25" style="height:25px;width:351px;">的Phusion热启动DNA聚合酶（2<html> U /μL） </td><td style="width:123px;"> 0.5微升</td><td style="width:121px;"> 0.02 U /μL </td></tr><tr height="25"><td height="25" style="height:25px;width:351px;">模板DNA </td><td style="width:123px;"> 0.5微升</td><td style="width:121px;"> 〜1纳克</td></tr></tbody></table> 表1。PCR反应混合物。该协议是用于Phusion DNA聚合酶进行了优化。项目应按此顺序添加。 <table border="0" cellpadding="0" cellspacing="0" width="525"><colgroup><col /><col /><col /><col /></colgroup><tbody><tr height="25"><td height="25" style="height:25px;width:159px;">循环步骤</td><td style="width:123px;">温度</td><td style="width:91px;">时间</td><td style="width:153px;">周期数</td></tr><tr height="28"><td height="28" style="height:28px;width:159px;">初始变性</td>"&gt; 95°C </td><td style="width:91px;"> 30秒</td><td style="width:153px;"> 1 </td></tr><tr height="28"><td height="28" style="height:28px;width:159px;">变性</td><td style="width:123px;"> 95°C </td><td style="width:91px;"> 5秒</td><td rowspan="3" style="width:153px;"> 30 </td></tr><tr height="28"><td height="28" style="height:28px;width:159px;">退火</td><td style="width:123px;"> 60°C </td><td style="width:91px;"> 10秒</td></tr><tr height="28"><td height="28" style="height:28px;width:159px;">延期</td><td style="width:123px;"> 72°C </td><td style="width:91px;"> 15秒/ KB </td></tr><tr height="28"><td height="56" rowspan="2" style="height:56px;width:159px;">最后延伸</td><td style="width:123px;"> 72°C </td><td style="width:91px;"> 5分钟</td><td rowspan="2" style="width:153px;"> 1 </td></tr><tr height="28"><td height="28" style="height:28px;width:123px;"> 4＆＃176，C </td><td style="width:91px;">持有</td></tr></tbody></table> 表2。PCR循环指令。退火温度根据引物的解链温度测定。延长时间是优化的Phusion DNA聚合酶。

Watch this Scientific Journal Video about Studying DNA Looping by Single-Molecule FRET at JoVE.com

Studying DNA Looping by Single-Molecule FRET

这项研究提出了一个详细的实验步骤，使用单分子荧光共振能量转移（FRET）来衡量的双链DNA循环动力学。该协议还描述了如何提取称为歼因子循环概率密度。

研究DNA环接了单分子荧光共振能量转移

Bending of double-stranded DNA (dsDNA) is associated with many important biological processes such as DNA-protein recognition and DNA packaging into ...

studying-dna-looping-by-single-molecule-fret

Universidad San Sebastian

Research

JoVE Journal

Biology

15.3K 次观看.  Georgia Institute of Technology. 这项研究提出了一个详细的实验步骤，使用单分子荧光共振能量转移（FRET）来衡量的双链DNA循环动力学。该协议还描述了如何提取称为歼因子循环概率密度。 

视频: Studying DNA Looping by Single-Molecule FRET

A Simple, Robust, and High Throughput Single Molecule Flow Stretching Assay Implementation for Studying Transport of Molecules Along DNA

We describe a simple, robust and high throughput single molecule flow-stretching assay for studying 1D diffusion of molecules along DNA. In this assay, glass coverslips are functionalized in a one-step reaction with silane-PEG-biotin. Flow cells are constructed by sandwiching an adhesive tape with pre-cut channels between a functionalized coverslip and a PDMS slab containing inlet and outlet holes. Multiple channels are integrated into one flow cell and the flow of reagents into each channel can be fully automated, which significantly increases the assay throughput and reduces hands-on time per assay. Inside each channel, biotin-&#955;-DNAs are immobilized on the surface and a laminar flow is applied to flow-stretch the DNAs. The DNA molecules are stretched to &#62;80% of their contour length and serve as spatially extended templates for studying the binding and transport activity of fluorescently labeled molecules. The trajectories of single molecules are tracked by time-lapse Total Internal Reflection Fluorescence (TIRF) imaging. Raw images are analyzed using streamlined custom single particle tracking software to automatically identify trajectories of single molecules diffusing along DNA and estimate their 1D diffusion constants.

This protocol demonstrates a simple, robust and high throughput single molecule flow-stretching assay for studying one-dimensional (1D) diffusion of molecules along DNA.

一种简单、健壮且高通量的单分子流动拉伸试验研究分子沿 DNA 运移的方法

We describe a simple, robust and high throughput single molecule flow-stretching assay for studying 1D diffusion of molecules along DNA. In this assay, ...

科研

生物化学

Probing The Structure And Dynamics Of Nucleosomes Using Atomic Force Microscopy Imaging

Chromatin, which is a long chain of nucleosome subunits, is a dynamic system that allows for such critical processes as DNA replication and transcription to take place in eukaryotic cells. The dynamics of nucleosomes provides access to the DNA by replication and transcription machineries, and critically contributes to the molecular mechanisms underlying chromatin functions. Single-molecule studies such as atomic force microscopy (AFM) imaging have contributed significantly to our current understanding of the role of nucleosome structure and dynamics. The current protocol describes the steps enabling high-resolution AFM imaging techniques to study the structural and dynamic properties of nucleosomes. The protocol is illustrated by AFM data obtained for the centromere nucleosomes in which H3 histone is replaced with its counterpart centromere protein A (CENP-A). The protocol starts with the assembly of mono-nucleosomes using a continuous dilution method. The preparation of the mica substrate functionalized with aminopropyl silatrane (APS-mica) that is used for the nucleosome imaging is critical for the AFM visualization of nucleosomes described and the procedure to prepare the substrate is provided. Nucleosomes deposited on the APS-mica surface are first imaged using static AFM, which captures a snapshot of the nucleosome population. From analyses of these images, such parameters as the size of DNA wrapped around the nucleosomes can be measured and this process is also detailed. The time-lapse AFM imaging procedure in the liquid is described for the high-speed time-lapse AFM that can capture several frames of nucleosome dynamics per second. Finally, the analysis of nucleosome dynamics enabling the quantitative characterization of the dynamic processes is described and illustrated.

Here, we present a protocol to characterize nucleosome particles at the single-molecule level using static and time-lapse atomic force microscopy (AFM) imaging techniques. The surface functionalization method described allows for the capture of the structure and dynamics of nucleosomes in high-resolution at the nanoscale.

原子力显微镜成像在核细胞结构和动力学研究中的应用

Chromatin, which is a long chain of nucleosome subunits, is a dynamic system that allows for such critical processes as DNA replication and transcription ...

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 ...

生物工程

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 ...

Dual DNA Rulers to Study the Mechanism of Ribosome Translocation with Single-Nucleotide Resolution

The ribosome translocation refers to the ribosomal movement on the mRNA by exactly three nucleotides (nt), which is the central step in protein synthesis. To investigate its mechanism, there are two essential technical requirements. First is single-nt resolution that can resolve normal translocation from frameshifting, during which the ribosome moves by other than 3 nt. The second is the capability to probe both the entrance and exit sides of mRNA in order to elucidate the whole picture of translocation. We report the dual DNA ruler assay that is based on the critical dissociation forces of DNA-mRNA duplexes, obtained by force-induced remnant magnetization spectroscopy (FIRMS).&#160; With 2-4 pN force resolution, the dual ruler assay is sufficient to distinguish different translocation steps. By implementing a long linker on the probing DNAs, they can reach the mRNA on the opposite side of the ribosome, so that the mRNA position can be determined for both sides. Therefore, the dual ruler assay is uniquely suited to investigate the ribosome translocation, and nucleic acid motion in general. We show representative results which indicated a looped mRNA conformation and resolved normal translocation from frameshifting.

Dual DNA ruler assay is developed to determine the mRNA position during ribosome translocation, which relies on the dissociation forces of the formed DNA-mRNA duplexes. With single-nucleotide resolution and capability of reaching both ends of mRNA, it can provide mechanistic insights for ribosome translocation and probe other nucleic acid displacements.

双DNA标尺研究单核苷酸分辨率的核糖体易位机制

The ribosome translocation refers to the ribosomal movement on the mRNA by exactly three nucleotides (nt), which is the central step in protein synthesis. ...

R-Loop Analysis by Dot-Blot

The three-stranded nucleic acid structure, R-loop, is increasingly recognized for its role in gene regulation. Initially, R-loops were thought to be the by-products of transcription; but recent findings of fewer R-loops in diseased cells made it clear that R-loops have functional roles in a variety of human cells. Next, it is critical to understand the roles of R-loops and how cells balance their abundance. A challenge in the field is the quantitation of R-loops since much of the work relies on the S9.6 monoclonal antibody whose specificity for RNA-DNA hybrids has been questioned. Here, we use dot-blots with the S9.6 antibody to quantify R-loops and show the sensitivity and specificity of this assay with RNase H, RNase T1, and RNase III that cleave RNA-DNA hybrids, single-stranded RNA, and double-stranded RNA, respectively. This method is highly reproducible, uses general laboratory equipment and reagents, and provides results within two days. This assay can be used in research and clinical settings to quantify R-loops and assess the effect of mutations in genes such as senataxin on R-loop abundance.

This protocol details a simple method that quantifies R-loop, a three-stranded nucleic acid structure that comprises of an RNA-DNA hybrid and a displaced DNA strand.

The three-stranded nucleic acid structure, R-loop, is increasingly recognized for its role in gene regulation. Initially, R-loops were thought to be the ...

遗传学

Optical Tweezers to Study RNA-Protein Interactions in Translation Regulation

RNA adopts diverse structural folds, which are essential for its functions and thereby can impact diverse processes in the cell. In addition, the structure and function of an RNA can be modulated by various trans-acting factors, such as proteins, metabolites or other RNAs. Frameshifting RNA molecules, for instance, are regulatory RNAs located in coding regions, which direct translating ribosomes into an alternative open reading frame, and thereby act as gene switches. They may also adopt different folds after binding to proteins or other trans-factors. To dissect the role of RNA-binding proteins in translation and how they modulate RNA structure and stability, it is crucial to study the interplay and mechanical features of these RNA-protein complexes simultaneously.&#160;This work illustrates how to employ single-molecule-fluorescence-coupled optical tweezers to explore the conformational and thermodynamic landscape of RNA-protein complexes at a high resolution. As an example, the interaction of the SARS-CoV-2 programmed ribosomal frameshifting element with the trans-acting factor short isoform of zinc-finger antiviral protein is elaborated. In addition, fluorescence-labeled ribosomes were monitored using the confocal unit, which would ultimately enable the study of translation elongation. The fluorescence coupled OT assay can be widely applied to explore diverse RNA-protein complexes or trans-acting factors regulating translation and could facilitate studies of RNA-based gene regulation.

This protocol presents a complete experimental workflow for studying RNA-protein interactions using optical tweezers. Several possible experimental setups are outlined including the combination of optical tweezers with confocal microscopy.

光学镊子研究翻译调控中的RNA-蛋白质相互作用

RNA adopts diverse structural folds, which are essential for its functions and thereby can impact diverse processes in the cell. In addition, the ...

Single-Molecule Imaging of EWS-FLI1 Condensates Assembling on DNA

The fusion genes resulting from chromosomal translocation have been found in many solid tumors or leukemia. EWS-FLI1, which belongs to the FUS/EWS/TAF15 (FET) family of fusion oncoproteins, is one of the most frequently involved fusion genes in Ewing sarcoma. These FET family fusion proteins typically harbor a low-complexity domain (LCD) of FET protein at their N-terminus and a DNA-binding domain (DBD) at their C-terminus. EWS-FLI1 has been confirmed to form biomolecular condensates at its target binding loci due to LCD-LCD and LCD-DBD interactions, and these condensates can recruit RNA polymerase II to enhance gene transcription. However, how these condensates are assembled at their binding sites remains unclear. Recently, a single-molecule biophysics method-DNA Curtains-was applied to visualize these assembling processes of EWS-FLI1 condensates. Here, the detailed experimental protocol and data analysis approaches are discussed for the application of DNA Curtains in studying the biomolecular condensates assembling on target DNA.

Here, we describe the use of the single-molecule imaging method, DNA Curtains, to study the biophysical mechanism of EWS-FLI1 condensates assembling on DNA.

EWS-FLI1凝聚物在DNA上组装的单分子成像

The fusion genes resulting from chromosomal translocation have been found in many solid tumors or leukemia. EWS-FLI1, which belongs to the FUS/EWS/TAF15 ...

<meta charset="utf8"></head><body>用于单分子研究的原位核小体重构

In Situ Nucleosome Assembly for Single-Molecule Correlative Force and Fluorescence Microscopy

Nucleosomes constitute the primary unit of eukaryotic chromatin and have been the focus of numerous informative single-molecule investigations regarding their biophysical properties and interactions with chromatin-binding proteins. Nucleosome reconstitution on DNA for these studies typically involves a salt dialysis procedure that provides precise control over the placement and number of nucleosomes formed along a DNA tether. However, this protocol is time-consuming and requires a substantial amount of DNA and histone octamers as inputs. To offer an alternative strategy, an in situ nucleosome reconstitution method for single-molecule force and fluorescence microscopy that utilizes the histone chaperone Nap1 is described. This method enables users to assemble nucleosomes on any DNA template without the need for strong nucleosome positioning sequences, adjust nucleosome density on demand, and use fewer reagents. In situ nucleosome formation occurs within seconds, offering a simpler experimental workflow and a convenient transition into single-molecule measurements. Examples of two downstream assays for probing nucleosome mechanics and visualizing the behavior of individual proteins on chromatin are further described.

<meta charset="utf8"></head><body>作者聚焦：用于单分子技术的高效核小体重构

This article presents a detailed experimental procedure for reconstituting nucleosome-containing DNA tethers for single-molecule correlative force and fluorescence microscopy. It further describes several downstream experiments that can be conducted to visualize the binding behavior of chromatin-interacting proteins and analyze changes in the physical properties of nucleosomes.

用于单分子相关力和荧光显微镜的原位核小体组装

Nucleosomes constitute the primary unit of eukaryotic chromatin and have been the focus of numerous informative single-molecule investigations regarding ...

研究DNA环接了单分子荧光共振能量转移

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此视频中的章节

一种简单、健壮且高通量的单分子流动拉伸试验研究分子沿 DNA 运移的方法

原子力显微镜成像在核细胞结构和动力学研究中的应用

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

双DNA标尺研究单核苷酸分辨率的核糖体易位机制

R-Loop Analysis by Dot-Blot

光学镊子研究翻译调控中的RNA-蛋白质相互作用

EWS-FLI1凝聚物在DNA上组装的单分子成像

用于单分子相关力和荧光显微镜的原位核小体组装

用于单分子相关力和荧光显微镜的原位核小体组装