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

This work was made possible by the&#160;NIH R01 grant (NS094721, K.A.J.).&#160;

1. In Vitro SHAPE
NOTE: The protocol is modified from the published protocol1.
<ol>
	<li>Preparing the RNA template 
	NOTE: The template for T7 transcription was ordered as a synthetic double-stranded DNA (dsDNA) and amplified by either insertion into an E. coli vector that carries a unique pair of EcoRI/BamHI restriction endonuclease sites, such as pET28a, or by PCR. The protocol for PCR amplification i...

<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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D., Varani, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+based+approaches+for+targeting+non-coding+RNAs+with+small+molecules.">Structure based approaches for targeting non-coding RNAs with small molecules.</a> Current Opinion in Structural Biology. 30, 79-88 (2015).</li><li>Gallego, J., Varani, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+RNA+with+Small-Molecule+Drugs:+Therapeutic+Promise+and+Chemical+Challenges.">Targeting RNA with Small-Molecule Drugs: Therapeutic Promise and Chemical Challenges.</a> Accounts of Chemical Research. 34, 836-843 (2001).</li><li>Rizvi, N. F., Smith, G. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA+as+a+small+molecule+druggable+target.">RNA as a small molecule druggable target.</a> Bioorganic &amp; Medicinal Chemistry Letters. 27, 5083-5088 (2017).</li><li>Connelly, C. M., Moon, M. H., Schneekloth, 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=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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In in vitro SHAPE, it is critical to use high quality homogeneous RNA template. T7 transcription, however, often yields heterogeneous sequences36. Especially, sequences with &#177;1 nucleotide at the 3&#39;-terminus with non-negligible yields36 are usually difficult to be removed by polyacrylamide gel purification. Heterogeneous RNA template can result in more than one set of the signal in the sequencing gel profiling of the primer extension product, which sometimes makes i...

Selective 2&#39;-hydroxyl acylation analyzed by primer extension (SHAPE) is a method of measuring the kinetics of each nucleotide in an RNA sequence of interest and elucidating the secondary structure at single-nucleotide resolution1. SHAPE methodologies, both in in vitro conditions2,3,4 (purified RNA in a defined buffer system) and in living mammalian cells5,6, have been developed to investigate the secondary structure of medium length RNA sequences (typically &#60;1,000 nucleotides for in-cell SHAPE and &#60;200 nucleotides for in vitro SHAPE). It is particularly useful to evaluate structural changes in receptor RNA upon binding to RNA-interacting small molecule metabolites2,4,7,8 and to study mechanistic actions of RNA-targeting molecules during drug development9,10.
RNA-targeting drug discovery has recently drawn attention in academic laboratories and the pharmaceutical industry11,12 via different approaches and strategies13,14,15,16. Recent examples of RNA-targeting small molecules for clinical use include two structurally distinct experimental drugs, LMI-07017 and RG-791618,19, for spinal muscular atrophy (SMA), which showed promising results in phase II clinical trials20. Both molecules were demonstrated to target survival of motor neuron (SMN) 2 pre-mRNA and regulate the splicing process of the SMN2 gene6,17,21. We previously demonstrated the application of in vitro and in-cell SHAPE in an examination of the target RNA structural changes in the presence of an analog of RG-7916 known as SMN-C26.
In principle, SHAPE measures the 2&#39;-OH acylation rate of each nucleotide of an RNA sequence in the presence of excess amounts of a self-quenching acylation reagent in an unbiased manner. The acylation reagent is not stable in water, with a short half-life of (e.g., T1/2 = 17 s for 1-methyl-7-nitroisatoic anhydride; or 1M7, ~20 min for 2-methylnicotinic acid imidazolide, or NAI)22 and insensitivity to the identity of the bases23. This results in a more favorable acylation of the 2&#39;-OH groups of flexible bases, which can be transformed into an accurate assessment of the dynamics of each nucleotide. Specifically, a nucleotide in a base-pair is usually less reactive than an unpaired one to a 2&#39;-OH modifying reagent, such as NAI and 1M7.
Looking at the source of the RNA template and where 2&#39;-OH acylation takes place, SHAPE can generally be categorized into in vitro and in-cell SHAPE. In vitro SHAPE uses purified T7 transcribed RNA and lacks a cellular context in experimental designs. In in-cell SHAPE, both the RNA template transcription and 2&#39;-OH acylation occur within living cells; therefore, the results can recapitulate the RNA structural model in a cellular context. In-cell SHAPE has been referred to as in vivo SHAPE for the SHAPE carried in living cells in the literature24. Since this experiment is not performed in an animal, we termed this experiment as in-cell SHAPE for accuracy.
The strategies for the primer extension stage of in vitro and in-cell SHAPE are also different. In in vitro SHAPE, reverse transcription stops at the 2&#39;-OH acylation position in the presence of Mg2+. A 32P-labled primer extension therefore appears as a band in polyacrylamide gel electrophoresis (PAGE) and the intensity of the band is proportional to the acylation rate1. In in-cell SHAPE, reverse transcription generates random mutations at the 2&#39;-OH adduct position in the presence of Mn2+. The mutational rate of each nucleotide can be captured by in depth next-generation sequencing, and the SHAPE reactivity at single-nucleotide resolution can then be calculated.
A potential problem for in-cell SHAPE is the low signal-to-noise ratio (i.e., a majority of the 2&#39;-OH groups is unmodified, while the unmodified sequences occupy most of the read in next-generation sequencing). Recently, a method to enrich the 2&#39;-OH modified RNA, referred to as in vivo click SHAPE (icSHAPE), was developed by the Chang laboratory25. This enrichment method may be advantageous in studying weak small molecules such as RNA interactions, especially in a transcriptome-wide interrogation.

<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

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.

We previously demonstrated that an RNA splicing modulator, SMN-C2, interacts with AGGAAG motif on exon 7 of the SMN2 gene&#39;s pre-mRNA, and used SHAPE to assess the RNA structural changes in the presence of SMN-C26. The binding site of SMN-C2 is distinct from the FDA-approved antisense oligonucleotide (ASO) for SMA, nusinersen, which binds and blocks the intronic splicing silencer (ISS) on intron 727,28 (...

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

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.

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

This video will provide detailed protocols for in vitro shape experiments to determine the secondary structure of RNA sequences of interest in the presence of an RNA targeting small molecule. In vitro shape is a cost effective method to understand the dynamics of each nucleotide on an amino-sized RNA element which is usually less than 200 nucleotides. After preparing the RNA template as described in the manuscript, radio label reverse transcription primers by adding a gamma 32P ATP, reversed transcription primer.10XPNK reaction buffer, polynucleotide kinase and RNA's free water in a 1.7 milliliter microcentrifuge tube in a total volume of 20 microliters. Then incubate at 37 degrees Celsius for one hour. You then activate the samples for 20 minutes since 65 degrees Celsius and then filter the samples in a desalting column and centrifuge at 1000 times G.Add 25 microliters of 2XTBE urea sample buffer and mix.Lower these labeled products into a 10%acrylamide gel and make sure to avoid bleeding the radioactivity into the top reservoirs. Run the gel at 20 watts for one hour. After the run, remove the glass plates of the gel cassette.Lay the gel on the piece of plastic wrap. Fold the plastic onto the top of the gel to make an air-tight sandwich. Place the gel sandwich on a a calcium-tungstate phosphor screen and expose with an imagining instrument.Image the gel again with regular light to know where the edges of the gel are. Use an image-processing software to overlay the two images and overlay the light and dark images. Then make the top image slightly opaque in order to see both images simultaneously.Make sure the printed image has the same size as the actual gel. Then align the gel on the printed image. Lastly, use a sterilized razor blade to cut the desired bends out of the gel and place each piece of gel in a separate 1.7 milliliter microcentrifuge tube.To recover the DNA from the gel slice by passive elution, add 0.3 milliliters of elution precipitation mix to each 1.7 milliliter tube. Incubate the tubes in a radioactive-safe container on a rotator at four degrees Celsius for 16 hours. After incubation, transfer the liquid into a new 1.7 milliliter tube.Add 2.5x ice cold 200 proof ethanol and invert the tubes six times to mix the liquid. Incubate the tubes at minus 80 degrees Celsius for a minimum of one hour. After centrifuging at 10, 000g and four degrees Celsius for 10 minutes, remove the supernatant carefully by pipetting.Add one milliliter of 80%ethanol to wash the pellet. Vortex for 15 seconds and centrifuge again under the same conditions. After removing the supernatant by pipetting, air-dry the DNA pellet for five minutes.Add 50 microliters of RNA's free water and mix by pipetting up and down 10 times. Normalize the DNA concentration to about 100, 000 counts per minute per microliter in a scintillation counter. To prepare four samples for chemical modification, add eight picomoles of RNA in 32 microliters of 0.5 XTE buffer to a 1.7 milliliter microcentrifuge tube.Heat at 80 degrees Celsius for two minutes and snap cool on ice for at least one minute. Then add eight microliters of 5x folding mix, mix an allocate nine microliters of this RNA mixture into each of the four PCR tubes, labeled from one to four. Add one microliter of 10%DMSO into the tube marked as one, one microliter of concentrated small molecule solution into tube two, one microliter of diluted small molecule solution into tube three and one microliter of water into tube four.Incubate all PCR tubes at 37 degrees Celsius in a thermal cycler for 30 minutes. Directly before the two prime OH modification reaction, dilute one microliter of two molar NAI stock solution in three microliters of DEPC-treated water to yield a 0.5 molar working solution. Immediately add one microliter of this solution into tubes one, two and three.Add one microliter of 25%DMSO into tube four and incubate in a thermal cycler at 37 degrees Celsius for 15 minutes. Prepare four fresh 1.7 milliliter microcentrifuge tubes and add 100 microliters of elution precipitation mix and two microliters of glycogen to each tube. Then into each of these tubes transfer all the liquid from the corresponding PCR tubes.Add 0.34 milliliters of ice-cold 200 proof ethanol into each tube and mix by vortexing for five seconds. Place the tubes in a minus 80 degrees Celsius freezer for at least one hour. Then centrifuge the tubes for 15 minutes at 14, 000 times G at four degrees Celsius.Without disturbing the RNA pellet, remove the supernatant from each tube. Add 0.5 milliliters of 80%ethanol per tube to wash the RNA pellet and vortex for 15 seconds. After centrifuging at the same conditions again, remove the supernatant and air-dry the RNA pellet for five minutes.Add nine microliters of RNA's free water and pipe that up and down 10 times to mix. Transfer the modified RNA into four new PCR tubes and label them one to four as in the previous step. Add two picomoles of RNA in seven microliters of DEPC-treated water to each of four additional PCR tubes and label them with numbers five to eight.Add two microliters of the recovered radiolabeled primer to each of the eight PCR tubes marked one to eight. After heating at 65 degrees Celsius for five minutes, immediately cool down to four degrees Celsius in the thermal cycler. Add four microliters of 5x RT buffer, one microliter of 0.1 molar DTT and one microliter of DNTP mix to each of the tubes.Then add two microliters of DEPC-treated water to tubes one to four. Add four microliters of five millimolar ddATP to tube five, four microliters of five millimolar ddTTP to tube six and four microliters of five millimolar ddCTP to tube seven and one microliter of five millimolar ddGTP and three microliters of DEPC-treated water to tube eight and pipette four times to mix. After heating all of the PCR tubes at 52 degrees Celsius for one minute in a thermal cycler, add one microliter of reverse transcriptase to each tube and pipe it up and down four times to mix.Incubate the reactions at 52 degrees Celsius for 20 minutes in the thermal cycler. To hydrolyze the RNA, add one microliter of five molar sodium hydroxide into each of the tubes and heat them at 95 degrees Celsius for five minutes. Then add five microliters of one molar hydrochloric acid to neutralize the reaction and repeat the ethanol precipitation as previously described.After air-drying the DNA pellet for five minutes, add 10 microliters of water and 10 microliters of 2xTBE urea sample loading buffer in 1.7 milliliter tubes and pipe it up and down 10 times to redissolve. Heat the samples at 70 degrees Celsius for five minutes, then place the tubes on ice, before loading onto the gel. Load 10 microliters of the sample that has been placed on ice onto 8%polyacrylamide sequencing gel.Run the gel at 65 watts for two hours or until the bottom dye reaches the 3/4ths of the gel. Remove the spacer and gently insert a spatula to remove the top siliconized glass plate. Cover the gel with a piece of large filter paper and gently press to allow the gel to adhere to the filter paper.Starting from the bottom end, lift the filter paper and remove the gel from the glass plate together with the paper. After transferring the gel to a wrapping paper as previously done, dry it at 70 degree Celsius for one hour with a vacuum, making sure to use several filter papers between the gel and the dryer. Transfer the gel sandwich onto a photo-bleached phosphor storage screen cassette and expose the radioactivity of the gel to the screen for four to 16 hours.Finally, place the phosphor storage screen on an imaging device and scan with medium resolution for approximately 30 minutes. After exposing polyacrylamide sequencing gel to the phosphor storage screen, a successful shape experiment showed a single and most intense bend at the top of the gel and bends throughout the gel at single nucleotide resolution without smear. The gel also showed that the intensity of some bends changed in the presence of small molecules, indicating changes of base pairing strength.A common problem in page analysis is that a smear region known as the salt front may appear in the middle of the gel, probably due to high concentration of salt, DMSO or other unwanted substance in the loading sample. That can be avoided by removing the unwanted substance by ethanol precipitation. A homogenous RNA template is preferable for in vitro shape.We use ribosomes at both five prime and three prime ends to obtain such an RNA. In-cell shape can also be performed to obtain RNA secondary structure in the cellular context. Remember, in vitro shape may not recapitulate in vivo RNA binding protein interactions.In vitro shape is a powerful method for detecting changes in RNA structure due to the presence of a small molecule. This is done by quantifying changes in the patterning strength of reverse transcriptase stops. Be cautious that all experiments described in this video be performed in an authorized radioactive workplace with appropriate personal protection and plexiglass shielding.

Research

JoVE Journal

Genetics

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