Name: protocol for the solid-phase synthesis of oligomers of rna containing a 2'-o-thiophenylmethyl modification and characterization via circular dichroism
Uploaded: 2017-07-28T15:00:00.000+00:00
Duration: 11 min 37 s
Description: Watch this Scientific Journal Video about Protocol for the Solid-phase Synthesis of Oligomers of RNA Containing a 2'-O-thiophenylmethyl Modification and Characterization via Circular Dichroism at JoVE

这份手稿的准备工作得到了科罗拉多州丹佛大学（JMRE）的启动资金的支持。 AF希望得到研究和创意活动奖（RaCAS，CU Denver）的支持。克罗地亚科罗拉多大学研究服务处资助出版费用得到承认。我们要感谢实验室成员Cassandra Herbert女士和Yannick K. Dzowo先生在视频部分的贡献。

1. RNA寡聚核苷酸的固相合成<ol><li> 制备含有亚磷酰胺的溶液（表1）。 <ol><li>计算核苷酸数量，并适合n + 1方程（其中n =核苷酸数目）对应于每个碱基，并填写缺失值的表格。体积= 0.15mL /核苷酸，浓度为0.1M，溶于无水乙腈。 </li><li>将每个亚磷酰胺称量到烘箱干燥的10毫升琥珀色瓶（隔垫顶部琥珀色394阿米申13毫米ID×20毫米OD顶部）中，并使用含有干燥剂的干燥器立即置于真空下。 </li><li>用干燥氩气填充干燥器，取出瓶子并立即用相应量的乙腈（无水）稀释，然后再固定在仪器上。 注意:确保使用气密注射器，并始终保持无水的气氛。 </li><li>从瓶子上取下隔垫并放在m上通过瓶子更换功能。 注意:该方法需要在合成之前补充每个溶液0.15mL（因此在上述n + 1方程中的过量体积）引出线。 </li></ol></li><li> 使用软件设置序列和固相合成（ 图3 ）。 <ol><li>单击软件图标（ 例如 ，OligoNet 1.0.1），然后选择仪器名称&gt;确定 。通过File&gt; New Synthesis&gt; Order创建一个新的综合。 </li><li>填写日期runid为;选择仪器;序列名称序列（5&#39;-to-3&#39;end）。在|循环下，分配先前创建的方法（ 图3 ）。选择结束程序（留下与CPG树脂结合的寡核苷酸）&gt;手册。 </li><li>选择DMT Off&gt;（TCA处理在最后一步获得5&#39;-OH基团合成结束） ;保存文件&gt;另存为并提供实验的名称。 </li><li>发送文件开始综合订单&gt;发送订单到合成器并选择列 1-4 。打开合成器窗口，选择三位一体监视器|选择功能|三位一体&gt;监控每一步 。 </li><li>根据需要合成的寡核苷酸数量，重复上述步骤（请注意，所有订单必须具有相同的方法， 即相同的耦合时间和事件顺序）。 </li><li>开始合成Synthesizer&gt;准备开始 。将所需的3&#39;末端柱放在仪器上指示的位置。在仪器上单击开始&gt;否 （用于ABI准备）。 </li><li>一旦合成完成，从仪器中取出色谱柱，放入圆底烧瓶中，然后减压干燥约0.5小时。 注意:建议检查在兰德里是否有深橙色om耦合（步骤1.2.4 - 1.2.7），以避免三位一体监视功能的潜在错误。 </li></ol></li><li> RNA寡核苷酸的脱保护和纯化。 <ol><li>通过扭转黑色盖子将白色树脂的所有（或一半，根据需要或目的））转移到1.6 mL离心管中，打开色谱柱。以1:1的比例加入0.5mL甲胺（40％水溶液）/氨（40％水溶液）。 </li><li>用石蜡膜固定离心管盖，并使用热块将其加热至60°C 1.5 h。 注意:重物可放置在管的顶部，以确保反应管内的氨浓度保持不变。 </li><li>从热块中取出并慢慢冷却至室温。将样品简单离心以旋转内容物，然后将上清液转移到新的离心管中。 </li><li>将试管浸入液氮（或干冰/乙醇浴）中冷冻样品，同时旋转干燥在离心机中减压。 </li><li>将固体重悬于0.4mL三乙胺/ N-甲基吡咯烷酮/三乙胺 - 三氢氟酸（2:2:3比例）溶液中。加热至60℃1.5小时，然后缓慢冷却至室温。 </li><li>加入0.04mL NaOAc溶液（3M，pH 5.5，用HCl调节），然后用移液管尖端轻轻搅拌。加入乙醇（1 mL），冷却至约-70℃（干冰/乙醇浴）15分钟。 </li><li>以15,000rpm和4℃离心10分钟。使用移液管提取等分试样，并在减压下干燥所得颗粒。 </li><li>将所得固体重新悬浮在加载缓冲液（0.2mL，90％甲酰胺，1mM EDTA）中，并混合直到混合物均匀。 </li><li>将悬浮液装载到预先制备的聚丙烯酰胺凝胶上（20％变性，孔尺寸:30cm×1mm） 22 。 </li><li>通过凝胶施加电流，直到溴酚蓝标记位于1 / 2-2 / 3之间凝胶（凝胶尺寸:约21.5厘米×30厘米）。 </li><li>从支架上取出凝胶，将凝胶内容物从玻璃转移到塑料包装（双面覆盖）上，放在覆盖有二氧化硅（含有254nm荧光染料）的薄层色谱（TLC）板上以观察条带使用紫外灯（ λmax = 254nm）。 </li><li>使用标记来描绘上方所在的位置，并使用新的剃须刀切割。将含有RNA（无塑料包装）的凝胶放入50mL锥形管中，并用玻璃棒压碎成小块。 </li><li>将凝胶残留物悬浮于NaCl水溶液中。溶液（2mM和1mM EDTA），并将悬浮液在37℃下摇动12小时。将锥形管离心10分钟。 </li><li>通过使用反相C18色谱柱脱盐。 <ol><li>使用10 mL注射器，通过以下方式洗涤，准备墨盒: 乙腈（10mL） H 2 O（两次，10mL） 5mM NH4氯化钠溶液（3mL） 含有RNA的溶液，注意不要倒入任何凝胶残留物。 </li><li>用H 2 O洗涤（3次，10 mL）。 </li><li>使用60％的水溶液从柱中洗脱。甲醇溶液（3mL） </li></ol></li><li>在减压下浓缩并重新溶解于无RNase的水（0.3mL） </li><li>准备稀释溶液（10μL），并在UV-vis仪器（ 如 Nanodrop）上沉积1μL，以测量紫外 - 可见光谱（200-450 nm）。 </li><li>使用啤酒朗伯定律来确定所得溶液的浓度: <img alt="公式1" src="/files/ftp_upload/56189/56189eq1.jpg" /> 其中A =获得的吸光度; ε =计算摩尔消光系数; c =浓度，对于1mm的路径长度， l = 0.1。 </li><li>计算寡核苷酸的摩尔消光系数;在这里用在线计算器计算使用DINAMelt软件（http://unafold.ra.albany.edu/?q=dinamelt）23 </li></ol></li><li> 通过MALDI-TOF进行浓度测定和表征。 <ol><li>将样品加载到MALDI平板上，使用装有C18端头的移液管末端进行脱盐，并点出每个寡核苷酸。 <ol><li>用50％乙腈（10μLx 2）清洗尖端。用0.1％三氟乙酸（TFA;10μL×2）平衡末端。加载样品（通常为100-150 pmol）。 </li><li>用0.1％TFA（10μLx 2），然后用水（10μLx 2）清洗末端。 </li><li>将样品洗脱到含有所需基质的溶液中;我们使用10μL25mM-2,4,6-三羟基苯乙酮一水合物（THAP），10mM柠檬酸铵和300mM氟化铵在50％乙腈中的溶液。 </li><li>通过沉积0.9μL（随后空气干燥）直接点到MALDI板上，并根据需要重复该过程。 注意:所有光谱均在反射器正模式下获得。 </li></ol></li></ol></li></ol> 2. 通过 CD进行RNA结构分析<ol><li> CD的解决方案的准备。 <ol><li>制备含有修饰的RNA [3μM]，NaCl [10mM]，磷酸钠缓冲液[10mM，pH7.2]和MgCl 2 [5mM]的0.25mL。样品准备好分析，特别是如果它代表单分子转换。 </li><li>如果目标是分析双相结构或双分子转换，则此时添加补体（如果适用，为1摩尔当量），或继续下面的步骤。 </li><li>将样品放在热块上，预热至90°C，关闭热量以控制缓慢冷却至室温（典型值为2-4h）。 </li></ol></li><li> 光谱采集 <ol><li>制备空白溶液（NaCl 10mM，磷酸钠10mM pH 7.3，MgCl 2 5mM），转移toa微量比色皿（1cm路径长度，250μL最小体积），并置于仪器内的样品更换器的保持器位置。 </li><li>将含有RNA的样品转移到另一个微量比色皿中，并置于样品更换器中。如果测量热变性转变，请小心地添加一层油，而不会中断水层，并使用一块特氟龙胶带固定比色杯盖。 </li><li>打开氮气罐以提供将位于仪器上的空气浮子移动到大约的流量。打开冷却器。 </li><li>打开仪器并打开软件Spectra Manager图标，然后打开采集窗口光谱测量 。 </li><li>在采集前用氮气吹扫仪器5分钟。 </li><li>使用以下参数测量&gt;参数获取光谱，并按如下所示调整参数: <ol><li>在常规下 ，选择:扫描200-350 nm;频道CD和HT:数据间距为0.1nm;扫描速度为100 nm / min;带宽为1nm;累计数量为5。 </li><li>在电池单元下 ，选择20°C。在控制下 ，选择快门自动打开和关闭。在信息下 ，选择名称，浓度，运算符。 </li><li>在数据下 ，浏览到所需的文件夹。单击确定 。 </li></ol></li><li>获取光谱测量&gt;样品测量 ，相应地识别样品转换器1-6中的比色杯的位置，单击确定 。 </li></ol></li><li> 如果进行热变性转变: <ol><li>关闭采集软件文件&gt;退出并打开新程序可变温度测量&gt;测量&gt;参数 。 </li><li>应用以下参数记录热转换，可根据需要进行调整: <ol><li>在温度下 ，选择开始温度:4°C;间隔:0.2; TArget:90°C;梯度:1℃/ min;等待0秒。在开始/结束下，根据需要选择启动条件和结束条件。 </li><li>在一般情况下 ，选择3个通道，CD / HT / Abs;带宽:1nm;波长:270nm（或根据需要）。在控制下 ，选择无。在" 信息"下 ，选择名称，浓度，运算符。 </li><li>在数据下 ，浏览到所需的文件夹。单击确定 。 </li></ol></li><li>将样品冷却至4°C，然后在此温度下等待5 min，然后再进行光谱测量。 </li></ol></li><li> 光谱数据处理。 <ol><li>使用软件上的功能，从目标采集中减去空白频谱，以考虑从使用的缓冲系统产生的背景信号: 光谱分析&gt;文件&gt;打开 。 </li><li>一旦打开了空白文件和光谱文件，请在View 1（屏幕左侧）下拖动View 2。 </li><li>点击Pr加权&gt;减法&gt;确定或交换数据&gt;确定 。 </li><li>将数据提取为ASCII文件: 文件&gt;导出并选择ASCII 。 </li><li>使用软件绘制相应的光谱。在信噪比低于预期的情况下，数据平滑是可选的。可以通过应用平滑数据选项来应用此转换。 </li></ol></li><li> 热变性转变的数据处理。 <ol><li>从JASCO文件中提取数据作为ASCII文件。 </li><li>绘制椭圆率作为温度的函数。通常，根据正在检查的波长，需要平滑数据。在一些情况下，使用210nm波长的图形显示出需要平滑的曲线之间的较大变化。然而，根据样品和浓度，使用原始数据进行了一些计算。 </li><li>计算热值固化转变（T m ）值，获得曲线的一阶导数。最大值或最小值是该参数的指示。如上一步，有些情况需要平滑数据以获得最准确的值。 注意:实验通常一式三份进行，这导致测量的相应平均值和标准偏差。 </li></ol></li></ol>

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E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biophysical+properties+and+thermal+stability+of+oligonucleotides+of+RNA+containing+7,8-dihydro-8-hydroxyadenosine.">Biophysical properties and thermal stability of oligonucleotides of RNA containing 7,8-dihydro-8-hydroxyadenosine.</a> Biopolymers. 103 (3), 167-174 (2015).</li><li>Maniatis, T., Fritsch, E. F., Sambrook, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Molecular cloning: a laboratory manual. 14, 173-185 (1987).</li><li>Markham, N. R., Zuker, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=UNAFold:+software+for+nucleic+acid+folding+and+hybridization.">UNAFold: software for nucleic acid folding and hybridization.</a> Methods Mol Biol. 453, 3-31 (2008).</li><li>Breslow, R., Huang, D. -. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+metal+ions+including+Mg2++and+lanthanides+on+the+cleavage+of+ribonucleotides+and+RNA+model+compounds.">Effects of metal ions including Mg2+ and lanthanides on the cleavage of ribonucleotides and RNA model compounds.</a> Proc Natl Acad Sci U S A. 88 (10), 4080-4083 (1991).</li><li>Blumberg, D. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Creating+a+ribonuclease-free+environment.">Creating a ribonuclease-free environment.</a> Methods Enzymol. 152 (2), 20-24 (1987).</li><li>AbouHaidar, M. G., Ivanov, I. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-enzymatic+RNA+promoted+by+the+combined+catalytic+activity+of+buffers+and+magnesium+ions.">Non-enzymatic RNA promoted by the combined catalytic activity of buffers and magnesium ions.</a> Z Naturforsch C. 54 (7-8), 542-548 (1999).</li><li>Farrell, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> RNA Methodologies (4th Ed): A laboratory guide for isolation and characterization. 2, 45-80 (2010).</li></ol>

作者没有什么可以披露的。

该手稿的目的是作为领域研究人员，初学者或专家的指导，成功实现或增强DNA或RNA寡核苷酸的合成。所描述的方法侧重于使用通过标准亚磷酰胺化学的自动化DNA / RNA合成仪的固相合成。该报告描述了RNA十二聚体的合成，纯化和表征的逐步描述。此外，使用CD来识别二级结构基序和热变性转变。 重要的是要注意，这项工作可以适应其他情况， 即固相合成中的各种品牌的?...

固相合成，以获得DNA的寡核苷酸/ RNA是一个功能强大的工具，已经使用亚磷酰胺构建模块4自1970年代以来1，2，3提供在各种领域中的应用的几个。其广泛的影响的例子包括:其在标记的影响（ 通过点击化学反应）5，结构探测6，和反义技术的图7，以及其生物机制8,9，源阐明作为遗传物质10，和的研究各种天然的和/或化学修饰11，12，许多others.The修改中，我们在这里使用代表了我们的努力，以获得含有RNA的寡核苷酸的第一步光活性探针，能够对这一重要生物聚合物的结构和功能进行时间​​控制。 具有序列5&#39; - [CUA CG G A AU CAU] -3&#39;/ 5&#39; - [AUG AUU CCG UAG] -3&#39;的RNA十二烷基化物的合成（下划线位置表示C2&#39;- O-硫代苯基甲基修饰）构成了本研究的重点。选择序列以使RNA链的量化和测量成为单链，或者作为其相应的双链体结构（没有其他二级结构被预测为热力学稳定的）。 CD用于建立结构参数， 即双相形成和热变性转变。 合成 获得这些寡核苷酸的总体程序如图1所示，并遵循逐步的方法:自动化固相合成→Deprotection→净化→定量→表征。 图2显示了该过程中必需的单体单元。 RNA的固相合成类似于DNA，因为它基于亚磷酰胺化学（ 图2左侧）和在G，A和C上用于亲核环状胺的碱不稳定保护基团， 例如 ，乙酰基，苯甲酰基，苯氧基乙酰基， 叔丁基或N ， N-二甲基甲酰胺（ 图2 ，右）。由于存在C2&#39;-OH基团（缺乏脱氧寡核苷酸生物聚合物），RNA需要考虑的另一个方面是额外的步骤，必须并入其中以保护和随后的脱亲维护该亲核位置。在这方面，硅基保护基由于其作为双正交部分的潜力而变得有吸引力的策略（特异性的在氟化物存在下脱保护）， 叔丁基二甲基甲硅烷基（TBDMS）和三异丙基甲硅烷氧基甲基 （TOM）作为流行选择（ 图2左下方）。 在这项工作中，自动合成在使用标准亚磷酰胺化学的DNA / RNA合成仪上进行。仪器上的制造商设置包括使用商业版本的亚磷酰胺用于DNA的自动稀释步骤，或者用户稀释的选项。然而，我们决定称量RNA亚磷酰胺并手动稀释，因为:1）RNA的规范亚磷酰胺的价格较高（在某些情况下高达50倍）; 2）经修饰的亚磷酰胺通常少量得到;和3）使用自动稀释步骤（由制造商设定）时浪费的材料的量很大。此外，我们使用:1）市售的固体支持物（ 例如 ，CPG），其含有受保护的核碱基以用作3&#39;-末端;和2）在C2&#39;- O位置用TBDMS基团保护的商业亚磷酰胺（规范核碱基）。合成步骤的详细列表在图3和表1中提供 ，以及针对RNA合成调整的步骤的进一步描述和评论。此外， 图4示出了在选择"三位一体监测器"选项之后对于每个步骤观察到的逐步产量，其定量从每个去离子化步骤释放的三苯甲基。 值得注意的是，在我们的经验中，限制因素是获得含有所需改性的亚磷酰胺。也就是说，开发一种允许在选定地点纳入修改的综合方法。在本报告中，我们专注于掺入修饰的核苷酸，我们已经建立了相应的合成方法，即C2&#39;- O-硫代苯甲基。该组的尺寸小，并且不以任何方式影响固相合成。由于已经报道了将该基团并入RNA的寡核苷酸中，连同结构和热力学参数4 ，本文将描述导致经修饰的亚磷酰胺的有机合成的任何方面。 脱保护，纯化和表征 环外胺和β-氰基乙基的去保护在与CPG-树脂切割相同的步骤中发生。我们应用了在AMA水溶液存在下加热所得树脂的常用条件，然后在氟离子存在下裂解C2&#39;- O-甲硅烷基，然后通过凝胶纯化电泳。虽然这些已经成为在许多情况下的标准条件，其是不稳定的碱性条件或氟离子可能要求更温和的条件13，14， 例如 ，碳酸甲醇/钾（甲醇/ K 2 CO 3），或丁胺修改。因此，在相应的亚磷酰胺上必须有一组不同的保护基团。此外，鉴于我们以前使用该方法的经验和缺乏其他仪器，我们选择电泳作为纯化脱保护的寡聚体的首选替代物。然而，HPLC可以替代地用作有效的方法15 。通过质谱法，基质辅助激光解吸/电离 - 飞行时间（MALDI-TOF）进行纯化寡核苷酸的表征，使用我们组16报道的方法。 结构表征和其他<html> 通过 CD进行获得的双链体的稳定性。具体地说，我们利用CD来测定经修饰和未修饰的RNA寡核苷酸的热变性转变，遵循在约束下带的椭圆率的降低。 270个纳米，以及带的消失（与负椭圆率）与λ 最大 210nm处。提供杂交前后的光谱比较，以说明其差异并提供所采用的方法的验证。 CD的使用在核酸和氨基酸17中的结构基序的测定中被广泛接受，因此可用作确定各种结构和热力学参数的工具18 ;然而，没有多少例子用于评估热变性过渡的技术。一些情况包括测定含有G-四链体DNA的DNA的热稳定性屁股="外部参照"&gt; 19,20或双链体RNA和21的发夹。 本报告旨在为非专家读者或观众提供一系列工具，以便顺利开始此类研究。这将有助于加强和比较其他研究实验室的方法和技术，这些研究实验室参与了这个令人兴奋的科学分支。本报告的内容从各种来源中增加了该技术的现有协议，并丰富和便利了每一步视觉辅助的体验。

<table><tbody><tr><td>AbsolveTM</td><td>PerkinElmer</td><td>6NE9711</td><td></td></tr><tr><td>Acetonitrile 99.9%, HPLC Grade</td><td>Fisher Scientific</td><td>75-05-8</td><td></td></tr><tr><td>Acetonitrile 99.9%, anhydrous for DNA sequencing</td><td>Fisher BioReagents</td><td>75-05-8</td><td></td></tr><tr><td>Acrylamide, 99+%</td><td>ACROS Organics</td><td>164850025</td><td></td></tr><tr><td>Ammonium chloride 98+%</td><td>Alfa Aesar</td><td>12125-02-9</td><td></td></tr><tr><td>Ammonium citrate, dibasic 98%</td><td>Sigma Aldrich</td><td>3012-65-5</td><td></td></tr><tr><td>Ammonium Fluoride&amp;nbsp; 98.0%, ACS grade</td><td>Alfa Aesar</td><td>12125-01-8</td><td></td></tr><tr><td>Ammonium hydroxide 28 - 30% in water, ACS Plus</td><td>Fisher Chemical</td><td>1336-21-6</td><td></td></tr><tr><td>Ammonium persulfate</td><td>ACROS Organics</td><td>1444</td><td></td></tr><tr><td>Argon-ultra high purity</td><td>&amp;nbsp;Airgas</td><td>7440-37-1</td><td></td></tr><tr><td>Bis-acrylamide Ultra pure</td><td>VWR-Amresco</td><td>172</td><td></td></tr><tr><td>Boric Acid</td><td>Fisher Scientific</td><td>A73-1</td><td></td></tr><tr><td>Diethyl pyrocarbonate, 97%</td><td>ACROS Organics</td><td>A0368487</td><td></td></tr><tr><td>Ethanol, anhydrous, histological grade</td><td>Fisher Chemical</td><td>64-17-5</td><td></td></tr><tr><td>Ethylenediamine tetraacetic acid, disodium salt dehydrate 100.2%</td><td>Fisher Chemical</td><td>6381-92-6</td><td></td></tr><tr><td>Formamide&amp;nbsp;</td><td>Thermo Scientific</td><td>75-12-7</td><td></td></tr><tr><td>Hydrochloric acid, 36.5 - 38.0%, Certified ACS Plus</td><td>Fisher Chemical</td><td>7647-01-0</td><td></td></tr><tr><td>Magnesium chloride hexahydrate, 99%</td><td>Fisher Scientific</td><td>7786-30-3</td><td></td></tr><tr><td>Methanol, 99.9%,&amp;nbsp; HPLC Grade</td><td>Fisher Chemical</td><td>67-56-1</td><td></td></tr><tr><td>Methylamine 40% in water</td><td>Sigma Aldrich</td><td>74-89-5</td><td></td></tr><tr><td>1-Methyl-2-pyrrolidinone, andhydrous, 99.5%</td><td>Aldrich</td><td>872-50-4</td><td></td></tr><tr><td>Opti-TOFTM 96 Well Insert (123 x 81 mm)&amp;nbsp;</td><td>MDS SCIEX</td><td>1020157</td><td></td></tr><tr><td>RNase Away</td><td>Molecular BioProducts</td><td>7005-11</td><td></td></tr><tr><td>Sodium acetate, anhydrous 99.2%, Certified ACS</td><td>Fisher Chemical</td><td>127-09-3</td><td></td></tr><tr><td>Sodium chloride, 100.5%, Certified ACS</td><td>Fisher Chemical</td><td>7647-14-5</td><td></td></tr><tr><td>Sodium phosphate monobasic dihydrate 99.0%</td><td>Sigma</td><td>13472-35-0</td><td></td></tr><tr><td>2&amp;rsquo;,4&amp;rsquo; Triethylamine, 99+%</td><td>Alfa Aesar</td><td>121-44-8</td><td></td></tr><tr><td>TEMED</td><td>Amresco</td><td>761</td><td></td></tr><tr><td>Triethylamine trihydrofluoride, 98%</td><td>Aldrich</td><td>73602-61-6</td><td></td></tr><tr><td>Trifluoroacetic acid, 99%</td><td>Alfa Aesar</td><td>76-05-1</td><td></td></tr><tr><td>6&amp;rsquo;-Trihydroxyacetophenone monohydrate 98%</td><td>Sigma Aldrich</td><td>480-66-0</td><td></td></tr><tr><td>Tris Base</td><td>Fisher Scientific</td><td>BP154-3</td><td></td></tr><tr><td>Urea</td><td>Fisher Scientific</td><td>U15-3</td><td></td></tr><tr><td>&lt;strong&gt;Reagents for the RNA synthesis:&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Deblocking mix, 3% trichloroacetic acid in dichloromethane</td><td>Glen Research</td><td>&amp;nbsp;40-4140-57</td><td></td></tr><tr><td>Cap Mix A, THF/Pyridine/Acetic anhydride</td><td>Glen Research</td><td>40-4110-52</td><td></td></tr><tr><td>Cap Mix B, 10% 1-methylimidazole in THF</td><td>Glen Research</td><td>40-4120-52</td><td></td></tr><tr><td>Activator, 0.25 M 5-ethylthio-1H-tetrazole in anhydrous acetonitrile</td><td>Glen Research</td><td>&amp;nbsp;30-3140-52</td><td></td></tr><tr><td>Oxidizing Solution, 0.02 M iodine in THF/Pyridine/Water</td><td>Glen Research</td><td>40-4330-52</td><td></td></tr><tr><td>U-RNA-CPG</td><td>Glen Research</td><td>20-3330-xx</td><td></td></tr><tr><td>Ac-G-RNA-CPG&amp;nbsp;</td><td>Glen Research</td><td>20-3324-xx</td><td></td></tr><tr><td>Ac-G-CE Phosphoramidite&amp;nbsp;</td><td>Glen Research</td><td>10-3025-xx</td><td></td></tr><tr><td>U-CE Phosphoramidite</td><td>Glen Research</td><td>10-3030-xx</td><td></td></tr><tr><td>Ac-C-CE Phosphoramidite</td><td>Glen Research</td><td>10-3015-xx</td><td></td></tr><tr><td>Bz-A-CE Phosphoramidite&amp;nbsp;</td><td>Glen Research</td><td>10-3003-xx</td><td></td></tr></tbody></table>

protocol for the solid-phase synthesis of oligomers of rna containing a 2'-o-thiophenylmethyl modification and characterization via circular dichroism

已经使用固相合成来获得特定于DNA或RNA的核酸的规范和修饰的聚合物，其已经成为各种领域和不同研究目的的应用的流行方法。本文所描述的过程集中于合成，纯化和RNA 5的十二聚体的表征" - [CUA CGG AAU CAU] -3"含位于C2&#39;-ø -位零个，一个或两个的修改。探针基于2-噻吩基甲基，通过标准有机合成并入RNA核苷酸，并通过其各自的亚磷酰胺引入相应的寡核苷酸。本报告通过四种典型的核碱基（尿苷（U），胞嘧啶（C），鸟苷（G），腺苷（A））以及在2&#39;- O-氨基酸修饰的2-噻吩基甲基官能化核苷酸，位置;然而，该方法适用于一个大的变量多年来一直发展起来的各种修改。在受控孔玻璃（CPG）载体上合成寡核苷酸，然后在标准条件下， 即氨和甲胺（AMA），然后是氟化氢/三乙胺/ N-甲基吡咯烷酮的混合物从树脂上裂解并脱保护。相应的寡核苷酸通过经由反相色谱（的Sep-Pak，C 18 -column），接着洗脱，脱盐，并进行隔离聚丙烯酰胺电泳（20％变性）纯化。通过紫外 - 可见（UV-vis）和圆二色性（CD）光度分析分别评估定量和结构参数。本报告旨在为有兴趣参与这一领域的初学者和专家研究人员提供资源和指导。随着新技术和方法的发展，预计将作为一项正在进行的工作。对thi内的方法和技术的描述s文件对应于使用亚磷酰胺化学的DNA / RNA合成仪（在2013年翻新并购买）。

描述了在C2&#39;- O-位上含有0,1或2个2-噻吩基甲基修饰的RNA十二烷基化物的合成以及其相应的纯化和表征。此外，还包括通过CD进行的结构分析的详细描述。 通过固相合成获得四条RNA（包括具有互补序列的链），随后每个寡核苷酸的纯度在300-700nmol之间。通过脱盐〜150 pmol的每个样品进行质谱，然后沉积在平板上用于MALDI-TOF...

Watch this Scientific Journal Video about Protocol for the Solid-phase Synthesis of Oligomers of RNA Containing a 2'-O-thiophenylmethyl Modification and Characterization via Circular Dichroism at JoVE

Protocol for the Solid-phase Synthesis of Oligomers of RNA Containing a 2'-O-thiophenylmethyl Modification and Characterization via Circular Dichroism

本文提供了在C2&#39;- O位上修饰的RNA的十二聚体的固相合成，纯化和表征的详细程序。 UV-vis和圆二色性光度分析用于量化和表征结构方面， 即单链或双链。

用于固相合成含有2&#39;-<em&gt; 0</em通过圆二色性修饰和表征

Solid-phase synthesis has been used to obtain canonical and modified polymers of nucleic acids, specifically of DNA or RNA, which has made it a popular ...

protocol-for-solid-phase-synthesis-oligomers-rna-containing-2-o

Universidad San Sebastian

Research

JoVE Journal

Chemistry

Protocol for the Solid-phase Synthesis of Oligomers of RNA Containing a 2'-O-thiophenylmethyl Modification and Characterization via Circular Dichroism

18.9K 次观看.  University of Colorado Denver. 本文提供了在C2'- O位上修饰的RNA的十二聚体的固相合成，纯化和表征的详细程序。 UV-vis和圆二色性光度分析用于量化和表征结构方面， 即单链或双链。 

视频: Protocol for the Solid-phase Synthesis of Oligomers of RNA Containing a 2'-O-thiophenylmethyl Modification and Characterization via Circular Dichroism

Nucleoside Triphosphates - From Synthesis to Biochemical Characterization

The traditional strategy for the introduction of chemical functionalities is the use of solid-phase synthesis by appending suitably modified phosphoramidite precursors to the nascent chain. However, the conditions used during the synthesis and the restriction to rather short sequences hamper the applicability of this methodology. On the other hand, modified nucleoside triphosphates are activated building blocks that have been employed for the mild introduction of numerous functional groups into nucleic acids, a strategy that paves the way for the use of modified nucleic acids in a wide-ranging palette of practical applications such as functional tagging and generation of ribozymes and DNAzymes. One of the major challenges resides in the intricacy of the methodology leading to the isolation and characterization of these nucleoside analogues.
	
	In this video article, we present a detailed protocol for the synthesis of these modified analogues using phosphorous(III)-based reagents. In addition, the procedure for their biochemical characterization is divulged, with a special emphasis on primer extension reactions and TdT tailing polymerization. This detailed protocol will be of use for the crafting of modified dNTPs and their further use in chemical biology.

The protocol described herein aims to explain and abridge the numerous obstacles in the way of the intricate route leading to modified nucleoside triphosphates. Consequently, this protocol facilitates both the synthesis of these activated building-blocks and their availability for practical applications.

核苷三磷酸 - 从合成到生化特征

The  traditional strategy for the introduction of chemical functionalities is the  use of solid-phase synthesis by appending suitably modified ...

科研

化学

Sequence-specific and Selective Recognition of Double-stranded RNAs over Single-stranded RNAs by Chemically Modified Peptide Nucleic Acids

RNAs are emerging as important biomarkers and therapeutic targets. Thus, there is great potential in developing chemical probes and therapeutic ligands for the recognition of RNA sequence and structure. Chemically modified Peptide Nucleic Acid (PNA) oligomers have been recently developed that can recognize RNA duplexes in a sequence-specific manner. PNAs are chemically stable with a neutral peptide-like backbone. PNAs can be synthesized relatively easily by the manual Boc-chemistry solid-phase peptide synthesis method. PNAs are purified by reverse-phase HPLC, followed by molecular weight characterization by matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF). Non-denaturing polyacrylamide gel electrophoresis (PAGE) technique facilitates the imaging of the triplex formation, because carefully designed free RNA duplex constructs and PNA bound triplexes often show different migration rates. Non-denaturing PAGE with ethidium bromide post staining is often an easy and informative technique for characterizing the binding affinities and specificities of PNA oligomers. Typically, multiple RNA hairpins or duplexes with single base pair mutations can be used to characterize PNA binding properties, such as binding affinities and specificities. 2-Aminopurine is an isomer of adenine (6-aminopurine); the 2-aminopurine fluorescence intensity is sensitive to local structural environment changes, and is suitable for the monitoring of triplex formation with the 2-aminopurine residue incorporated near the PNA binding site. 2-Aminopurine fluorescence titration can also be used to confirm the binding selectivity of modified PNAs towards targeted double-stranded RNAs (dsRNAs) over single-stranded RNAs (ssRNAs). UV-absorbance-detected thermal melting experiments allow the measurement of the thermal stability of PNA-RNA duplexes and PNA&#183;RNA2 triplexes. Here, we describe the synthesis and purification of PNA oligomers incorporating modified residues, and describe biochemical and biophysical methods for characterization of the recognition of RNA duplexes by the modified PNAs.

We report the protocols for the synthesis and purification of Peptide Nucleic Acid (PNA) oligomers incorporating modified residues. The biochemical and biophysical methods for the characterization of the recognition of RNA duplexes by the modified PNAs are described.

化学修饰肽核酸对单链 rna 双链 rna 的序列特异和选择性识别

RNAs are emerging as important biomarkers and therapeutic targets. Thus, there is great potential in developing chemical probes and therapeutic ligands ...

遗传学

Using In Vitro and In-cell SHAPE to Investigate Small Molecule Induced Pre-mRNA Structural Changes

In the process of drug development of RNA-targeting small molecules, elucidating the structural changes upon their interactions with target RNA sequences is desired. We herein provide a detailed in vitro and in-cell selective 2&#39;-hydroxyl acylation analyzed by primer extension (SHAPE) protocol to study the RNA structural change in the presence of an experimental drug for spinal muscular atrophy (SMA), survival of motor neuron (SMN)-C2, and in exon 7 of the pre-mRNA of the SMN2 gene. In in vitro SHAPE, an RNA sequence of 140 nucleotides containing SMN2 exon 7 is transcribed by T7 RNA polymerase, folded in the presence of SMN-C2, and subsequently modified by a mild 2&#39;-OH acylation reagent, 2-methylnicotinic acid imidazolide (NAI). This 2&#39;-OH-NAI adduct is further probed by a 32P-labeled primer extension and resolved by polyacrylamide gel electrophoresis (PAGE). Conversely, 2&#39;-OH acylation in in-cell SHAPE takes place in situ with SMN-C2 bound cellular RNA in living cells. The pre-mRNA sequence of exon 7 in the SMN2 gene, along with SHAPE-induced mutations in the primer extension, was then amplified by PCR and subject to next-generation sequencing. Comparing the two methodologies, in vitro SHAPE is a more cost-effective method and does not require computational power to visualize results. However, the in vitro SHAPE-derived RNA model sometimes deviates from the secondary structure in a cellular context, likely due to the loss of all interactions with RNA-binding proteins. In-cell SHAPE does not need a radioactive material workplace and yields a more accurate RNA secondary structure in the cellular context. Furthermore, in-cell SHAPE is usually applicable for a larger range of RNA sequences (~1,000 nucleotides) by utilizing next-generation sequencing, compared to in vitro SHAPE (~200 nucleotides) that usually relies on PAGE analysis. In case of exon 7 in SMN2 pre-mRNA, the in vitro and in-cell SHAPE derived RNA models are similar to each other.

Detailed protocols for both in vitro and in-cell selective 2&#39;-hydroxyl acylation analyzed by primer extension (SHAPE) experiments to determine the secondary structure of pre-mRNA sequences of interest in the presence of an RNA-targeting small molecule are presented in this article.

利用体外和细胞内形状研究小分子诱导的预 mrna 结构变化

In the process of drug development of RNA-targeting small molecules, elucidating the structural changes upon their interactions with target RNA sequences ...

Artificial RNA Polymerase II Elongation Complexes for Dissecting Co-transcriptional RNA Processing Events

Eukaryotic mRNA synthesis is a complex biochemical process requiring transcription of a DNA template into a precursor RNA by the multi-subunit enzyme RNA polymerase II and co-transcriptional capping and splicing of the precursor RNA to form the mature mRNA. During mRNA synthesis, the RNA polymerase II elongation complex is a target for regulation by a large collection of transcription factors that control its catalytic activity, as well as the capping, splicing, and 3&#8217;-processing enzymes that create the mature mRNA. Because of the inherent complexity of mRNA synthesis, simpler experimental systems enabling isolation and investigation of its various co-transcriptional stages have great utility.
In this article, we describe one such simple experimental system suitable for investigating co-transcriptional RNA capping. This system relies on defined RNA polymerase II elongation complexes assembled from purified polymerase and artificial transcription bubbles. When immobilized via biotinylated DNA, these RNA polymerase II elongation complexes provide an easily manipulable tool for dissecting co-transcriptional RNA capping and mechanisms by which the elongation complex recruits and regulates capping enzyme during co-transcriptional RNA capping. We anticipate this system could be adapted for studying recruitment and/or assembly of proteins or protein complexes with roles in other stages of mRNA maturation coupled to the RNA polymerase II elongation complex.

Here, we describe the assembly of RNA polymerase II (Pol II) elongation complexes requiring only short synthetic DNA and RNA oligonucleotides and purified Pol II. These complexes are useful for studying mechanisms underlying co-transcriptional processing of transcripts associated with the Pol II elongation complex.

用于分离共转录RNA处理事件的人工RNA聚合酶II伸长复合物

Eukaryotic mRNA synthesis is a complex biochemical process requiring transcription of a DNA template into a precursor RNA by the multi-subunit enzyme RNA ...

2D-HELS MS Seq: A General LC-MS-Based Method for Direct and de novo Sequencing of RNA Mixtures with Different Nucleotide Modifications

Mass spectrometry (MS)-based sequencing approaches have been shown to be useful in&#160;direct&#160;sequencing&#160;RNA without the need for a complementary DNA (cDNA) intermediate. However, such approaches are rarely applied as a de novo RNA sequencing method, but used mainly as a tool that can assist in quality assurance for confirming known sequences of purified single-stranded RNA samples. Recently, we developed&#160;a direct RNA sequencing method by integrating a 2-dimensional mass-retention time hydrophobic end-labeling strategy into MS-based sequencing (2D-HELS MS Seq). This method is capable of accurately sequencing single RNA sequences as well as mixtures containing up to 12 distinct RNA sequences. In addition to the four canonical ribonucleotides (A, C, G, and U), the method has the capacity to sequence RNA oligonucleotides containing modified nucleotides. This is possible because the modified nucleobase either has an intrinsically unique mass that can help in its identification and its location in the RNA sequence, or can be converted into a product with a unique mass. In this study, we have used RNA, incorporating two representative modified nucleotides&#160;(pseudouridine (&#936;) and 5-methylcytosine (m5C)), to illustrate the application of the method for the de novo sequencing of a single RNA oligonucleotide&#160;as well as a mixture of RNA oligonucleotides, each with a different sequence and/or modified nucleotides. The procedures and protocols described here to sequence these model RNAs will be applicable to other short RNA samples (&#60;35 nt) when using a standard high-resolution LC-MS system, and can also be used for&#160;sequence verification of modified therapeutic RNA oligonucleotides.&#160;In the future, with the development of more robust algorithms and with better instruments, this method could allow sequencing of more complex biological samples.&#160;

<meta charset="utf8"></head><body>2D-HELS MS Seq:一种基于 LC-MS 的通用方法，用于对具有不同核苷酸修饰的 RNA 混合物进行直接和从头测序

Here, we describe a detailed protocol for an LC-MS-based sequencing method that can be used as a direct method to sequence short RNA (&#60;35 nt per run) without a cDNA intermediate, and as a general method to sequence different nucleotide modifications in a single study at single-base precision.

2D-HELS MS Seq:一种基于 LC-MS 的通用方法，用于对具有不同核苷酸修饰的 RNA 混合物进行直接和从头测序

Mass spectrometry (MS)-based sequencing approaches have been shown to be useful in&#160;direct&#160;sequencing&#160;RNA without the need for a ...

DNAzyme-dependent Analysis of rRNA 2&#8217;-O-Methylation

Guide box C/D small nucleolar RNAs (snoRNAs) catalyze 2&#8217;-O-methylation of ribosomal and small nuclear RNA. However, a large number of snoRNA in higher eukaryotes may promiscuously recognize other RNA species and 2&#8217;-O-methylate multiple targets. Here, we provide step-by-step guide for the fast and non-expensive analysis of the site-specific 2&#8217;-O-methylation using a well-established method employing short DNA oligonucleotides called DNAzymes. These DNA fragments contain catalytic sequences which cleave RNA at specific consensus positions, as well as variable homology arms directing DNAzyme to its RNA targets. DNAzyme activity is inhibited by 2-&#8217;O-methylation of the nucleotide adjacent to the cleavage site in the RNA. Thus, DNAzymes, limited only by the consensus of the cleaved sequence, are perfect tools for the quick analysis of snoRNA-mediated RNA 2&#8217;-O-methylation. We analyzed snoRNA snR13- and snR47-guided 2&#8217;-O-methylation of 25S ribosomal RNA in Saccharomyces cerevisiae to demonstrate the simplicity of the technique and to provide a detailed protocol for the DNAzyme-dependent assay.

Here we present a protocol for DNAzyme-dependent cleavage of RNA. This enables fast and site-dependent analysis of RNA 2&#8217;-O-methylation. This approach can be used for the preliminary or major assessment of snoRNA activity.

rRNA 2'-O-甲基化的脱氧核糖核酸依赖性分析

Guide box C/D small nucleolar RNAs (snoRNAs) catalyze 2&#8217;-O-methylation of ribosomal and small nuclear RNA. However, a large number of snoRNA in ...

发育生物学

Practical Aspects of Sample Preparation and Setup of 1H R1&#961; Relaxation Dispersion Experiments of RNA

RNA is a highly flexible biomolecule, wherein changes in structures play crucial roles in the functions that RNA molecules execute as cellular messengers and modulators. While these dynamic states remain hidden to most structural methods, R1&#961; relaxation dispersion (RD) spectroscopy allows the study of conformational dynamics in the micro- to millisecond regime at atomic resolution. The use of 1H as the observed nucleus further expands the time regime covered and gives direct access to hydrogen bonds and base pairing.
The challenging steps in such a study are high-purity and high-yield sample preparation, potentially 13C- and 15N-labeled, as well as setup of experiments and fitting of data to extract population, exchange rate, and secondary structure of the previously invisible state. This protocol provides crucial hands-on steps in sample preparation to ensure the preparation of a suitable RNA sample and setup of 1H R1&#961; experiments with both isotopically labeled and unlabeled RNA samples.

We present a protocol to measure micro- to millisecond dynamics on 13C/15N-labeled and unlabeled RNA with 1H R1&#961; relaxation dispersion nuclear magnetic resonance (NMR) spectroscopy. The focus of this protocol lies in high-purity sample preparation and setup of NMR experiments.

1H R1 + RNA 放松分散实验样品制备和设置的实用方面

RNA is a highly flexible biomolecule, wherein changes in structures play crucial roles in the functions that RNA molecules execute as cellular messengers ...

生物化学

Generation of RNA/DNA Hybrids in Genomic DNA by Transformation using RNA-containing Oligonucleotides

Synthetic short nucleic acid polymers, oligonucleotides (oligos), are the most functional and widespread tools of molecular biology. Oligos can be produced to contain any desired DNA or RNA sequence and can be prepared to include a wide variety of base and sugar modifications. Moreover, oligos can be designed to mimic specific nucleic acid alterations and thus, can serve as important tools to investigate effects of DNA damage and mechanisms of repair. We found that Thermo Scientific Dharmacon RNA-containing oligos with a length between 50 and 80 nucleotides can be particularly suitable to study, in vivo, functions and consequences of chromosomal RNA/DNA hybrids and of ribonucleotides embedded into DNA. RNA/DNA hybrids can readily form during DNA replication, repair and transcription, however, very little is known about the stability of RNA/DNA hybrids in cells and to which extent these hybrids can affect the genetic integrity of cells. RNA-containing oligos, therefore, represent a perfect vector to introduce ribonucleotides into chromosomal DNA and generate RNA/DNA hybrids of chosen length and base composition. Here we present the protocol for the incorporation of ribonucleotides into the genome of the eukaryotic model system yeast /Saccharomyces cerevisiae/. Yet, our lab has utilized Thermo Scientific Dharmacon RNA-containing oligos to generate RNA/DNA hybrids at the chromosomal level in different cell systems, from bacteria to human cells.

This work shows how to form an RNA/DNA hybrid at the chromosomal level and reveal transfer of genetic information from RNA to genomic DNA in yeast cells.

改造使用含RNA寡核苷酸基因组DNA的RNA / DNA杂交一代

Synthetic short nucleic acid polymers, oligonucleotides (oligos), are the most functional and widespread tools of molecular biology. Oligos can be ...

生物学

Chemical Triphosphorylation of Oligonucleotides

The 5&#8242;-triphosphate is an essential nucleic acid modification found throughout all life and increasingly used as a functional modification of oligonucleotides in biotechnology and synthetic biology. Oligonucleotide 5&#8242;-triphosphates have historically been prepared in vitro by enzymatic methods. However, these methods are limited to natural RNA oligonucleotides, have strong sequence preferences, and tend to produce heterogeneous products. New methods of chemical triphosphorylation complement both the reduced cost of automated oligonucleotide synthesis by phosphoramidite chemistry and the diverse range of nucleotide modifications now available. Thus, the synthesis of oligonucleotide triphosphates of arbitrary sequence and length, and optionally containing various nonnatural modifications, is now accessible.
This paper presents the appropriate methods and techniques for chemical triphosphorylation of oligonucleotides using salicyl phosphorochloridite and pyrophosphate. This method uses commercially available reagents, is compatible with most oligonucleotides prepared by standard solid-phase synthesis methods, and can be completed in 2 h following oligonucleotide synthesis, before deprotection and purification. Two uses of chemically triphosphorylated oligonucleotides as substrates for catalytic RNA enzymes are demonstrated, including the synthesis of a mirror-image version of the hammerhead ribozyme from nonbiological L-RNA triphosphates.

Oligonucleotide 5&#8242;-triphosphates are ubiquitous components in essential biological pathways and have seen increasing use in biotechnology applications. Here, we describe techniques for the routine synthesis and purification of oligonucleotide 5&#8242;-triphosphates, starting from oligonucleotides prepared by standard automated synthesis techniques.

寡核苷酸的化学三磷酸化

The 5&#8242;-triphosphate is an essential nucleic acid modification found throughout all life and increasingly used as a functional modification of ...

用于固相合成含有2'-

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核苷三磷酸 - 从合成到生化特征

化学修饰肽核酸对单链 rna 双链 rna 的序列特异和选择性识别

利用体外和细胞内形状研究小分子诱导的预 mrna 结构变化

用于分离共转录RNA处理事件的人工RNA聚合酶II伸长复合物

2D-HELS MS Seq:一种基于 LC-MS 的通用方法，用于对具有不同核苷酸修饰的 RNA 混合物进行直接和从头测序

2D-HELS MS Seq:一种基于 LC-MS 的通用方法，用于对具有不同核苷酸修饰的 RNA 混合物进行直接和从头测序

rRNA 2'-O-甲基化的脱氧核糖核酸依赖性分析

¹H R_{1 +} RNA 放松分散实验样品制备和设置的实用方面

改造使用含RNA寡核苷酸基因组DNA的RNA / DNA杂交一代

寡核苷酸的化学三磷酸化