This work acknowledges a Faculty Early Career Grant from the University of Newcastle (CM).

注意:实验涉及使用放射性同位素32 p和没有工作应该进行，直到所有适当的安全条件已经具备。一般人员都必须参加安全课程，并接受之前，使用放射性试剂的实验监督的做法。请佩戴个人防护装备进行反应时（热释光剂量计，防护眼镜，手套，放射室实验室外套，全长裤，封闭趾鞋）。 1.试验材料的制备<ol><li>获取所需的蛋白质转录分析。 <ol><li>对于这里所描述的实验中，过量产生和纯化大肠杆菌大肠杆菌 RNA聚合酶在实验室中描述11，或获得自商业来源（材料清单）。 注意:两个提供类似的结果。对于天然或亲和标记的RNA聚合酶的纯化的其他方法也可以使用12,13。适马/努沙登加​​因素可为d获得旁切在以前的工作14,15。 </li></ol></li><li>焦碳酸二乙酯的制备（DEPC）处理的水 注意:可替换地，可以从商业来源（材料清单）获得无RNase水。 <ol><li>加入1ml焦碳酸二乙酯（DEPC），以1升纯化水（0.1％体积/体积）并混合过夜。 </li><li>高压灭菌DEPC水和提示两次。 </li></ol></li><li> DNA模板制备<ol><li>使用质粒纯化试剂盒根据制造商的说明（材料清单）纯化质粒pSG1154 16和/或pIA226 17。 </li><li>以放大决胜转录测定的模板（360 bp的片段包含在P XYL启动子区域），装配在冰上的PCR反应包括25微升2×PCR混合物（材料清单），每个引物2微升（5μM） :5&#39;-CAAAGCCTGTCGGAATTGG-3&#39;（正向）和5&#39;-CCCATTAACATCACCATC-3&#39;（反向），1微升pSG1154质粒公关eparation（250μM）和20μl纯化水）。 <ol><li>或者可以选择，对于单轮转录实验，扩增447 bp的片段从pIA226 17涵盖了λP R启动区。设置用25微升2×PCR混合物，2微升的各引物（5μM）在冰上的PCR反应:5&#39;-GTGCTCATACGTTAAATCT-3&#39;（正向）和5&#39;-CAGTTCCCTACTCTCGCATG-3&#39;（反向），1微升pIA226 17质粒制备（250μM）和20μl纯化水）。 注意:此模板缺乏胞嘧啶，直到+26 NT位置，这是从转录起始位点的26 日核苷酸。因此，在使用该模板将允许启动，启动子间隙和在+26核苷酸失速，以形成一个稳定的转录延伸复合物（EC），当只有ATP，UTP和GTP存在于反应转录。 </li></ol></li><li>设置了满足以下条件的PCR反应:95℃;℃，30秒，50℃，30秒，72℃30秒，重复30个循环。 </li><li>纯化使用根据一个PCR清理试剂盒（材料清单）制造商的指导产品并在用于转录分析100μl的转录缓冲液洗脱。 </li><li>确定使用fluorospectrometer（材料清单）模板DNA的浓度。 </li></ol></li><li> NTP准备 注意:的NTPs可从不同的商业来源（材料列表）来获得。 <ol><li>溶解于DEPC处理过的水的NTPs（≥99％纯），以使1M的库存，等分试样1ml溶液进入1.5毫升管（50微升在每个管），并存储在-20℃。 </li><li>为了α-32 p UTP（3000居里/毫摩尔）在实验前32 P具有14.29天的半衰期短。 注意:使用阿尔法 - 标记的UTP避免比较不同长度带的强度。 </li></ol></li><li> RNA梯子准备<ol><li>组装后续在室温下ING反应:0.5微克的RNA模板混合（材料清单），80毫米的HEPES缓冲液pH7.5的12毫氯化镁 ，10毫摩尔NaCl，10毫摩尔DTT，2毫摩尔ATP / CTP / GTP，0.5mM的UTP，2微升α-32 p UTP，0.5微升T7 RNA聚合酶（材料清单），DEPC处理过的水到20微升。 </li><li>允许反应在37℃进行1小时。立即使用或在-80℃保存。 </li></ol></li></ol> 2. RNA测序凝胶的制备<ol><li>凝胶制备<ol><li>清洗从测序凝胶装置（材料清单）用70％的乙醇与纯净水两次板，并用梳子和两个间隔件组装的单元格。 注意:一个盘可以与硅化试剂（材料列表）进行包衣，以促进在步骤4.2凝胶去除。 </li><li>溶解33.6克脲在22ml水和16.4 5倍的Tris /硼酸盐/ EDTA（TBE）缓冲液（54克亚磷酸三（羟甲基）氨基甲烷的溶液27.5克硼酸10毫升1M的乙二胺四乙酸（EDTA），pH为7.4在1L水）用磁力搅拌器。 </li><li>将16毫升40％丙烯酰胺/双丙烯酰胺溶液（19:1）的尿素溶液，并通过0.45μm的过滤器进行过滤。 </li><li>添加514微升10％（重量/体积）新鲜制备的过硫酸铵在水中，随后51.4微升N，N，N&#39;，N&#39;四甲基乙二胺（TEMED），以过滤后的溶液。 </li><li>轻轻颠倒凝胶溶液以避免过度通气和立即顺利注入从底部喷射孔的水平定位预包装测序细胞。注意避免在注射过程中制作气泡。 </li><li>允许1-1.5小时，直到凝胶在转录实验使用前完全设置。当与在测序细胞的顶部水的湿纸巾包装的凝胶可以在室温下贮存两年或三天。 </li></ol></li></ol> 3.转录反应<ol><li>转录延长暂停试验<ol><li>一个转录延伸复合物的形成<ol><li>加入1μlRNAP核心酶（转录缓冲1μM股票）以1.5 ml管。 </li><li>加入1μl纯化的Σ-70因子（σ70;在转录缓冲液为3μM股票）的RNA聚合酶，并在冰上孵育10分钟，以形成RNA聚合酶全酶。 </li><li>添加在转录缓冲液3微升500nM的纯化的模板DNA（来自pIA226 PCR产物λP R启动）到RNAP全酶，并在37ºC孵育5分钟，以形成开放的复杂。 </li><li>制备反应成分的混合物中，用5微升10×转录缓冲液（材料清单另外150毫升管:400毫的Tris-HCl，1.6M的氯化钾，100mM的MgCl 2的，10毫摩尔DTT，50％甘油，pH值7.9中DEPC处理水），2微升250μM每个GTP和UTP，1微升1毫米ATP和1微升α-32 p UTP（3000居里/毫摩尔）的。 DEPC处理水加至41微升。 没有德:RNase抑制剂（材料清单）可以被用于最小化RNase污染。 </li><li>该混合物溶液转移到含有开放的复杂的管，并在37ºC孵育15分钟，以形成转录延伸复杂在+26停止核苷酸。 </li><li>移动至反应至室温（〜21℃）并加入1微升的2.5毫克/毫升利福平至50微克/毫升的最终浓度。 </li><li>加入1微升0.1mM的纯化的NusA到反应混合物中的孵育在室温下5分钟。 </li><li>添加2微升250微米的CTP到反应混合物中，以使50微升的最终体积，并使用定时器测量的反应时间。 </li><li>转移5μl反应溶液加入到2微升的RNA凝胶上样缓冲液（95％甲酰胺，0.05％溴酚蓝和0.05％二甲苯青），在30，60，120，240，480，和1200秒以猝灭反应。 </li><li>处置的放射性废物的续32磷UTP提示和管ainer。 </li></ol></li></ol></li><li>转录起始含量<ol><li>在开放的复杂的形成<ol><li> σ70稀释纯化至0.5微米。 </li><li>重复步骤3.1.1.1至3.1.1.3使用0.5微米（而不是3μM）σ70精制后，DNA模板（从pSG1154 PCR产物在P XYL启动子区）。 </li></ol></li><li>转录起始试验的抑制<ol><li>添加旨在防止RNA聚合酶全酶形成到RNAP核心酶之前加入σ70（步骤3.1.1.1之后）抑制剂，接着步骤3.1.1.2和3.1.1.3检查抑制剂是否能够破坏全酶形成。 </li><li>另外，步骤3.1.1.2后，添加抑制剂来检查它是否能够竞争性抑制σ70结合RNA聚合酶。 </li><li>使用二甲基亚砜，抑制剂的溶剂相同量，而不是抑制剂小号滴答控制的反应。 </li></ol></li><li>在反应完成<ol><li>溶于水肝素到在-20℃的储备浓度为1mg / ml和库存。 注意:肝素是DNA的竞争者能够螯合尚未形成一个开放的复杂RNA聚合酶。一旦开放的复杂形成，肝素不能进入RNA聚合酶的DNA结合位点，并从预形成的开放的复杂抑制转录。因此，肝素只需要单轮试验;它并不需要用于多轮测定。 </li><li>制备使用5微升的10×转录缓冲液（材料清单），1微升的1毫克/毫升肝素溶液，1微升10毫每个ATP，GTP，和CTP，2.5mM的UTP的，和1μl的α-32的反应混合物P UTP（3000居里/毫摩尔）。 DEPC处理水加至45微升。 </li><li>混合与转录开放的复杂，脉冲自旋反应混合物中并在37℃孵育20分钟。 </li><li>转移反应的10微升这样lution入5微升的RNA凝胶上样缓冲液（95％甲酰胺，0.05％溴酚蓝和0.05％二甲苯青），以淬灭反应。 </li></ol></li></ol></li></ol> 4. RNA凝胶的运行与发展<ol><li>运行变性聚丙烯酰胺凝胶<ol><li>运行在50ºC在1×TBE缓冲液中的凝胶和50瓦为约1.5小时，直至凝胶的温度和缓冲液是稳定的，在50ºC。 </li><li>在90ºC30秒热样品并且将它们移动到冰之前装载到凝胶上。 </li><li>负载样品插入凝胶的顶部旁边含有2微升的RNA梯一个车道的孔（1:100稀释）。 </li><li>在50瓦和50ºC在1×TBE缓冲液中运行该凝胶直到溴酚蓝染料到达〜2/3的向下的凝胶。 </li></ol></li><li>凝胶处理<ol><li>打开缓慢而仔细地上部板从测序细胞中分离出来。采取非常谨慎，以避免破坏凝胶。 </li><li>切chromatograpHY纸（材料清单）从顶部至溴酚蓝染料匹配凝胶尺寸。用一个盖革计数器确认放射性标记的RNA转录物的大致位置。 </li><li>小心盖上滤纸上的凝胶，然后用力按压，直到凝胶粘附在滤纸。切断并丢弃凝胶底部放射性废物的未掺入的NTPs运行到凝胶底部，它可以是非常热。 </li><li>放置在真空干燥机的干燥床的类似大小的一片滤纸，并小心地放置凝胶涂布纸到它与凝胶的一面朝上。 </li><li>覆盖保鲜膜和干燥在60℃下1.5小时的凝胶。 </li><li>用保鲜膜包住凝胶涂覆的滤纸的另一侧，并将其放置于在成像情况下的光激励荧光体板，用适当的曝光时间，这可以从1小时变化到过夜根据反应的效率。 </li><li>图像使用荧光板根据制造商的说明的成像器扫描仪。决定根据软件说明书使用ImageJ软件相对带强度。 </li><li>用一个盖革计数器来检查放射性污染在工作区，擦拭可疑物品和面积与组织和清洗剂和处置的组织中的处置容器。 </li></ol></li></ol>

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The authors have nothing to disclose.

在所有生物中，转录是一严格调节的过程。 在体外已经开发转录测定，以用于测试的转录因子，小分子和转录抑制剂的影响提供了平台。在该方法中纸张，被描述为一般细菌转录的测定法。因为它们允许沿时间线中的所有转录产物可视化转录实验与成绩单的测序凝胶电泳结合是机理研究非常重要。其结果是，这种方法是能够识别的在反应条件下小的变化的影响，并露出动作的一个可行的机...

转录是在其中RNA是从一个特定的DNA模板合成的过程。在真核细胞中有三个不同的RNAPs:RNA聚合酶我转录rRNA的前体，RNA聚合酶II负责mRNA和某些小核RNA的合成，和5S rRNA基因和tRNA的合成是通过RNA聚合酶III进行的。在细菌中，有负责RNA的所有类的转录只有一个RNAP。有转录三个阶段:起始，延伸和终止。转录是在细胞中最高度调节过程中的一个。 在转录周期各阶段表示基因表达1的调节一个检查点。为起始，RNA聚合酶具有与σ因子相关联，以形成全酶，这是需要以引导酶特定网站的所谓启动子2以形成一个开放的启动子复合物。随后，大套房的转录因子负责监管Ô在延伸和终止阶段˚FRNA聚合酶活性。这里研究的转录因子是高度保守的和必需的蛋白质，的NusA。它参与rRNA的合成3-5中调节转录暂停和终止，以及抗终止。 体外转录测定法已被开发作为有力工具转录6中学习复杂的调节步骤。在一般情况下，DNA的包括一个启动子区的线性片段是必需的作为模板进行转录。 DNA模板通常是通过PCR或通过线性化的质粒产生的。纯化的蛋白质和的NTPs（包括一个放射性标记的NTP为检测的目的），然后加入和产品分析培养所需要的时间以下。使用相应的模板和反应条件，一直在使用这种方法，它使得transcr详细的分子特征研究转录的各个阶段iption在过去的半个世纪7。在与RNA聚合酶的三维结构信息的组合，它也是可能探测由抗生素和抗生素引线转录抑制的分子机制，以及在新的，改进的药物8-10开发使用此信息。 在这项工作中，我们提供的转录分析如何可以被用来确定由转录延伸/终止因子的NusA，以及如何能够确定的一个新的转录起始抑制剂引线的作用机制的调节机制的例子。

<table><tbody><tr><td>Obtain the proteins required for transcription assay</td><td></td><td></td><td></td></tr><tr><td>&lt;em&gt;E. coli&lt;/em&gt; RNAP</td><td>Epicentre</td><td>S90250</td><td></td></tr><tr><td>Preparation of DEPC-treated water</td><td></td><td></td><td></td></tr><tr><td>diethyl pyrocarbonate (DEPC)&amp;nbsp;</td><td>Sigma-Aldrich&amp;nbsp;</td><td>D5758</td><td></td></tr><tr><td>RNase-free water&amp;nbsp;</td><td></td><td></td><td></td></tr><tr><td>Ambion Nuclease-Free Water</td><td>ThermoFisher</td><td>AM9937</td><td></td></tr><tr><td>DNA template preparation</td><td></td><td></td><td></td></tr><tr><td>Wizard&amp;nbsp;Plus&amp;nbsp;SV Minipreps DNA Purification System</td><td>Promega</td><td>A1330</td><td></td></tr><tr><td>ACCUZYME Mix</td><td>Bioline</td><td>BIO-25028</td><td></td></tr><tr><td>PCR primers</td><td></td><td></td><td></td></tr><tr><td>Wizard SV Gel and PCR Clean-Up System&amp;nbsp;</td><td>Promega</td><td>A9281</td><td></td></tr><tr><td>NanoDrop 3300 fluorospectrometer</td><td>Thermo Scientific</td><td>ND-3300</td><td></td></tr><tr><td>NTP Preparation</td><td></td><td></td><td></td></tr><tr><td>ATP</td><td>Sigma-Aldrich&amp;nbsp;</td><td>A6559</td><td></td></tr><tr><td>UTP</td><td>Sigma-Aldrich&amp;nbsp;</td><td>U1006</td><td></td></tr><tr><td>GTP</td><td>Sigma-Aldrich&amp;nbsp;</td><td>G3776</td><td></td></tr><tr><td>CTP</td><td>Sigma-Aldrich&amp;nbsp;</td><td>C9274</td><td></td></tr><tr><td>High Purity rNTPs</td><td>GE Healthcare</td><td>27-2025-01</td><td></td></tr><tr><td>&amp;alpha;-&lt;sup&gt;32&lt;/sup&gt;P UTP&amp;nbsp;</td><td>PerkinElmer&amp;nbsp;&amp;nbsp;</td><td>BLU007C001MC</td><td>Radioactive compound</td></tr><tr><td>RNA ladder preparation&amp;nbsp;</td><td></td><td></td><td></td></tr><tr><td>Novagen Perfect RNA Marker Template Mix 0.1&amp;ndash;1 kb</td><td>Millipore</td><td>69003</td><td></td></tr><tr><td>HEPES</td><td>Sigma-Aldrich&amp;nbsp;</td><td>H7006</td><td></td></tr><tr><td>Sodium chloride</td><td>Sigma-Aldrich&amp;nbsp;</td><td>S7653</td><td></td></tr><tr><td>Magnesium chloride</td><td>Sigma-Aldrich&amp;nbsp;</td><td>M8266</td><td></td></tr><tr><td>DTT</td><td>Sigma-Aldrich</td><td>DTT-RO</td><td></td></tr><tr><td>T7 RNAP</td><td>Promega</td><td>P2075</td><td></td></tr><tr><td>Gel preparation</td><td></td><td></td><td></td></tr><tr><td>Sequi-Gen GT nucleic acid sequencing cell&amp;nbsp;</td><td>Bio-Rad&amp;nbsp;&amp;nbsp;</td><td>165-3804</td><td></td></tr><tr><td>Sigmacote&amp;nbsp;&amp;nbsp;</td><td>Sigma-Aldrich&amp;nbsp;</td><td>SL2</td><td></td></tr><tr><td>urea&amp;nbsp;&amp;nbsp;</td><td>Sigma-Aldrich&amp;nbsp;</td><td>U6504</td><td></td></tr><tr><td>tris(hydroxymethyl)aminomethane&amp;nbsp;&amp;nbsp;</td><td>Sigma-Aldrich&amp;nbsp;</td><td>154563</td><td></td></tr><tr><td>boric acid&amp;nbsp;</td><td>Sigma-Aldrich&amp;nbsp;</td><td>B7901</td><td></td></tr><tr><td>ethylenediaminetetraacetic acid&amp;nbsp;</td><td>Sigma-Aldrich&amp;nbsp;</td><td>ED</td><td></td></tr><tr><td>40% Acrylamide/bis-acrylamide&amp;nbsp;</td><td>Sigma-Aldrich&amp;nbsp;</td><td>A9926</td><td></td></tr><tr><td>ammonium persulfate&amp;nbsp;</td><td>Sigma-Aldrich&amp;nbsp;</td><td>A3678</td><td></td></tr><tr><td>&lt;em&gt;N,N,N&#039;,N&#039;&lt;/em&gt;-Tetramethylethylenediamine (TEMED)</td><td>Sigma-Aldrich </td><td>T9281</td><td></td></tr><tr><td>Transcription Assay</td><td></td><td></td><td></td></tr><tr><td>Potassium chloride</td><td>Sigma-Aldrich&amp;nbsp;</td><td>P9541</td><td></td></tr><tr><td>glycerol&amp;nbsp;&amp;nbsp;</td><td>Sigma-Aldrich&amp;nbsp;</td><td>G5516</td><td></td></tr><tr><td>rifampicin&amp;nbsp;&amp;nbsp;</td><td>Sigma-Aldrich&amp;nbsp;</td><td>R3501</td><td></td></tr><tr><td>formamide&amp;nbsp;&amp;nbsp;</td><td>Sigma-Aldrich&amp;nbsp;</td><td>F9037</td><td></td></tr><tr><td>bromophenol blue&amp;nbsp;</td><td>Sigma-Aldrich&amp;nbsp;</td><td>B0126</td><td></td></tr><tr><td>xylene cyanol&amp;nbsp;</td><td>Sigma-Aldrich&amp;nbsp;</td><td>X4126</td><td></td></tr><tr><td>heparin&amp;nbsp;</td><td>Sigma-Aldrich&amp;nbsp;</td><td>84020</td><td></td></tr><tr><td>RNasin Ribonuclease Inhibitor</td><td>Promega</td><td>N2511</td><td></td></tr><tr><td>Transcription buffer&amp;nbsp;</td><td></td><td></td><td></td></tr><tr><td>Tris base</td><td>Sigma-Aldrich&amp;nbsp;</td><td>T1503</td><td></td></tr><tr><td>Potassium chloride</td><td>Sigma-Aldrich&amp;nbsp;</td><td>P9541</td><td></td></tr><tr><td>Magnesium chloride</td><td>Sigma-Aldrich&amp;nbsp;</td><td>M2393</td><td></td></tr><tr><td>DTT&amp;nbsp;&amp;nbsp;</td><td>Sigma-Aldrich&amp;nbsp;</td><td>DTT-RO</td><td></td></tr><tr><td>glycerol&amp;nbsp;&amp;nbsp;</td><td>Sigma-Aldrich&amp;nbsp;</td><td>G5516</td><td></td></tr><tr><td>Filter paper</td><td></td><td></td><td></td></tr><tr><td>Whatman&amp;nbsp;3MM Chr Chromatography Paper</td><td>Fisher Scientific</td><td>05-714-5</td><td></td></tr><tr><td>Radioactive decontaminant</td><td></td><td></td><td></td></tr><tr><td>Decon 90</td><td>decon</td><td>decon90</td><td></td></tr><tr><td>Gel Treatment</td><td></td><td></td><td></td></tr><tr><td>Typhoon Trio+ imager</td><td>GE Healthcare Life Sciences</td><td>63-0055-89</td><td></td></tr></tbody></table>

in vitro transcription assays and their application in drug discovery

体外转录测定法已被开发并广泛使用了许多年，以研究参与转录的分子机制。这个过程需要多亚基DNA依赖性RNA聚合酶（RNAP）等一系列的，其作用的基因表达过程中调节RNA聚合酶的活性转录因子。放射性成绩单的测序凝胶电泳用于提供关于如何转录所得，哪些参数可以影响它的详细机理信息。在本文中，我们描述了协议来研究必不可少延伸因子的NusA如何调控转录暂停，以及一个以确定抗菌剂通过抑制RNA聚合酶全酶形成指定转录起始方法。这些方法可用于一个作为平台的额外方法的发展探索转录因子的作用机制仍然不清楚，以及新的抗菌阿根TS靶向转录这是在抗生素研发的未充分利用的药物靶标。

转录效率可以通过测量在不同时间点的频段辐射水平来确定。的暂停检测来测试启用暂停，终止的可视化的NusA因子的功能，和径流制品（ 图1A）。在的NusA的N-末端结构域的存在（的NusA NTD;氨基酸残基1-137）时，RNA产物的外观相比缺乏的NusA对照实验显著延迟。的的NusA片段，或改变的一系列氨基酸的丙氨酸的氨基酸残基104-137（螺旋3）缺失（残基K36，K37，R104，Q10...

Watch this Scientific Journal Video about In Vitro Transcription Assays and Their Application in Drug Discovery at JoVE.com

In Vitro Transcription Assays and Their Application in Drug Discovery

In this manuscript, we describe a protocol to functionally examine transcription and the inhibitory activity of antibacterial agents targeting bacterial transcription.

体外转录测定及其在药物研究中的应用

In vitro transcription assays have been developed and widely used for many years to study the molecular mechanisms involved in transcription. This process ...

in-vitro-transcription-assays-and-their-application-in-drug-discovery

Research

JoVE Journal

Genetics

In Vitro Transcription Assays and Their Application in Drug Discovery

15.1K 次观看.  University of Newcastle. In this manuscript, we describe a protocol to functionally examine transcription and the inhibitory activity of antibacterial agents targeting bacterial transcription.

视频: In Vitro Transcription Assays and Their Application in Drug Discovery

An In Vitro Assay to Detect tRNA-Isopentenyl Transferase Activity

N6-isopentenyladenosine RNA modifications are functionally diverse and highly conserved among prokaryotes and eukaryotes. One of the most highly conserved N6-isopentenyladenosine modifications occurs at the A37 position in a subset of tRNAs. This modification improves translation efficiency and fidelity by increasing the affinity of the tRNA for the ribosome. Mutation of enzymes responsible for this modification in eukaryotes are associated with several disease states, including mitochondrial dysfunction and cancer. Therefore, understanding the substrate specificity and biochemical activities of these enzymes is important for understanding of normal and pathologic eukaryotic biology. A diverse array of methods has been employed to characterize i6A modifications. Herein is described a direct approach for the detection of isopentenylation by Mod5. This method utilizes incubation of RNAs with a recombinant isopentenyl transferase, followed by RNase T1 digestion, and 1-dimensional gel electrophoresis analysis to detect i6A modifications. In addition, the potential adaptability of this protocol to characterize other RNA-modifying enzymes is discussed.

An In Vitro Assay to Detect tRNA-Isopentenyl Transferase Activity

Here, we describe a protocol for the biochemical characterization of the yeast RNA-modifying enzyme, Mod5, and discuss how this protocol could be applied to other RNA-modifying enzymes.

体外检测 tRNA-Isopentenyl 转移酶活性的实验研究

N6-isopentenyladenosine RNA modifications are functionally diverse and highly conserved among prokaryotes and eukaryotes. One of the most highly conserved ...

科研

生物化学

Identification of Transcription Factor Regulators using Medium-Throughput Screening of Arrayed Libraries and a Dual-Luciferase-Based Reporter

Transcription factors can alter the expression of numerous target genes that influence a variety of downstream processes making them good targets for anti-cancer therapies. However, directly targeting transcription factors is often difficult and can cause adverse side effects if the transcription factor is necessary in one or more adult tissues. Identifying upstream regulators that aberrantly activate transcription factors in cancer cells offers a more feasible alternative, particularly if these proteins are easy to drug. Here, we describe a protocol that can be used to combine arrayed medium-scale lentiviral libraries and a dual-luciferase-based transcriptional reporter assay to identify novel regulators of transcription factors in cancer cells. Our approach offers a quick, easy, and inexpensive way to test hundreds of genes in a single experiment. To demonstrate the use of this approach, we performed a screen of an arrayed lentiviral RNAi library containing several regulators of Yes-associated protein (YAP) and transcriptional co-activator with PDZ-binding motif (TAZ), two transcriptional co-activators that are the downstream effectors of the Hippo pathway. However, this approach could be modified to screen for regulators of virtually any transcription factor or co-factor and could also be used to screen CRISPR/CAS9, cDNA, or ORF libraries.

To identify novel regulators of transcription factors, we developed an approach to screen arrayed lentiviral or retroviral RNAi libraries using a dual-luciferase-based transcriptional reporter assay. This approach offers a quick and relatively inexpensive way to screen hundreds of candidates in a single experiment.

使用阵列库的中等通量筛选和基于双 Luciferase 的分录报告器识别转录因子稳压器

Transcription factors can alter the expression of numerous target genes that influence a variety of downstream processes making them good targets for ...

癌症研究

A Multiplexed Luciferase-based Screening Platform for Interrogating Cancer-associated Signal Transduction in Cultured Cells

Genome-scale interrogation of gene function using RNA interference (RNAi) holds tremendous promise for the rapid identification of chemically tractable cancer cell vulnerabilities. Limiting the potential of this technology is the inability to rapidly delineate the mechanistic basis of phenotypic outcomes and thus inform the development of molecularly targeted therapeutic strategies. We outline here methods to deconstruct cellular phenotypes induced by RNAi-mediated gene targeting using multiplexed reporter systems that allow monitoring of key cancer cell-associated processes. This high-content screening methodology is versatile and can be readily adapted for the screening of other types of large molecular libraries.

Achieving a systems level understanding of cellular processes is a goal of modern-day cell biology. We describe here strategies for multiplexing luciferase reporters of various cellular function end-points to interrogate gene function using genome-scale RNAi libraries.

多路复用讯问癌症相关的信号转导在培养的细胞中的荧光素酶的筛选平台

Genome-scale interrogation of gene function using RNA interference (RNAi)  holds tremendous promise for the rapid identification of chemically tractable  ...

医学

Rapid Screening of HIV Reverse Transcriptase and Integrase Inhibitors

Although a number of anti HIV drugs have been approved, there are still problems with toxicity and drug resistance. This demonstrates a need to identify new compounds that can inhibit infection by the common drug resistant HIV-1 strains with minimal toxicity. Here we describe an efficient assay that can be used to rapidly determine the cellular cytotoxicity and efficacy of a compound against WT and mutant viral strains.
The desired target cell line is seeded in a 96-well plate and, after a 24 hr incubation, serially dilutions of the compounds to be tested are added. No further manipulations are necessary for cellular cytotoxicity assays; for anti HIV assays a predetermined amount of either a WT or drug resistant HIV-1 vector that expresses luciferase is added to the cells. Cytotoxicity is measured by using an ATP dependent luminescence assay and the impact of the compounds on infectivity is measured by determining the amount of luciferase in the presence or the absence of the putative inhibitors.
This screening assay takes 4 days to complete and multiple compounds can be screened in parallel. Compounds are screened in triplicate and the data are normalized to the infectivity/ATP levels in absence of target compounds. This technique provides a quick and accurate measurement of the efficacy and toxicity of potential anti HIV compounds.

Here we describe cellular cytotoxicity and single round infectivity assays that allow for the rapid and accurate screening of compounds to determine their cellular cytotoxicity (CC50) and IC50 values against WT and drug resistant HIV-1.

HIV逆转录酶和整合酶抑制剂的快速筛选

Although a number of anti HIV drugs have been approved, there are still problems with toxicity and drug resistance. This demonstrates a need to identify ...

免疫与感染

Implementation of In Vitro Drug Resistance Assays: Maximizing the Potential for Uncovering Clinically Relevant Resistance Mechanisms

Although targeted therapies are initially effective, resistance inevitably emerges. Several methods, such as genetic analysis of resistant clinical specimens, have been applied to uncover these resistance mechanisms to facilitate follow-up care. Although these approaches have led to clinically relevant discoveries, difficulties in attaining the relevant patient material or in deconvoluting the genomic data collected from these specimens have severely hampered the path towards a cure. To this end, we here describe a tool for expeditious discovery that may guide improvement in first-line therapies and alternative clinical management strategies. By coupling preclinical in vitro or in vivo drug selection with next-generation sequencing, it is possible to identify genomic structural variations and/or gene expression alterations that may serve as functional drivers of resistance. This approach facilitates the spontaneous emergence of alterations, enhancing the probability that these mechanisms may be observed in the patients. In this protocol we provide guidelines to maximize the potential for uncovering single nucleotide variants that drive resistance using adherent lines.

Implementation of In Vitro Drug Resistance Assays: Maximizing the Potential for Uncovering Clinically Relevant Resistance Mechanisms

Drug resistance to targeted therapeutics is widespread and the need to identify mechanisms of resistance--prior to or following clinical onset--is critical for guiding alternative clinical management strategies. Here, we present a protocol to couple derivation of drug-resistant lines in vitro with sequencing to expedite discovery of these mechanisms.

实施<em&gt;体外</em&gt;耐药性检测:最大化的潜力，揭开临床有关的耐药机制

Although targeted therapies are initially effective, resistance inevitably emerges. Several methods, such as genetic analysis of resistant clinical ...

Optimization of In vitro Transcription Reaction for mRNA Production Using Chromatographic At-Line Monitoring

In vitro transcription reaction (IVT) is a complex, multi-component enzymatic synthesis of mRNA from a linear DNA template, catalyzed by an RNA polymerase, e.g. T7. Due to the high cost of IVT reagents,&#160; IVT is a critical step in the mRNA drug substance production process and has been the focus of intense optimization in the field. To decrease the cost of mRNA production, reagents must be utilized optimally. Effective optimization necessitates a comprehensive understanding of the impact of individual reagents on reaction kinetics, i.e., the consumption of nucleoside triphosphates (NTPs) and the production of mRNA. Traditionally, low-throughput, end-point analytical techniques have been used for the analysis of mRNA. Though such methods give valuable information on mRNA content, at-line, near real-time analytics is needed to fully understand IVT reaction. We demonstrate how a liquid chromatography analytical method that separates NTPs, as well as pDNA and mRNA, can be used in near real-time to study IVT reaction kinetics. With the knowledge of the influence of different IVT components on kinetics and yield, rapid chromatographic analysis can be used to convert batch IVT reaction into a fed-batch mode that further increases productivity and reduces the overall cost of the reaction.

Optimizing in vitro transcription (IVT) is critical for cost-effective mRNA production. This protocol details an analytical chromatographic method for at-line analysis of IVT reaction, monitoring NTP depletion and mRNA production in batch or fed-batch modes, applicable to various RNA modalities, enhancing productivity and reducing costs.

使用色谱旁在线监测优化用于 mRNA 生产的 体外 转录反应

In vitro transcription reaction (IVT) is a complex, multi-component enzymatic synthesis of mRNA from a linear DNA template, catalyzed by an RNA ...

In Vitro Transcribed RNA-based Luciferase Reporter Assay to Study Translation Regulation in Poxvirus-infected Cells

Every poxvirus mRNA transcribed after viral DNA replication has an evolutionarily conserved, non-templated 5&#39;-poly(A) leader in the 5&#39;-UTR. To dissect the role of 5&#39;-poly(A) leader in mRNA translation during poxvirus infection we developed an in vitro transcribed RNA-based luciferase reporter assay. This reporter assay comprises of four core steps: (1) PCR to amplify the DNA template for in vitro transcription; (2) in vitro transcription to generate mRNA using T7 RNA polymerase; (3) Transfection to introduce in vitro transcribed mRNA into cells; (4) Detection of luciferase activity as the indicator of translation. The RNA-based luciferase reporter assay described here circumvents issues of plasmid replication in poxvirus-infected cells and cryptic transcription from the plasmid. This protocol can be used to determine translation regulation by cis-elements in an mRNA including 5&#39;-UTR and 3&#39;-UTR in systems other than poxvirus-infected cells. Moreover, different modes of translation initiation like cap-dependent, cap-independent, re-initiation, and internal initiation can be investigated using this method.

We present a protocol to study mRNA translation regulation in poxvirus-infected cells using in vitro Transcribed RNA-based luciferase reporter assay. The assay can be used for studying translation regulation by cis-elements of an mRNA, including 5&#8217;-untranslated region (UTR) and 3&#8217;-UTR. Different translation initiation modes can also be examined using this method.

体外转录 rna 基荧光素酶记者法研究类病毒感染细胞的翻译规律

Every poxvirus mRNA transcribed after viral DNA replication has an evolutionarily conserved, non-templated 5&#39;-poly(A) leader in the 5&#39;-UTR. To ...

In Vitro Selection of Engineered Transcriptional Repressors for Targeted Epigenetic Silencing

Gene inactivation is instrumental to study gene function and represents a promising strategy for the treatment of a broad range of diseases. Among traditional technologies, RNA interference suffers from partial target abrogation and the requirement for life-long treatments. In contrast, artificial nucleases can impose stable gene inactivation through induction of a DNA double strand break (DSB), but recent studies are questioning the safety of this approach. Targeted epigenetic editing via engineered transcriptional repressors (ETRs) may represent a solution, as a single administration of specific ETR combinations can lead to durable silencing without inducing DNA breaks.
ETRs are proteins containing a programmable DNA-binding domain (DBD) and effectors from naturally occurring transcriptional repressors. Specifically, a combination of three ETRs equipped with the KRAB domain of human ZNF10, the catalytic domain of human DNMT3A and human DNMT3L, was shown to induce heritable repressive epigenetic states on the ETR-target gene. The hit-and-run nature of this platform, the lack of impact on the DNA sequence of the target, and the possibility to revert to the repressive state by DNA demethylation on demand, make epigenetic silencing a game-changing tool. A critical step is the identification of the proper ETRs&#39; position on the target gene to maximize on-target and minimize off-target silencing. Performing this step in the final ex vivo or in vivo preclinical setting can be cumbersome.
Taking the CRISPR/catalytically dead Cas9 system as a paradigmatic DBD for ETRs, this paper describes a protocol consisting of the in vitro screen of guide RNAs (gRNAs) coupled to the triple-ETR combination for efficient on-target silencing, followed by evaluation of the genome-wide specificity profile of top hits. This allows for reduction of the initial repertoire of candidate gRNAs to a short list of promising ones, whose complexity is suitable for their final&#160;evaluation in the therapeutically relevant setting of interest.

In Vitro Selection of Engineered Transcriptional Repressors for Targeted Epigenetic Silencing

Here, we present a protocol for the in vitro selection of engineered transcriptional repressors (ETRs) with high, long-term, stable, on-target silencing efficiency and low genome-wide, off-target activity. This workflow allows for reducing an initial, complex repertoire of candidate ETRs to a short list, suitable for further evaluation in therapeutically relevant settings.

体外研究 用于靶向表观遗传沉默的工程化转录抑制因子的选择

Gene inactivation is instrumental to study gene function and represents a promising strategy for the treatment of a broad range of diseases. Among ...

生物工程

A Quantitative Assay to Study Protein:DNA Interactions, Discover Transcriptional Regulators of Gene Expression, and Identify Novel Anti-tumor Agents

Many DNA-binding assays such as electrophoretic mobility shift assays (EMSA), chemiluminescent assays, chromatin immunoprecipitation (ChIP)-based assays, and multiwell-based assays are used to measure transcription factor activity. However, these assays are nonquantitative, lack specificity, may involve the use of radiolabeled oligonucleotides, and may not be adaptable for the screening of inhibitors of DNA binding. On the other hand, using a quantitative DNA-binding enzyme-linked immunosorbent assay (D-ELISA) assay, we demonstrate nuclear protein interactions with DNA using the RUNX2 transcription factor that depend on specific association with consensus DNA-binding sequences present on biotin-labeled oligonucleotides. Preparation of cells, extraction of nuclear protein, and design of double stranded oligonucleotides are described. Avidin-coated 96-well plates are fixed with alkaline buffer and incubated with nuclear proteins in nucleotide blocking buffer. Following extensive washing of the plates, specific primary antibody and secondary antibody incubations are followed by the addition of horseradish peroxidase substrate and development of the colorimetric reaction. Stop reaction mode or continuous kinetic monitoring were used to quantitatively measure protein interaction with DNA. We discuss appropriate specificity controls, including treatment with non-specific IgG or without protein or primary antibody. Applications of the assay are described including its utility in drug screening and representative positive and negative results are discussed.

We developed a quantitative DNA-binding, ELISA-based assay to measure transcription factor interactions with DNA. High specificity for the RUNX2 protein was achieved with a consensus DNA-recognition oligonucleotide and specific monoclonal antibody. Colorimetric detection with an enzyme-coupled antibody substrate reaction was monitored in real time.

定量检测，以研究蛋白质：DNA的相互作用，发现转录基因表达的调节，并确定新的抗肿瘤剂

Many DNA-binding assays such as electrophoretic mobility shift assays (EMSA), chemiluminescent assays, chromatin immunoprecipitation (ChIP)-based assays, ...

生物学

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 ...

体外转录测定及其在药物研究中的应用

摘要

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此视频中的章节

体外检测 tRNA-Isopentenyl 转移酶活性的实验研究

使用阵列库的中等通量筛选和基于双 Luciferase 的分录报告器识别转录因子稳压器

多路复用讯问癌症相关的信号转导在培养的细胞中的荧光素酶的筛选平台

HIV逆转录酶和整合酶抑制剂的快速筛选

实施

使用色谱旁在线监测优化用于 mRNA 生产的体外转录反应

体外转录 rna 基荧光素酶记者法研究类病毒感染细胞的翻译规律

体外研究用于靶向表观遗传沉默的工程化转录抑制因子的选择

定量检测，以研究蛋白质：DNA的相互作用，发现转录基因表达的调节，并确定新的抗肿瘤剂

高通量筛选转录后调控基因的化学调节剂

体外转录测定及其在药物研究中的应用

摘要

探索更多视频

此视频中的章节

体外检测 tRNA-Isopentenyl 转移酶活性的实验研究

使用阵列库的中等通量筛选和基于双 Luciferase 的分录报告器识别转录因子稳压器

多路复用讯问癌症相关的信号转导在培养的细胞中的荧光素酶的筛选平台

HIV逆转录酶和整合酶抑制剂的快速筛选

实施

使用色谱旁在线监测优化用于 mRNA 生产的 体外 转录反应

体外转录 rna 基荧光素酶记者法研究类病毒感染细胞的翻译规律

体外研究 用于靶向表观遗传沉默的工程化转录抑制因子的选择

定量检测，以研究蛋白质：DNA的相互作用，发现转录基因表达的调节，并确定新的抗肿瘤剂

高通量筛选转录后调控基因的化学调节剂

使用色谱旁在线监测优化用于 mRNA 生产的体外转录反应

体外研究用于靶向表观遗传沉默的工程化转录抑制因子的选择