院内研究计划的美国国家癌症研究所，美国国立卫生研究院，美国，JW，KJ Purzycka的J. Sztuba Solinska S. Lusvarghi，劳施和SFJ格莱斯支持。

引物设计和RNA的3&#39;端延伸要通过高通量的形状的长链核酸分析，应选择这样，他们属于（i）分离的〜300个核苷酸，（ⅱ）是20-30个核苷酸的长度，及（iii）RNA /引物杂交位点的一系列DNA杂交所产生的DNA退火站点有一个预期的熔融温度&gt; 50℃。此外，高度结构化的，预测的核糖核酸的分类应该被避免，虽然作出这样的判断需要一定的预知的RNA结构，这通常是不可用的。 DNA引物杂交到这些网站，然后应设计，照顾，以确保他们预计不会形成稳定的二聚体或链内二级结构。 一旦设计，引物必须购买（ 例如集成DNA技术，爱荷华州Ames）或合成24,25。 5&#39;-标记引物用Cy5，Cy5.5标记，WellRedD2（Beckman Coulter公司），IRDye800（Lycor）/ WellRedD1（Beckman Coulter公司）的最适合于Beckman Coulter公司8000 CEQ，提供良好的信号强度，同时最大限度地减少串扰。标记的寡核苷酸可无限期存储小，10微米等分在-20°C，避免反复冷冻/解冻周期。 通过使用以这种方式设计的引物，它是寻找任意长度的一个完整的RNA未能获得形状数据。但是，该序列的RNA的3&#39;末端处或附近总是无法访问SHAPE，除非工程包含（ 例如，一个“结构的磁带盒”）的3&#39;末端延伸的引物可以杂交的RNA。 通过毛细管电泳RNA制备虽然从生物样品中的RNA可被用于高通量的形状，这里给出的协议优化，通过体外转录产生的RNA。商业TRA如nscription包MegaShortScript（Ambion公司）与MegaClear RNA纯化柱（Ambion公司）结合使用是非常适合产生大量的纯RNA。应贮存在-20℃和-80℃下在TE缓冲液中的RNA为了获得最好的效果，应该出现的RNA均匀双方​​变性和非变性聚丙烯酰胺凝胶电泳。 1。 RNA折叠<ol><li>在0.5毫升离心管中，稀释12 pmol的RNA至18微升与水和10X复性缓冲液中加入2μl。拌匀。 </li><li>加热到85°下进行1分钟，然后冷却至4℃下在一个速率为0.1℃/秒。 </li><li>添加100μl的水和30微升5X折叠缓冲液。 </li><li>在37℃下孵育30-60分钟，这取决于被折叠的RNA。在一般情况下，镁离子依赖的更长的时间，更加结构化的RNA折叠需要更长的温育时间。 </li><li> 72微升等分转移两个0.5毫升离心管：修饰ED（+）和控制（ - ）。 </li></ol> 2。化学修饰的RNA 好，其特征在于形状亲电试剂包括靛红酸酐（IA），N-methylisatoic的的酸酐（NMIA），1 -甲基-7 -硝基靛红酸酐（1M7）26苯甲酰氰（BZCN）的27。 ，其中最常用的高通量形状1M7 NMIA，并且只有后者是市售的（Life Technologies公司）。改性剂的最终浓度，必须优化为每个RNA取得“单次击中”修改动力学， 即一次，该区域的RNA，分析了11个条件，其中大多数在溶液中的RNA被修改。该最佳浓度可确定进行多个反应，在该反应中，各不相同的试剂的浓度在下文第2.1节中的表所示的范围内（次）。使用的试剂浓度，很容易产生一个检测信号，而分imizing长和短的DNA的合成的产品（ 例如 图3）之间的信号强度的差异。 <img alt="图3" fo:content-width="6in" fo:src="/files/ftp_upload/50243/50243fig3highres.jpg" src="/files/ftp_upload/50243/50243fig3.jpg" /> 图3。从〜360 nt的RNA（A）0（B）2.5毫米或（C）10毫米1M7治疗的形状产生的电泳，电泳显示在相同的规模。蓝色，绿色，红色和黑色的痕迹对应于（+）的反应产物（Cy5标记），（ - ）的反应产物（Cy5.5标记），和两个的测序梯（的D2 WellRed和IRDye800）。用来制作图像（B）的RNA已用1M7的最佳数量，展示良好的峰值分辨率和强度，在整个跟踪（左）与最小的信号衰减。阅读在这些条件下的最大长度。与此相反，没有介质的int密度，很好地解决高峰期（A），表明次优浓度1M7。相反，（C）中的信号衰减明显表明，单一冲击动力学观测不到时，RNA是改性。在这种情况下，尤其是当RT预计不会遇到的RNA模板的5&#39;末端，读取长度将是次优的。 <ol><li>准备形状的试剂（NMIA或1M7）10X股票。这是最好的实现通过添加少量试剂到1.5 ml微量离心管中，然后加入DMSO，以达到所需的浓度。 注意：形状试剂溶液必须保持无水，直到与RNA混合。商店DMSO在干燥器中在室温下，立即准备股票方案之前使用，以尽量减少暴露到环境的水汽。 <table border="1"><tbody><tr><td> 试剂 </td><td> 最佳的10倍的浓度（在DMSO中） </td><td> 时间完全降解试剂27 </td></tr><tr><td> NMIA </td><td> 10-100毫</td><td> 〜20分钟</td></tr><tr><td> 1M7 </td><td> 10-50毫米</td><td> 70秒</td></tr></tbody></table> 表1中。亲电试剂，用于RNA的变形例。 </li><li>加入8微升10X NMIA/1M7或无水DMSO中，改性的（+）和（ - ）分别混合， 注：2.5毫米已被证明是一个有效的起始浓度为两个NMIA 1M7，无论所分析的RNA。 </li><li>在37℃下孵育50分钟（NMIA）或5分钟（1M7），视情况而定。 </li><li>通过加入8微升0.1体积的3M醋酸钠（pH值5.2），8微升的100mM EDTA，240微升（3体积）的冷无水乙醇和1μl的10毫克/毫升糖原沉淀RNA。冷藏2小时，然后于4℃离心30分钟14,000 XG用冷的70％乙醇洗涤沉淀两次。 注意：重要的是要为了尽量减少共沉淀的盐，因为这可能会产生不利影响峰分辨率电泳过程中，尽量减少制冷时间，离心时间和速度。 </li><li>取出上清液用微量，5分钟，在室温空气干燥颗粒。 </li><li>沉淀的RNA溶解于10μl的TE缓冲液中，并在室温下孵育5分钟。这是足以溶解的RNA为两个反转录。储存于-20℃下的警告：未使用的部分机械再悬浮沉淀通常是没有必要的，并且有可能损坏的RNA。 </li></ol> 3。反转录此步骤生成的荧光标记的cDNA的产品是用于间接识别RNA核苷酸在何种程度上已被修改的形状试剂。形状，标III（Invitrogen公司）RT表现优于所有其他RTS久经考验，是选择使用此酶协议。用Cy5，Cy5.5标记标记的寡核苷酸被用于素数的（+）和（ - ）反应。对于较短的RNA引物杂交原生RNA的3&#39;端延伸（ 如 “盒式结构”），以获得信息的3&#39;末端4。 注意：从这个角度通过CE，样品应受到保护光。 <ol><li>准备（+）和（ - ）样品在0.5ml微量离心管中进行反转录。对于RT反应（+），混合修饰的RNA（+）5微升，6μl水和1微升Cy5标记引物（10微米）的RT反应（ - ），混合5微升控制RNA（ - ）， 6μl水，1微升Cy5标记引物（10微米）。 注意：扎尔施泰特PCR管（REF 72.735.002）推荐此应用程序。 </li><li>将管子放在一个热循环仪，退火引物进行反转录RNA和准备运用下面的程序：85℃，1分钟，60°C，5分钟;35℃，5分钟，50℃，保持。 </li><li>在退火工序中，准备足够的2.5X RT混合的数目以进行反应，加50％（ 例如，2（+）和两个（ - ）反应，规模的4.5倍）。一个反应需要8微升，如下所示：4微升5×RT缓冲液，1微升的100mM DTT，1.5μl水，1微升的10mM dNTPs浓度，加入0.5μl的SuperScript III RT。保持在冰上。 注意：5X RT缓冲区和100毫米的DTT上标III RT。 </li><li>一旦温度达到50℃退火混合，加入8微升的2.5X RT混合（+）和（ - ）反应。 建议：暖RT混合至37°C 5分钟，然后将它添加到反应。 </li><li>孵育在50℃下50分钟，然后冷却至4℃和/或放置在冰上注：长于50分钟，孵化的反转录（RT）反应，可能会导致异常的cDNA产物。 </li><li>水解RNA，加入1微升4M的NaOH中和加热至95℃下<html> 3分钟。酷冰，然后中和反应，通过加入2微升2 M盐酸。 注意：此步骤省略cDNA的产品质量差，分离结果。 </li><li>相结合（+）和（ - ）反应，并通过加入0.1体积的3M醋酸钠，0.1体积的100mM EDTA，1.5体积的冷乙醇，1微升10毫克/毫升糖原沉淀的cDNA。冷藏2小时，然后以14,000 xg离心30分钟，在4°C。两次，用冷的70％乙醇洗涤沉淀。 注意：离心以更高的速率，或一段较长时间，结果在重悬沉淀（次）的困难。 </li><li> 40微升去离子甲酰胺重悬浮颗粒的cDNA通过加热到65°C为10分钟，超过30分钟剧烈的震荡。 注意：颗粒可能是无形的。可能缺乏的信号或信号弱的下面电泳未能充分溶解沉淀在这个阶段的结果。 </li></ol>E“&gt; 4。制备测序梯测序梯子作为标记的核苷酸位置确定在数据处理过程中。这些生成的使用一个USB循环测序试剂盒（＃78500），具有相同的序列的RNA正在研究的DNA，引物标记的与WellRed D2或D1/Lycor 800。通常情况下，该反应中的DNA将使用中的RNA作为转录模板。尽管反应这里介绍的协议非常相似，试剂盒生产商推荐的，将反应缩放数倍。虽然DDA和DDT被用作链终止剂，在下文描述的反应中，任何一对终端装置可用于产生测序梯。 <ol><li>混合40微升的的DDA终止混合，5 pmol的DNA模板，4.6微升10X测序酶缓冲液，10μl的WellRed D2标记的引物，4.6微升测序酶和水，以使总体积为82μl。加入测序最后。准备工作重的第二测序反应中相同的方式，利用DDT和LICOR IR800标记的引物，而不是。 </li><li>继续使用USB推荐条件进行PCR扩增。 注意：不需要也不推荐添加矿物油的协议/热循环利用热盖。 </li><li> DDA和滴滴涕结合成一个1.5毫升离心管（164微升总）测序反应。 </li><li>沉淀DNA，如下所示：...添加16微升3 M醋酸钠（pH 5.2）中，16微升的100mM EDTA，1微升10毫克/毫升糖原，和480微升95％乙醇。拌匀，孵育4°C为30分钟，离心30分钟14,000 XG在4°C。 </li><li>重悬在100微升去离子甲酰胺，加热至65℃至少30分钟，然后剧烈振荡10分钟沉淀的cDNA。 </li></ol> 5。毛细管电泳反应产物经分馏毛细管电泳同时允许从四个反应汇集成一个单一的样本分离，cDNA合成产品。八个样本同时进行分馏，而多达96个样品可以在一个单一的运行（ 图2）进行分馏。 <ol><li>混合汇集形状 ​​样品40微升10微升的汇集测序梯子，并转移到96孔样品板。 注意：当务之急是贝克曼试剂板（包括的LPA-I凝胶，运行缓冲液，矿物油，样品加载溶液和样品和缓冲液板）一起使用的Beckman-Coulter公司CEQ 8000遗传分析仪。 </li><li>计划和准备毛细管电泳仪，并启动运行，按照制造商的说明。 注：对于样品的最佳分辨率，使用先前公布的中央美院方法参数28。 </li></ol>在理想的情况下，外引物和强一站式峰，信号的每个峰在所有四个电泳吨种族应该在线性范围内，逐步减少关闭信号是可以接受的。然而，有时大峰（站）是显而易见的，即使在控制反应，这些可能会干扰后续的数据处理。这些峰产生的截短的cDNA，可以是反转录过程中（ 例如，RNA二级结构），或RNA降解的一种天然障碍的结果。在前者的情况下，可能会改善的添加剂，例如甜菜碱RT的进行性降低RT暂停/过早终止。 数据处理 ShapeFinder软件允许用户可视化和改造CE痕迹，并将其转换成SHAPE反应谱18。一旦反应值列，他们被归进口入RNAStructure（V5.3）的生成和完善二级结构模型。 6。 ShapeFinder软件跟踪处理平台的延伸BaseFinder29的形式，出版版本的ShapeFinder是自由可用于非商业用途18。详细说明数据处理ShapeFinder软件文档。 <ol><li>电泳进口CEQ ShapeFinder，在那里他们被调整，以纠正有关（i）荧光背景，（二），（三）流动性的变化赋予不同标记的引物，荧光通道之间的光谱重叠（四）荧光强度差异常见的产品用不同的荧光标记，反转录过早终止（五）导致信号衰减。 </li><li> “设置”自动分配功能的“调整与整合”工具ShapeFinder个别峰的身份和相关的RNA序列所定义的用户输入和两个测序梯子。虽然初始分配一般都是不完善的，错误可以得到纠正使用“修改”功能手动相同的工具。最后，“飞度”功能，计算领域的下对齐（+）和（ - ）反应峰，伴随着相应的核苷酸制表符分隔的文本文件，并列出这些反应值。 </li></ol> 注意：分析的数据是关键的准确性形状，和一些考虑因素是非常重要的，在此分析中，包括： <ul><li>信号 - 噪声：该signal-to-noise比是这样的，各个峰应该是很容易识别的，即使为低反应性的位置。虽然ShapeFinder提供了数据平滑选项，这种替代应该使用非常谨慎，因为它可以歪斜后续分析。 </li><li>的国家或地区的分析：通常情况下，可以可靠的数据获得的cDNA的300〜600个核苷酸长，从引物3&#39;末端40-80个核苷酸的区域处开始和结束的信号衰减的水平很难从背景噪声中区分。使用多辉底漆集将需要分析绵延较长的RNA。在这种情况下，它是建议取值范围为30-50核苷酸引物组之间的可靠的信号重叠。在较短的RNA，逆转录经常到达终点的RNA模板，必须谨慎采取排除那些峰信噪比受DNA合成强停止。 </li><li>信号衰减：信号衰减有关的核糖核酸修饰的程度，以及在实验过程中的不完善的进行性的RT。在理想的情况下，单命中动力学相对于所分析的RNA的区域应该达到的，以便最大限度地读取长度。 Shapefinder包含一个工具，是有效校正信号衰减的，然而，因为这往往会引入到分析错误-尤其是当没有观察到单发命中动力学，它是最好的时使用的信号衰减最小（ 即分布峰是一致的单发命中母牛抽动）。最近转化信号，信号衰减，提高了算法已出版了30，并应进行调查，如果信号衰减特别关注在一个特定的实验。 </li><li>信号缩放。可以说是最任意的形状数据处理步骤，控制配置文件应缩放峰强度之间的最低限度的反应（+）和（ - ）的痕迹，都是平等的。缩放控制跟踪太大的程度，将导致大量的负反应值在第一四分位数（见下文数据标准化）。在这种情况下，应相应地减少的比例因子，重新集成的数据。 </li><li>峰分配。在一般情况下，峰值指定的自动化版本的效果很好。然而，当该过程将失败，用户必须确保所有的峰已确认由软件实现，特别是当该信号对噪声的比率低。肩宽峰，例如，并不总是检测，和富G-SEquences经常被压缩。 </li></ul> 7。数据标准化核苷酸的反应性的档案纳入到由RNAStructure（v5.3的）软件使用的算法的二级结构，和/或密切相关的RNA的档案进行比较，形状数据必须归一化在一个标准化方式12。这包括（一）所有的反应值除以不包括后续计算的离群，（二）确定的“有效的最大”的反应（ 即平均反应值最高的8％，野值剔除），及（iii）正常化“有效的最大”，如下所示： <ol><li>打开对齐和整合后产生制表符分隔的文本文件，将其内容复制到一个Excel电子表格。最右边的列的文件（RX.area BG.area）的包含所计算出的绝对的形状，对每个碱基的RNA的反应性价值。最左边的列涉及的RNA SEQU的反应高效。 </li><li>计算和存储第一和三分之二的四分位数（ 即第25和75百分位）值（RX.area BG.area）使用Excel函数“=四分位数（ 数组 ， 夸脱 ）” </li><li>计算和存储的四分位数间距的差异“= QUARTILE（ 阵列 ，3）-QUARTILE（ 阵列 ，1）” </li><li>计算和存储“离群值的临界值”使用公式“=（四分位数， 数组 ，3）+1.5 *（（四分位数（ 数组 ，3）四分位数（ 阵列 ，1））”所有的反应值大于该值被排除在随后的计算。 </li><li>的反应值（RX.area BG.area）复制并粘贴到相邻的空列，然后这些值进行排序，最大的是在列的顶部。 </li><li>在新创建的“排序值列”，删除值大于的离群截止值。 </li><li>计算和存储的平均最大8％的反应性剩余值中的“排序的值双子星MN“这个值是”有效的最大“反应。 </li><li> “最大有效”反应值划分的无序（RX​​.area BG.area）的每个核苷酸（包括异常值）获得“标准化反应值”。这些资料储存在一个空列，在左边留下一个空列。然后，在左边的表中复制的核苷酸号码，并将其粘贴到空列直接到左边的“归一化反应值”。 </li><li>核苷酸位置归一化反应值对复制并粘贴到一个文本编辑器。 </li><li>消除值低于-0.09（ 即 ，留下空白的空格），因为这些都是有可能的结果RT暂停期间cDNA合成化学修饰的模板以外的原因。此外，任何反应性值核苷酸强暂停上观察到的未经修改的模板（所确定的“对齐和集成的”ShapeFinder档案中通过目测），应排除在外。 </li><li>萨VE文件使用结构分析与RNAstructure（V5.3）软件。形状延长。 </li></ol> 8。数据建模 RNAstructure（V5.3）软件被用来，预测实验支持的RNA二级结构（S）使用伪自由能约束来自形状分析19。该软件提供了图形表示的最低能量2D RNA结构，以及这些结构点支架符号文字表述。后者可以被导入的RNA结构产生出版物品质的图像查看器，用户的偏好， 例如 Pseudoviewer 23或22瓦尔纳。 注：考虑由RNAstructure（V5.3）软件结构时，必须小心。例如，该软件可以无法解决三级相互作用，如假结和亲吻循环，也不能区分是否缺乏在目标区域中的反应性是由于所结合的蛋白质的碱基配对或空间位阻保护。作为一个结果，这些因素，以及报告个别结构的能量，必须考虑出现一个明确的结构模型。

<ol>
	<li>Scott, W. G., Martick, M., Chi, Y. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+and+function+of+regulatory+RNA+elements:+ribozymes+that+regulate+gene+expression.">Structure and function of regulatory RNA elements: ribozymes that regulate gene expression.</a> Biochim. Biophys. Acta. 1789, 634-641 (2009).</li><li>Moore, P. B., Steitz, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+roles+of+RNA+in+the+synthesis+of+protein.">The roles of RNA in the synthesis of protein.</a> Cold Spring Harb. Perspect. Biol. 3, a003780 (2011).</li><li>Wilkinson, K. 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=High-throughput+SHAPE+analysis+reveals+structures+in+HIV-1+genomic+RNA+strongly+conserved+across+distinct+biological+states.">High-throughput SHAPE analysis reveals structures in HIV-1 genomic RNA strongly conserved across distinct biological states.</a> Plos Biol. 6, 883-899 (2008).</li><li>Merino, E. J., Wilkinson, K. A., Coughlan, J. L., Weeks, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA+structure+analysis+at+single+nucleotide+resolution+by+selective+2+'-hydroxyl+acylation+and+primer+extension+(SHAPE).">RNA structure analysis at single nucleotide resolution by selective 2 '-hydroxyl acylation and primer extension (SHAPE).</a> J. Am. Chem. Soc. 127, 4223-4231 (2005).</li><li>Watts, J. M., 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=Architecture+and+secondary+structure+of+an+entire+HIV-1+RNA+genome.">Architecture and secondary structure of an entire HIV-1 RNA genome.</a> Nature. 460, 711-716 (2009).</li><li>Xu, W., Bolduc, F., Hong, N., Perreault, 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=The+use+of+a+combination+of+computer-assisted+structure+prediction+and+SHAPE+probing+to+elucidate+the+secondary+structures+of+five+viroids.">The use of a combination of computer-assisted structure prediction and SHAPE probing to elucidate the secondary structures of five viroids.</a> Mol. Plant Pathol. , (2012).</li><li>Novikova, I. V., Hennelly, S. P., Sanbonmatsu, K. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+architecture+of+the+human+long+non-coding+RNA,+steroid+receptor+RNA+activator.">Structural architecture of the human long non-coding RNA, steroid receptor RNA activator.</a> Nucleic Acids Res. 40, 5034-5051 (2012).</li><li>Leshin, J. A., Heselpoth, R., Belew, A. T., Dinman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+structural+analysis+of+yeast+ribosomes+using+hSHAPE.">High-throughput structural analysis of yeast ribosomes using hSHAPE.</a> RNA Biol. 8, 478-487 (2011).</li><li>Souliere, M. F., Haller, A., Rieder, R., Micura, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+powerful+approach+for+the+selection+of+2-aminopurine+substitution+sites+to+investigate+RNA+folding.">A powerful approach for the selection of 2-aminopurine substitution sites to investigate RNA folding.</a> J. Am. Chem. Soc. 133, 16161-16167 (2011).</li><li>Wilkinson, K. A., Merino, E. J., Weeks, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+2+'-hydroxyl+acylation+analyzed+by+primer+extension+(SHAPE):+quantitative+RNA+structure+analysis+at+single+nucleotide+resolution.">Selective 2 '-hydroxyl acylation analyzed by primer extension (SHAPE): quantitative RNA structure analysis at single nucleotide resolution.</a> Nat. Protoc. 1, 1610-1616 (2006).</li><li>McGinnis, J. L., Duncan, C. D. S., Weeks, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-Throughput+Shape+and+Hydroxyl+Radical+Analysis+of+Rna+Structure+and+Ribonucleoprotein+Assembly.">High-Throughput Shape and Hydroxyl Radical Analysis of Rna Structure and Ribonucleoprotein Assembly.</a> Method Enzymol. 468, 67-89 (2009).</li><li>Low, J. T., Weeks, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SHAPE-directed+RNA+secondary+structure+prediction.">SHAPE-directed RNA secondary structure prediction.</a> Methods. 52, 150-158 (2010).</li><li>Das, R., Laederach, A., Pearlman, S. M., Herschlag, D., Altman, R. B. S. A. F. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Semi-automated+footprinting+analysis+software+for+high-throughput+quantification+of+nucleic+acid+footprinting+experiments.">Semi-automated footprinting analysis software for high-throughput quantification of nucleic acid footprinting experiments.</a> Rna-a Publication of the Rna Society. 11, 344-354 (2005).</li><li>Kertesz, M., 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=Genome-wide+measurement+of+RNA+secondary+structure+in+yeast.">Genome-wide measurement of RNA secondary structure in yeast.</a> Nature. 467, 103-107 (2010).</li><li>Underwood, J. G., 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=FragSeq:+transcriptome-wide+RNA+structure+probing+using+high-throughput+sequencing.">FragSeq: transcriptome-wide RNA structure probing using high-throughput sequencing.</a> Nat. Methods. 7, 995-1001 (2010).</li><li>Mauger, D. M., Weeks, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toward+global+RNA+structure+analysis.">Toward global RNA structure analysis.</a> Nat. Biotechnol. 28, 1178-1179 (2010).</li><li>Lucks, 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=Multiplexed+RNA+structure+characterization+with+selective+2'-hydroxyl+acylation+analyzed+by+primer+extension+sequencing+(SHAPE-Seq).">Multiplexed RNA structure characterization with selective 2'-hydroxyl acylation analyzed by primer extension sequencing (SHAPE-Seq).</a> Proc. Natl. Acad. Sci. USA. 108, 11063-11068 (2011).</li><li>Vasa, S. M., Guex, N., Wilkinson, K. A., Weeks, K. M., Giddings, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ShapeFinder:+a+software+system+for+high-throughput+quantitative+analysis+of+nucleic+acid+reactivity+information+resolved+by+capillary+electrophoresis.">ShapeFinder: a software system for high-throughput quantitative analysis of nucleic acid reactivity information resolved by capillary electrophoresis.</a> RNA. 14, 1979-1990 (2008).</li><li>Reuter, J. S., Mathews, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNAstructure:+software+for+RNA+secondary+structure+prediction+and+analysis.">RNAstructure: software for RNA secondary structure prediction and analysis.</a> BMC Bioinformatics. 11, 129 (2010).</li><li>Pang, P. S., Elazar, M., Pham, E. A., Glenn, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simplified+RNA+secondary+structure+mapping+by+automation+of+SHAPE+data+analysis.">Simplified RNA secondary structure mapping by automation of SHAPE data analysis.</a> Nucleic Acids Res. 39, e151 (2011).</li><li>Deigan, K. E., Li, T. W., Mathews, D. H., Weeks, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accurate+SHAPE-directed+RNA+structure+determination.">Accurate SHAPE-directed RNA structure determination.</a> Proc. Natl. Acad. Sci. USA. 106, 97-102 (2009).</li><li>Darty, K., Denise, A., Ponty, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=VARNA:+Interactive+drawing+and+editing+of+the+RNA+secondary+structure.">VARNA: Interactive drawing and editing of the RNA secondary structure.</a> Bioinformatics. 25, 1974-1975 (2009).</li><li>Byun, Y., Han, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PseudoViewer:+web+application+and+web+service+for+visualizing+RNA+pseudoknots+and+secondary+structures.">PseudoViewer: web application and web service for visualizing RNA pseudoknots and secondary structures.</a> Nucleic Acids Res. 34, 416-422 (2006).</li><li>Brown, T., Brown, D. J. S., Eckstein, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Oligonucleotides and Analogues - A Practical Approach. , 20 (1990).</li><li>Legiewicz, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+RNA+Transport+Element+of+the+Murine+musD+Retrotransposon+Requires+Long-range+Intramolecular+Interactions+for+Function.">The RNA Transport Element of the Murine musD Retrotransposon Requires Long-range Intramolecular Interactions for Function.</a> J. Biol. Chem. 285, 42097-42104 (2010).</li><li>Steen, K., Siegfried, N. A., Weeks, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Syntheis+of+1-methyl-8-nitroisatoic+anhydride+(1M7).">Syntheis of 1-methyl-8-nitroisatoic anhydride (1M7).</a> Protocol Exchange. , (2011).</li><li>Mortimer, S. A., Weeks, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+fast-acting+reagent+for+accurate+analysis+of+RNA+secondary+and+tertiary+structure+by+SHAPE+chemistry.">A fast-acting reagent for accurate analysis of RNA secondary and tertiary structure by SHAPE chemistry.</a> J. Am. Chem. Soc. 129, 4144-4145 (2007).</li><li>Mitra, S., Shcherbakova, I. V., Altman, R. B., Brenowitz, M., Laederach, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+single-nucleotide+structural+mapping+by+capillary+automated+footprinting+analysis.">High-throughput single-nucleotide structural mapping by capillary automated footprinting analysis.</a> Nucleic Acids Res. 36, e63 (2008).</li><li>Giddings, M. C., Severin, J., Westphall, M., Wu, J., Smith, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+software+system+for+data+analysis+in+automated+DNA+sequencing.">A software system for data analysis in automated DNA sequencing.</a> Genome Res. 8, 644-665 (1998).</li><li>Aviran, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modeling+and+automation+of+sequencing-based+characterization+of+RNA+structure.">Modeling and automation of sequencing-based characterization of RNA structure.</a> Proc. Natl. Acad. Sci. USA. 108, 11069-11074 (2011).</li></ol>

没有利益冲突的声明。

我们在座的详细协议为高吞吐量形状，一种技术，使任何规模的RNA的单核苷酸分辨率为二级结构的测定。此外，耦合形状实验数据与二级结构预测算法有利于RNA二维模型生成具有更高的准确度可能比单独的任一方法。荧光标记的引物和自动CE的结合，提供了显着优于传统的基于凝胶的形状在一次实验中，有利于解决长的RNA序列，以及相当高的速度和通过多个实验。权宜之计此方法，并提供合适的?...

要了解催化和非编码RNA的功能，参与调控拼接，翻译，病毒的复制和癌症，RNA结构的详细知识是必需的1,2。不幸的是，准确预测RNA折叠提出了一个严峻的挑战。古典探测剂遭受许多缺点，如毒性，不完整覆盖核苷酸和/或每100-150个核苷酸实验吞吐量有限。肉眼二级结构预测算法同样是不利的，因为不准确，致使他们无法有效区分大力相似的结构。特别大的RNA也常耐火材料，如X-射线晶体学和核磁共振（NMR）谱，由于它们的构象灵活性和大量的这些技术所需的高纯度的样品的三维结构的测定方法。 ħIGH-吞吐量形状可以解决许多这些问题提供一个有效的，简单的方法来探测的大RNA的结构，单核苷酸分辨率。此外，该试剂用于形状是安全的，易于处理，在大多数其他化学探测试剂相比，所有四个核糖反应。这些试剂也可以穿透细胞膜，使得有可能探测的RNA在其体内上下文（次）3。最初4周实验室开发的，形状已经用于分析各种各样的RNA〜9 KB HIV-1 RNA基因组的完整的二级结构的测定，最显着的例子。其他显着的成果，使用形状感染类病毒6，人长非编码RNA 7，酵母核糖8，9和核糖开关以及确定蛋白质结合位点的病毒体相关的HIV-1 RNA的结构，包括澄清。瓦其他地方10-12法兰西岛大区的原件和高通量的变化形状协议已经公布，目前的工作提供了详细的说明，通过高通量的RNA二级结构的测定采用荧光寡核苷酸，贝克曼CEQ 8000遗传分析仪， SHAPEfinder RNAStructure（V5.3）软件。先前未公开的技术细节和故障排除建议也包括在内。 形状变化的本质的外型及其变化是暴露的RNA在水溶液中电酸酐选择性酰化2&#39;-羟基（2&#39;-OH）的核糖基生产笨重的加成物在网站修改。这种化学反应作为一种手段，询问当地的RNA结构动力学，单链核苷酸更容易采取有利于电攻击这些试剂的构象，而碱基配对或建筑构造ained核苷酸少或不反应10。加合物的形成的位点被检测到通过反转录启动从荧光或放射性标记的引物杂交到特定站点上的修饰的RNA（“（+）”引物延伸反应）。当逆转录酶（RT）失败遍历酰化核糖核苷酸cDNA的产品生产，游泳池，其长度，配合网站修改。一控“（ - ）”引扩展利用RNA尚未暴露试剂的反应也表现（ 即 “停止”）由于DNA合成RNA结构，非特异性的RNA链断裂， 等等 ，可提前终止通过化学修饰暂时停止生产加以区分。最后，两个二脱氧测序反应，启动从相同的引物被用来作为标记，电泳后的RNA一级序列相关联反应的核苷酸。 在原来的应用SHAPE，在相同的32个P-末端标记的引物被用于（+），（ - ），和两个测序反应。这些反应的产品装入相邻的井，在5％至8％的聚丙烯酰胺平板凝胶，并通过变性聚丙烯酰胺凝胶电泳（PAGE， 图1）分馏。可以执行常规形状所产生的凝胶图像的定量分析，使用SAFA，半自动化的足迹分析软件13。 与此相反，高通量的形状采用荧光标记的引物和自动化的毛细管电泳。具体而言，对每个区域的RNA的调查中，一组四个的DNA引物，具有一个共同的序列，但不同的5&#39;端荧光标记必须被合成或购买。这些不同的标记的寡核苷酸素两个形状的反应和两个测序反应，汇集和分离/检测自动毛细管电泳（CE）产品服务。到哪里去EAS 100-150个核苷酸的RNA的反应档案可从一组四个反应，使用原始的方法，高通量的形状允许从单一样本3的分辨率为300-600个核苷酸。最多8台反应同时进行分馏，而多达96个样品可以在连续12个CE运行（ 图2）的过程中分馏制备。此外，的SHAPEfinder软件，开发新兴CEQ和其他遗传分析仪的数据处理和分析，更加自动化，需要用户干预的少得多，比SAFA 13或其他凝胶分析软件包。 最近出现了更先进的高通量方法如PARS（平行的RNA结构分析）14和片段序列（片段测序）15，它使用结构特异性的酶，而不是配合新一代测序技术获得烷基化试剂的资n关于RNA的结构。尽管这些技术的吸引力，核酸探测的许多固有的局限性仍然16。这些问题是可以规避在SHAPE的测序（形状序列）17的协议，进行常规形状类似的方式通过化学修饰的RNA的反转录前面下一代测序。虽然这些方法可能代表了未来的RNA结构测定，重要的是要记住，下一代测序是非常昂贵的，并且仍然无法许多实验室。 形状数据分析在遗传分析仪产生的数据的形式的电泳，其特征在于，所述的样品的荧光强度（次）流经毛细管探测器的对一个索引的迁移时间作图。此图的形式重叠痕迹对应于四个荧光通道用来在不同检测不同的荧光基团，其中每个轨迹是由对应于个体的cDNA或测序反应产物的峰。电泳数据是从遗传分析仪为制表符分隔的文本文件导出和导入ShapeFinder转换和分析软件18。 ShapeFinder最初用于执行一系列的数据的数学变换，以确保准确地反映迁移时间和峰卷的身份和数量的反应产物分别。峰，然后对准和集成，并与主要的RNA序列的结果列在一起。减去控制值的（+）的值与每个RNA核苷酸，和归一化数据（如下文所述）得到的RNA相关的段A“反应性配置文件”。此配置文件导入到软件19,20 RNAstructure（V5.3），归一化反应的val转换UES成伪能量纳入RNA二级结构折叠算法的约束。配合化学探测和折叠的算法以这种方式，显着地提高了单独12,21的任一方法相比，结构预测的准确度。输出的RNAstructure（V5.3）包括最低能量的图像颜色编码的形状反应曲线（S），以及文字圆点括号表示法中的相同结构的RNA二级结构。随后，后者可被导出RNA二级结构（如瓦尔纳22 PseudoViewer 23）的图形显示的专用软件。 <img alt="图1" fo:content-width="4.8in" fo:src="/files/ftp_upload/50243/50243fig1highres.jpg" src="/files/ftp_upload/50243/50243fig1.jpg" /> 图1。 （A）+ RNA米流程图形状4,10 RNA结构测定通过。获得（B）根据讯号源，RNA折叠或以其他方式处理和修改形状试剂（C）反转录使用荧光或放射性标记的引物（D）cDNA产物是从生物样品或通过体外转录。分馏通过毛细管或平板凝胶电泳（E）片段分析（F）RNA结构预测<a href="http://www.jove.com/files/ftp_upload/50243/50243fig1large.jpg" target="_blank">点击这里查看大图。</a> <img alt="图2" fo:content-width="6in" fo:src="/files/ftp_upload/50243/50243fig2highres.jpg" src="/files/ftp_upload/50243/50243fig2.jpg" /> 图2。 （A）基于CE的形状允许高吞吐量的字符的多个RNA的快速分析，和/或相同的RNA的多个段。 </st荣&gt;表示的RNA可以被划分为300-600核苷酸部分（编码的颜色为绿色，蓝色和红色），（B）的被探测的RNA部分独立地使用不同组的荧光引物（黑色箭头）（C）设置反应池井A1，B1，C1 等 ，分别装入，提供完整的覆盖〜3 kb的基因组RNA1。的RNA 2，3，4，等反应产物可能会同样分馏连续电泳奔跑的准备。 <a href="http://www.jove.com/files/ftp_upload/50243/50243fig2large.jpg" target="_blank">点击这里查看大图。</a>

<table border="1">
	<tbody>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>REAGENTS</td>
		</tr>
		<tr>
			<td>N-methylisatoic anhydride (NMIA)</td>
			<td>Life technologies</td>
			<td>M25</td>
			<td>Dissolve in anhydrous DMSO</td>
		</tr>
		<tr>
			<td>1-methyl-t-nitroisatoic anhydride (1M7)</td>
			<td>see ref. 22</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Superscript III Reverse Transcriptase</td>
			<td>Life technologies</td>
			<td>18080044</td>
			<td>10,000 units</td>
		</tr>
		<tr>
			<td>Thermo sequenase cycle sequencing kit</td>
			<td>Affymetrix</td>
			<td>78500</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Materials provided by the user</td>
		</tr>
		<tr>
			<td>RNA of interest</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>6 pmol per reaction (the limit of detection will be determined by the instrument)</td>
		</tr>
		<tr>
			<td>Sets of four 5' labeled primers (Cy5, Cy5.5, WellRed D2 and WellRed D1/Licor IR800)</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Primers are complementary to the RNA and are used in reverse transcription and sequencing reactions. The listed fluorophores are optimal for the Beckman Coulter 8000 CEQ. Primers may be purchased or synthesized in house.</td>
		</tr>
		<tr>
			<td>DNA template</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>DNA is used for sequencing reactions, and must contain the sequence of the RNA being studied - including any 3'terminal extension, if present. Where applicable, it is often convenient to use the RNA transcription template.</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Buffers</td>
		</tr>
		<tr>
			<td>10x RNA renaturation buffer</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>100 mM Tris-HCl pH 8.0, 1 M KCl, 1 mM EDTA</td>
		</tr>
		<tr>
			<td>5X RNA folding buffer</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>200 mM Tris-HCl pH 8.0, 25 mM MgCl2, 2.5 mM EDTA, 650 mM KCl. (This buffer might be changed depending on the case (e.g. pH, EDTA, Mg, RNase inhibitor)</td>
		</tr>
		<tr>
			<td>2.5X RT mix</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>4 &mu;l 5X buffer, 1 &mu;l 100 mM DTT, 1.5 &mu;l water,1 &mu;l 10 mM dNTPs, 0.5 &mu;l SuperScript III. Note that the 5X buffer and 100 mM DTT are provided with purchase of SuperScript III (Invitrogen).</td>
		</tr>
		<tr>
			<td>GenomeLab Sample Loading Solution (Beckman Coulter)</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Attention: Avoid multiple freeze-thaw cycles</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>EQUIPMENT</td>
		</tr>
		<tr>
			<td>Capillary electrophoresis</td>
			<td>Beckman</td>
			<td>CEQ8000</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Thermocycler</td>
			<td>varies</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

rna secondary structure prediction using high-throughput shape

了解RNA的功能涉及生物过程，需要全面了解的RNA结构。为此，该方法被称为“引物延伸”，或形状分析的高通量选择性的2&#39;羟基的酰化，允许预测RNA二级结构的单核苷酸分辨率。这种方法利用化学探测剂，优先酰化的RNA在水溶液中的单链或柔性区域。化学修饰的位点被检测到的修饰的RNA的反转录，并在此反应中的产品是通过自动毛细管电泳（CE）分馏。由于修改后由SHAPE试剂在这些RNA的核苷酸逆转录酶暂停，由此产生的cDNA文库间接映射的上下文中的折叠的RNA的核糖核苷酸的单链。使用的电泳软件ShapeFinder，产生的自动化CE的处理和转换成菜单核苷酸反应表转换成伪能量约束在RNAStructure（V5.3）预测算法。二维形状探测在硅片 RNA二级结构预测相结合所获得的RNA结构被认为是比单独使用这两种方法得到的结构更准确。

RNA含有HIV-1转应答元件（RRE）和一个3&#39;端结构的盒4从线性化的质粒进行体外转录，之后，将其折叠通过加热，冷却，和孵育在37℃下，在存在的MgCl 2。 RNA的暴露NMIA，然后从5&#39;-末端标记的DNA杂交的引物的3&#39;末端结构的磁带盒反转录。由此产生的形状的cDNA文库，连同控制和定序的反应，然后分馏使用Beckman Coulter公司CEQ 8000自动毛细管电泳系统，以产生图4中...

Watch this Scientific Journal Video about RNA Secondary Structure Prediction Using High-throughput SHAPE at JoVE.com

RNA Secondary Structure Prediction Using High-throughput SHAPE

的高通量选择性2&#39;羟基的酰化反应分析的引物延伸（SHAPE）利用了一种新的化学探测技术，反转录，毛细管电泳确定结构的RNA二级结构预测软件从几百到几千个核苷酸，单核苷酸分辨率。

使用高通量形状的RNA二级结构预测

Understanding the function of RNA involved in biological processes requires a thorough knowledge of RNA structure. Toward this end, the methodology dubbed ...

rna-secondary-structure-prediction-using-high-throughput-shape

Research

Education

JoVE Journal

JoVE Core

Molecular Biology

Biology

Unit 1

Transcription: DNA to RNA

SCIENTISTS IN ACTION

31.2K 次观看.  Frederick National Laboratory for Cancer Research. 的高通量选择性2'羟基的酰化反应分析的引物延伸（SHAPE）利用了一种新的化学探测技术，反转录，毛细管电泳确定结构的RNA二级结构预测软件从几百到几千个核苷酸，单核苷酸分辨率。

视频: RNA Secondary Structure Prediction Using High-throughput SHAPE

High-throughput Yeast Plasmid Overexpression Screen

The budding yeast, Saccharomyces cerevisiae, is a powerful model system for defining fundamental mechanisms of many important cellular processes, including those with direct relevance to human disease. Because of its short generation time and well-characterized genome, a major experimental advantage of the yeast model system is the ability to perform genetic screens to identify genes and pathways that are involved in a given process. Over the last thirty years such genetic screens have been used to elucidate the cell cycle, secretory pathway, and many more highly conserved aspects of eukaryotic cell biology 1-5. In the last few years, several genomewide libraries of yeast strains and plasmids have been generated 6-10. These collections now allow for the systematic interrogation of gene function using gain- and loss-of-function approaches 11-16. Here we provide a detailed protocol for the use of a high-throughput yeast transformation protocol with a liquid handling robot to perform a plasmid overexpression screen, using an arrayed library of 5,500 yeast plasmids. We have been using these screens to identify genetic modifiers of toxicity associated with the accumulation of aggregation-prone human neurodegenerative disease proteins. The methods presented here are readily adaptable to the study of other cellular phenotypes of interest.

Here we describe a plasmid overexpression screen in Saccharomyces cerevisiae, using an arrayed plasmid library and a high-throughput yeast transformation protocol with a liquid handling robot.

高通量酵母质粒过表达屏幕

The budding yeast, Saccharomyces cerevisiae, is a powerful model system for defining fundamental mechanisms of many important cellular processes, ...

科研

生物学

A Protocol for Computer-Based Protein Structure and Function Prediction

Genome sequencing projects have ciphered millions of protein sequence, which require knowledge of their structure and function to improve the understanding of their biological role. Although experimental methods can provide detailed information for a small fraction of these proteins, computational modeling is needed for the majority of protein molecules which are experimentally uncharacterized. The I-TASSER server is an on-line workbench for high-resolution modeling of protein structure and function. Given a protein sequence, a typical output from the I-TASSER server includes secondary structure prediction, predicted solvent accessibility of each residue, homologous template proteins detected by threading and structure alignments, up to five full-length tertiary structural models, and structure-based functional annotations for enzyme classification, Gene Ontology terms and protein-ligand binding sites. All the predictions are tagged with a confidence score which tells how accurate the predictions are without knowing the experimental data. To facilitate the special requests of end users, the server provides channels to accept user-specified inter-residue distance and contact maps to interactively change the I-TASSER modeling; it also allows users to specify any proteins as template, or to exclude any template proteins during the structure assembly simulations. The structural information could be collected by the users based on experimental evidences or biological insights with the purpose of improving the quality of I-TASSER predictions. The server was evaluated as the best programs for protein structure and function predictions in the recent community-wide CASP experiments. There are currently &gt;20,000 registered scientists from over 100 countries who are using the on-line I-TASSER server.

Guidelines for computer based structural and functional characterization of protein using the I-TASSER pipeline is described. Starting from query protein sequence, 3D models are generated using multiple threading alignments and iterative structural assembly simulations. Functional inferences are thereafter drawn based on matches to proteins with known structure and functions.

基于计算机的蛋白质结构与功能预测议定书

Genome sequencing projects have ciphered millions of protein sequence, which require knowledge of their structure and function to improve the ...

High-throughput Crystallization of Membrane Proteins Using the Lipidic Bicelle Method

Membrane proteins (MPs) play a critical role in many physiological processes such as pumping specific molecules across the otherwise impermeable membrane bilayer that surrounds all cells and organelles. Alterations in the function of MPs result in many human diseases and disorders; thus, an intricate understanding of their structures remains a critical objective for biological research. However, structure determination of MPs remains a significant challenge often stemming from their hydrophobicity. 
MPs have substantial hydrophobic regions embedded within the bilayer. Detergents are frequently used to solubilize these proteins from the bilayer generating a protein-detergent micelle that can then be manipulated in a similar manner as soluble proteins. Traditionally, crystallization trials proceed using a protein-detergent mixture, but they often resist crystallization or produce crystals of poor quality. These problems arise due to the detergent&prime;s inability to adequately mimic the bilayer resulting in poor stability and heterogeneity. In addition, the detergent shields the hydrophobic surface of the MP reducing the surface area available for crystal contacts. To circumvent these drawbacks MPs can be crystallized in lipidic media, which more closely simulates their endogenous environment, and has recently become a de novo technique for MP crystallization.
Lipidic cubic phase (LCP) is a three-dimensional lipid bilayer penetrated by an interconnected system of aqueous channels1. Although monoolein is the lipid of choice, related lipids such as monopalmitolein and monovaccenin have also been used to make LCP2. MPs are incorporated into the LCP where they diffuse in three dimensions and feed crystal nuclei. A great advantage of the LCP is that the protein remains in a more native environment, but the method has a number of technical disadvantages including high viscosity (requiring specialized apparatuses) and difficulties in crystal visualization and manipulation3,4. Because of these technical difficulties, we utilized another lipidic medium for crystallization-bicelles5,6 (Figure 1). Bicelles are lipid/amphiphile mixtures formed by blending a phosphatidylcholine lipid (DMPC) with an amphiphile (CHAPSO) or a short-chain lipid (DHPC). Within each bicelle disc, the lipid molecules generate a bilayer while the amphiphile molecules line the apolar edges providing beneficial properties of both bilayers and detergents. Importantly, below their transition temperature, protein-bicelle mixtures have a reduced viscosity and are manipulated in a similar manner as detergent-solubilized MPs, making bicelles compatible with crystallization robots. 
Bicelles have been successfully used to crystallize several membrane proteins5,7-11 (Table 1). This growing collection of proteins demonstrates the versatility of bicelles for crystallizing both alpha helical and beta sheet MPs from prokaryotic and eukaryotic sources. Because of these successes and the simplicity of high-throughput implementation, bicelles should be part of every membrane protein crystallographer&prime;s arsenal. In this video, we describe the bicelle methodology and provide a step-by-step protocol for setting up high-throughput crystallization trials of purified MPs using standard robotics.

Bicelles are lipid/amphiphile mixtures that maintain membrane proteins (MPs) within a lipid bilayer but have unique phase behavior that facilitates high-throughput screening by crystallization robots. This technique has successfully produced a number of high-resolution structures from both prokaryotic and eukaryotic sources. This video describes protocols for generating the lipidic bicelle mixture, incorporating MPs into the bicelle mixture, setting up crystallizations trials (manually as well as robotically) and harvesting crystals from the medium.

使用油脂全线Bicelle方法的膜蛋白的高通量结晶

Membrane proteins (MPs) play a critical role in many physiological processes such as pumping specific molecules across the otherwise impermeable membrane ...

High-throughput Physical Mapping of Chromosomes using Automated in situ Hybridization

Projects to obtain whole-genome sequences for 10,000 vertebrate species1 and for 5,000 insect and related arthropod species2 are expected to take place over the next 5 years. For example, the sequencing of the genomes for 15 malaria mosquitospecies is currently being done using an Illumina platform3,4. This Anopheles species cluster includes both vectors and non-vectors of malaria. When the genome assemblies become available, researchers will have the unique opportunity to perform comparative analysis for inferring evolutionary changes relevant to vector ability. However, it has proven difficult to use next-generation sequencing reads to generate high-quality de novo genome assemblies5. Moreover, the existing genome assemblies for Anopheles gambiae, although obtained using the Sanger method, are gapped or fragmented4,6.
Success of comparative genomic analyses will be limited if researchers deal with numerous sequencing contigs, rather than with chromosome-based genome assemblies. Fragmented, unmapped sequences create problems for genomic analyses because: (i) unidentified gaps cause incorrect or incomplete annotation of genomic sequences; (ii) unmapped sequences lead to confusion between paralogous genes and genes from different haplotypes; and (iii) the lack of chromosome assignment and orientation of the sequencing contigs does not allow for reconstructing rearrangement phylogeny and studying chromosome evolution. Developing high-resolution physical maps for species with newly sequenced genomes is a timely and cost-effective investment that will facilitate genome annotation, evolutionary analysis, and re-sequencing of individual genomes from natural populations7,8.
Here, we present innovative approaches to chromosome preparation, fluorescent in situ hybridization (FISH), and imaging that facilitate rapid development of physical maps. Using An. gambiae as an example, we demonstrate that the development of physical chromosome maps can potentially improve genome assemblies and, thus, the quality of genomic analyses. First, we use a high-pressure method to prepare polytene chromosome spreads. This method, originally developed for Drosophila9, allows the user to visualize more details on chromosomes than the regular squashing technique10. Second, a fully automated, front-end system for FISH is used for high-throughput physical genome mapping. The automated slide staining system runs multiple assays simultaneously and dramatically reduces hands-on time11. Third, an automatic fluorescent imaging system, which includes a motorized slide stage, automatically scans and photographs labeled chromosomes after FISH12. This system is especially useful for identifying and visualizing multiple chromosomal plates on the same slide. In addition, the scanning process captures a more uniform FISH result. Overall, the automated high-throughput physical mapping protocol is more efficient than a standard manual protocol.

High-throughput Physical Mapping of Chromosomes using Automated in situ Hybridization

Genome assemblies based on massively parallel DNA sequencing technologies are usually highly fragmented. The development of physical chromosome maps can potentially improve genome assemblies. Here, we demonstrate innovative approaches to chromosome preparation, fluorescent in situ hybridization, and imaging that significantly increase throughput of the physical map development.

高通量使用自动染色体的物理图谱<em&gt;原位</em&gt;杂交

Projects to obtain whole-genome sequences for 10,000 vertebrate species1 and for 5,000 insect and related arthropod species2 are expected to take place ...

High-throughput Functional Screening using a Homemade Dual-glow Luciferase Assay

We present a rapid and inexpensive high-throughput screening protocol to identify transcriptional regulators of alpha-synuclein, a gene associated with Parkinson&#39;s disease. 293T cells are transiently transfected with plasmids from an arrayed ORF expression library, together with luciferase reporter plasmids, in a one-gene-per-well microplate format. Firefly luciferase activity is assayed after 48 hr to determine the effects of each library gene upon alpha-synuclein transcription, normalized to expression from an internal control construct (a hCMV promoter directing Renilla luciferase). This protocol is facilitated by a bench-top robot enclosed in a biosafety cabinet, which performs aseptic liquid handling in 96-well format. Our automated transfection protocol is readily adaptable to high-throughput lentiviral library production or other functional screening protocols requiring triple-transfections of large numbers of unique library plasmids in conjunction with a common set of helper plasmids. We also present an inexpensive and validated alternative to commercially-available, dual luciferase reagents which employs PTC124, EDTA, and pyrophosphate to suppress firefly luciferase activity prior to measurement of Renilla luciferase. Using these methods, we screened 7,670 human genes and identified 68 regulators of alpha-synuclein. This protocol is easily modifiable to target other genes of interest.

We present a rapid and inexpensive screening method for identifying transcriptional regulators using high-throughput robotic transfections and a homemade dual-glow luciferase assay. This protocol rapidly generates direct side-by-side functional data for thousands of genes and is easily modifiable to target any gene of interest.

使用自制双辉光萤光素酶检测的高通量筛选功能

We present a rapid and inexpensive high-throughput screening protocol to identify transcriptional regulators of alpha-synuclein, a gene associated with ...

A Manual Small Molecule Screen Approaching High-throughput Using Zebrafish Embryos

Zebrafish have become a widely used model organism to investigate the mechanisms that underlie developmental biology and to study human disease pathology due to their considerable degree of genetic conservation with humans. Chemical genetics entails testing the effect that small molecules have on a biological process and is becoming a popular translational research method to identify therapeutic compounds. Zebrafish are specifically appealing to use for chemical genetics because of their ability to produce large clutches of transparent embryos, which are externally fertilized. Furthermore, zebrafish embryos can be easily drug treated by the simple addition of a compound to the embryo media. Using whole-mount in situ hybridization (WISH), mRNA expression can be clearly visualized within zebrafish embryos. Together, using chemical genetics and WISH, the zebrafish becomes a potent whole organism context in which to determine the cellular and physiological effects of small molecules. Innovative advances have been made in technologies that utilize machine-based screening procedures, however for many labs such options are not accessible or remain cost-prohibitive. The protocol described here explains how to execute a manual high-throughput chemical genetic screen that requires basic resources and can be accomplished by a single individual or small team in an efficient period of time. Thus, this protocol provides a feasible strategy that can be implemented by research groups to perform chemical genetics in zebrafish, which can be useful for gaining fundamental insights into developmental processes, disease mechanisms, and to identify novel compounds and signaling pathways that have medically relevant applications.

The zebrafish is an excellent experimental organism to study vertebrate developmental processes and model human disease. Here, we describe a protocol on how to perform a manual high-throughput chemical screen in zebrafish embryos with a whole-mount in situ hybridization (WISH) read-out.

手册小分子屏幕接近高通量使用斑马鱼胚胎

Zebrafish have become a widely used model organism to investigate the mechanisms that underlie developmental biology and to study human disease pathology ...

Highly Efficient Ligation of Small RNA Molecules for MicroRNA Quantitation by High-Throughput Sequencing

MiRNA cloning and high-throughput sequencing, termed miR-Seq, stands alone as a transcriptome-wide approach to quantify miRNAs with single nucleotide resolution. This technique captures miRNAs by attaching 3&#8217; and 5&#8217; oligonucleotide adapters to miRNA molecules and allows de novo miRNA discovery. Coupling with powerful next-generation sequencing platforms, miR-Seq has been instrumental in the study of miRNA biology. However, significant biases introduced by oligonucleotide ligation steps have prevented miR-Seq from being employed as an accurate quantitation tool. Previous studies demonstrate that biases in current miR-Seq methods often lead to inaccurate miRNA quantification with errors up to 1,000-fold for some miRNAs1,2. To resolve these biases imparted by RNA ligation, we have developed a small RNA ligation method that results in ligation efficiencies of over 95% for both 3&#8217; and 5&#8242; ligation steps. Benchmarking this improved library construction method using equimolar or differentially mixed synthetic miRNAs, consistently yields reads numbers with less than two-fold deviation from the expected value. Furthermore, this high-efficiency miR-Seq method permits accurate genome-wide miRNA profiling from in vivo total RNA samples2.

MicroRNAs (miRNAs) are a widely conserved class of regulatory molecules. Here we describe a miRNA cloning method that relies upon two potent ligation steps followed by high-throughput sequencing. Our method permits accurate genome-wide quantitation of miRNAs.

小RNA分子的高效结扎的微小RNA定量高通量测序

MiRNA cloning and high-throughput sequencing, termed miR-Seq, stands alone as a transcriptome-wide approach to quantify miRNAs with single nucleotide ...

High-throughput Screening for Chemical Modulators of Post-transcriptionally Regulated Genes

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays focus on transcriptional regulation, being essentially aimed at the identification of promoter-targeting molecules. As a result, post-transcriptional mechanisms are largely uncovered by gene expression targeted drug development. Here we describe a cell-based assay aimed at investigating the role of the 3&#39; untranslated region (3&#8217; UTR) in the modulation of the fate of its mRNA, and at identifying compounds able to modify it. The assay is based on the use of a luciferase reporter construct containing the 3&#8217; UTR of a gene of interest stably integrated into a disease-relevant cell line. The protocol is divided into two parts, with the initial focus on the primary screening aimed at the identification of molecules affecting luciferase activity after 24 hr of treatment. The second part of the protocol describes the counter-screening necessary to discriminate compounds modulating luciferase activity specifically through the 3&#8217; UTR. In addition to the detailed protocol and representative results, we provide important considerations about the assay development and the validation of the hit(s) on the endogenous target. The described cell-based reporter gene assay will allow scientists to identify molecules modulating protein levels via post-transcriptional mechanisms dependent on a 3&#8217; UTR.

Here we describe a cell-based reporter gene assay as a valuable tool to screen chemical libraries for compounds modulating post-transcriptional control mechanisms exerted through 3&#8217; UTR.

高通量筛选转录后调控基因的化学调节剂

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays ...

Detection of miRNA Targets in High-throughput Using the 3'LIFE Assay

Luminescent Identification of Functional Elements in 3&#8217;UTRs (3&#8217;LIFE) allows the rapid identification of targets of specific miRNAs within an array of hundreds of queried 3&#8217;UTRs. Target identification is based on the dual-luciferase assay, which detects binding at the mRNA level by measuring translational output, giving a functional readout of miRNA targeting. 3&#8217;LIFE uses non-proprietary buffers and reagents, and publically available reporter libraries, making genome-wide screens feasible and cost-effective. 3&#8217;LIFE can be performed either in a standard lab setting or scaled up using liquid handling robots and other high-throughput instrumentation. We illustrate the approach using a dataset of human 3&#8217;UTRs cloned in 96-well plates, and two test miRNAs, let-7c and miR-10b. We demonstrate how to perform DNA preparation, transfection, cell culture and luciferase assays in 96-well format, and provide tools for data analysis. In conclusion 3&#39;LIFE is highly reproducible, rapid, systematic, and identifies high confidence targets.

Detection of miRNA Targets in High-throughput Using the 3&#039;LIFE Assay

Luminescent identification of functional elements in 3&#8217; untranslated regions (3&#8217;UTRs) (3&#8217;LIFE) is a technique to identify functional regulation in 3&#8217;UTRs by miRNAs or other regulatory factors. This protocol utilizes high-throughput methodology such as 96-well transfection and luciferase assays to screen hundreds of putative interactions for functional repression.

检测miRNA靶在高通量的使用3&#39;LIFE分析

Luminescent Identification of Functional Elements in 3&#8217;UTRs (3&#8217;LIFE) allows the rapid identification of targets of specific miRNAs within an ...

Identification of Kinase-substrate Pairs Using High Throughput Screening

We have developed a screening platform to identify dedicated human protein kinases for phosphorylated substrates which can be used to elucidate novel signal transduction pathways. Our approach features the use of a library of purified GST-tagged human protein kinases and a recombinant protein substrate of interest. We have used this technology to identify MAP/microtubule affinity-regulating kinase 2 (MARK2) as the kinase for a glucose-regulated site on CREB-Regulated Transcriptional Coactivator 2 (CRTC2), a protein required for beta cell proliferation, as well as the Axl family of tyrosine kinases as regulators of cell metastasis by phosphorylation of the adaptor protein ELMO. We describe this technology and discuss how it can help to establish a comprehensive map of how cells respond to environmental stimuli.

Protein phosphorylation is a central feature of how cells interpret and respond to information in their extracellular milieu. Here, we present a high throughput screening protocol using kinases purified from mammalian cells to rapidly identify kinases that phosphorylate a substrate(s) of interest.

激酶底物对鉴定使用高通量筛选


We have developed a screening platform to identify dedicated human protein kinases for phosphorylated substrates which can be used to elucidate novel ...