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。 </p...

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 border="0" cellpadding="0" cellspacing="0" width="793">
	<tbody>
		<tr height="17">
			<td height="17" style="height:17px;width:351px;">Small DNA FRAG Extract Kit-100PR</td>
			<td style="width:124px;">VWR</td>
			<td style="width:135px;">97060-558</td>
			<td style="width:183px;"></td>
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		<tr height="17">
			<td height="17" style="height:17px;width:351px;">Acrylamide 40% solution 500 mL</td>
			<td style="width:124px;">VWR</td>
			<td style="width:135px;">97064-522</td>
			<td style="width:183px;"></td>
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		<tr height="17">
			<td height="17" style="height:17px;">Bis-acrylamide 2% (w/v) solution 500 mL</td>
			<td style="width:124px;">VWR</td>
			<td style="width:135px;">97063-948</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:351px;">GeneRuler 100 bp DNA Ladder, 100-1000 bp</td>
			<td style="width:124px;">Fermentas</td>
			<td>SM0241</td>
			<td></td>
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		<tr height="17">
			<td height="17" style="height:17px;width:351px;">Mini Vertical PAGE System</td>
			<td style="width:124px;">VWR</td>
			<td style="width:135px;">89032-300</td>
			<td></td>
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		<tr height="17">
			<td height="17" style="height:17px;width:351px;">Syringe filter 0.2um CS50</td>
			<td style="width:124px;">VWR</td>
			<td style="width:135px;">A2666</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:351px;">Trolox</td>
			<td style="width:124px;">Sigma-Aldrich</td>
			<td style="width:135px;">238813-1G</td>
			<td>triplet state quencher</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:351px;">Protocatechuic acid (PCA)</td>
			<td style="width:124px;">Sigma-Aldrich</td>
			<td style="width:135px;">08992-50MG</td>
			<td>oxygen scavenging system</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:351px;">Protocatechuate 3,4-Dioxygenase (PCD)</td>
			<td style="width:124px;">Sigma-Aldrich</td>
			<td style="width:135px;">P8279-25UN</td>
			<td>oxygen scavenging system</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:351px;">mPEG-silane, MW 2000 1g</td>
			<td style="width:124px;">Laysan Bio</td>
			<td style="width:135px;">MPEG-SIL-2000-1g</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:351px;">Biotin-PEG-Silane, MW 3400</td>
			<td style="width:124px;">Laysan Bio</td>
			<td style="width:135px;">Biotin-PEG-SIL-3400-1g</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:351px;">Avidin, NeutrAvidin Biotin-binding Protein</td>
			<td style="width:124px;">Invitrogen</td>
			<td style="width:135px;">A2666</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:351px;">Phusion Hot Start High-Fidelity DNA Polymerase</td>
			<td style="width:124px;">New England Biolabs</td>
			<td style="width:135px;">F-540L</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Gel/PCR DNA Fragments Extraction Kit</td>
			<td>IBI Scientific</td>
			<td>IB47020</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:351px;">Premium plain glass microscope slides</td>
			<td style="width:124px;">Fisher Scientific</td>
			<td style="width:135px;">12-544-1</td>
			<td></td>
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		<tr height="17">
			<td height="17" style="height:17px;width:351px;">VWR micro cover glass, rectangular, no. 1</td>
			<td style="width:124px;">VWR</td>
			<td style="width:135px;">48404-456</td>
			<td></td>
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		<tr height="17">
			<td height="17" style="height:17px;width:351px;">Fisher Scientific Isotemp 1006s Recirculating Chiller/Heater</td>
			<td style="width:124px;">Fisher Scientific</td>
			<td style="width:135px;"></td>
			<td>temperature control</td>
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		<tr height="17">
			<td height="17" style="height:17px;width:351px;">Objective Cooling Collar</td>
			<td style="width:124px;">Bioptechs</td>
			<td style="width:135px;">150303</td>
			<td>temperature control</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:351px;">KMI53 Biological Micrometer Measuring Stage</td>
			<td style="width:124px;">Semprex</td>
			<td style="width:135px;">KMI53</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:351px;">High Performance DPSS Laser 532nm 50mW&#160;</td>
			<td style="width:124px;">Edmund optics</td>
			<td style="width:135px;">NT66-968</td>
			<td>Cy3 excitation</td>
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		<tr height="24">
			<td height="24" style="height:24px;width:351px;">CUBE Fiber Pigtailed 640 nm, 30mW, Fiber, FC/APC Connector</td>
			<td style="width:124px;">Coherent</td>
			<td style="width:135px;">1139604</td>
			<td>Cy5 excitation</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:351px;">650 nm BrightLine Dichroic Beamsplitter</td>
			<td style="width:124px;">Semrock</td>
			<td style="width:135px;">FF650-Di01-25x36</td>
			<td>splitting dichroic</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:351px;">LaserMUX Beam Combiner, reflects 514.5, 532, &#38; 543.5 nm lasers, 25 mm</td>
			<td style="width:124px;">Semrock</td>
			<td style="width:135px;">LM01-552-25</td>
			<td>combining dichroic</td>
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		<tr height="17">
			<td height="17" style="height:17px;width:351px;">Brightline Fluorescence Filter 593/40</td>
			<td style="width:124px;">Semrock</td>
			<td>FF01-593/40-25</td>
			<td>Cy3 emission filter</td>
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		<tr height="17">
			<td height="17" style="height:17px;width:351px;">635 nm EdgeBasic LWP longpass Filter, 25 mm</td>
			<td style="width:124px;">Semrock</td>
			<td style="width:135px;">BLP01-635R-25</td>
			<td>Cy5 emission filter</td>
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		<tr height="17">
			<td height="17" style="height:17px;width:351px;">EMCCD iXon+</td>
			<td style="width:124px;">Andor Technology</td>
			<td style="width:135px;">DU-897E-CS0-#BV</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:351px;">IX51 inverted microscope frame</td>
			<td style="width:124px;">Olympus</td>
			<td style="width:135px;"></td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:351px;">Objective UApo N 100x/1.49 Oil TIRF</td>
			<td style="width:124px;">Olympus</td>
			<td style="width:135px;"></td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:351px;">Immersion oil type-F for fluorescence microscopy</td>
			<td style="width:124px;">Olympus</td>
			<td style="width:135px;">IMMOIL-F30CC</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">2mm Diameter 45&#176; Rod Lens Aluminum Coated&#160;</td>
			<td style="width:124px;">Edmund optics</td>
			<td style="width:135px;">54-092</td>
			<td>miniature mirror</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">1/4&#34; Travel Single-Axis Translation Stage</td>
			<td style="width:124px;">Thorlabs</td>
			<td style="width:135px;">MS-1</td>
			<td>translation of miniature mirror</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">&#216;1&#34; Achromatic Doublet, ARC: 400-700 nm, f=200 mm</td>
			<td style="width:124px;">Thorlabs</td>
			<td style="width:135px;">AC254-200-A</td>
			<td>focusing lens</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:351px;">Adjustable Mechanical Slit</td>
			<td style="width:124px;">Thorlabs</td>
			<td>VA100</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:351px;">Dielectric Mirror</td>
			<td style="width:124px;">Thorlabs</td>
			<td style="width:135px;">BB1-E02</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:351px;">&#216;1&#34; Achromatic Doublet, f = 100 mm</td>
			<td style="width:124px;">Thorlabs</td>
			<td style="width:135px;">AC254-100-A</td>
			<td>relay lens</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:351px;">Lens Mount for &#216;1&#34; Optics</td>
			<td style="width:124px;">Thorlabs</td>
			<td style="width:135px;">LMR1</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:351px;">Dichroic Filter Mount</td>
			<td style="width:124px;">Thorlabs</td>
			<td style="width:135px;">FFM1</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:351px;">Fixed Cage Cube Platform</td>
			<td style="width:124px;">Thorlabs</td>
			<td style="width:135px;">B3C</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:351px;">Kinematic Mount for &#216;1&#34; Optics</td>
			<td style="width:124px;">Thorlabs</td>
			<td style="width:135px;">KM100</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:351px;">N-BK7 Plano-Convex Lens, &#216;1&#34;, f = 40 mm</td>
			<td style="width:124px;">Thorlabs</td>
			<td style="width:135px;">LA1422-A</td>
			<td>collimating lens</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:351px;">N-BK7 Plano-Convex Lense, &#216;6.0 mm, f = 15 mm</td>
			<td style="width:124px;">Thorlabs</td>
			<td style="width:135px;">LA1222-A</td>
			<td>telescope lens</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:351px;">N-BK7 Plano-Convex Lense, &#216;6.0 mm, f = 150 mm</td>
			<td style="width:124px;">Thorlabs</td>
			<td style="width:135px;">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?...

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

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

Single Molecule Methods for Monitoring Changes in Bilayer Elastic Properties

Membrane protein function is regulated by the cell membrane lipid composition.  This regulation is due to a combination of specific lipid-protein interactions and more general lipid bilayer-protein interactions. These interactions are particularly important in pharmacological research, as many current pharmaceuticals on the market can alter the lipid bilayer material properties, which can lead to altered membrane protein function.   The formation of gramicidin channels are dependent on conformational changes in gramicidin subunits which are in turn dependent on the properties of the lipid.  Hence the gramicidin channel  current is a reporter of altered properties of the bilayer due to certain compounds.  

Membrane protein function is regulated by the cell membrane lipid composition. This video-article details how to form a patch using bilayer patch electrodes, as well as how to use gramicidin channels as reporters of altered membrane properties.

双层弹性性能的监测变化的单分子方法

Membrane protein function is regulated by the cell membrane lipid composition.  This regulation is due to a combination of specific lipid-protein ...

科研

生物学

Combining QD-FRET and Microfluidics to Monitor DNA Nanocomplex Self-Assembly in Real-Time

Advances in genomics continue to fuel the development of therapeutics that can target pathogenesis at the cellular and molecular level. Typically functional inside the cell, nucleic acid-based therapeutics require an efficient intracellular delivery system. One widely adopted approach is to complex DNA with a gene carrier to form nanocomplexes via electrostatic self-assembly, facilitating cellular uptake of DNA while protecting it against degradation. The challenge lies in the rational design of efficient gene carriers, since premature dissociation or overly stable binding would be detrimental to the cellular uptake and therapeutic efficacy. Nanocomplexes synthesized by bulk mixing showed a diverse range of intracellular unpacking and trafficking behavior, which was attributed to the heterogeneity in size and stability of nanocomplexes. Such heterogeneity hinders the accurate assessment of the self-assembly kinetics and adds to the difficulty in correlating their physical properties to transfection efficiencies or bioactivities. We present a novel convergence of nanophotonics (i.e. QD-FRET) and microfluidics to characterize the real-time kinetics of the nanocomplex self-assembly under laminar flow. QD-FRET provides a highly sensitive indication of the onset of molecular interactions and quantitative measure throughout the synthesis process, whereas microfluidics offers a well-controlled microenvironment to spatially analyze the process with high temporal resolution (~milliseconds). For the model system of polymeric nanocomplexes, two distinct stages in the self-assembly process were captured by this analytic platform. The kinetic aspect of the self-assembly process obtained at the microscale would be particularly valuable for microreactor-based reactions which are relevant to many micro- and nano-scale applications. Further, nanocomplexes may be customized through proper design of microfludic devices, and the resulting QD-FRET polymeric DNA nanocomplexes could be readily applied for establishing structure-function relationships. 

We present a novel and powerful integration of nanophotonics (QD-FRET) and microfluidics to investigate the formation of polyelectrolyte polyplexes, which is expected to provide better control and synthesis of uniform and customizable polyplexes for future nucleic acid-based therapeutics.

结合QD - FRET技术和微流体实时监测DN​​A纳米复合自组装

Advances in genomics continue to fuel the development of therapeutics that can target pathogenesis at the cellular and molecular level. Typically ...

Visualizing Single-molecule DNA Replication with Fluorescence Microscopy

We describe a simple fluorescence microscopy-based real-time method for observing DNA replication at the single-molecule level. A circular, forked DNA template is attached to a functionalized glass coverslip and replicated extensively after introduction of replication proteins and nucleotides (Figure 1). The growing product double-strand DNA (dsDNA) is extended with laminar flow and visualized by using an intercalating dye. Measuring the position of the growing DNA end in real time allows precise determination of replication rate (Figure 2). Furthermore, the length of completed DNA products reports on the processivity of replication. This experiment can be performed very easily and rapidly and requires only a fluorescence microscope with a reasonably sensitive camera.


This protocol demonstrates a simple single-molecule fluorescence microscopy technique for visualizing DNA replication by individual replisomes in real time.

可视化的单分子荧光显微镜下的DNA复制

We describe a simple fluorescence microscopy-based real-time method for observing DNA replication at the single-molecule level. A circular, forked DNA ...

Automated System for Single Molecule Fluorescence Measurements of Surface-immobilized Biomolecules

Fluorescence Resonance Energy Transfer (FRET) microscopy has been widely used to study the structure and dynamics of molecules of biological interest, such as nucleic acids and proteins. Single molecule FRET (sm-FRET) measurements on immobilized molecules permit long observations of the system -effectively until both dyes photobleach- resulting in time-traces that report on biomolecular dynamics with a broad range of timescales from milliseconds to minutes. To facilitate the acquisition of large number of traces for statistical analyses, the process must be automated and the sample environment should be tightly controlled over the entire measurement time (~12 hours). This is accomplished using an automated scanning confocal microscope that allows the interrogation of thousands of single molecules overnight, and a microfluidic cell that permits the controlled exchange of buffer, with restricted oxygen content and maintains a constant temperature throughout the entire measuring period. Here we show how to assemble the microfluidic device and how to activate its surface for DNA immobilization. Then we explain how to prepare a buffer to maximize the photostability and lifetime of the fluorophores. Finally, we show the steps involved in preparing the setup for the automated acquisition of time-resolved single molecule FRET traces of DNA molecules.

In this article we describe how we obtain FRET traces from individual DNA molecules immobilized to a surface using an automated scanning confocal microscope.

表面固定生物分子的单分子荧光检测的自动化系统

Fluorescence Resonance Energy Transfer (FRET) microscopy has been widely used to study the structure and dynamics of molecules of biological interest, ...

Direct Observation of Enzymes Replicating DNA Using a Single-molecule DNA Stretching Assay

We describe a method for observing real time replication of individual DNA molecules mediated by proteins of the bacteriophage replication system. Linearized &lambda; DNA is modified to have a biotin on the end of one strand, and a digoxigenin moiety on the other end of the same strand. The biotinylated end is attached to a functionalized glass coverslip and the digoxigeninated end to a small bead. The assembly of these DNA-bead tethers on the surface of a flow cell allows a laminar flow to be applied to exert a drag force on the bead. As a result, the DNA is stretched close to and parallel to the surface of the coverslip at a force that is determined by the flow rate (Figure 1). The length of the DNA is measured by monitoring the position of the bead. Length differences between single- and double-stranded DNA are utilized to obtain real-time information on the activity of the replication proteins at the fork. Measuring the position of the bead allows precise determination of the rates and processivities of DNA unwinding and polymerization (Figure 2).

We describe a method for observing real time replication of individual DNA molecules mediated by proteins of the bacteriophage replication system.

使用单分子DNA拉伸含量直接观察DNA复制的酶

We describe a method for observing real time replication of individual DNA molecules mediated by proteins of the bacteriophage replication system. ...

Single-Molecule Imaging of Nuclear Transport

The utility of single molecule fluorescence microscopy approaches has been proven to be of a great avail in understanding biological reactions over the last decade. The investigation of molecular interactions with high temporal and spatial resolutions deep within cells has remained challenging due to the inherently weak signals arising from individual molecules. Recent works by Yang et al. demonstrated that narrow-field epifluorescence microscopy allows visualization of nucleocytoplasmic transport at the single molecule level. By the single molecule approach, important kinetics, such as nuclear transport time and efficiency, for signal-dependent and independent cargo molecules have been obtained. Here we described a protocol for the methodological approach with an improved spatiotemporal resolution of 0.4 ms and 12 nm. The improved resolution enabled us to capture transient active transport and passive diffusion events through the nuclear pore complexes (NPC) in semi-intact cells. We expect this method to be used in elucidating other binding and trafficking events within cells.

Single molecule microscopy approch provided novel insights into nuclear transport.

单分子成像核运输

The utility of single molecule fluorescence microscopy approaches has been proven to be of a great avail in understanding biological reactions over the ...

Monitoring Kinase and Phosphatase Activities Through the Cell Cycle by Ratiometric FRET

F&ouml;rster resonance energy transfer (FRET)-based reporters1 allow the assessment of endogenous kinase and phosphatase activities in living cells. Such probes typically consist of variants of CFP and YFP, intervened by a phosphorylatable sequence and a phospho-binding domain. Upon phosphorylation, the probe changes conformation, which results in a change of the distance or orientation between CFP and YFP, leading to a change in FRET efficiency (Fig 1). Several probes have been published during the last decade, monitoring the activity balance of multiple kinases and phosphatases, including reporters of PKA2, PKB3, PKC4, PKD5, ERK6, JNK7, Cdk18, Aurora B9 and Plk19. Given the modular design, additional probes are likely to emerge in the near future10. 
Progression through the cell cycle is affected by stress signaling pathways 11. Notably, the cell cycle is regulated differently during unperturbed growth compared to when cells are recovering from stress12.Time-lapse imaging of cells through the cell cycle therefore requires particular caution. This becomes a problem particularly when employing ratiometric imaging, since two images with a high signal to noise ratio are required to correctly interpret the results. Ratiometric FRET imaging of cell cycle dependent changes in kinase and phosphatase activities has predominately been restricted to sub-sections of the cell cycle8,9,13,14.
Here, we discuss a method to monitor FRET-based probes using ratiometric imaging throughout the human cell cycle. The method relies on equipment that is available to many researchers in life sciences and does not require expert knowledge of microscopy or image processing.

FRET-based reporters are increasingly used to monitor kinase and phosphatase activities in live cells. Here we describe a method on how to use FRET-based reporters to assess cell cycle-dependent changes in target phosphorylation.

通过细胞周期激酶和磷酸酶活性成比例的FRET监测

F&ouml;rster resonance energy transfer (FRET)-based reporters1 allow the assessment of endogenous kinase and phosphatase activities in living cells. Such ...

Single-molecule Imaging of Gene Regulation In vivo Using Cotranslational Activation by Cleavage (CoTrAC)

We describe a fluorescence microscopy method, Co-Translational Activation by Cleavage (CoTrAC) to image the production of protein molecules in live cells with single-molecule precision without perturbing the protein's functionality. This method makes it possible to count the numbers of protein molecules produced in one cell during sequential, five-minute time windows. It requires a fluorescence microscope with laser excitation power density of ~0.5 to 1 kW/cm2, which is sufficiently sensitive to detect single fluorescent protein molecules in live cells. The fluorescent reporter used in this method consists of three parts: a membrane targeting sequence, a fast-maturing, yellow fluorescent protein and a protease recognition sequence. The reporter is translationally fused to the N-terminus of a protein of interest. Cells are grown on a temperature-controlled microscope stage. Every five minutes, fluorescent molecules within cells are imaged (and later counted by analyzing fluorescence images) and subsequently photobleached so that only newly translated proteins are counted in the next measurement.
Fluorescence images resulting from this method can be analyzed by detecting fluorescent spots in each image, assigning them to individual cells and then assigning cells to cell lineages. The number of proteins produced within a time window in a given cell is calculated by dividing the integrated fluorescence intensity of spots by the average intensity of single fluorescent molecules. We used this method to measure expression levels in the range of 0-45 molecules in single 5 min time windows. This method enabled us to measure noise in the expression of the &lambda; repressor CI, and has many other potential applications in systems biology.

Single-molecule Imaging of Gene Regulation In vivo Using Cotranslational Activation by Cleavage CoTrAC

We describe a fluorescence microscopy method, Co-Translational Activation by Cleavage (CoTrAC), to image the production of protein molecules in live cells with single-molecule precision without perturbing the protein's functionality. This method has been used to follow the stochastic expression dynamics of a transcription factor, the &lambda; repressor CI 1.

单分子成像的基因调控<em在体内</em使用转位激活裂解（CoTrAC）

We describe a fluorescence microscopy method, Co-Translational Activation by Cleavage (CoTrAC) to image the production of protein molecules in live cells ...

Combining Single-molecule Manipulation and Imaging for the Study of Protein-DNA Interactions

The paper describes the combination of optical tweezers and single molecule fluorescence detection for the study of protein-DNA interaction. The method offers the opportunity of investigating interactions occurring in solution (thus avoiding problems due to closeby surfaces as in other single molecule methods), controlling the DNA extension and tracking interaction dynamics as a function of both mechanical parameters and DNA sequence. The methods for establishing successful optical trapping and nanometer localization of single molecules are illustrated. We illustrate the experimental conditions allowing the study of interaction of lactose repressor (lacI), labeled with Atto532, with a DNA molecule containing specific target sequences (operators) for LacI binding. The method allows the observation of specific interactions at the operators, as well as one-dimensional diffusion of the protein during the process of target search. The method is broadly applicable to the study of protein-DNA interactions but also to molecular motors, where control of the tension applied to the partner track polymer (for example actin or microtubules) is desirable.

Here we describe the instrumentation and methods for detecting single fluorescently-labeled protein molecules interacting with a single DNA molecule suspended between two optically trapped microspheres.

结合单分子操纵和成像蛋白-DNA相互作用的研究

The paper describes the combination of optical tweezers and single molecule fluorescence detection for the study of protein-DNA interaction. The method ...

FRET Imaging in Three-dimensional Hydrogels

Imaging of F&#246;rster resonance energy transfer (FRET)&#160;is a powerful tool for examining cell biology in real-time. Studies utilizing FRET commonly employ two-dimensional (2D) culture, which does not mimic the three-dimensional (3D) cellular microenvironment. A method to perform quenched emission FRET imaging using conventional widefield epifluorescence microscopy of cells within a 3D hydrogel environment is presented. Here an analysis method for ratiometric FRET probes that yields linear ratios over the probe activation range is described. Measurement of intracellular cyclic adenosine monophosphate (cAMP) levels is demonstrated in chondrocytes under forskolin stimulation using a probe for EPAC1 activation (ICUE1) and the ability to detect differences in cAMP signaling dependent on hydrogel material type, herein a photocrosslinking hydrogel (PC-gel, polyethylene glycol dimethacrylate) and a thermoresponsive hydrogel (TR-gel). Compared with 2D FRET methods, this method requires little additional work. Laboratories already utilizing FRET imaging in 2D can easily adopt this method to perform cellular studies in a 3D microenvironment. It can further be applied to high throughput drug screening in engineered 3D microtissues. Additionally, it is compatible with other forms of FRET imaging, such as anisotropy measurement and fluorescence lifetime imaging (FLIM), and with advanced microscopy platforms using confocal, pulsed, or modulated illumination.

F&#246;rster resonance energy transfer (FRET) imaging is a powerful tool for real-time cell biology studies. Here a method for FRET imaging cells in physiologic three-dimensional (3D) hydrogel microenvironments using conventional epifluorescence microscopy is presented. An analysis for ratiometric FRET probes that yields linear ratios over the activation range is described.

FRET成像的三维水凝胶

Imaging of F&#246;rster resonance energy transfer (FRET)&#160;is a powerful tool for examining cell biology in real-time. Studies utilizing FRET commonly ...