Name: using in vitro and in-cell shape to investigate small molecule induced pre-mrna structural changes
Uploaded: 2019-01-30T15:00:00
Description: Watch this Scientific Journal Video about Using In Vitro and In-cell SHAPE to Investigate Small Molecule Induced Pre-mRNA Structural Changes at JoVE.com

这项工作是由国家卫生研究院 r01 赠款 (ns094721, k. a. j.) 促成的。

1. 体外形状
请注意:从已发布的协议1修改协议.
<ol><li>准备 rna 模板 请注意:t7 转录模板被排序为合成双链 dna (dsdna), 并通过插入大肠杆菌载体进行扩增 , 该载体携带独特的一对 ecrori/bamhi 限制性内切酶位点, 如 pet28a, 或通过 pcr 进行扩增。pcr 扩增协议如下图所示。<ol>
<li>混合以下材料: 50μl pcr 主混合物 (见?...

<ol>
	<li>Wilkinson, K. A., Merino, E. J., Weeks, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+2'-hydroxyl+acylation+analyzed+by+primer+extension+(SHAPE):+Quantitative+RNA+structure+analysis+at+single+nucleotide+resolution.">Selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE): Quantitative RNA structure analysis at single nucleotide resolution.</a> Nature Protocols. 1, 1610-1616 (2006).</li><li>Trausch, J. J., 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=Structural+basis+for+diversity+in+the+SAM+clan+of+riboswitches.">Structural basis for diversity in the SAM clan of riboswitches.</a> Proceedings of the National Academy of Sciences. 111, 6624-6629 (2014).</li><li>Souliere, M. F., 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=Tuning+a+riboswitch+response+through+structural+extension+of+a+pseudoknot.">Tuning a riboswitch response through structural extension of a pseudoknot.</a> Proceedings of the National Academy of Sciences. 110, E3256-E3264. , E3256-E3264 (2013).</li><li>Rice, G. M., Busan, S., Karabiber, F., Favorov, O. V., Weeks, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SHAPE+Analysis+of+Small+RNAs+and+Riboswitches.">SHAPE Analysis of Small RNAs and Riboswitches.</a> Methods in Enzymology. , 165-187 (2014).</li><li>Spitale, R. 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=RNA+SHAPE+analysis+in+living+cells.">RNA SHAPE analysis in living cells.</a> Nature Chemical Biology. 9, 18-20 (2013).</li><li>Wang, J., Schultz, P. G., Johnson, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanistic+studies+of+a+small-molecule+modulator+of+SMN2+splicing.">Mechanistic studies of a small-molecule modulator of SMN2 splicing.</a> Proceedings of the National Academy of Sciences. 115, E4604-E4612 (2018).</li><li>Price, I. R., Gaballa, A., Ding, F., Helmann, J. 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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=Development+of+Small+Molecules+with+a+Noncanonical+Binding+Mode+to+HIV-1+Trans+Activation+Response+(TAR)+RNA.">Development of Small Molecules with a Noncanonical Binding Mode to HIV-1 Trans Activation Response (TAR) RNA.</a> Journal of Medicinal Chemistry. 59, 11148-11160 (2016).</li><li>Shortridge, M. D., 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=A+Macrocyclic+Peptide+Ligand+Binds+the+Oncogenic+MicroRNA-21+Precursor+and+Suppresses+Dicer+Processing.">A Macrocyclic Peptide Ligand Binds the Oncogenic MicroRNA-21 Precursor and Suppresses Dicer Processing.</a> ACS Chemical Biology. 12, 1611-1620 (2017).</li><li>Warner, K. D., Hajdin, C. E., Weeks, K. 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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Emerging+Role+of+RNA+as+a+Therapeutic+Target+for+Small+Molecules.">The Emerging Role of RNA as a Therapeutic Target for Small Molecules.</a> Cell Chemical Biology. 23, 1077-1090 (2016).</li><li>Palacino, J., 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=SMN2+splice+modulators+enhance+U1-pre-mRNA+association+and+rescue+SMA+mice.">SMN2 splice modulators enhance U1-pre-mRNA association and rescue SMA mice.</a> Nature Chemical Biology. 11, 511-517 (2015).</li><li>Ratni, H., 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=Discovery+of+Risdiplam,+a+Selective+Survival+of+Motor+Neuron-2+(+SMN2+)+Gene+Splicing+Modifier+for+the+Treatment+of+Spinal+Muscular+Atrophy+(SMA).">Discovery of Risdiplam, a Selective Survival of Motor Neuron-2 ( SMN2 ) Gene Splicing Modifier for the Treatment of Spinal Muscular Atrophy (SMA).</a> Journal of Medicinal Chemistry. 61, 6501-6517 (2018).</li><li>Naryshkin, N., 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=SMN2+splicing+modifiers+improve+motor+function+and+longevity+in+mice+with+spinal+muscular+atrophy.">SMN2 splicing modifiers improve motor function and longevity in mice with spinal muscular atrophy.</a> Science. 345, (2014).</li><li>Chiriboga, 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=Preliminary+Evidence+for+Pharmacodynamics+Effects+of+RG7916+in+JEWELFISH,+a+Study+in+Patients+with+Spinal+Muscular+Atrophy+who+Previously+Participated+in+a+Study+with+Another+SMN2-Splicing+Targeting+Therapy+(S46.003).">Preliminary Evidence for Pharmacodynamics Effects of RG7916 in JEWELFISH, a Study in Patients with Spinal Muscular Atrophy who Previously Participated in a Study with Another SMN2-Splicing Targeting Therapy (S46.003).</a> Neurology. 90, (2018).</li><li>Sivaramakrishnan, M., 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=Binding+to+SMN2+pre-mRNA-protein+complex+elicits+specificity+for+small+molecule+splicing+modifiers.">Binding to SMN2 pre-mRNA-protein complex elicits specificity for small molecule splicing modifiers.</a> Nature Communications. 8, (2017).</li><li>Lee, 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=Comparison+of+SHAPE+reagents+for+mapping+RNA+structures+inside+living+cells.">Comparison of SHAPE reagents for mapping RNA structures inside living cells.</a> RNA. 23, 169-174 (2017).</li><li>Weeks, K. M., Mauger, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exploring+RNA+Structural+Codes+with+SHAPE+Chemistry.">Exploring RNA Structural Codes with SHAPE Chemistry.</a> Accounts of Chemical Research. 44, 1280-1291 (2011).</li><li>Smola, M. J., 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=SHAPE+reveals+transcript-wide+interactions+,+complex+structural+domains+,+and+protein+interactions+across+the+Xist+lncRNA+in+living+cells.">SHAPE reveals transcript-wide interactions , complex structural domains , and protein interactions across the Xist lncRNA in living cells.</a> Proceedings of the National Academy of Sciences. 113, 10322-10327 (2016).</li><li>Flynn, R. 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=Transcriptome-wide+interrogation+of+RNA+secondary+structure+in+living+cells+with+icSHAPE.">Transcriptome-wide interrogation of RNA secondary structure in living cells with icSHAPE.</a> Nature Protocols. 11, 273-290 (2016).</li><li>Smola, M. J., Rice, G. M., Busan, S., Siegfried, N. A., Weeks, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+2+&#8242;+-hydroxyl+acylation+analyzed+by+primer+extension+and+mutational+profiling+(SHAPE-MaP)+for+direct+,+versatile+and+accurate+RNA+structure+analysis.">Selective 2 &#8242; -hydroxyl acylation analyzed by primer extension and mutational profiling (SHAPE-MaP) for direct , versatile and accurate RNA structure analysis.</a> Nature Protocols. 10, 1643-1669 (2015).</li><li>Hua, 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=Antisense+correction+of+SMN2+splicing+in+the+CNS+rescues+necrosis+in+a+type+III+SMA+mouse+model.">Antisense correction of SMN2 splicing in the CNS rescues necrosis in a type III SMA mouse model.</a> Genes &amp; Development. 24, 1634-1644 (2010).</li><li>Hua, 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=Peripheral+SMN+restoration+is+essential+for+long-term+rescue+of+a+severe+spinal+muscular+atrophy+mouse+model.">Peripheral SMN restoration is essential for long-term rescue of a severe spinal muscular atrophy mouse model.</a> Nature. 478, 123-126 (2011).</li><li>Wee, C. D., Havens, M. A., Jodelka, F. M., Hastings, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+SR+proteins+improves+SMN+expression+in+spinal+muscular+atrophy+cells.">Targeting SR proteins improves SMN expression in spinal muscular atrophy cells.</a> PloS One. 9, e115205 (2014).</li><li>Hammond, J. A., Rambo, R. P., Filbin, M. E., Kieft, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+and+functional+implications+of+the+3D+architectures+of+viral+tRNA-like+structures.">Comparison and functional implications of the 3D architectures of viral tRNA-like structures.</a> RNA. 15, 294-307 (2009).</li><li>Singh, N. N., Singh, R. N., Androphy, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulating+role+of+RNA+structure+in+alternative+splicing+of+a+critical+exon+in+the+spinal+muscular+atrophy+genes.">Modulating role of RNA structure in alternative splicing of a critical exon in the spinal muscular atrophy genes.</a> Nucleic Acids Research. 35, 371-389 (2007).</li><li>Deigan, K. E., Li, T. W., Mathews, D. H., Weeks, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accurate+SHAPE-directed+RNA+structure+determination.">Accurate SHAPE-directed RNA structure determination.</a> Proceedings of the National Academy of Sciences. 106, 97-102 (2009).</li><li>Qi, H., 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=.">.</a> Preparation of pyridopyrimidine derivatives and related compounds for treating spinal muscular atrophy. , (2013).</li><li>Lorson, C. L., Hahnen, E., Androphy, E. J., Wirth, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+single+nucleotide+in+the+SMN+gene+regulates+splicing+and+is+responsible+for+spinal+muscular+atrophy.">A single nucleotide in the SMN gene regulates splicing and is responsible for spinal muscular atrophy.</a> Proceedings of the National Academy of Sciences of the United States of America. 96, 6307-6311 (1999).</li><li>Siegfried, N. A., Busan, S., Rice, G. M., Nelson, J. A. E., Weeks, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA+motif+discovery+by+SHAPE+and+mutational+profiling+(SHAPE-MaP).">RNA motif discovery by SHAPE and mutational profiling (SHAPE-MaP).</a> Nature Methods. 11, 959 (2014).</li><li>Milligan, J. F., Groebe, D. R., Witherell, G. W., Uhlenbeck, O. 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G., Hennig, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA+structure+determination+by+NMR.">RNA structure determination by NMR.</a> Methods in Molecular Biology. 452, 29-61 (2008).</li></ol>

在体外形状中, 使用高质量的均质 rna 模板至关重要。然而, t7 转录通常会产生异质序列36。特别是在 3 '-末端有±1核苷酸的序列, 产量不可忽略36 , 通常很难通过聚丙烯酰胺凝胶纯化去除。异构 rna 模板可以在引物延伸产物的测序凝胶分析中产生多组信号, 这有时会使其难以解释结果。rna 模板表达盒的 5 '-和 3 ' 端的核酶将使两端同质化。
无论...

选择性 2 '-羟基酰化分析引物延伸 (shape) 是一种测量每个核苷酸在感兴趣的 rna 序列中的动力学的方法, 并阐明单核苷酸分辨率 1下的二级结构。在体外条件 2,3,4 (纯化 rna 在一个定义的缓冲系统) 和在活哺乳动物细胞5, 6,已经开发了 shape 方法来研究继发中长 rna 序列的结构 (通常和 lt;1,000 核苷酸用于细胞内形状和 lt;200 核苷酸体外形状)。它是特别有用的评估的结构变化后, 结合 rna 相互作用的小分子代谢物2,4,7,8 和研究机械作用在药物开发过程中的 rna 靶向分子 9,10。
rna 靶向药物发现最近通过不同的方法和策略引起了学术实验室和制药业的关注 11、 12,分别为 13、14、15 ,16。最近针对临床使用的小分子的例子包括两种结构不同的实验药物, lmi-070 17 和 rg-7916 18,19, 用于脊髓肌肉萎缩 (sma), 这表明很有希望在第二阶段临床试验20。这两种分子都被证明是针对运动神经元 (smn) 2 的存活, 并调节 smn2 基因 6、17、21 的剪接过程.我们之前展示了体外和细胞内形状在检测目标 rna 结构变化的应用, 在存在的类似的 rg-7916 被称为 smn-c26.
原则上, shape 以无偏见的方式测量 rna 序列的每个核苷酸在存在过量的自淬火酰化试剂的情况下的 2 '-oh 酰化速率。酰化试剂在水中不稳定, 对 1-甲基-7-亚硝基二酸酐的半衰期较短 (例如, 1 -甲基-7-亚硝基二氢化物的半衰期为 17; 1 m7, 2-甲基烟酸咪唑 (nai) 22 的半衰期较短,对其同一性不敏感, 对其同一性不敏感。基地23。这将导致更有利的酰化 2 '-oh 组的柔性碱基, 这可以转化为一个准确的评估动态的每个核苷酸。具体而言, 基对中的核苷酸通常比未配对的核苷酸对 2 '-oh 改性试剂 (如 nai 和 1m7) 的反应性较低。
查看 rna 模板的来源和 2 '-oh 酰化发生的位置, shape 通常可分为体外和细胞内形状。体外 shape 使用纯化的 t7 转录 rna, 在实验设计中缺乏细胞环境。在细胞内形态中, rna 模板转录和 2 '-oh 酰化都发生在活细胞内;因此, 研究结果可以在细胞环境中重述 rna 结构模型。在文献24中, 细胞内形状被称为活体形状, 用于活细胞中携带的形状。由于这个实验不是在动物身上进行的, 我们把这个实验称为细胞内形状的准确性。
体外引物延伸期和细胞内形状的策略也不同。在体外形态中, 逆转录酶在 mg2+存在的情况下停止在 2 '-oh 酰化位置。因此, 在聚丙烯酰胺凝胶电泳 (page) 中, 一个32p-ld 引物延伸率显示为一个带, 该带的强度与酰化速率1成正比。在细胞内形态中, 逆转录酶在 mn2 +存在的情况下, 在 2 '-oh 加合物位置产生随机突变。通过深度下一次测序可以捕获每个核苷酸的突变速率, 然后可以计算单核苷酸分辨率下的 shape 反应性。
细胞内 shape 的一个潜在问题是低信噪比 (即, 大多数 2 '-oh 组未被修改, 而未修改的序列占据了下一代测序中的大部分读数)。最近, 由 chang 实验室25开发了一种浓缩2-oh 改性 rna 的方法, 该方法被称为体内点击 shape (icshape)。这种浓缩方法可能有利于研究弱小分子, 如 rna 相互作用, 特别是在转录全范围的审讯中。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>DNA oligonucleotide</td>
			<td>IDT</td>
			<td></td>
			<td>gBlock for &#62; 200 bp DNA synthesis</td>
		</tr>
		<tr>
			<td>Phusion Green Hot Start II High-Fidelity PCR Master Mix</td>
			<td>Thermo Fisher</td>
			<td>F566S</td>
			<td></td>
		</tr>
		<tr>
			<td>NucleoSpin gel and PCR clean-up kit</td>
			<td>Takara</td>
			<td>740609.50</td>
			<td></td>
		</tr>
		<tr>
			<td>MegaScript T7 transcription kit</td>
			<td>Thermo Fisher</td>
			<td>AM1333</td>
			<td>Contains 10X reaction buffer, T7 enzyme, NTP and Turbo DNase</td>
		</tr>
		<tr>
			<td>DEPC-treated water</td>
			<td>Thermo Fisher</td>
			<td>750023</td>
			<td></td>
		</tr>
		<tr>
			<td>2X TBE-urea sample buffer</td>
			<td>Thermo Fisher</td>
			<td>LC6876</td>
			<td></td>
		</tr>
		<tr>
			<td>40% acrylamide/ bisacrylamide solution (29:1)</td>
			<td>Bio-Rad</td>
			<td>1610146</td>
			<td></td>
		</tr>
		<tr>
			<td>10X TBE buffer</td>
			<td>Thermo Fisher</td>
			<td>15581044</td>
			<td></td>
		</tr>
		<tr>
			<td>Nalgene Rapid-Flow&#8482; Filter Unit</td>
			<td>Thermo Fisher</td>
			<td>166-0045</td>
			<td></td>
		</tr>
		<tr>
			<td>Kimwipe</td>
			<td>Kimberly-Clark</td>
			<td>34133</td>
			<td></td>
		</tr>
		<tr>
			<td>TEMED</td>
			<td>Thermo Fisher</td>
			<td>17919</td>
			<td></td>
		</tr>
		<tr>
			<td>SYBR-Safe dye</td>
			<td>Thermo Fisher</td>
			<td>S33102</td>
			<td></td>
		</tr>
		<tr>
			<td>6 % TBE-urea mini-gel</td>
			<td>Thermo Fisher</td>
			<td>EC6865BOX</td>
			<td></td>
		</tr>
		<tr>
			<td>ChemiDoc</td>
			<td>Bio-Rad</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>T4 PNK</td>
			<td>NEB</td>
			<td>M0201S</td>
			<td></td>
		</tr>
		<tr>
			<td>&#947;-32P-ATP</td>
			<td>Perkin Elmer</td>
			<td>NEG035C005MC</td>
			<td></td>
		</tr>
		<tr>
			<td>Hyperscreen&#8482; Intensifying Screen</td>
			<td>GE Healthcare</td>
			<td>RPN1669</td>
			<td>calcium tungstate phosphor screen</td>
		</tr>
		<tr>
			<td>phosphor storage screen</td>
			<td>Molecular Dynamics</td>
			<td>BAS-IP MS 3543 E</td>
			<td></td>
		</tr>
		<tr>
			<td>Amersham Typhoon</td>
			<td>GE Healthcare</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>NAI (2M)</td>
			<td>EMD Millipore</td>
			<td>03-310</td>
			<td></td>
		</tr>
		<tr>
			<td>GlycoBlue</td>
			<td>Thermo Fisher</td>
			<td>AM9515</td>
			<td></td>
		</tr>
		<tr>
			<td>SuperScript IV Reverse Transcriptase</td>
			<td>Thermo Fisher</td>
			<td>18090010</td>
			<td>Contains 5X RT buffer, SuperScript IV</td>
		</tr>
		<tr>
			<td>dNTP mix (10 mM)</td>
			<td>Thermo Fisher</td>
			<td>R0192</td>
			<td></td>
		</tr>
		<tr>
			<td>ddNTP set (5mM)</td>
			<td>Sigma</td>
			<td>GE27-2045-01</td>
			<td></td>
		</tr>
		<tr>
			<td>large filter paper</td>
			<td>Whatman</td>
			<td>1001-917</td>
			<td></td>
		</tr>
		<tr>
			<td>Gel dryer</td>
			<td>Hoefer</td>
			<td>GD 2000</td>
			<td></td>
		</tr>
		<tr>
			<td>QIAamp DNA Blood Mini Kit</td>
			<td>Qiagen</td>
			<td>51104</td>
			<td>Also contains RNase A and protease K</td>
		</tr>
		<tr>
			<td>SMN2 minigene34</td>
			<td>Addgene</td>
			<td>72287</td>
			<td></td>
		</tr>
		<tr>
			<td>Heat inactivated FBS</td>
			<td>Thermo Fisher</td>
			<td>10438026</td>
			<td></td>
		</tr>
		<tr>
			<td>Pen-Strep</td>
			<td>Thermo Fisher</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>Opti-MEM I</td>
			<td>Thermo Fisher</td>
			<td>31985062</td>
			<td></td>
		</tr>
		<tr>
			<td>FuGene HD</td>
			<td>Promega</td>
			<td>E2311</td>
			<td></td>
		</tr>
		<tr>
			<td>TrpLE</td>
			<td>Thermo Fisher</td>
			<td>12605010</td>
			<td></td>
		</tr>
		<tr>
			<td>DPBS without Ca/ Mg</td>
			<td>Thermo Fisher</td>
			<td>14190250</td>
			<td></td>
		</tr>
		<tr>
			<td>TRIzol</td>
			<td>Thermo Fisher</td>
			<td>15596018</td>
			<td></td>
		</tr>
		<tr>
			<td>RNeasy mini column</td>
			<td>Qiagen</td>
			<td>74104</td>
			<td>Also contains RW1, RPE buffer</td>
		</tr>
		<tr>
			<td>RNase-Free DNase Set</td>
			<td>Qiagen</td>
			<td>79254</td>
			<td>Contains DNase I and RDD buffer</td>
		</tr>
		<tr>
			<td>Deionized formamide</td>
			<td>Thermo Fisher</td>
			<td>AM9342</td>
			<td></td>
		</tr>
		<tr>
			<td>MnCl2&#8226;4H2O</td>
			<td>Sigma-Aldrich</td>
			<td>M3634</td>
			<td></td>
		</tr>
		<tr>
			<td>random nonamer</td>
			<td>Sigma-Aldrich</td>
			<td>R7647</td>
			<td></td>
		</tr>
		<tr>
			<td>SuperScript First-Strand Synthesis System</td>
			<td>Thermo Fisher</td>
			<td>11904-018</td>
			<td>Contains 10X RT buffer, SuperScript II reverse transcriptase</td>
		</tr>
		<tr>
			<td>AccuPrime pfx DNA polymerse</td>
			<td>Thermo Fisher</td>
			<td>12344024</td>
			<td></td>
		</tr>
		<tr>
			<td>NextSeq500</td>
			<td>Illumina</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>NucAway column</td>
			<td>Thermo Fisher</td>
			<td>AM10070</td>
			<td>for desalting purpose</td>
		</tr>
	</tbody>
</table>

using in vitro and in-cell shape to investigate small molecule induced pre-mrna structural changes

在针对小分子的药物开发过程中, 需要阐明它们与目标 rna 序列相互作用的结构变化。本文提供了一个详细的体外和细胞内选择性 2 '-羟基酰化分析引物延伸 (shape) 协议, 以研究 rna 结构的变化, 在存在的实验药物存在的脊髓肌肉萎缩 (sma), 生存运动神经元 (smn)-c2, 以及 smn2 基因的前 mrna 的外显子7。在体外形态中, 含有 smn2 外显子7的140个核苷酸的 rna 序列通过 t7 rna 聚合酶转录, 在 smn-c2 存在下折叠, 然后用温和的 2 '-oh 酰化试剂 2-甲基咪唑 (nai) 进行改性。这种 2 '-oh-nai 加合物通过32p 标记引物延伸进一步探测, 并通过聚丙烯酰胺凝胶电泳 (page) 进行求解。相反, 2 '-oh 酰化在细胞内的形状发生在原位与 smn-c2 结合细胞 rna 在活细胞。然后, pcr 扩增了 smn2 基因中外显子7的 mrna 前序列, 以及 shape 诱导的引物延伸突变, 并进行了下一代测序。通过对这两种方法的比较, 体外 shape 是一种更具成本效益的方法, 不需要计算能力来可视化结果。然而, 体外 shape 衍生 rna 模型有时会偏离细胞环境中的二级结构, 这可能是由于与 rna 结合蛋白的所有相互作用的丧失。细胞内形状不需要放射性物质工作场所, 并在细胞环境中产生更准确的 rna 二级结构。此外, 细胞内 shape 通常适用于更大范围的 rna 序列 (~ 1, 000个核苷酸), 利用下一代测序, 而体外 shape (~ 200 核苷酸) 通常依赖于 page 分析。在 smn2 前 mrna 中的外显子7的情况下, 体外和细胞内形状 rna 模型是相似的。

我们之前证明, rna 拼接调节剂 smn-c2 与 smn2 基因前 mrna 的外显子7上的 aggaag 母题相互作用, 并使用 shape 评估 smn-c26 存在时的 rna 结构变化。smn-c2 的结合位点不同于 fda 批准的 sma、nusinersen 反义寡核苷酸 (aso), 它在内含子 7、28 (图 1 a) 上结合和阻断了内含子上的内含子拼接消声器 (iss)。大多?...

Watch this Scientific Journal Video about Using In Vitro and In-cell SHAPE to Investigate Small Molecule Induced Pre-mRNA Structural Changes at JoVE.com

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

通过引物扩展 (shape) 实验分析的体外和细胞内选择性 2 '-羟基酰化的详细方案, 以确定在存在 rna 靶向小分子的情况下, mrna 前序列的二级结构。本文中介绍。

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 ...

本视频将为体外形状实验提供详细的方案，以确定RNA序列的二级结构，即RNA针对小分子的存在。体外形状是一种经济有效的方法来了解每个核苷酸在氨基酸大小的RNA元素上的动态，该元素通常少于200个核苷酸。在准备手稿中描述的RNA模板后，通过添加伽玛32P ATP，逆转转录底法，对转录底剂进行放射性标签逆转转录底剂。10XPNK反应缓冲液、多核苷酸激酶和RNA自由水在1.7毫升微离心管中，总体积为20微升。然后在37摄氏度下孵育一小时。然后，从 65 摄氏度开始激活样品 20 分钟，然后在脱盐柱中过滤样品，然后以 1000 倍 G.添加 25 微升 2XTBE 尿素样品缓冲液和混合物进行离心机。将这些贴有标签的产品降入 10%丙烯酰胺凝胶中，并确保避免将放射性出血到顶部储液罐中。以 20 瓦的功率运行凝胶一小时。运行后，取出凝胶盒的玻璃板。将凝胶放在塑料包装上。将塑料折叠到凝胶顶部，制作一个气密三明治。将凝胶三明治放在钙通态荧光屏上，用想象仪器曝光。用常规光线再次对凝胶进行成像，以了解凝胶的边缘在哪里。使用图像处理软件覆盖两个图像并覆盖浅色和深色图像。然后使顶部图像稍微不透明，以便同时查看两个图像。确保打印的图像与实际凝胶具有相同的大小。然后将凝胶对齐在打印的图像上。最后，使用消毒剃须刀刀片将所需的弯曲从凝胶中切出，将每块凝胶放在单独的 1.7 毫升微离心管中。要通过被动洗脱从凝胶片中恢复DNA，每1.7毫升管中加入0.3毫升的洗脱沉淀混合物。在4摄氏度的旋转器上将管子在放射性安全容器中孵育16小时。孵育后，将液体转移到新的1.7毫升管中。加入2.5倍冰冷200防乙醇，并反转管子六次混合液体。在零下80摄氏度下孵育管子至少一小时。在10000克和4摄氏度的离心10分钟后，通过移液小心地取出上一液。加入一毫升80%乙醇来清洗颗粒。涡流15秒，在相同条件下再次离心。通过移液去除上流水器后，风干DNA颗粒五分钟。加入50微升RNA自由水，通过上下移液10次混合。在闪烁计数器中，DNA浓度标准化为每分钟约10万个微升计数。要准备四个用于化学修饰的样品，在 32 微升 0.5 XTE 缓冲液中加入 8 个 RNA 图片，加入 1.7 毫升微离心管。在80摄氏度下加热两分钟，在冰上加热至少一分钟。然后加入8个5倍折叠混合微升，将这种RNA混合物的9微升混合到四个PCR管中，从1个到4个。将一微升10%DMSO加入管中标为一，一微升浓缩小分子溶液入管二，一微升稀释小分子溶液放入管三和一微升水到管四。在热循环器中，在 37 摄氏度下孵育所有 PCR 管 30 分钟。直接在两个主 OH 改性反应之前，在三微升 DEPC 处理水中稀释两个摩尔 NAI 库存溶液的一微升，产生 0.5 摩尔工作溶液。立即将溶液的一微升添加到管一、二和三中。将 25%DMSO 的微升加入第四管，在 37 摄氏度的热循环器中孵育 15 分钟。准备四个新鲜的1.7毫升微离心管，并在每个管中加入100微升的洗脱沉淀混合物和两个微升的糖原。然后，从相应的PCR管中转移所有液体。在每个管中加入0.34毫升的冰冷200证明乙醇，通过涡流混合5秒钟。将管子放在零下80摄氏度的冰柜中至少一小时。然后在摄氏4度的14，000倍G下将管离心15分钟。在不干扰RNA颗粒的情况下，从每个管中去除上一液。每管加入0.5毫升80%乙醇，清洗RNA颗粒和漩涡15秒。在相同条件下再次离心后，取出上经液并风干RNA颗粒五分钟。加入9微升RNA的免费水，并上下管10次混合。将修改后的RNA转移到四个新的PCR管中，并像上一步一样将它们贴上一到四个标签。在 7 微升 DEPC 处理水中加入两个 RNA 图片，每个微升的 PCR 管中加入两个 RNA，并标记数字 5 至 8。在标有一至八的八个 PCR 管中，每个管中加入两个回收的无线电标记底材的微升。在65摄氏度加热5分钟后，在热循环器中立即冷却至4摄氏度。在每个管中加入四个 5x RT 缓冲液的微升、一个 0.1 摩尔 DTT 的微升和一个 DNTP 混合的微升。然后将两升DEPC处理的水加入一至四管。在管五中加入四微升五毫摩尔 ddATP，将四微升五毫摩尔 ddTTTP 添加到管六和四微升五毫摩尔 ddCTP 到管七和一微升五毫摩尔 ddGTP 和三微升五毫摩尔 ddGTP 和三微升 DEPC 处理水到管八和移液器四次混合。在热循环器中，所有PCR管在52摄氏度下加热一分钟后，在每个管中加入一微升的逆转录酶，然后上下管四次混合。在热循环器中，在52摄氏度下孵育反应20分钟。要对RNA进行水解，将一升五摩尔氢氧化钠的微升加入每个管中，并在95摄氏度下加热5分钟。然后加入五微升一摩尔盐酸，以中和反应，并重复乙醇沉淀，如前面所述。在风干DNA颗粒5分钟后，在1.7毫升管中加入10微升水和10微升2xTBE尿素样品加载缓冲液，上下管10次，重新溶解。在70摄氏度下加热样品5分钟，然后将管子放在冰上，然后装入凝胶。将放在冰上的样品的10微升加载到8%聚丙烯酰胺测序凝胶上。以 65 瓦的功率运行凝胶两小时，或直到底部染料达到凝胶的 3/4。拆下垫片，轻轻插入铲子以拆下顶部硅化玻璃板。用一块大滤纸盖住凝胶，轻轻按压，使凝胶粘附在滤纸上。从底端开始，提起滤纸，将凝胶与纸张一起从玻璃板上取出。将凝胶转移到包装纸之前一样，用真空在 70 摄氏度下干燥一小时，确保在凝胶和烘干机之间使用几个滤纸。将凝胶三明治转移到光漂白的荧光荧光储存盒上，将凝胶的放射性暴露到屏幕上 4 到 16 小时。最后，将荧光粉存储屏幕放在成像设备上，以中等分辨率扫描约 30 分钟。将聚丙烯酰胺测序凝胶暴露至荧光粉储存屏幕后，一个成功的形状实验显示，凝胶顶部有一个最强烈的弯曲，并且以单核苷酸分辨率弯曲整个凝胶，无需涂抹。凝胶还显示，一些弯曲的强度在小分子的存在下发生变化，表明碱基配对强度的变化。页面分析中的一个常见问题是，称为盐面的涂片区域可能出现在凝胶的中间，可能是由于加载样品中盐、DMSO 或其他不需要的物质浓度高。这可以通过乙醇沉淀去除不需要的物质来避免。同质RNA模板更可用于体外形状。我们使用五个质端和三个质端的核糖体来获得这样的RNA。也可以执行细胞内形状，以获得细胞环境中的RNA二级结构。请记住，体外形状可能不会重述体内RNA结合蛋白相互作用。体外形状是一种强大的方法，用于检测RNA结构中由于存在小分子而发生的变化。这是通过量化逆转录酶停止模式强度的变化。请小心，本视频中描述的所有实验都在经过授权的放射性工作场所进行，并提供适当的个人保护和有机玻璃屏蔽。

Research

JoVE Journal

Genetics

Optogenetic Random Mutagenesis Using Histone-miniSOG in C. elegans

Optogenetic Random Mutagenesis Using Histone-miniSOG in C. elegans

光遗传学随机突变用组蛋白miniSOG在<em&gt;℃。线虫</em

Forward genetic screening in model organisms is the workhorse to discover functionally important genes and pathways in many biological processes. In most ...

科研

遗传学

Analysis of the Ambient Particulate Matter-induced Chromosomal Aberrations Using an In Vitro System

Analysis of the Ambient Particulate Matter-induced Chromosomal Aberrations Using an In Vitro System

使用了大气颗粒物引起的染色体畸变分析<em&gt;体外</em&gt;系统

Exposure to particulate matter (PM) is a major world health concern, which may damage various cellular components, including the nuclear genetic material. ...

In Vitro Bioluminescence Assay to Characterize Circadian Rhythm in Mammary Epithelial Cells

In Vitro Bioluminescence Assay to Characterize Circadian Rhythm in Mammary Epithelial Cells

体外生物荧光法对乳腺上皮细胞昼夜节律的表征

The circadian rhythm is a fundamental physiological process present in all organisms that regulates biological processes ranging from gene expression to ...

Real-time Analysis of Transcription Factor Binding, Transcription, Translation, and Turnover to Display Global Events During Cellular Activation

转录因子结合、转录、翻译和营业额的实时分析在细胞激活过程中显示全球事件

Upon activation, cells rapidly change their functional programs and, thereby, their gene expression profile. Massive changes in gene expression occur, for ...

An Optogenetic Method to Control and Analyze Gene Expression Patterns in Cell-to-cell Interactions

细胞间相互作用中基因表达模式的控制与分析的 Optogenetic 方法

Cells should respond properly to temporally changing environments, which are influenced by various factors from surrounding cells. The Notch signaling ...

Single Molecule Fluorescence In Situ Hybridization (smFISH) Analysis in Budding Yeast Vegetative Growth and Meiosis

Single Molecule Fluorescence In Situ Hybridization smFISH Analysis in Budding Yeast Vegetative Growth and Meiosis

单分子荧光原位杂交 (smFISH) 在发芽酵母营养生长和减数分裂中的分析

Single molecule fluorescence in situ hybridization (smFISH) is a powerful technique to study gene expression in single cells due to its ability to detect ...

Dissection of Enhancer Function Using Multiplex CRISPR-based Enhancer Interference in Cell Lines

基于多重 CRISPR 增强器的细胞系干扰增强功能解剖

Multiple enhancers often regulate a given gene, yet for most genes, it remains unclear which enhancers are necessary for gene expression, and how these ...

Quantitative Analysis of Alternative Pre-mRNA Splicing in Mouse Brain Sections Using RNA In Situ Hybridization Assay

Quantitative Analysis of Alternative Pre-mRNA Splicing in Mouse Brain Sections Using RNA In Situ Hybridization Assay

RNA原位杂交法定量分析小鼠脑切片中的替代前 mRNA 拼接方法

Alternative splicing (AS) occurs in more than 90% of human genes. The expression pattern of an alternatively spliced exon is often regulated in a cell ...