The authors would like to thank members of the Yi laboratory especially Zhaojie Zhang for fruitful discussions regarding linker design and ligation efficiencies, as well as the American Cancer Society for supporting this work through a postdoctoral fellowship (#125209) to J.E.L. Research reported in this publication was also supported by the National Institute of Arthritis and Musculoskeletal and Skin Diseases of the National Institutes of Health under Award Number R01AR059697 (to R.Y.) and a research grant from the Linda Crnic Institute for Down Syndrome. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

注：关键是要在整个过程中保持无RNA酶的条件。 1.腺苷酸化的3&#39;连接器<ol><li>稀释设计用于3&#39;连接反应至100μM在不含核酸酶的H 2 O的DNA寡核苷酸 注：该寡核苷酸应具有一个5&#39;磷酸基团，将在5随机化的核苷酸&#39;末端和3&#39;dideoxycytosine。 5&#39;磷酸基团是第M个酶的高效5的规定&#39;腺苷酸化10。磷酸盐可以通过预处理寡核苷酸的添加与多核苷酸激酶，或排序，直接修改。随机核苷酸在5&#39;端是用于最小化序列偏好其中的RNA连接酶表现出对于其他6某些端到端连接的反应很重要。在3的寡核苷酸吨端&#39;dideoxycytosine在3放置一个阻挡元件“ø抑制的接头-连接体多联体在随后的连接步骤的形成和还用于阻止连接反应11 3&#39;形成于3的端部与miRNA -接头分子的端部“。选择用于无偏的miR-SEQ方法的寡核苷酸接头序列如下：5&#39;PO 4 NNTGGAATTCTCGGGTGCCAAGG-dideoxyC3&#39;。 </li><li>组装下面腺苷酸化反应：200pmol的3&#39;接头寡核苷酸，4微升10×5&#39;腺苷酸化的DNA缓冲液，4μl1mM的ATP，4μl的第M个 RNA连接酶，核酸酶游离H 2 O至40微升的最终体积。 </li><li>孵育在65℃下反应1小时。终止反应，加热至85℃5分钟。 </li><li>通过加入2.5倍体积的100％乙醇和1/3体积的10M乙酸铵沉淀腺苷酸化的连接体，以除去残留的ATP，并防止在将来的反应未预料到的连接反应。 <ol><li>简单地说，混合人cohol，盐，并在4℃下进行20分钟的寡核苷酸溶液至均匀，以全速在微量离心纺丝（〜15000×g离心）。洗70％的乙醇，空气干燥，悬浮颗粒在核酸游离H 2 O的适当体积，以产生50-100微米寡腺苷酸化的。 </li></ol></li><li>通过运行在18％聚丙烯酰胺凝胶确认腺苷酸化。 <ol><li>通过混合制备凝胶下面一起6.3克尿素，6.75毫升40％的丙烯酰胺，和1.5毫升10×TBE。加入H 2 O操作15毫升。 </li><li>混合直到尿素完全溶解，并添加150μl的10％（重量/体积）过硫酸铵（APS）和15微升tetramethlyethylenediamine（TEMED）。 </li><li>一旦凝胶已设定，预运行30分钟，在凝胶宽度的24伏/厘米用1×TBE缓冲液在室温下。一旦30分钟可达冲洗井用电泳缓冲液。 </li><li>拌5皮摩尔腺苷酸化寡核苷酸与和的2倍变性的RNA上样缓冲液等体积​​（95％（体积/体积）甲酰胺，0.02％（重量/体积）SDS，0.02％溴酚蓝（重量/体积），0.01％（重量/体积）二甲苯蓝，1.0mM的EDTA）中，热简要至75℃，并搭扣在冰上冷却。分配整个样品进入凝胶的孔中。还包括一个相同的量的非腺苷化控制的，以及低分子量大小标准。 </li><li>运行该凝胶在24伏/厘米，直到溴酚蓝的方式向凝胶的底部¾。以在1×TBE缓冲液1×SYBR -金溶液拆卸装置，后染色凝胶和可视化的紫外透射仪箱（ 图2）。 注：凝胶纯化寡腺苷酸化在类似于上面描述的，如果“没有输入的RNA”控件都导致高的量的PCR产物的方式相比，与输入的RNA样品。 </li></ol></li></ol> 2.连接器结扎到的miRNA的3&#39;末端<ol><li> 3&#39;结扎<ol><li>装配在冰上下列组分按所列顺序：1.0微升50％（重量/体积）的PEG 8000，0.5＆＃956;微升10x RNA连接酶缓冲液（500毫摩尔的Tris-HCl pH为7.5，100毫摩尔的MgCl 2，10毫摩尔DTT），1.0微升腺苷酸化的3&#39;连接器（100μM），0.5μl的RNase抑制剂，0.5-8.0微克总RNA， 0.5微升T4 RNA连接酶2，无核酸酶的H 2 O操作的5微升的最终体积。 </li><li>混合通过移液反应的溶液太粘稠，旋涡，由于高浓度的PEG 8000。一旦彻底混合，将用加热盖在热循环仪中进行反应。设置块℃至16℃，并保温4小时。 注：该反应可以按比例增加至20微升的最终体积，而不在连接效率的任何损失。反应4小时，在环境温度下过夜或在4℃下，以产生基本上相同的连接效率。 </li></ol></li><li>凝胶净化<ol><li>继3&#39;结扎，拌上2×RNA上样缓冲液等体积​​反应。热至70℃5分钟，将其卡在冰上冷却。 </li&gt; <li>运行用于PAGE纯化15％聚丙烯酰胺凝胶中的样品。 <ol><li>制备含有7M尿素的15％聚丙烯酰胺凝胶，在类似于步骤1.5先前描述的方式。 </li><li>运行前的凝胶在凝胶宽度的24伏/厘米至少30分钟。关闭电源，冲洗水井，负载样品和凝胶运行在相同的电压预运行。 </li><li>装载样品和运行页面，直至溴酚蓝刚退出凝胶。所需产物将迁移靠近二甲苯蓝。 </li></ol></li></ol></li><li>染色，可视化，并切除<ol><li>在干净的托盘1X电泳缓冲液（TBE），1X SYBR金，和摇滚15分钟拆卸胶设备和场所凝胶。 </li><li>从染色皿中取出凝胶和可视化的紫外透射盒子盖上干净的保鲜膜。连接产物应该是大约45个核苷酸长度。 </li><li>切下含有连接产物的区域用一块干净的razo- [R刀片和地点在0.5ml微离心管中。 </li></ol></li><li>产品分离和降水<ol><li> 0.5 ml离心管刺穿底部与地下30针，然后将穿孔的管中的第二清洁，低滞留，1.5 ml离心管。离心5分钟，在〜15,000×克。 </li><li>目视确认该整个凝胶切片已经通过在所述内管的孔移动。如果有必要，用干净的枪头一些凝胶碎片距离拉得更近了洞。 </li><li>弃去空0.5毫升管中，加入400μl的高盐柱缓冲液（HSCB 400 mM氯化钠，的25mM的Tris-HCl pH为7.5，0.1％SDS）中的丙烯酰胺的浆液。岩管（多个）在4℃下过夜。 </li><li>使用宽孔移液管尖端传送浆料到0.22微米醋酸纤维素离心柱上。离心3分钟，在室温下以〜15000×g离心，收集所有的液体远离丙烯酰胺凝胶基质。 </li><li>液体转移到一个干净的，低保留，1.5毫升试管中含有荷兰国际集团1微升20mg / ml的糖原。添加异丙醇等体积的1/10体积乙酸钠。通过倒转试管几次混合溶液和沉淀在-80℃下冷冻20分钟。 </li><li>离心机以全速在4℃下进行20分钟以沉淀核酸。除去上清，洗涤在70％乙醇中。空气干燥沉淀物，在室温下10分钟，并直接进行5&#39;连接步骤。 </li></ol></li></ol> 3.连接器连接以5&#39;端的miRNA的3&#39;混合连接器<ol><li>以沉淀核酸，添加以下组分：2微升PEG 8000（50％重量/体积），0.5微升10×RNA连接酶缓冲液，0.5微升ATP（10毫摩尔），0.5微升NN-RNA的寡核苷酸（100μM）和H 2 O 4微升的最终体积。 注：“NN-RNA的寡核苷酸”的组合物是如下5&#39;-倒置脱氧TCUACAGUCCGACGAUCNN3&#39;和由生产经HPLC进行纯化。 </li><li>混此示例吹打上下反复。这样做一段时间（最长5分钟），以确保颗粒被完全重新溶解。如果再溶解是有问题的，加热样品简短（3-5分钟）至37℃，并继续移液混合。 </li><li>一旦粒料是早在溶液中，并将内容物转移到含有1微升1干净的PCR管中：RNA酶抑制剂和T4 RNA连接酶1（5个单位）的1:1混合物。通过上下吹打再次混合。 </li><li>孵育在热循环仪中，反应在37℃下搅拌4小时。立即进行cDNA合成。 </li></ol> 4.反转录/ cDNA合成<ol><li>将以下组件添加到5&#39;连接反应：2微升5×RT缓冲液，0.75微升100mM的DTT，1微升Illumina公司的RT引物5&#39;-GCCTTGGCACCCGAGAATTCCA-3&#39;（1μM），和0.5微升的dNTPs（10毫摩尔）。 </li><li>通过移液和热拌至65℃5分钟。然后慢慢冷却在板凳吨同前。 </li><li>一旦反应冷却至室温，放置​​管在冰上并添加每个逆转录酶和RNA酶抑制剂0.5微升。通过移液并将其放置在用于制造商的指示所指示的延伸步骤的热循环仪中混合。 </li><li>一旦完成添加5微升不含核酸酶的H 2澳带来的cDNA文库的最终体积达到15微升。 </li></ol> 5.图书馆准备<ol><li>为索引的PCR，装配在冰上下列反应：6微升5倍的Phusion缓冲液，1微升的dNTPs（10毫摩尔），0.9微升的DMSO，0.75μl的5&#39;引物（25μM），0.75μl的3&#39;引物（25μM）， 3微升cDNA文库（取决于输入的量可变浓度），加入0.5μlPhusion聚合酶（1单位），无核酸酶的H 2 O操作的30微升的最终体积。选择的引物序列，以确保与模板和测序平台的兼容性的选择。 <ol><li>运行，使用以下设置将PCR反应：在98℃预变性3分钟，在第4个周期，变性98℃15秒，退火50℃15秒，72℃延伸15秒。 注：根据所输入的材料的量，改变总的PCR循环数。典型地，对于1-2微克总RNA的一个输入端，13个总PCR循环足以产生所述的miR-SEQ库。 </li><li>进行随后的周期中， 例如，循环5-12中，在退火和延伸循环相结合，并在70℃下30秒和变性的两步方式是再次98℃持续15秒。 注：反应体积为30微升，以允许暂停PCR程序，并删除一个10微升等分试样在预先确定的周期数来监视外观所需产物。通常情况下，循环10，13，和16被选择，用于从总RNA样品的测试。 </li></ol></li><li>准备通过混合2毫升40％的丙烯酰胺，加入1ml的10×TBE，和H 2 O至10ml 8％非变性聚丙烯酰胺凝胶。然后加入100微升10％APS和10微升TEMED的。倒入凝胶，让集。 <ol><li>运行前的凝胶在凝胶宽度的12伏/厘米30分钟。同花顺井。加入1微升10×加样缓冲液（15％聚蔗糖-400，66毫摩尔EDTA，20毫摩尔的Tris-HCl pH 8.0的，0.1％（重量/体积）SDS，0.01％（重量/体积）溴酚兰）中，添加10微升的样品尺寸的各孔5毫米×1毫米×15毫米。典型地，每个孔可以加载多达30微升的样品。运行示例凝胶在凝胶宽度在1×TBE缓冲液中10伏/厘米，直至溴酚蓝染料刚刚跑出了凝胶的底部。 </li><li>染色，如在步骤2.3中所述可视化凝胶。 </li><li>消费税的146碱基对带和洗脱过夜0.25毫升HSCB在类似于在2.4节中描述的一种时尚。在异丙醇中的等体积的1/10体积乙酸钠使DNA沉淀。洗涤沉淀在70％Ethanol并悬浮在TE缓冲液。 </li><li>使用的PicoGreen根据制造商的建议进行量化的库。 </li></ol></li></ol>

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The authors declare no competing financial interests.

本文所描述的方法利用了几个关键变量最大化连接效率，即高浓度的PEG，用随机接头的，与高浓度的接头2,6,9的。这种方法可以从总RNA样品2可靠的定量测序文库。我们所进行的输入的RNA多步滴定，并得出结论认为，前面的方法是最适合的总RNA的量在1-8微克的范围（数据未显示）。当在10-500纳克范围的量使用，大部分的读取空间由适配器多联体，并从其中所述RNA连接酶被纯化细菌序...

高通量测序基础的方法已被广泛应用到许多生物样品近年来大大扩大我们的生物系统3,4的分子复杂性的理解。然而，制备RNA样品进行高通量测序的常赋予固有的就业方法的具体偏差，限制了这些强大的技术的潜在效用。这些具体的方法偏差得到了很好的证明了结扎为主，小RNA库准备1,2,5,6。这些偏见导致1000倍的变化读取号码等摩尔合成的miRNA，使得miRNA的丰推断，从测序数据疯狂可变且容易出错。 研究着重于噬菌体衍生的T4 RNA连接酶的性质已经证明，酶表现出的基于核苷酸的偏好7，它体现在高通量测序实验作为偏置库<sUP&gt; 1,2,8。为了最小化被RNA连接酶所赋予的偏见，多种策略已经使用;大分子拥挤9中 ，随机化是近端结扎部位6的适配器上的碱基序列，以及使用高浓度的结扎适配器2。通过这三种方法的组合，我们开发了一个工作流的小RNA文库的高通量测序（ 图1）兼容的无偏制剂。对于目前的协议和我们的优化方法进行直接的比较，请参阅最近的一份报告2。这种优化的方法产生的大于95％的在两个3&#39;和5&#39;步骤结扎的效率，并允许小分子RNA合成和生物样品2的无偏结扎。

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			<td style="width:280px;">Comments/Description</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:261px;">3&#39; Linker (5&#39; phosphorylated, 3&#39; blocked)</td>
			<td style="width:175px;">Integrated DNA Technologies</td>
			<td style="width:157px;">custom</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:261px;">5&#39; Linker</td>
			<td style="width:175px;">Integrated DNA Technologies</td>
			<td style="width:157px;">custom</td>
			<td>5&#39; blocked, HPLC Purified</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">T4RNL2 (1-249 K227Q)</td>
			<td style="width:175px;">New England Biolabs</td>
			<td style="width:157px;">M0351S</td>
			<td>Specialized for ligation of pre-adenylated DNA adapters</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">10X Ligation Buffer (without ATP)</td>
			<td style="width:175px;">New England Biolabs</td>
			<td style="width:157px;">Included with M0351S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">10X Ligation Buffer (with ATP)</td>
			<td style="width:175px;">New England Biolabs</td>
			<td style="width:157px;">Included with M0204L</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">RNaseOUT&#160;</td>
			<td style="width:175px;">Invitrogen</td>
			<td style="width:157px;">10777-019</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Polyethylene Glycol (mol. Wt. 8000)</td>
			<td style="width:175px;">New England Biolabs</td>
			<td style="width:157px;">Included with M0204L</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Nuclease-free water</td>
			<td style="width:175px;">Ambion</td>
			<td style="width:157px;">AM9937</td>
			<td>We have found water collected from a distillation apparatus to be of equvalent quality.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">T4RNL1</td>
			<td style="width:175px;">New England Biolabs</td>
			<td style="width:157px;">M0204L</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Superscript III RT kit</td>
			<td style="width:175px;">Invitrogen</td>
			<td style="width:157px;">18080-051&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Phusion PCR kit</td>
			<td style="width:175px;">New England Biolabs</td>
			<td style="width:157px;">M0530S</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:261px;">Illumina RP1 Primer</td>
			<td style="width:175px;">Integrated DNA Technologies</td>
			<td style="width:157px;">custom</td>
			<td>Sequence information available from Illumina</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:261px;">Illumina RT Primer</td>
			<td style="width:175px;">Integrated DNA Technologies</td>
			<td style="width:157px;">custom</td>
			<td>Sequence information available from Illumina</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:261px;">Illumina Index Primer(s)</td>
			<td style="width:175px;">Integrated DNA Technologies</td>
			<td style="width:157px;">custom</td>
			<td>Sequence information available from Illumina</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">40% Acrylamide</td>
			<td style="width:175px;">Fisher Scientific</td>
			<td style="width:157px;">BP14081</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Urea</td>
			<td style="width:175px;">Sigma Aldrich</td>
			<td style="width:157px;">U6504</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Ammonium persulfate</td>
			<td style="width:175px;">Sigma Aldrich</td>
			<td style="width:157px;">A3678</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Tetramethyethylenediamine (TEMED)</td>
			<td style="width:175px;">Sigma Aldrich</td>
			<td style="width:157px;">T9281</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">2X Denaturing RNA loading buffer</td>
			<td style="width:175px;">New England Biolabs</td>
			<td style="width:157px;">Included with M0351S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Razor blades</td>
			<td style="width:175px;">VWR</td>
			<td style="width:157px;">55411-050</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">SpinX Centricon Tubes</td>
			<td style="width:175px;">Costar</td>
			<td style="width:157px;">CLS8161</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Low Retention Microfuge tubes</td>
			<td style="width:175px;">Fisher Scientific</td>
			<td style="width:157px;">02-681-320</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Sybr Gold</td>
			<td style="width:175px;">Invitrogen</td>
			<td style="width:157px;">S-11494</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Adenylation Kit</td>
			<td style="width:175px;">New England Biolabs</td>
			<td style="width:157px;">E2610L</td>
			<td></td>
		</tr>
	</tbody>
</table>

highly efficient ligation of small rna molecules for microrna quantitation by high-throughput sequencing

miRNA的克隆和高通量测序，所谓的miR-SEQ，独树一帜的全基因组范围的方法来量化的miRNA与单核苷酸分辨率。这种技术捕获的miRNAs通过连接3&#39;和5&#39;寡核苷酸衔接头到miRNA分子，并允许从头 miRNA的发现。具有强大的新一代测序平台的耦合中，miR-SEQ一直在miRNA的生物学研究。然而，寡核苷酸连接的步骤介绍显著的偏见阻碍的miR-SEQ被用作一个精确的定量工具。以往的研究表明，在当前的miR-SEQ方法的偏见往往会导致不准确的miRNA定量与错误的高达1000倍的某些miRNA的1,2。要解决这些偏见，通过RNA结扎传授，我们已经开发出一种小RNA结扎方法导致95％以上的为3&#39;和5&#39;连接步骤连接效率。这个基准测试IM证明文库构建方法，使用等摩尔或差分混合的合成的miRNA，始终如一地产生具有与预期值小于2倍的偏差读出的数字。此外，这种高效率的miR-SEQ方法允许从体内总RNA样品2精确的全基因组miRNA表达谱。

预期的结果为前述方法最初应观测的DNA寡核苷酸，这是受腺苷酰化通过第M个 RNA连接酶（ 图2）的尺寸的单核苷酸移位（增加）的。下面的3&#39;连接，将丙烯酰胺凝胶的可视化表示（ 见图3）尖锐的高分子量条带在凝胶中的100-300核苷酸区域明显。这表明，总RNA样品使用是高品质（未劣化）。其次应该看到一个非常明亮的信号凝胶，这是多余的25个核苷酸区域，未连接...

Watch this Scientific Journal Video about Highly Efficient Ligation of Small RNA Molecules for MicroRNA Quantitation by High-Throughput Sequencing at JoVE.com

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

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 ...

highly-efficient-ligation-small-rna-molecules-for-microrna

Research

JoVE Journal

Biology

11.8K 次观看.  University of Colorado, Boulder. 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. 

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

Gramicidin-based Fluorescence Assay; for Determining Small Molecules Potential for Modifying Lipid Bilayer Properties

Many drugs and other small molecules used to modulate biological function are amphiphiles that adsorb at the bilayer/solution interface and thereby alter lipid bilayer properties. This is important because membrane proteins are energetically coupled to their host bilayer by hydrophobic interactions. Changes in bilayer properties thus alter membrane protein function, which provides an indirect way for amphiphiles to modulate protein function and a possible mechanism for "off-target" drug effects. We have previously developed an electrophysiological assay for detecting changes in lipid bilayer properties using linear gramicidin channels as probes 3,12. Gramicidin channels are mini-proteins formed by the transbilayer dimerization of two non-conducting subunits. They are sensitive to changes in their membrane environment, which makes them powerful probes for monitoring changes in lipid bilayer properties as sensed by bilayer spanning proteins. We now demonstrate a fluorescence assay for detecting changes in bilayer properties using the same channels as probes. The assay is based on measuring the time-course of fluorescence quenching from fluorophore-loaded large unilamellar vesicles due to the entry of a quencher through the gramicidin channels. We use the fluorescence indicator/quencher pair 8-aminonaphthalene-1,3,6-trisulfonate (ANTS)/Tl+ that has been successfully used in other fluorescence quenching assays 5,13. Tl+ permeates the lipid bilayer slowly 8 but passes readily through conducting gramicidin channels 1,14. The method is scalable and suitable for both mechanistic studies and high-throughput screening of small molecules for bilayer-perturbing, and potential "off-target", effects. We find that results using this method are in good agreement with previous electrophysiological results 12.

We introduce a fast fluorescence-based assay that monitors the rate of fluorescence quenching as a measure of gramicidin channel activity. The gramicidin channels are used as molecular force transducers to monitor changes in lipid bilayer properties as sensed by bilayer spanning proteins.

短杆菌肽为基础的荧光分析法，确定小分子的修改脂质双层属性的潜在

Many drugs and other small molecules used to modulate biological function are amphiphiles that adsorb at the bilayer/solution interface and thereby alter ...

科研

生物学

High-throughput Screening for Small-molecule Modulators of Inward Rectifier Potassium Channels

Specific members of the inward rectifier potassium (Kir) channel family are postulated drug targets for a variety of disorders, including hypertension, atrial fibrillation, and pain1,2. For the most part, however, progress toward understanding their therapeutic potential or even basic physiological functions has been slowed by the lack of good pharmacological tools. Indeed, the molecular pharmacology of the inward rectifier family has lagged far behind that of the S4 superfamily of voltage-gated potassium (Kv) channels, for which a number of nanomolar-affinity and highly selective peptide toxin modulators have been discovered3. The bee venom toxin tertiapin and its derivatives are potent inhibitors of Kir1.1 and Kir3 channels4,5, but peptides are of limited use therapeutically as well as experimentally due to their antigenic properties and poor bioavailability, metabolic stability and tissue penetrance. The development of potent and selective small-molecule probes with improved pharmacological properties will be a key to fully understanding the physiology and therapeutic potential of Kir channels.
The Molecular Libraries Probes Production Center Network (MLPCN) supported by the National Institutes of Health (NIH) Common Fund has created opportunities for academic scientists to initiate probe discovery campaigns for molecular targets and signaling pathways in need of better pharmacology6. The MLPCN provides researchers access to industry-scale screening centers and medicinal chemistry and informatics support to develop small-molecule probes to elucidate the function of genes and gene networks. The critical step in gaining entry to the MLPCN is the development of a robust target- or pathway-specific assay that is amenable for high-throughput screening (HTS).
Here, we describe how to develop a fluorescence-based thallium (Tl+) flux assay of Kir channel function for high-throughput compound screening7,8,9,10.The assay is based on the permeability of the K+ channel pore to the K+ congener Tl+. A commercially available fluorescent Tl+ reporter dye is used to detect transmembrane flux of Tl+ through the pore. There are at least three commercially available dyes that are suitable for Tl+ flux assays: BTC, FluoZin-2, and FluxOR7,8. This protocol describes assay development using FluoZin-2. Although originally developed and marketed as a zinc indicator, FluoZin-2 exhibits a robust and dose-dependent increase in fluorescence emission upon Tl+ binding. We began working with FluoZin-2 before FluxOR was available7,8 and have continued to do so9,10. However, the steps in assay development are essentially identical for all three dyes, and users should determine which dye is most appropriate for their specific needs. We also discuss the assay's performance benchmarks that must be reached to be considered for entry to the MLPCN. Since Tl+ readily permeates most K+ channels, the assay should be adaptable to most K+ channel targets.

Methods for developing and validating a quantitative fluorescence assay for measuring the activity of inward rectifier potassium (Kir) channels for high-throughput compound screening is presented.

内向整流钾通道的小分子调节剂的高通量筛选

Specific members of the inward rectifier potassium (Kir) channel family are postulated drug targets for a variety of disorders, including hypertension, ...

Substrate Generation for Endonucleases of CRISPR/Cas Systems

The interaction of viruses and their prokaryotic hosts shaped the evolution of bacterial and archaeal life. Prokaryotes developed several strategies to evade viral attacks that include restriction modification, abortive infection and CRISPR/Cas systems. These adaptive immune systems found in many Bacteria and most Archaea consist of clustered regularly interspaced short palindromic repeat (CRISPR) sequences and a number of CRISPR associated (Cas) genes (Fig. 1) 1-3. Different sets of Cas proteins and repeats define at least three major divergent types of CRISPR/Cas systems 4. The universal proteins Cas1 and Cas2 are proposed to be involved in the uptake of viral DNA that will generate a new spacer element between two repeats at the 5' terminus of an extending CRISPR cluster 5. The entire cluster is transcribed into a precursor-crRNA containing all spacer and repeat sequences and is subsequently processed by an enzyme of the diverse Cas6 family into smaller crRNAs 6-8. These crRNAs consist of the spacer sequence flanked by a 5' terminal (8 nucleotides) and a 3' terminal tag derived from the repeat sequence 9. A repeated infection of the virus can now be blocked as the new crRNA will be directed by a Cas protein complex (Cascade) to the viral DNA and identify it as such via base complementarity10. Finally, for CRISPR/Cas type 1 systems, the nuclease Cas3 will destroy the detected invader DNA 11,12 .
These processes define CRISPR/Cas as an adaptive immune system of prokaryotes and opened a fascinating research field for the study of the involved Cas proteins. The function of many Cas proteins is still elusive and the causes for the apparent diversity of the CRISPR/Cas systems remain to be illuminated. Potential activities of most Cas proteins were predicted via detailed computational analyses. A major fraction of Cas proteins are either shown or proposed to function as endonucleases 4.
Here, we present methods to generate crRNAs and precursor-cRNAs for the study of Cas endoribonucleases. Different endonuclease assays require either short repeat sequences that can directly be synthesized as RNA oligonucleotides or longer crRNA and pre-crRNA sequences that are generated via in vitro T7 RNA polymerase run-off transcription. This methodology allows the incorporation of radioactive nucleotides for the generation of internally labeled endonuclease substrates and the creation of synthetic or mutant crRNAs. Cas6 endonuclease activity is utilized to mature pre-crRNAs into crRNAs with 5'-hydroxyl and a 2',3'-cyclic phosphate termini.

CRISPR/Cas systems mediate adaptive immunity in Bacteria and Archaea. Many Cas proteins are proposed to act as endoribonucleases acting on crRNA precursors of varying length. Here we illustrate three different approaches to generate pre-crRNA substrates for the biochemical analysis of Cas endonuclease activity.

CRISPR / CAS系统内切酶的底物生成

The  interaction of viruses and their prokaryotic hosts shaped the evolution of  bacterial and archaeal life. Prokaryotes developed several strategies to ...

RNA Secondary Structure Prediction Using High-throughput SHAPE

Understanding the function of RNA involved in biological processes requires a thorough knowledge of RNA structure. Toward this end, the methodology dubbed "high-throughput selective 2' hydroxyl acylation analyzed by primer extension", or SHAPE, allows prediction of RNA secondary structure with single nucleotide resolution. This approach utilizes chemical probing agents that preferentially acylate single stranded or flexible regions of RNA in aqueous solution. Sites of chemical modification are detected by reverse transcription of the modified RNA, and the products of this reaction are fractionated by automated capillary electrophoresis (CE). Since reverse transcriptase pauses at those RNA nucleotides modified by the SHAPE reagents, the resulting cDNA library indirectly maps those ribonucleotides that are single stranded in the context of the folded RNA. Using ShapeFinder software, the electropherograms produced by automated CE are processed and converted into nucleotide reactivity tables that are themselves converted into pseudo-energy constraints used in the RNAStructure (v5.3) prediction algorithm. The two-dimensional RNA structures obtained by combining SHAPE probing with in silico RNA secondary structure prediction have been found to be far more accurate than structures obtained using either method alone.

High-throughput selective 2' hydroxyl acylation analyzed by primer extension (SHAPE) utilizes a novel chemical probing technology, reverse transcription, capillary electrophoresis and secondary structure prediction software to determine the structures of RNAs from several hundred to several thousand nucleotides at single nucleotide resolution.

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 ...

Generation of High Quality Chromatin Immunoprecipitation DNA Template for High-throughput Sequencing (ChIP-seq)

ChIP-sequencing (ChIP-seq) methods directly offer whole-genome coverage, where combining chromatin immunoprecipitation (ChIP) and massively parallel sequencing can be utilized to identify the repertoire of mammalian DNA sequences bound by transcription factors in vivo. "Next-generation" genome sequencing technologies provide 1-2 orders of magnitude increase in the amount of sequence that can be cost-effectively generated over older technologies thus allowing for ChIP-seq methods to directly provide whole-genome coverage for effective profiling of mammalian protein-DNA interactions.
For successful ChIP-seq approaches, one must generate high quality ChIP DNA template to obtain the best sequencing outcomes. The description is based around experience with the protein product of the gene most strongly implicated in the pathogenesis of type 2 diabetes, namely the transcription factor transcription factor 7-like 2 (TCF7L2). This factor has also been implicated in various cancers.
Outlined is how to generate high quality ChIP DNA template derived from the colorectal carcinoma cell line, HCT116, in order to build a high-resolution map through sequencing to determine the genes bound by TCF7L2, giving further insight in to its key role in the pathogenesis of complex traits.

Generation of High Quality Chromatin Immunoprecipitation DNA Template for High-throughput Sequencing ChIP-seq

The combination of chromatin immunoprecipitation and ultra-high-throughput sequencing (ChIP-seq) can identify and map protein-DNA interactions in a given tissue or cell line. Outlined is how to generate a high quality ChIP template for subsequent sequencing, using experience with the transcription factor TCF7L2 as an example.

代高通量测序技术（ChIP-seq的高品质的染色质免疫沉淀DNA模板）

ChIP-sequencing (ChIP-seq) methods directly  offer whole-genome coverage, where combining chromatin immunoprecipitation  (ChIP) and massively parallel ...

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 ...

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 ...

Automated Modular High Throughput Exopolysaccharide Screening Platform Coupled with Highly Sensitive Carbohydrate Fingerprint Analysis

Many microorganisms are capable of producing and secreting exopolysaccharides (EPS), which have important implications in medical fields, food applications or in the replacement of petro-based chemicals. We describe an analytical platform to be automated on a liquid handling system that allows the fast and reliable analysis of the type and the amount of EPS produced by microorganisms. It enables the user to identify novel natural microbial exopolysaccharide producers and to analyze the carbohydrate fingerprint of the corresponding polymers within one day in high-throughput (HT). Using this platform, strain collections as well as libraries of strain variants that might be obtained in engineering approaches can be screened. The platform has a modular setup, which allows a separation of the protocol into two major parts. First, there is an automated screening system, which combines different polysaccharide detection modules: a semi-quantitative analysis of viscosity formation via a centrifugation step, an analysis of polymer formation via alcohol precipitation and the determination of the total carbohydrate content via a phenol-sulfuric-acid transformation. Here, it is possible to screen up to 384 strains per run. The second part provides a detailed monosaccharide analysis for all the selected EPS producers identified in the first part by combining two essential modules: the analysis of the complete monomer composition via ultra-high performance liquid chromatography coupled with ultra violet and electrospray ionization ion trap detection (UHPLC-UV-ESI-MS) and the determination of pyruvate as a polymer substituent (presence of pyruvate ketal) via enzymatic oxidation that is coupled to a color formation. All the analytical modules of this screening platform can be combined in different ways and adjusted to individual requirements. Additionally, they can all be handled manually or performed with a liquid handling system. Thereby, the screening platform enables a huge flexibility in order to identify various EPS.

We present an automated modular high-throughput-method for the identification and characterization of microbial exopolysaccharides in small scale. This method combines a fast preselection to analyze the total amount of secreted polysaccharides with a detailed carbohydrate fingerprint to enable the fast screening of newly isolated bacterial strains or entire strain collections.

再加上高灵敏度指纹碳水化合物自动分析模块化高通量筛选胞外多糖平台

Many microorganisms are capable of producing and secreting exopolysaccharides (EPS), which have important implications in medical fields, food ...

DamID-seq: Genome-wide Mapping of Protein-DNA Interactions by High Throughput Sequencing of Adenine-methylated DNA Fragments

The DNA adenine methyltransferase identification (DamID) assay is a powerful method to detect protein-DNA interactions both locally and genome-wide. It is an alternative approach to chromatin immunoprecipitation (ChIP). An expressed fusion protein consisting of the protein of interest and the E. coli DNA adenine methyltransferase can methylate the adenine base in GATC motifs near the sites of protein-DNA interactions. Adenine-methylated DNA fragments can then be specifically amplified and detected. The original DamID assay detects the genomic locations of methylated DNA fragments by hybridization to DNA microarrays, which is limited by the availability of microarrays and the density of predetermined probes. In this paper, we report the detailed protocol of integrating high throughput DNA sequencing into DamID (DamID-seq). The large number of short reads generated from DamID-seq enables detecting and localizing protein-DNA interactions genome-wide with high precision and sensitivity. We have used the DamID-seq assay to study genome-nuclear lamina (NL) interactions in mammalian cells, and have noticed that DamID-seq provides a high resolution and a wide dynamic range in detecting genome-NL interactions. The DamID-seq approach enables probing NL associations within gene structures and allows comparing genome-NL interaction maps with other functional genomic data, such as ChIP-seq and RNA-seq.

We describe herein an assay by coupling DNA adenine methyltransferase identification (DamID) to high throughput sequencing (DamID-seq). This improved method provides a higher resolution and a wider dynamic range, and allows analyzing DamID-seq data in conjunction with other high throughput sequencing data such as ChIP-seq, RNA-seq, etc.

DamID-SEQ：蛋白质-DNA相互作用的全基因组映射腺嘌呤甲基化的DNA片段通过高通量测序

The DNA adenine methyltransferase identification (DamID) assay is a powerful method to detect protein-DNA interactions both locally and genome-wide. It is ...

A Protocol for Functional Assessment of Whole-Protein Saturation Mutagenesis Libraries Utilizing High-Throughput Sequencing

Site-directed mutagenesis has long been used as a method to interrogate protein structure, function and evolution. Recent advances in massively-parallel sequencing technology have opened up the possibility of assessing the functional or fitness effects of large numbers of mutations simultaneously. Here, we present a protocol for experimentally determining the effects of all possible single amino acid mutations in a protein of interest utilizing high-throughput sequencing technology, using the 263 amino acid antibiotic resistance enzyme TEM-1 &#946;-lactamase as an example. In this approach, a whole-protein saturation mutagenesis library is constructed by site-directed mutagenic PCR, randomizing each position individually to all possible amino acids. The library is then transformed into bacteria, and selected for the ability to confer resistance to &#946;-lactam antibiotics. The fitness effect of each mutation is then determined by deep sequencing of the library before and after selection. Importantly, this protocol introduces methods which maximize sequencing read depth and permit the simultaneous selection of the entire mutation library, by mixing adjacent positions into groups of length accommodated by high-throughput sequencing read length and utilizing orthogonal primers to barcode each group. Representative results using this protocol are provided by assessing the fitness effects of all single amino acid mutations in TEM-1 at a clinically relevant dosage of ampicillin. The method should be easily extendable to other proteins for which a high-throughput selection assay is in place.

We present a protocol for the functional assessment of comprehensive single-site saturation mutagenesis libraries of proteins utilizing high-throughput sequencing. Importantly, this approach uses orthogonal primer pairs to multiplex library construction and sequencing. Representative results using TEM-1 &#946;-lactamase selected at a clinically relevant dosage of ampicillin are provided.

经过一蛋白饱和诱变图书馆的功能评价规范利用高通量测序

Site-directed mutagenesis has long been used as a method to interrogate protein structure, function and evolution. Recent advances in massively-parallel ...