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
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...

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	<li>Matteucci, M. D., Caruthers, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+synthesis+of+oligodeoxypyrimidines+on+a+polymer+support.">The synthesis of oligodeoxypyrimidines on a polymer support.</a> Tetrahedron Lett. 21 (8), 719-722 (1980).</li><li>Beaucage, S. L., Caruthers, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deoxynucleoside+phosphoramidites-A+new+class+of+key+intermediates+for+deoxypolynucleotides.">Deoxynucleoside phosphoramidites-A new class of key intermediates for deoxypolynucleotides.</a> Tetrahedron Lett. 22 (20), 1859-1862 (1981).</li><li>Caruthers, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+synthesis+machines:+DNA+Chemistry+and+its+uses.">Gene synthesis machines: DNA Chemistry and its uses.</a> Science. 230 (4723), 281-285 (1985).</li><li>Nguyen, J. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis,+Thermal+Stability,+Biophysical+Properties,+and+Molecular+Modeling+of+Oligonucleotides+of+RNA+Containing+2'-O-2-Thiophenylmethyl+Groups.">Synthesis, Thermal Stability, Biophysical Properties, and Molecular Modeling of Oligonucleotides of RNA Containing 2'-O-2-Thiophenylmethyl Groups.</a> J Org Chem. 81 (19), 8947-8958 (2016).</li><li>El-Sagheer, A. H., Brown, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Click+chemistry+with+DNA.">Click chemistry with DNA.</a> Chem Soc Rev. 39, 1388-1405 (2010).</li><li>Puffer, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=5-Fluoro+pyrimidines:+labels+to+probe+DNA+and+RNA+secondary+structures+by+1D+19F+NMR+spectroscopy.">5-Fluoro pyrimidines: labels to probe DNA and RNA secondary structures by 1D 19F NMR spectroscopy.</a> Nucleic Acids Res. 37 (22), 7728-7740 (2009).</li><li>Wan, W. B., Seth, P. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+medicinal+chemistry+of+therapeutic+oligonucleotides.">The medicinal chemistry of therapeutic oligonucleotides.</a> J Med Chem. 59 (21), 9645-9667 (2016).</li><li>Zhou, C., Greenberg, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+Damage+by+histone+radicals+in+nucleosome+core+particles.">DNA Damage by histone radicals in nucleosome core particles.</a> J Am Chem Soc. 136 (18), 6562-6565 (2014).</li><li>Zhang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=UV-Induced+proton-coupled+electron+transfer+in+cyclic+DNA+miniduplexes.">UV-Induced proton-coupled electron transfer in cyclic DNA miniduplexes.</a> J Am Chem Soc. 138 (23), 7395-7401 (2016).</li><li>Anosova, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+structural+diversity+of+artificial+genetic+polymers.">The structural diversity of artificial genetic polymers.</a> Nucleic Acids Res. 44 (3), 1007-1021 (2016).</li><li>Riml, C., Micura, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis+of+5-Hydroxymethylcytidine-+and+5-Hydroxymethyl-uridine-Modified+RNA.">Synthesis of 5-Hydroxymethylcytidine- and 5-Hydroxymethyl-uridine-Modified RNA.</a> Synthesis. 48, 1108-1116 (2016).</li><li>Anderson, B. A., Hrdlicka, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Merging+Two+Strategies+for+Mixed-Sequence+Recognition+of+Double-Stranded+DNA:+Pseudocomplementary+Invader+Probes.">Merging Two Strategies for Mixed-Sequence Recognition of Double-Stranded DNA: Pseudocomplementary Invader Probes.</a> J Org Chem. 81 (8), 3335-3346 (2016).</li><li>Beaucage, S. L., Iyer, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis+of+oligonucleotides+by+the+phosphoramidite+approach.">Synthesis of oligonucleotides by the phosphoramidite approach.</a> Tetrahedron. 48 (12), 2223-2311 (1992).</li><li>Gillet, L. C. J., Alzeer, J., Sch&#228;rer, O. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Site-specific+incorporation+of+N-(deoxyguanosin-8-yl)-2-acetylaminofluorene+(dG-AAF)+into+oligonucleotides+using+modified+'ultra-mild'+DNA+synthesis.">Site-specific incorporation of N-(deoxyguanosin-8-yl)-2-acetylaminofluorene (dG-AAF) into oligonucleotides using modified 'ultra-mild' DNA synthesis.</a> Nucleic Acids Res. 33 (6), 1961-1969 (2005).</li><li>Shiba, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical+synthesis+of+a+very+long+oligoribonucleotide+with+2-cyanoethoxymethyl+(CEM)+as+the+2'-O.-protecting+group:+structural+identification+and+biological+activity+of+a+synthetic+110mer+precursor-microRNA+candidate.">Chemical synthesis of a very long oligoribonucleotide with 2-cyanoethoxymethyl (CEM) as the 2'-O.-protecting group: structural identification and biological activity of a synthetic 110mer precursor-microRNA candidate.</a> Nucleic Acids Res. 35 (10), 3287-3296 (2007).</li><li>Choi, Y. J., Gibala, K. S., Ayele, T., Deventer, K. D., Resendiz, M. J. 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,+thermal+stability+and+functional+impact+of+8-oxo-7,8-dihydroguanine+on+oligonucleotides+of+RNA+-+a+study+of+duplex,+hairpins+and+the+aptamer+for+preQ1+as+models.">Biophysical properties, thermal stability and functional impact of 8-oxo-7,8-dihydroguanine on oligonucleotides of RNA - a study of duplex, hairpins and the aptamer for preQ1 as models.</a> Nucleic Acids Res. 45 (4), 2099-2111 (2017).</li><li>Ranjbar, B., Gill, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circular+dichroism+techniques:+Biomolecular+and+nanostructural+analyses+-+A+review.">Circular dichroism techniques: Biomolecular and nanostructural analyses - A review.</a> Chem Biol Drug Des. 74 (2), 101-120 (2009).</li><li>Mergny, J. -. L., Lacroix, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+thermal+melting+curves.">Analysis of thermal melting curves.</a> Oligonucleotides. 13 (6), 515-537 (2003).</li><li>Jin, R., Breslauer, K. J., Jones, R. A., Gaffney, B. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tetraplex+formation+of+a+guanine-containing+nonameric+DNA+fragment.">Tetraplex formation of a guanine-containing nonameric DNA fragment.</a> Science. 250 (4980), 543-546 (1990).</li><li>Virgilio, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=5-Hydroxymethyl-2'-deoxyuridine+residues+in+the+thrombin+binding+aptamer:+Investigating+anticoagulant+activity+by+making+a+tiny+chemical+modification.">5-Hydroxymethyl-2'-deoxyuridine residues in the thrombin binding aptamer: Investigating anticoagulant activity by making a tiny chemical modification.</a> CHEMBIOCHEM. 15 (16), 2427-2434 (2014).</li><li>Chauca-Diaz, A. M., Choi, Y. J., Resendiz, M. J. 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>

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。

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			<td height="20" style="height:20px;width:445px;">AbsolveTM</td>
			<td style="width:152px;">PerkinElmer</td>
			<td style="width:117px;">6NE9711</td>
			<td style="width:64px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Acetonitrile 99.9%, HPLC Grade</td>
			<td>Fisher Scientific</td>
			<td>75-05-8</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Acetonitrile 99.9%, anhydrous for DNA sequencing</td>
			<td>Fisher BioReagents</td>
			<td>75-05-8</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Acrylamide, 99+%</td>
			<td>ACROS Organics</td>
			<td>164850025</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ammonium chloride 98+%</td>
			<td>Alfa Aesar</td>
			<td>12125-02-9</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ammonium citrate, dibasic 98%</td>
			<td>Sigma Aldrich</td>
			<td>3012-65-5</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ammonium Fluoride&#160; 98.0%, ACS grade</td>
			<td>Alfa Aesar</td>
			<td>12125-01-8</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ammonium hydroxide 28-30% in water, ACS Plus</td>
			<td>Fisher Chemical</td>
			<td>1336-21-6</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ammonium persulfate</td>
			<td>ACROS Organics</td>
			<td>1444</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Argon-ultra high purity</td>
			<td>&#160;Airgas</td>
			<td>7440-37-1</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Bis-acrylamide Ultra pure</td>
			<td>VWR-Amresco</td>
			<td>172</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Boric Acid</td>
			<td>Fisher Scientific</td>
			<td>A73-1</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Diethyl pyrocarbonate, 97%</td>
			<td>ACROS Organics</td>
			<td>A0368487</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ethanol, anhydrous, histological grade</td>
			<td>Fisher Chemical</td>
			<td>64-17-5</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ethylenediamine tetraacetic acid, disodium salt dehydrate 100.2%</td>
			<td>Fisher Chemical</td>
			<td>6381-92-6</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Formamide&#160;</td>
			<td>Thermo Scientific</td>
			<td>75-12-7</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Hydrochloric acid, 36.5-38.0%, Certified ACS Plus</td>
			<td>Fisher Chemical</td>
			<td>7647-01-0</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Magnesium chloride hexahydrate, 99%</td>
			<td>Fisher Scientific</td>
			<td>7786-30-3</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Methanol, 99.9%,&#160; HPLC Grade</td>
			<td>Fisher Chemical</td>
			<td>67-56-1</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Methylamine 40% in water</td>
			<td>Sigma Aldrich</td>
			<td>74-89-5</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">1-Methyl-2-pyrrolidinone, andhydrous, 99.5%</td>
			<td>Aldrich</td>
			<td>872-50-4</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Opti-TOFTM 96 Well Insert (123 x 81 mm)&#160;</td>
			<td>MDS SCIEX</td>
			<td>1020157</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">RNase Away</td>
			<td>Molecular BioProducts</td>
			<td>7005-11</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sodium acetate, anhydrous 99.2%, Certified ACS</td>
			<td>Fisher Chemical</td>
			<td>127-09-3</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sodium chloride, 100.5%, Certified ACS</td>
			<td>Fisher Chemical</td>
			<td>7647-14-5</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sodium phosphate monobasic dihydrate 99.0%</td>
			<td>Sigma</td>
			<td>13472-35-0</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">2&#8217;,4&#8217; Triethylamine, 99+%</td>
			<td>Alfa Aesar</td>
			<td>121-44-8</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">TEMED</td>
			<td>Amresco</td>
			<td>761</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Triethylamine trihydrofluoride, 98%</td>
			<td>Aldrich</td>
			<td>73602-61-6</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Trifluoroacetic acid, 99%</td>
			<td>Alfa Aesar</td>
			<td>76-05-1</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">6&#8217;-Trihydroxyacetophenone monohydrate 98%</td>
			<td>Sigma Aldrich</td>
			<td>480-66-0</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tris Base</td>
			<td>Fisher Scientific</td>
			<td>BP154-3</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Urea</td>
			<td>Fisher Scientific</td>
			<td>U15-3</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Reagents for the RNA synthesis:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Deblocking mix, 3% trichloroacetic acid in dichloromethane</td>
			<td>Glen Research</td>
			<td>&#160;40-4140-57</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cap Mix A, THF/Pyridine/Acetic anhydride</td>
			<td>Glen Research</td>
			<td>40-4110-52</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cap Mix B, 10% 1-methylimidazole in THF</td>
			<td>Glen Research</td>
			<td>40-4120-52</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Activator, 0.25 M 5-ethylthio-1H-tetrazole in anhydrous acetonitrile</td>
			<td>Glen Research</td>
			<td>&#160;30-3140-52</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Oxidizing Solution, 0.02 M iodine in THF/Pyridine/Water</td>
			<td>Glen Research</td>
			<td>40-4330-52</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">U-RNA-CPG</td>
			<td>Glen Research</td>
			<td>20-3330-xx</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ac-G-RNA-CPG&#160;</td>
			<td>Glen Research</td>
			<td>20-3324-xx</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ac-G-CE Phosphoramidite&#160;</td>
			<td>Glen Research</td>
			<td>10-3025-xx</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">U-CE Phosphoramidite</td>
			<td>Glen Research</td>
			<td>10-3030-xx</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ac-C-CE Phosphoramidite</td>
			<td>Glen Research</td>
			<td>10-3015-xx</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Bz-A-CE Phosphoramidite&#160;</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 ...

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

Split-and-pool Synthesis and Characterization of Peptide Tertiary Amide Library

分裂和池合成及肽叔酰胺图书馆表征

Peptidomimetics are great sources of protein ligands. The oligomeric nature of these compounds enables us to access large synthetic libraries on solid ...

科研

化学

Synthesis and Characterization of Functionalized Metal-organic Frameworks

功能化的合成与表征金属 - 有机骨架

Metal-organic frameworks have attracted extraordinary amounts of research attention, as they are attractive candidates for numerous industrial and ...

Synthetic Methodology for Asymmetric Ferrocene Derived Bio-conjugate Systems via Solid Phase Resin-based Methodology

合成方法通过固相树脂为基础的方法不对称二茂铁产生的生物共轭系统

Early detection is a key to successful treatment of most diseases, and is particularly imperative for the diagnosis and treatment of many types of cancer. ...

The Synthesis, Characterization and Reactivity of a Series of Ruthenium N-triphosPh Complexes

The Synthesis, Characterization and Reactivity of a Series of Ruthenium N-triphosPh Complexes

一系列钌的合成，表征和反应<em&gt;Ñ</em&gt; -triphos<sup&gt;博士</sup&gt;配合

Herein we report the synthesis of a tridentate phosphine ligand N(CH2PPh2)3 (N-triphosPh) (1) via a phosphorus based Mannich reaction of the ...

A Direct, Early Stage Guanidinylation Protocol for the Synthesis of Complex Aminoguanidine-containing Natural Products

一个直接的，早期胍基协议的复杂含氨基胍，天然产物合成

The guanidine functional group, displayed most prominently in the amino acid arginine, one of the fundamental building blocks of life, is an important ...

Protocol for the Synthesis of Ortho-trifluoromethoxylated Aniline Derivatives

Protocol for the Synthesis of Ortho-trifluoromethoxylated Aniline Derivatives

协议的合成<em&gt;邻</em&gt; -trifluoromethoxylated苯胺类化合物

Molecules bearing trifluoromethoxy (OCF3) group often show desired pharmacological and biological properties. However, facile synthesis of ...

Synthesis and Characterization of Supramolecular Colloids

超分子胶体的合成与表征

Control over colloidal assembly is of utmost importance for the development of functional colloidal materials with tailored structural and mechanical ...

Synthesis of Protein Bioconjugates via Cysteine-maleimide Chemistry

Synthesis of Protein Bioconjugates via Cysteine-maleimide Chemistry

蛋白质生物缀合物的合成<em&gt;通过</em&gt;半胱氨酸马来酰亚胺化学

The chemical linking or bioconjugation of proteins to fluorescent dyes, drugs, polymers and other proteins has a broad range of applications, such as the ...

Microfluidic Dry-spinning and Characterization of Regenerated Silk Fibroin Fibers

微流控干纺及再生丝素纤维的表征

The protocol demonstrates a method for mimicking the spinning process of silkworm. In the native spinning process, the contracting spinning duct enables ...