Name: a novel saturation mutagenesis approach: single step characterization of regulatory protein binding sites in rna using phosphorothioates
Uploaded: 2018-08-21T14:00:00
Description: Watch this Scientific Journal Video about A Novel Saturation Mutagenesis Approach: Single Step Characterization of Regulatory Protein Binding Sites in RNA Using Phosphorothioates at JoVE.com

作者感谢国立卫生研究院过去的资助和感谢迈克尔 r. 格林合成寡核苷酸。

注:图 1提供了硫代核苷酸突变的概述, 并总结了过程中的关键步骤。
1. 用非野生型核苷酸生成突变体掺杂 DNA 模板的库
<ol><li>合成 T7 底漆 (5 '-GTAATACGACTCACTATAG-3 ') 通过化学合成的 DNA 合成物。</li>
	<li>在与蛋白质结合部位相对应的 DNA 合成器上合成掺杂的寡核苷酸 (互补链)。在化学合成过程中使用适当的 phosphoramidites 混合物 (X 以下), 其比例为 90% a 作为野?...

<ol>
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L., 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=Expression+of+msl-2+causes+assembly+of+dosage+compensation+regulators+on+the+X+chromosomes+and+female+lethality+in+Drosophila.">Expression of msl-2 causes assembly of dosage compensation regulators on the X chromosomes and female lethality in Drosophila.</a> Cell. 81 (6), 867-877 (1995).</li><li>Hager, J., Cline, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+female+Sex-lethal+RNA+splicing+in+male+germ+cells:+implications+for+Drosophila+germline+sex+determination.">Induction of female Sex-lethal RNA splicing in male germ cells: implications for Drosophila germline sex determination.</a> Development. 124 (24), 5033-5048 (1997).</li><li>Vied, C., Halachmi, N., Salzberg, A., Horabin, J. 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K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sex-lethal+enables+germline+stem+cell+differentiation+by+down-regulating+Nanos+protein+levels+during+Drosophila+oogenesis.">Sex-lethal enables germline stem cell differentiation by down-regulating Nanos protein levels during Drosophila oogenesis.</a> Proc Natl Acad Sci U S A. 109 (24), 9465-9470 (2012).</li><li>Singh, R., Valcarcel, J., Green, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+binding+specificities+and+functions+of+higher+eukaryotic+polypyrimidine+tract-binding+proteins.">Distinct binding specificities and functions of higher eukaryotic polypyrimidine tract-binding proteins.</a> Science. 268 (5214), 1173-1176 (1995).</li><li>Singh, R., Banerjee, H., Green, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+recognition+of+the+polypyrimidine-tract+by+the+general+splicing+factor+U2AF65+and+the+splicing+repressor+sex-lethal.">Differential recognition of the polypyrimidine-tract by the general splicing factor U2AF65 and the splicing repressor sex-lethal.</a> RNA. 6 (6), 901-911 (2000).</li><li>Sakashita, E., Sakamoto, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+RNA+binding+specificity+of+the+Drosophila+sex-+lethal+protein+by+in+vitro+ligand+selection.">Characterization of RNA binding specificity of the Drosophila sex- lethal protein by in vitro ligand selection.</a> Nucleic Acids Res. 22 (20), 4082-4086 (1994).</li><li>Kanaar, R., Lee, A. L., Rudner, D. Z., Wemmer, D. E., Rio, D. 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突变一直被用来表征蛋白质结合部位。首先, 一系列突变体可以构造和单独测试的结合检测, 以分析其对结合亲和性的影响。虽然标准突变方法提供了一种分析多个序列的方法, 但标准方法中涉及的多个步骤, 如构造突变体和对每个变种人执行一系列绑定反应, 都是费力和耗时的, 可能不会允许饱和诱变, 特别是对较长的序列。第二, 序列可以是随机的和池用于多周期的结合和聚合酶链反应 (PCR) 放大?...

基因调控在生物学中起着重要的作用。基因可以调节在转录水平, 前 mRNA 剪接, 3 ' 末端形成, RNA 出口, 翻译, mRNA 定位, 衰变, 后转化/稳定性等. DNA 和 RNA 结合蛋白在基因中起关键作用调节。虽然分子遗传学分析已经确定了许多调控蛋白, 但它们中只有一小部分被完全用于细胞功能或体内的结合部位。系统进化序列分析和突变提供了互补的方法来表征 DNA 或 RNA-蛋白质相互作用。
RNA 结合蛋白在发育过程中很重要, 包括性分化。果蝇蛋白性致死 (SXL) 或主性开关蛋白不在男性, 但存在于女性。它能识别在体细胞1,2 中, 苷的序列或嘧啶-在下游前 mRNA 目标 (变压器,性致死和男性特定的 lethal2) 附近的特定剪接点. ,3,4。此外, 它通过结合苷丰富的 polyadenylation 增强器序列在基本(e (r)) 成绩单5,6中约束 polyadenylation 站点切换。SXL 可能对仍有待确定的女性生殖中的其他目标进行调节, 1、7、8、9、10、11、12,13。
通常, 绑定站点的特征包括突变, 例如, 通过删除或替代单个或多个核苷酸。每个突变结合点, 相对于野生类型的 RNA 序列, 然后分析使用一系列的蛋白质浓度, 以确定其结合亲和性 (Kd 或平衡离解常数) 的蛋白质的利益;Kd 是获得 50% RNA 结合所需的蛋白质浓度。这一劳动密集型的详细诱变过程涉及对众多突变体的产生和分析, 即三种非野生型核苷酸用于结合部位的每个位置。因此, 需要一种替代的方法, 以更快, 更简单, 廉价的饱和突变的蛋白质结合点在 RNA。
在这里, 我们描述了一步方法, 允许在 RNA 的蛋白质结合点的饱和诱变。它涉及掺杂 DNA 模板与非野生型核苷酸在结合部位, 合成单独 rna 与每个硫代核苷酸核苷酸, 并分离的束缚分数后孵化与蛋白质。非野生型核苷酸的干扰会导致它们在蛋白质束缚分数上的优先排斥。这是由凝胶电泳监测后, 选择性化学分裂与碘磷酸二酯债券含有 phosphorothioates (硫代核苷酸诱变或 PTM)。这种单步饱和诱变方法适用于 RNA 中任何蛋白质结合部位的表征。

<table>
	<tbody>
		<tr>
			<td>Uridine 5&#8217; a-thio triphosphate</td>
			<td>NEN (Boston, Massachusetts)</td>
			<td>NLP-017</td>
			<td></td>
		</tr>
		<tr>
			<td>Adenosine 5&#8217; a-thio triphosphate</td>
			<td>NEN (Boston, Massachusetts)</td>
			<td>NLP-016</td>
			<td></td>
		</tr>
		<tr>
			<td>Vacuum manifold</td>
			<td>Fisher Scientific</td>
			<td>XX1002500</td>
			<td>Millipore&#160;25&#160;mm Glass Microanalysis Vacuum Filter</td>
		</tr>
		<tr>
			<td>Vacuum manifold</td>
			<td>Millipore</td>
			<td>XX2702552</td>
			<td>1225 Sampling Vacuum Manifold</td>
		</tr>
		<tr>
			<td>Nitrocellulose</td>
			<td>Millipore</td>
			<td>HAWP</td>
			<td></td>
		</tr>
		<tr>
			<td>Nitrocellulose</td>
			<td>Schleicher &#38; Schuell</td>
			<td>PROTRAN</td>
			<td></td>
		</tr>
		<tr>
			<td>Dephosphorlyation Kit</td>
			<td>NEB</td>
			<td>M0508</td>
			<td></td>
		</tr>
		<tr>
			<td>T4 Polynucleotide Kinase</td>
			<td>NEB</td>
			<td>M0201S</td>
			<td></td>
		</tr>
		<tr>
			<td>Proteinase K</td>
			<td>NEB</td>
			<td>P8107S</td>
			<td></td>
		</tr>
		<tr>
			<td>T7 RNA polymerase</td>
			<td>NEB</td>
			<td>M0251S</td>
			<td></td>
		</tr>
		<tr>
			<td>RNasin</td>
			<td>Promega</td>
			<td></td>
			<td>RNase inhibitor</td>
		</tr>
		<tr>
			<td>Glass Plates</td>
			<td>Standard</td>
			<td>Standard</td>
			<td></td>
		</tr>
		<tr>
			<td>Gel Electrophoresis equipment</td>
			<td>Standard</td>
			<td>Standard</td>
			<td></td>
		</tr>
		<tr>
			<td>X-ray films</td>
			<td>Standard</td>
			<td>Standard</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyacrylamide gel solutions</td>
			<td>Standard</td>
			<td>Standard</td>
			<td></td>
		</tr>
	</tbody>
</table>

a novel saturation mutagenesis approach: single step characterization of regulatory protein binding sites in rna using phosphorothioates

基因调控在发展中起着重要的作用。大量的 DNA 和 RNA 结合蛋白结合其靶序列的高特异性来控制基因表达。这些调控蛋白控制基因表达无论是在 DNA 水平 (转录) 或在 RNA 水平 (前 mRNA 剪接, polyadenylation, mrna 传输, 衰变和翻译)。规范序列的识别不仅有助于了解基因是如何开启或关闭的, 而且还能理解哪些下游基因是由特定的调控蛋白调控的。在这里, 我们描述了一步方法, 允许在 RNA 的蛋白质结合点的饱和诱变。它涉及掺杂 DNA 模板与非野生型核苷酸在结合部位, 合成单独 rna 与每个硫代核苷酸核苷酸, 并分离的束缚分数后孵化与蛋白质。非野生型核苷酸的干扰会导致它们在蛋白质束缚分数上的优先排斥。这是由凝胶电泳监测后, 选择性化学分裂与碘磷酸二酯债券含有 phosphorothioates (硫代核苷酸诱变或 PTM)。这种单步饱和诱变方法适用于 RNA 中任何蛋白质结合部位的表征。

利用掺杂的饱和诱变原理:
对于野生型和其他核苷酸的适当摩尔比, 如果只分析一个位置, 则使用所有四核苷酸的相等混合物。但是, 如果同时对多个位置进行分析, 则必须调整非野类型与野型核苷酸的比值,即减少。否则, 除了单一的替代, 这是需要的, 也将有多个非野生型核苷酸在一个分子的模板, 排除了对单核苷酸替代效?...

Watch this Scientific Journal Video about A Novel Saturation Mutagenesis Approach: Single Step Characterization of Regulatory Protein Binding Sites in RNA Using Phosphorothioates at JoVE.com

A Novel Saturation Mutagenesis Approach: Single Step Characterization of Regulatory Protein Binding Sites in RNA Using Phosphorothioates

结合特定 RNA 序列的蛋白质在基因表达中扮演重要角色。对这些结合部位的详细描述对于我们理解基因调控至关重要。本文介绍了 RNA 蛋白质结合部位饱和诱变的单步法方法。这种方法与 RNA 中的所有蛋白结合部位有关。

一种新的饱和诱变方法: 利用 Phosphorothioates 对 RNA 调控蛋白结合位点的单步表征

Gene regulation plays an important role in development. Numerous DNA- and RNA-binding proteins bind their target sequences with high specificity to ...

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