我们非常感谢分享DNA双螺旋分子动力学模拟产生的坐标吉日Šponer的。我们也承认纳达Spackova为协助下载这些结构。通过USPHS研究资助支持这项工作GM34809和GM096889表示感谢。

1。安装软件包<ol><li>连接到的追梦3DNA在<a href="http://x3dna.org" target="_blank">http://x3dna.org</a>网站和单击链接到3DNA论坛。在论坛内，选择"注册"链接，并按照说明进行操作，以创建一个新的帐户。 </li><li>以下说明详细安装一个基于OS X的Macintosh电脑，一个默认的bash&#39;壳上的软件。 Linux或Windows（Cygwin的的MinGW / MSYS）系统常用shell（包括&#39;tcsh的&#39;）的程序内发现3DNA论坛。 </li><li>一旦你已经建立了用户名和密码，登录进入论坛。欢迎部分，在新页面上选择"下载"链接选择&#39;3 DNA的下载"。这会带给你追梦的下载页面，可以下载各种版本的软件。 </li><li>选择Mac OS X的英特尔链接下载一个压缩的文件（tar.gz的）包含软件。 </li><li>双集团公司k对（tar.gz）文件来创建一个文件夹名为-V2.1 x3dna的。这个文件夹，其中包含的3DNA软件套件，可以移动到任何位置。对于这个食谱的目的，我们将它拖放到"应用程序"目录。在这个阶段，你已完成的tar.gz文件，可以删除它。 </li><li>打开终端应用程序，通常发现在"实用工具"下，更改到V2.1 x3dna的第5步中创建的目录，使用以下命令（在这里结束，下面的说明一个回车）: CD / Applications/x3dna-v2.1的</li><li> 3DNA正常打字需要运行安装脚本: ./bin/x3dna_setup </li><li>将步骤7中印两线出口设置。如果用户已经在"应用程序"目录中放置软件，两行应显示如下: 出口X3DNA = / Applications/x3dna-v2.1号 出口PATH = / Applications/x3dna-v2.1/bin:$ PATH 一个不同的&#39;应用&#39;字，除了将APPEAR在出口命令，如果用户选择安装的软件在不同的目录。此备用目录名称会取代在上述两个命令的&#39;应用&#39;。请注意，如果默认的shell是tcsh的&#39;，而不是&#39;庆典&#39;，两行的内容将是:的setenv X3DNA / Applications/x3dna-v2.1和SETENV路径/ Applications/x3dna-v2.1/bin:$ PATH中。 </li><li>请在"搜索"看，如果一个文件名为&#39;。bashrc&#39;中已经存在你的电脑上。 </li><li>打开TextEdit应用程序在"应用程序"目录下的"格式"菜单中选择"纯文本"。 </li><li>如果&#39;。bashrc&#39;中存在文件从第9步，在打开文件TextEdit和导出设置粘贴步骤8至文件末尾。如果没有&#39;。bashrc中的文件退出，粘贴导出设置到一个空白文件，并从第8步保存的文件名&#39;。bashrc中&#39;在你的home目录。 （主目录是由"房子"在Macintosh上的图标表示。） </li><li>为了让新的设置到bê，你必须运行新保存&#39;。bashrc&#39;中文件内的终端程序，使用下面的命令: bashrc中源〜/。 </li><li>追梦的套房现在已经安装在你的系统内的终端程序，您可以执行协议2-5。协议6，一个网络接口使用w3DNA追梦软件选择的特征。 </li></ol> 2。晶体结构分析<ol><li>我们说明使用莱姆病螺旋体 8 HBB蛋白结合的DNA的核酸结构的分析。与这个结构的原子坐标文件，可以发现蛋白质数据银行13（PDB <a href="http://www.rcsb.org/" target="_blank">http://www.rcsb.org</a> ），它是在结构标识符2np2的分配。随附的补充资料包括一份这个文件，也可以找到<a href="http://forum.x3dna.org/jove" target="_blank">http://forum</a> 3DNA论坛<a href="http://forum.x3dna.org/jove" target="_blank">。</a>x3dna.org/jove。 </li><li>追梦的结构分析的第一步是创建一个文件，该文件列出了所有的配对基地。这是通过"find_pair程序通过键入以下内容: find_pair 2np2.pdb 2np2.bps 这里2np2.pdb文件的原子坐标上述2np2.bps的是输入输出的文本文件，其中包含的碱基配对信息。后者碱基对文件由用户如果有必要，可以检查和编辑。 </li><li>在分析中的下一个步骤是确定所特有的结构的几何参数。这些包括标准的化学扭转角14，pseudorotation角度描述的褶皱的糖环15，刚体参数指定基地和连续的碱基对16等措施，立体化学结构的安排。命令&#39;分析&#39;作为输入碱基对步骤2中生成的文件，并创建几个OU的TPUT文件，出现在当前工作目录。计算这些值是通过输入以下命令: 分析2np2.bps， 用在这里的输出文件包括的2np2.out，其中包含计算的参数的概述，bp_step.par，其中包含刚体上述参数的列表。后者的文件可以方便地阅读到电子表格程序，作进一步的分析和策划。代表结果（ 图1）的一个例子。 </li></ol> 3。从硬体参数构建的DNA结构<ol><li>除了一个核酸结构的分析，追梦的软件提供的能力，建立一个结构模型刚体使用&#39;重建&#39;命令的参数。在这个协议中，我们将展示如何构建两个DNA螺旋模型，首先用刚体参数计算协议2 HBB-DNA复合物，然后使用修改后的参数设置在w一族选定的滚动角被改变。侧倾角测量连续的碱基对的DNA进入槽16的弯曲程度。轧辊的正的值相关联的与缩小的大沟和负值的小沟变窄。的是标准基准帧17所通 ​​过3DNA和其他流行的核酸分析软件， 例如的曲线+ 18，可确保数值的一致性Watson-Crick碱基对中的计算参数。 </li><li> 3DNA"重建"功能默认情况下，将创建只包含含氮碱基中的原子坐标的精确的原子模型。为了创造一个近似的原子模型，其中包括骨干和基础原子的坐标，输入以下命令: x3dna_utils cp_std BDNA 此命令指示3DNA刚体参数引入一个标准的B型DNA骨干构建设模式。 </li><li>裴LD的双螺旋模型，刚体HBB-DNA结构中找到的参数，键入以下内容: 重建原子bp_step.par 2np2_rbld_org.pdb 在这里，&#39;原子&#39;指定的全原子模型的建设与所有重型（非氢）原子的原子坐标，&#39;bp_step.par&#39;议定书2第3步中生成的文件，其中包含了输入的刚体参数，&#39;2 np2_rbld_org.pdb&#39;是输出文件创建的结构的原子坐标。后者按照同规格的PDB文件格式，从而终止。PDB&#39;。 </li><li>可编辑&#39;bp_step.par的文件，生成一个修改过的结构。在Macintosh上的变化可以用TextEdit应用程序中所描述的协议1步骤10。 </li><li>在这里，我们修改两个站点的最大弯曲极端辊形成的夹角。值，以度表示，从64.95的值改为0，第17行和600.93第26行。我们用一个新名称保存修改后的文件，&#39;bp_step_roll0.par&#39;，关闭的TextEdit程序。 </li><li>我们建立的结构，在步骤3中，通过打字输入文件与修改后的一套步骤参数: 重建原子bp_step_roll0.par 2np2_rbld_roll0.pdb </li><li>可以被视为在一个标准的分子观者如PyMOL的（ <a href="http://www.pymol.org/" target="_blank">www.pymol.org</a> ），这两个模型，使用生成的坐标文件（的。pdb）作为输入。请参阅图像重建结构的代表性的结果（ 图2）。 </li></ol> 4。多模型结构文件分析<ol><li>协议2，在此我们分析一个单一的含核酸结构，在这里我们将展示如何分析结构大合奏。我们研究了随着时间的推移沿着一系列的14个碱基对的DNA链得到的分子动力学（MD）模拟11的刚体参数的变化</sup&gt;。分析认为，4500结构，间距为100皮秒的增量在MD计算，4,500单独的文件保存在。随附的补充数据包括这些文件的副本，以及反向的DNA序列（见碱基的确切顺序的介绍性文字）模拟了一套4500的其他文件。两套文件和一个较小的数据子集，也可以发现3DNA在<a href="http://forum.x3dna.org/jove" target="_blank">http://forum.x3dna.org/jove</a>论坛上。 </li><li>在分析中的第一个步骤是通过运行的&#39;find_pair程序进行协议2，识别配对的碱基相同。由于整个组的结构股相同的碱基配对方案一需要运行&#39;find_pair&#39;，只有一次有代表性的文件。在这里，我们选择的第1001个结构在DNA链含中央步骤，的文件名md_AT_set1.pdb.1001的模拟。下面的指令属韭碱基配对信息，并将其存储在文件中md_AT_set1.bps: find_pair md_AT_set1.pdb.1001 md_AT_set1.bps 用户可以检查所确定的碱基配对和知识结构的基础上在文件中进行手动更改。 </li><li>下一个步骤是确定整组使用"x3dna_ensemble分析"命令的结构的几何参数。该程序使用作为输入碱基对生成的文件在步骤2中（md_AT_set1.bps）以及一个文件包含要分析的结构的列表。后面的文件，这里称为md_AT_set1.list，包含4500个文件进行分析，在个别行上输入的文件名中的顺序生成的分子动力学模拟。这个文件的副本包含在补充材料，也提供3DNA论坛。用户应注意，由于大量的文件，计算的时间会很长（约20分钟AMD羿龙II X4 965处理器，每1000个文件）。钍e命令运行通过键入以下内容: x3dna_ensemble分析 - B md_AT_set1.bps的-L md_AT_set1.list邻md_AT_set1.out 在上面的例子&#39;-B&#39;涉及到碱基对在第2步中创建的文件，"-L"指定的文件列表，&#39;邻&#39;允许用户指定一个名称到输出文件中。也有很多选择&#39;x3dna_ensemble分析"命令，可以配合使用，如具体型号的选择。用户可以键入&#39;x3dna_ensemble分析-H"来获取有关这些选项的更多信息。 </li><li>下一步骤是提取所需的刚体参数从概述的输出文件，md_AT_set1.out，在步骤3中产生。这是使用"x3dna_ensemble提取物"命令执行。一上市可用的几何参数，可以发现输入&#39;x3dna_ensemble提取L&#39;。在这里，我们提取的滚动角，并创建一个以逗号分隔的文本文件（CSV）的侧倾角度用以下命令: x3dna_ensemble提取物 - 对滚-S，-F md_AT_set1.out邻md_AT_set1_roll.csv &#39;-P卷在此命令中指定要提取的参数，"-S"表示一个逗号分隔的文件，&#39;-F&#39;表示合奏分析命令的输出文件，和&#39;邻&#39;允许用户在所有分析的结构的DNA命名为CSV输出文件中的侧滚角沿。这个文件可以读取到其它程序中作进一步的分析和策划。请参阅代表结果（ 图3），在上述两个MD生成的数据集和用于生成这些图像在MATLAB脚本3DNA论坛辊的变化进行分析。 </li></ol> 5。多模型结构的叠加到一个共同的参考框架<ol><li>追梦（2.1版）的最新版本有一个新功能，它允许用户在多个结构从一个普通的角度看。 &#39;x3dna_ensemble重新定位"命令相关结构的叠加集合上课在一个共同的碱基对或碱基对的步骤。以下协议适用于这种能力O3 DNA运营商绑定在Lac阻遏蛋白12的头饰核磁共振派生结构。下的标识符2kek的多模式结构的文件，该文件包含了所有的结构模型在一个文件中，而不是单独的文件中，例如用于每个协议4的4500型号，这些结构被存储在PDB中。随附的补充资料包括一份这个文件，也可以找到3DNA在<a href="http://forum.x3dna.org/jove" target="_blank">http://forum.x3dna.org/jove</a>论坛。 </li><li>正如在先前的协议中，第一步是计算的DNA的碱基配对可与&#39;find_pair"程序获得。在这种情况下，我们使用输入文件kek.pdb &#39;2&#39;，并键入以下内容: find_pair 2kek.pdb 2kek.bps 碱基配对产生PDB文件使用的第一款车型。手动更正的输出bASE对的文件，&#39;2 kek.bps"，可以进行知识结构的基础上。 </li><li>该方案的x3dna_ensemble重新定向&#39;对齐在一个多模型结构的一个共同的参考帧的单独的模型。此程序需要pdb文件和碱基配对的文件作为输入，并从以前的步骤，运行以下命令: 重新调整-B 2kek.bps x3dna_ensemble F 1-E 2kek.pdb邻2kek_fr1.pdb 在这里&#39;-B&#39;选项指定的碱基配对文件，参数"-f 1&#39;表示的碱基对上所有的结构是一致的，在这种情况下，碱基对的1序列息链的5&#39;-末端&#39;-E&#39;指定的输入集合文件，"-o"参数允许用户提供一个输出文件名，在这种情况下，&#39;kek_fr1.pdb&#39;。有关命令选项的更多信息，可以通过键入&#39;x3dna_ensemble重新调整H&#39;。请参阅代表结果（ 图4）对齐的结构的讨论。 </li></ol> 6。工程业CTION的蛋白质修饰过的DNA分子<ol><li>的w3DNA Web界面包括一些流行的功能的3DNA软件包。在这里，我们提请注意的能力，构建三维模型与Web服务器的蛋白质修饰过的DNA。结合的DNA片段采用刚体参数所选择的蛋白质-DNA复合物，这里的值HBB 2协议分析的蛋白质的DNA结合。未绑定的DNA区域，可以采取三个用户选定的螺旋形式（A，B或C）之一。 A，B和C是指有三个典型的DNA模型，从早期的光纤衍射实验，分别为11，10，每回合的双螺旋19-21和9个碱基对。 </li><li>构建蛋白质修饰过的DNA模型的第一步是位于<a href="http://w3DNA.rutgers.edu" target="_blank">http://w3dna.rutgers.edu，</a>参观w3DNA网站。从菜单中选择"重建"的顶部左侧的页面激活vates在线模式建设能力。 </li><li>下一个步骤是选择在浅色阴影的框中，然后在新的页面的中间发现的&#39;束缚的蛋白质与DNA模板"链接。此选择将激活一个下拉菜单，该菜单允许用户指定结合的蛋白质的数量。在这里，我们选择了两个结合蛋白，并单击"继续"按钮。请注意，模型的大小受限制，以1,000个碱基对，或2000个核苷酸，和第三结合蛋白。 </li><li>结合的蛋白质的数量的选择导致在图5中所示的类似的规范页面。在该页面允许用户指定的DNA序列，螺旋形式的未结合的DNA结合蛋白的身份和位置的。 </li><li>用户类型或粘贴在中间的规格页中的文本框（ 图5，标签1）按照期望的顺序。注意唯一标准的残留物 - A，T，C，G，U - 被接受。在这里，我们进入了81个碱基对的坎opolymer，完全的A·T对组成序列息链中的A。 </li><li>有一个下拉菜单（ 图5，标签2），下面的文本框中，选择未绑定的螺旋构象的DNA区域。在这个例子中，我们选择的B型构象。 </li><li>底部进入的规格页的装订位置（s）和文件名 ​​（S）结合蛋白（S）（ 图5，标签3）附近有一个文本框。的结合位置指定的蛋白质结合的片段的DNA序列上的中心的位置。如果结合的DNA包含奇数个碱基对，如六溴代二苯DNA结构中的结合位置表示的位置的中间的碱基对的片段。 HBB-DNA结合的情况下，这中间的碱基对是T·T mispair。在这个例子中，在这里我们将两个拷贝的贫血症状的蛋白质的DNA模板，放置在位置20和63的81个碱基对的DNA序列中的T·T对序列。请注意，绑定的片段的序列不具有配合在步骤5输入。如果蛋白质结合的区域包含偶数数目的碱基对中的结合位置表示的位置上的DNA碱基对的中间步骤。也就是说，中央结合的片段的碱基对的步骤，n和n +1，其中n是沿段的指定数目的碱基对之间。另外请注意，用户可以与任何蛋白质修饰的DNA，只要其与DNA的复合物的结构是已知的并存储在标准PDB格式13。 </li><li>在规范的页的底部是一个盒子，它允许用户生成的蛋白结合的DNA（ 图5，标签4）的预览。在这里，我们请选中该复选框并单击"继续"按钮。这个动作会产生一个查看页面，列出所选的参数，以及可能出现的任何错误，从选定的结合位点的重叠。用户可以LECT"返回"按钮，如有需要作出改变或&#39;建立&#39;按钮继续。 </li><li>翻页显示的静态图像的DNA-蛋白质复合物在其大多数扩展的安排，并允许上线交互式可视化通过Jmol的Webmol的。用户还可以下载一个坐标（PDB）文件，按照同规格的PDB格式。这是通过选择"重建pdb文件生成的图像下面的链接下载。到一个分子的进一步可视化的图形程序，后者可以方便地阅读文件。代表结果的的贫血症状装饰DNA的例子（ 图6）如上所述，一个稍微长链（86个碱基对的）在位置20和67的中心的HBB。 </li></ol>

<ol>
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L., Grunwaldt, E., Davis, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lyme+disease-a+tick-borne+spirochetosis?.">Lyme disease-a tick-borne spirochetosis?.</a> Science. 216, 1317-1319 (1982).</li><li>Benach, J. L., Bosler, E. M., Hanrahan, J. P., Coleman, J. L., Habicht, G. S., Bast, T. F., Cameron, D. J., Ziegler, J. L., Barbour, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spirochetes+isolated+from+the+blood+of+two+patients+with+Lyme+disease.">Spirochetes isolated from the blood of two patients with Lyme disease.</a> N. Engl. J. 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R., Diekmann, S., Hunter, W. N., Kennard, O., von Kitzing, E., Lavery, R., Nelson, H. C. M., Olson, W. K., 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=Definitions+and+nomenclature+of+nucleic+acid+structure+parameters.">Definitions and nomenclature of nucleic acid structure parameters.</a> J. Mol. Biol. 205, 787-791 (1989).</li><li>Olson, W. K., Bansal, M., Burley, S. K., Dickerson, R. E., Gerstein, M., Harvey, S. C., Heinemann, U., Lu, X. -. J., Neidle, S., Shakked, Z., 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=A+standard+reference+frame+for+the+description+of+nucleic+acid+base-pair+geometry.">A standard reference frame for the description of nucleic acid base-pair geometry.</a> J. Mol. Biol. 313, 229-237 (2001).</li><li>Lavery, R., Moakher, M., Maddocks, J. H., Petkeviciute, D., Zakrzewska, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conformational+analysis+of+nucleic+acids+revisited:+Curves+.">Conformational analysis of nucleic acids revisited: Curves+.</a> Nucleic Acids Res. 37, 5917-5929 (2009).</li><li>Franklin, R. E., Gosling, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+configuration+in+sodium+thymonucleate.">Molecular configuration in sodium thymonucleate.</a> Nature. 171, 740-741 (1953).</li><li>Watson, J. D., Crick, F. H. 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K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modeling+the+Lac+repressor-operator+assembly:+the+influence+of+DNA+looping+on+Lac+repressor+conformation.">Modeling the Lac repressor-operator assembly: the influence of DNA looping on Lac repressor conformation.</a> Proc. Natl. Acad. Sci. U.S.A. 103, 9879-9884 (2006).</li><li>Czapla, L., Swigon, D., Olson, W. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+the+nucleoid+protein+HU+on+the+structure,+flexibility,+and+ring-closure+properties+of+DNA+deduced+from+Monte-Carlo+simulations.">Effects of the nucleoid protein HU on the structure, flexibility, and ring-closure properties of DNA deduced from Monte-Carlo simulations.</a> J. Mol. Biol. 382, 353-370 (2008).</li><li>Czapla, L., Peters, J. P., Rueter, E. M., Olson, W. K., Maher, L. 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没有利益冲突的声明。

在这篇文章中提出的协议集，只触及3DNA程序套件的能力。的工具，可以适用核糖核酸结构识别非经典的碱基对，以确定的二级结构的上下文中，会发生这样的配对，量化螺旋片段的空间配置，测量沿链主链的碱基重叠，等等。 rebuild命令允许用户构建简单和翔实的基地和基地对像中的插图所示， 图1块表示。建设工具还包括在一个给定的结构化模板功能，以"线"不同的序列，产生无数?...

了解三维结构的DNA，RNA和蛋白，药物和其他配体的配合物，用于解密其不同的生物功能是至关重要的，允许设计合理的治疗学。这种结构的探索需要三个单独的，但密切相关的部分组成:分析（提取型态的形状和相互作用），建模（评估能量和分子动力学），和可视化。结构分析和模型建设基本上同一枚硬币的两面，他们两个补充和可视化。 3DNA一套计算机程序，是一个日益流行的结构与功能的生物信息学工具包，分析，构建和可视化三维核酸结构。早期出版物概述软件1，提供的食谱执行所选任务2的功能，介绍了基于Web的界面软件的流行特点，结构特点数据库收集使用 3DNA 4，5和示出的软件的实用程序在分析中的DNA和RNA结构6，7。 本文目的是把的3DNA软件套件实验室科学家和其他人的利益和/或需要调查的DNA和RNA的空间组织与国家的最先进的计算工具。这里提出的协议包括一步一步的说明（一）下载并安装一个Mac OS X系统上的软件，（II-III）DNA碱基对组成的步骤，水平结构分析和修改（ IV-V），以分析和相关的DNA结构调整套，及（vi）与用户友好的w3DNA Web界面蛋白质修饰过的DNA链构建模型。软件具有分析能力，解决个别结构，利用X射线晶体学方法以及大型利用核磁共振（NMR）方法测定结构或计算机仿真技术所产生的合奏。 这里的结构检查包括（i）的高分辨率晶体结构的DNA势必到HBB蛋白从伯氏疏螺旋体 （蜱传播的细菌，导致莱姆病在人类9，10），（二）两个大组顺序相关的DNA分子与分子模拟11 - 4500快照d的（GGCAAAATTTTGCC）的，2和d（CCGTTTTAAAACGG）2在100皮秒为单位收集，在计算过程中，及（iii）O3 DNA操作员基于NMR的结构的一个小的合奏绑定的头饰在大肠杆菌 lac阻遏蛋白12。下面的说明包括有关如何访问这些结构，以及如何使用3DNA（此文件的一个副本被发现相关文件的原子坐标信息3DNA在<a href="http://forum.x3dna.org/jove" target="_blank">http://forum.x3dna.org/jove</a>论坛）审查和修改这些结构。

analyzing and building nucleic acid structures with 3dna

追梦的软件包是一个流行和通用的生物信息学工具与功能分析，构建，可视化三维核酸结构。本文提出了详细的协议的一个子集3DNA提供新的和流行的特点，适用于个别结构和相关结构的合奏。协议1列出下载并安装该软件所需要的指令的集合。其次，协议2，通过分析的核酸结构，包括碱基对的分配和确定刚体参数描述结构，协议3，由一个原子的重建的说明模型的结构从它的刚体参数。追梦，版本2.1，最新版本的结构歌舞团的分析和操作的新功能，如核磁共振（NMR）测量和分子动力学（MD推导出）模拟，这些功能在协议中的4和5。除了 ​​追梦的独立软件包，w3DNA位于<a href="http://w3dna.rutgers.edu" target="_blank">http://w3dna.rutgers.edu</a> ， web服务器，提供了一个友好的用户界面功能的软件选择。协议6演示了一个新的功能的网站建设模型长的DNA结合蛋白的分子修饰在用户指定的位置。

常规使用的的3DNA软件工具来分析核酸结构。例如，碱基对的身份和特征在双螺旋片段的碱基的DNA和RNA结构的安排刚体参数自动计算，为每一个新的条目存储在核酸数据库22，世界范围内的存储库核酸的结构信息。刚体参数确定协议2的值轻易表露在三维结构的扭曲，如极端的DNA大沟，大正滚动角（64.95°，60.93°）弯曲成两个网站，发现在AT·AT与伯氏疏螺旋体 HBB蛋白8（ ?...

Watch this Scientific Journal Video about Analyzing and Building Nucleic Acid Structures with 3DNA at JoVE.com

Analyzing and Building Nucleic Acid Structures with 3DNA

追梦的软件包是一个流行和通用的生物信息学工具与功能分析，构建，可视化三维核酸结构。本文提出了详细的协议的一个子集3DNA提供新的和流行的特点，适用于个别结构和相关结构的合奏。

核酸结构分析及建设3DNA

The 3DNA software package is a popular and versatile bioinformatics tool with capabilities to analyze, construct, and visualize three-dimensional nucleic ...

analyzing-and-building-nucleic-acid-structures-with-3dna

Research

JoVE Journal

Biology

20.4K 次观看.  Rutgers - The State University of New Jersey. 追梦的软件包是一个流行和通用的生物信息学工具与功能分析，构建，可视化三维核酸结构。本文提出了详细的协议的一个子集3DNA提供新的和流行的特点，适用于个别结构和相关结构的合奏。

视频: Analyzing and Building Nucleic Acid Structures with 3DNA

AC Electrokinetic Phenomena Generated by Microelectrode Structures

The field of AC electrokinetics is rapidly growing due to its ability to perform dynamic fluid and particle manipulation on the micro- and nano-scale, which is essential for Lab-on-a-Chip applications. AC electrokinetic phenomena use electric fields to generate forces that act on fluids or suspended particles (including those made of dielectric or biological material) and cause them to move in astonishing ways1, 2. Within a single channel, AC electrokinetics can accomplish many essential on-chip operations such as active micro-mixing, particle separation, particle positioning and micro-pattering. A single device may accomplish several of those operations by simply adjusting operating parameters such as frequency or amplitude of the applied voltage. Suitable electric fields can be readily created by micro-electrodes integrated into microchannels. It is clear from the tremendous growth in this field that AC electrokinetics will likely have a profound effect on healthcare diagnostics3-5, environmental monitoring6 and homeland security7.

In general, there are three AC Electrokinetic phenomena (AC electroosmosis, dielectrophoresis and AC electrothermal effect) each with unique dependencies on the operating parameters. A change in these operating parameters can cause one phenomena to become dominant over another, thus changing the particle or fluid behavior. 

It is difficult to predict the behavior of particles and fluids due to the complicated physics that underlie AC electrokinetics. It is the goal of this publication to explain the physics and elucidate particle and fluid behavior. Our analysis also covers how to fabricate the electrode structures that generate them, and how to interpret a wide number of experimental observations using several popular device designs. This video article will help scientists and engineers understand these phenomena and may encourage them to start using AC Electrokinetics in their research.

Manipulating fluids and suspended particles in the micro- and nano-scale is becoming more of a reality as enabling technologies, like AC electrokinetics, continue to develop. Here, we discuss the physics behind AC electrokinetics, how to fabricate these devices and how to interpret the experimental observations.

微电极结构产生的交流电动现象

The field of AC electrokinetics is rapidly growing due to its ability to perform dynamic fluid and particle manipulation on the micro- and nano-scale, ...

科研

生物学

Staining of Proteins in Gels with Coomassie G-250 without Organic Solvent and Acetic Acid

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents (Methanol, Ethanol or 2-Propanol) and acetic acid are used for fixation, staining and destaining of proteins in a gel after SDS-PAGE. To speed up the procedure, heating the staining solution in the microwave oven for a short time is frequently used. This usually results in evaporation of toxic or hazardous Methanol, Ethanol or 2-Propanol and a strong smell of acetic acid in the lab which should be avoided due to safety considerations. In a protocol originally published in two patent applications by E.M. Wondrak (US2001046709 (A1), US6319720 (B1)), an alternative composition of the staining solution is described in which no organic solvent or acid is used. The CBB is dissolved in bidistilled water (60-80mg of CBB G-250 per liter) and 35 mM HCl is added as the only other compound in the staining solution. The CBB staning of the gel is done after SDS-PAGE and thorough washing of the gel in bidistilled water. By heating the gel during the washing and staining steps, the process can be finished faster and no toxic or hazardous compunds are evaporating. The staining of proteins occurs already within 1 minute after heating the gel in staining solution and is fully developed after 15-30 min with a slightly blue background that is destained completely by prolonged washing of the stained gel in bidistilled water, without affecting the stained protein bands.

A short protocol for protein staining with Coomassie Brilliant Blue (CBB) G-250 in polyacrylamide gels is described without using organic solvents or acetic acid as in the classical staining procedures with CBB.

无有机溶剂和醋酸，蛋白质与考马斯亮蓝G - 250凝胶染色

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents ...

Fluorescent Labeling of Drosophila Heart Structures

The Drosophila melanogaster dorsal vessel, or heart, is a tubular structure comprised of a single layer of contractile cardiomyocytes, pericardial cells that align along each side of the heart wall, supportive alary muscles and, in adults, a layer of ventral longitudinal muscle cells. The contractile fibers house conserved constituents of the muscle cytoarchitecture including densely packed bundles of myofibrils and cytoskeletal/submembranous protein complexes, which interact with homologous components of the extracellular matrix. Here we describe a protocol for the fixation and the fluorescent labeling of particular myocardial elements from the hearts of dissected larvae and semi-intact adult Drosophila. Specifically, we demonstrate the labeling of sarcomeric F-actin and of &alpha;-actinin in larval hearts. Additionally, we perform labeling of F-actin and &alpha;-actinin in myosin-GFP expressing adult flies and of &alpha;-actinin and pericardin, a type IV extracellular matrix collagen, in wild type adult hearts. Particular attention is given to a mounting strategy for semi-intact adult hearts that minimizes handling and optimizes the opportunity for maintaining the integrity of the cardiac tubes and the associated tissues. These preparations are suitable for imaging via fluorescent and confocal microscopy. Overall, this procedure allows for careful and detailed analysis of the structural characteristics of the heart from a powerful genetically tractable model system.

Fluorescent Labeling of Drosophila Heart Structures

Here we describe a basic protocol for fluorescent labeling of different elements of heart tubes from larva and adult Drosophila melanogaster. These specimens are well-suited for imaging via fluorescent or confocal microscopy. This technique permits detailed structural analysis of the features of the hearts from a powerful model organism.

荧光标记的果蝇心结构

The Drosophila melanogaster dorsal vessel, or heart, is a tubular structure comprised of a single layer of contractile cardiomyocytes, pericardial cells ...

NanoDrop Microvolume Quantitation of Nucleic Acids

Biomolecular assays are continually being developed that use progressively smaller amounts of material, often precluding the use of conventional cuvette-based instruments for nucleic acid quantitation for those that can perform microvolume quantitation.
The NanoDrop microvolume sample retention system (Thermo Scientific NanoDrop Products) functions by combining fiber optic technology and natural surface tension properties to capture and retain minute amounts of sample independent of traditional containment apparatus such as cuvettes or capillaries. Furthermore, the system employs shorter path lengths, which result in a broad range of nucleic acid concentration measurements, essentially eliminating the need to perform dilutions. Reducing the volume of sample required for spectroscopic analysis also facilitates the inclusion of additional quality control steps throughout many molecular workflows, increasing efficiency and ultimately leading to greater confidence in downstream results.
The need for high-sensitivity fluorescent analysis of limited mass has also emerged with recent experimental advances. Using the same microvolume sample retention technology, fluorescent measurements may be performed with 2 &mu;L of material, allowing fluorescent assays volume requirements to be significantly reduced. Such microreactions of 10 &mu;L or less are now possible using a dedicated microvolume fluorospectrometer.
Two microvolume nucleic acid quantitation protocols will be demonstrated that use integrated sample retention systems as practical alternatives to traditional cuvette-based protocols. First, a direct A260 absorbance method using a microvolume spectrophotometer is described. This is followed by a demonstration of a fluorescence-based method that enables reduced-volume fluorescence reactions with a microvolume fluorospectrometer. These novel techniques enable the assessment of nucleic acid concentrations ranging from 1 pg/ &mu;L to 15,000 ng/ &mu;L with minimal consumption of sample.

The use of NanoDrop microvolume systems as practical and efficient alternatives to traditional nucleic acid quantitation methodology is described through the demonstration of two microvolume nucleic acid quantitation protocols.

NanoDrop微量核酸的定量

Biomolecular assays are continually being developed that use progressively smaller amounts of material, often precluding the use of conventional ...

Electricity-Free, Sequential Nucleic Acid and Protein Isolation

Traditional and emerging pathogens such as Enterohemorrhagic Escherichia coli (EHEC), Yersinia pestis, or prion-based diseases are of significant concern for governments, industries and medical professionals worldwide. For example, EHECs, combined with Shigella, are responsible for the deaths of approximately 325,000 children each year and are particularly prevalent in the developing world where laboratory-based identification, common in the United States, is unavailable 1. The development and distribution of low cost, field-based, point-of-care tools to aid in the rapid identification and/or diagnosis of pathogens or disease markers could dramatically alter disease progression and patient prognosis. We have developed a tool to isolate nucleic acids and proteins from a sample by solid-phase extraction (SPE) without electricity or associated laboratory equipment 2. The isolated macromolecules can be used for diagnosis either in a forward lab or using field-based point-of-care platforms. Importantly, this method provides for the direct comparison of nucleic acid and protein data from an un-split sample, offering a confidence through corroboration of genomic and proteomic analysis.
Our isolation tool utilizes the industry standard for solid-phase nucleic acid isolation, the BOOM technology, which isolates nucleic acids from a chaotropic salt solution, usually guanidine isothiocyanate, through binding to silica-based particles or filters 3. CUBRC's proprietary solid-phase extraction chemistry is used to purify protein from chaotropic salt solutions, in this case, from the waste or flow-thru following nucleic acid isolation4.
By packaging well-characterized chemistries into a small, inexpensive and simple platform, we have generated a portable system for nucleic acid and protein extraction that can be performed under a variety of conditions. The isolated nucleic acids are stable and can be transported to a position where power is available for PCR amplification while the protein content can immediately be analyzed by hand held or other immunological-based assays. The rapid identification of disease markers in the field could significantly alter the patient's outcome by directing the proper course of treatment at an earlier stage of disease progression. The tool and method described are suitable for use with virtually any infectious agent and offer the user the redundancy of multi-macromolecule type analyses while simultaneously reducing their logistical burden.

A tool and chemistries are described to sequentially isolate nucleic acids followed by proteins from a sample without the need for electricity. The tool consists of a sorbent held within a transfer pipette while the isolation chemistries are based on solid-phase extraction principles. The isolated macromolecules can be analyzed by immuno-based and PCR-based assays.

免电力，顺序的核酸和蛋白质的分离

Traditional and emerging pathogens such as Enterohemorrhagic Escherichia coli (EHEC), Yersinia pestis, or prion-based diseases are of significant concern ...

Simultaneous Multicolor Imaging of Biological Structures with Fluorescence Photoactivation Localization Microscopy

Localization-based super resolution microscopy can be applied to obtain a spatial map (image) of the distribution of individual fluorescently labeled single molecules within a sample with a spatial resolution of tens of nanometers. Using either photoactivatable (PAFP) or&#160;photoswitchable (PSFP) fluorescent proteins fused to proteins of interest, or organic dyes conjugated to antibodies or other molecules of interest, fluorescence photoactivation localization microscopy (FPALM) can simultaneously image multiple species of molecules within single cells. By using the following approach, populations of large numbers (thousands to hundreds of thousands) of individual molecules are imaged in single cells and localized with a precision of ~10-30 nm. Data obtained can be applied to understanding the nanoscale spatial distributions of multiple protein types within a cell. One primary advantage of this technique is the dramatic increase in spatial resolution: while diffraction limits resolution to ~200-250 nm in conventional light microscopy, FPALM can image length scales more than an order of magnitude smaller. As many biological hypotheses concern the spatial relationships among different biomolecules, the improved resolution of FPALM can provide insight into questions of cellular organization which have previously been inaccessible to conventional fluorescence microscopy. In addition to detailing the methods for sample preparation and data acquisition, we here describe the optical setup for FPALM. One additional consideration for researchers wishing to do super-resolution microscopy is cost: in-house setups are significantly cheaper than most commercially available imaging machines. Limitations of this technique include the need for optimizing the labeling of molecules of interest within cell samples, and the need for post-processing software to visualize results. We here describe the use of PAFP and PSFP expression to image two protein species in fixed cells. Extension of the technique to living cells is also described.

We demonstrate the use of fluorescence photo activation localization microscopy (FPALM) to simultaneously image multiple types of fluorescently labeled molecules&#160;within cells. The techniques described yield the localization of thousands to hundreds of thousands of individual fluorescent labeled proteins, with a precision of tens of nanometers&#160;within single cells.

Localization-based super resolution microscopy can be applied to obtain a spatial map (image) of the distribution of individual fluorescently labeled ...

The Fastest Western in Town: A Contemporary Twist on the Classic Western Blot Analysis

The Western blot techniques that were originally established in the late 1970s are still actively utilized today. However, this traditional method of Western blotting has several drawbacks that include low quality resolution, spurious bands, decreased sensitivity, and poor protein integrity. Recent advances have drastically improved numerous aspects of the standard Western blot protocol to produce higher qualitative and quantitative data. The Bis-Tris gel system, an alternative to the conventional Laemmli system, generates better protein separation and resolution, maintains protein integrity, and reduces electrophoresis to a 35 min run time. Moreover, the iBlot dry blotting system, dramatically improves the efficacy and speed of protein transfer to the membrane in 7 min, which is in contrast to the traditional protein transfer methods that are often more inefficient with lengthy transfer times. In combination with these highly innovative modifications, protein detection using infrared fluorescent imaging results in higher-quality, more accurate and consistent data compared to the standard Western blotting technique of chemiluminescence. This technology can simultaneously detect two different antigens on the same membrane by utilizing two-color near-infrared dyes that are visualized in different fluorescent channels. Furthermore, the linearity and broad dynamic range of fluorescent imaging allows for the precise quantification of both strong and weak protein bands. Thus, this protocol describes the key improvements to the classic Western blotting method, in which these advancements significantly increase the quality of data while greatly reducing the performance time of this experiment.

This protocol explores the latest advancements in performing Western blot analyses.&#160;These novel modifications employ a Bis-Tris gel system with a 35&#160;min electrophoresis run time, a 7&#160;min dry blotting transfer system, and infrared fluorescent protein detection and imaging that generates higher resolution, quality, sensitivity, and improved accuracy of Western data.

最快在西镇:对经典蛋白质印迹分析现代风味

The Western blot techniques that were originally established in the late 1970s are still actively utilized today. However, this traditional method of ...

Sequence-specific Labeling of Nucleic Acids and Proteins with Methyltransferases and Cofactor Analogues

S-Adenosyl-l-methionine (AdoMet or SAM)-dependent methyltransferases (MTase) catalyze the transfer of the activated methyl group from AdoMet to specific positions in DNA, RNA, proteins and small biomolecules. This natural methylation reaction can be expanded to a wide variety of alkylation reactions using synthetic cofactor analogues. Replacement of the reactive sulfonium center of AdoMet with an aziridine ring leads to cofactors which can be coupled with DNA by various DNA MTases. These aziridine cofactors can be equipped with reporter groups at different positions of the adenine moiety and used for Sequence-specific Methyltransferase-Induced Labeling of DNA (SMILing DNA). As a typical example we give a protocol for biotinylation of pBR322 plasmid DNA at the 5&#8217;-ATCGAT-3&#8217; sequence with the DNA MTase M.BseCI and the aziridine cofactor 6BAz in one step. Extension of the activated methyl group with unsaturated alkyl groups results in another class of AdoMet analogues which are used for methyltransferase-directed Transfer of Activated Groups (mTAG). Since the extended side chains are activated by the sulfonium center and the unsaturated bond, these cofactors are called double-activated AdoMet analogues. These analogues not only function as cofactors for DNA MTases, like the aziridine cofactors, but also for RNA, protein and small molecule MTases. They are typically used for enzymatic modification of MTase substrates with unique functional groups which are labeled with reporter groups in a second chemical step. This is exemplified in a protocol for fluorescence labeling of histone H3 protein. A small propargyl group is transferred from the cofactor analogue SeAdoYn to the protein by the histone H3 lysine 4 (H3K4) MTase Set7/9 followed by click labeling of the alkynylated histone H3 with TAMRA azide. MTase-mediated labeling with cofactor analogues is an enabling technology for many exciting applications including identification and functional study of MTase substrates as well as DNA genotyping and methylation detection.

DNA and proteins are sequence-specifically labeled with affinity or fluorescent reporter groups using DNA or protein methyltransferases and synthetic cofactor analogues. Depending on the cofactor specificity of the enzymes, aziridine or double activated cofactor analogues are employed for one- or two-step labeling.

核酸，蛋白质与甲基转移和辅酶类似物序列特异性标签

S-Adenosyl-l-methionine (AdoMet or SAM)-dependent methyltransferases (MTase) catalyze the transfer of the activated methyl group from AdoMet to specific ...

Simple Bulk Readout of Digital Nucleic Acid Quantification Assays

Digital assays are powerful methods that enable detection of rare cells and counting of individual nucleic acid molecules. However, digital assays are still not routinely applied, due to the cost and specific equipment associated with commercially available methods. Here we present a simplified method for readout of digital droplet assays using a conventional real-time PCR instrument to measure bulk fluorescence of droplet-based digital assays.
We characterize the performance of the bulk readout assay using synthetic droplet mixtures and a droplet digital multiple displacement amplification (MDA) assay. Quantitative MDA particularly benefits from a digital reaction format, but our new method&#160;applies to any digital assay. For established digital assay protocols such as digital PCR, this method serves to speed up and simplify assay readout.
Our bulk readout methodology brings the advantages of partitioned assays without the need for specialized readout instrumentation. The principal limitations of the bulk readout methodology are reduced dynamic range compared with droplet-counting platforms and the need for a standard sample, although the requirements for this standard are less demanding than for a conventional real-time experiment. Quantitative whole genome amplification (WGA) is used to test for contaminants in WGA reactions and is the most sensitive way to detect the presence of DNA fragments with unknown sequences, giving the method great promise in diverse application areas including pharmaceutical quality control and astrobiology.

We describe an endpoint digital assay for quantifying nucleic acids with a simplified (analog) readout. We measure bulk fluorescence of droplet-based digital assays using a standard qPCR machine rather than specialized instrumentation and confirm our results by microscopy.

数字核酸定量实验分析的简单堆积读数

Digital assays are powerful methods that enable detection of rare cells and counting of individual nucleic acid molecules. However, digital assays are ...

Efficient Nucleic Acid Extraction and 16S rRNA Gene Sequencing for Bacterial Community Characterization

There is a growing appreciation for the role of microbial communities as critical modulators of human health and disease. High throughput sequencing technologies have allowed for the rapid and efficient characterization of bacterial communities using 16S rRNA gene sequencing from a variety of sources. Although readily available tools for 16S rRNA sequence analysis have standardized computational workflows, sample processing for DNA extraction remains a continued source of variability across studies. Here we describe an efficient, robust, and cost effective method for extracting nucleic acid from swabs. We also delineate downstream methods for 16S rRNA gene sequencing, including generation of sequencing libraries, data quality control, and sequence analysis. The workflow can accommodate multiple samples types, including stool and swabs collected from a variety of anatomical locations and host species. Additionally, recovered DNA and RNA can be separated and used for other applications, including whole genome sequencing or RNA-seq. The method described allows for a common processing approach for multiple sample types and accommodates downstream analysis of genomic, metagenomic and transcriptional information.

We describe an efficient, robust, and cost effective method for extracting nucleic acid from swabs for characterization of bacterial communities using 16S rRNA gene amplicon sequencing. The method allows for a common processing approach for multiple sample types and accommodates a number of downstream analytic processes.

高效的核酸提取和16S rRNA基因序列的细菌群落特征

There is a growing appreciation for the role of microbial communities as critical modulators of human health and disease. High throughput sequencing ...