Name: in vitro biochemical assays using biotin labels to study protein-nucleic acid interactions
Uploaded: 2019-07-17T14:00:00
Description: Watch this Scientific Journal Video about In Vitro Biochemical Assays using Biotin Labels to Study Protein-Nucleic Acid Interactions at JoVE.com

We thank P. Jeremy Wang (University of Pennsylvania) for helpful edits and discussions. We also thank Sigrid Eckardt for language editing. K. Z. was supported by National Key R&#38;D Program of China (2016YFA0500902, 2018YFC1003500) and National Natural Science Foundation of China (31771653). L. Y. was supported by National Natural Science Foundation of China (81471502, 31871503) and Innovative and Entrepreneurial Program of Jiangsu Province.&#160;J. N. was supported by Zhejiang Medical Science and Technology Project (2019KY499).&#160;M. L. was supported by grants of National Natural Science Foundation of China (31771588) and the 1000 Youth Talent Plan.

1. Protein preparation
<ol>
	<li>MEIOB and MOV10 expression constructs
	<ol>
		<li>Generate cDNA expression constructs encoding mouse MEIOB-A, C, and E (Figure 1A) and MOV10.
		<ol>
			<li>Set up the polymerase chain reaction (PCR) reactions for each fragment. Mix 1 &#956;L of mouse cDNA (from C57BL/6 mouse testis), 1 &#956;L of dNTP, 2 &#956;L of 10 &#956;M forward primer, 2 &#956;L of 10 &#956;M reverse primer, 1 &#956;L of DNA polymerase, 25 &#956;L of 2x PCR buffer, an...

<ol>
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W., Leng, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid+and+sensitive+high-throughput+screening+method+to+identify+compounds+targeting+protein-nucleic+acids+interactions.">A rapid and sensitive high-throughput screening method to identify compounds targeting protein-nucleic acids interactions.</a> Nucleic Acids Research. 43 (8), 52 (2015).</li><li>Hwang, H., Myong, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+induced+fluorescence+enhancement+(PIFE)+for+probing+protein-nucleic+acid+interactions.">Protein induced fluorescence enhancement (PIFE) for probing protein-nucleic acid interactions.</a> Chemical Society Reviews. 43 (4), 1221-1229 (2014).</li><li>Gustafsdottir, S. 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=In+vitro+analysis+of+DNA-protein+interactions+by+proximity+ligation.">In vitro analysis of DNA-protein interactions by proximity ligation.</a> Proceedings of the National Academy of Sciences of the United States of America. 104 (9), 3067-3072 (2007).</li><li>Li, Y., Jiang, Z., Chen, H., Ma, W. 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+modified+quantitative+EMSA+and+its+application+in+the+study+of+RNA--protein+interactions.">A modified quantitative EMSA and its application in the study of RNA--protein interactions.</a> Journal of Biochemical and Biophysical Methods. 60 (2), 85-96 (2004).</li><li>Fahrer, J., Kranaster, R., Altmeyer, M., Marx, A., Burkle, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+analysis+of+the+binding+affinity+of+poly(ADP-ribose)+to+specific+binding+proteins+as+a+function+of+chain+length.">Quantitative analysis of the binding affinity of poly(ADP-ribose) to specific binding proteins as a function of chain length.</a> Nucleic Acids Research. 35 (21), 143 (2007).</li><li>Hsieh, Y. W., Alqadah, A., Chuang, C. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+Optimized+Protocol+for+Electrophoretic+Mobility+Shift+Assay+Using+Infrared+Fluorescent+Dye-labeled+Oligonucleotides.">An Optimized Protocol for Electrophoretic Mobility Shift Assay Using Infrared Fluorescent Dye-labeled Oligonucleotides.</a> Journal of Visualized Experiments. (117), (2016).</li><li>Yan, G., 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=Orphan+Nuclear+Receptor+Nur77+Inhibits+Cardiac+Hypertrophic+Response+to+Beta-Adrenergic+Stimulation.">Orphan Nuclear Receptor Nur77 Inhibits Cardiac Hypertrophic Response to Beta-Adrenergic Stimulation.</a> Molecular and Cellular Biology. 35 (19), 3312-3323 (2015).</li><li>Hellman, L. M., Fried, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrophoretic+mobility+shift+assay+(EMSA)+for+detecting+protein-nucleic+acid+interactions.">Electrophoretic mobility shift assay (EMSA) for detecting protein-nucleic acid interactions.</a> Nature Protocols. 2 (8), 1849-1861 (2007).</li><li>Fillebeen, C., Wilkinson, N., Pantopoulos, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrophoretic+mobility+shift+assay+(EMSA)+for+the+study+of+RNA-protein+interactions:+the+IRE/IRP+example.">Electrophoretic mobility shift assay (EMSA) for the study of RNA-protein interactions: the IRE/IRP example.</a> Journal of Visualized Experiments. (94), (2014).</li><li>Nishida, K. 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=Hierarchical+roles+of+mitochondrial+Papi+and+Zucchini+in+Bombyx+germline+piRNA+biogenesis.">Hierarchical roles of mitochondrial Papi and Zucchini in Bombyx germline piRNA biogenesis.</a> Nature. 555 (7695), 260-264 (2018).</li><li>Anders, C., Jinek, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+enzymology+of+Cas9.">In vitro enzymology of Cas9.</a> Methods in Enzymology. 546, 1-20 (2014).</li><li>Zhao, H., Zheng, J., Li, Q. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+plant+in+vitro+assay+system+for+pre-mRNA+cleavage+during+3'-end+formation.">A novel plant in vitro assay system for pre-mRNA cleavage during 3'-end formation.</a> Plant Physiology. 157 (3), 1546-1554 (2011).</li><li>Vourekas, 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=The+RNA+helicase+MOV10L1+binds+piRNA+precursors+to+initiate+piRNA+processing.">The RNA helicase MOV10L1 binds piRNA precursors to initiate piRNA processing.</a> Genes &amp; Development. 29 (6), 617-629 (2015).</li><li>Gregersen, L. 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=MOV10+Is+a+5'+to+3'+RNA+helicase+contributing+to+UPF1+mRNA+target+degradation+by+translocation+along+3'+UTRs.">MOV10 Is a 5' to 3' RNA helicase contributing to UPF1 mRNA target degradation by translocation along 3' UTRs.</a> Molecular Cell. 54 (4), (2014).</li><li>Talwar, T., 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+DEAD-box+protein+DDX43+(HAGE)+is+a+dual+RNA-DNA+helicase+and+has+a+K-homology+domain+required+for+full+nucleic+acid+unwinding+activity.">The DEAD-box protein DDX43 (HAGE) is a dual RNA-DNA helicase and has a K-homology domain required for full nucleic acid unwinding activity.</a> The Journal of Biological Chemistry. 292 (25), 10429-10443 (2017).</li><li>Nagy, N. M., Konya, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Study+of+fast+and+slow+consecutive+processes+by+heterogeneous+isotope+exchange+using+P-32+radiotracer.">Study of fast and slow consecutive processes by heterogeneous isotope exchange using P-32 radiotracer.</a> Journal of Radioanalytical And Nuclear Chemistry. 318 (3), 2349-2353 (2018).</li><li>Wilson, D. L., Beharry, A. A., Srivastava, A., O'Connor, T. R., Kool, E. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+Probes+for+ALKBH2+Allow+the+Measurement+of+DNA+Alkylation+Repair+and+Drug+Resistance+Responses.">Fluorescence Probes for ALKBH2 Allow the Measurement of DNA Alkylation Repair and Drug Resistance Responses.</a> Angewandte Chemie. 57 (39), 12896-12900 (2018).</li><li>Wilchek, M., Bayer, E. A., Livnah, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Essentials+of+biorecognition:+the+(strept)avidin-biotin+system+as+a+model+for+protein-protein+and+protein-ligand+interaction.">Essentials of biorecognition: the (strept)avidin-biotin system as a model for protein-protein and protein-ligand interaction.</a> Immunology Letters. 103 (1), 27-32 (2006).</li><li>Trippier, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthetic+strategies+for+the+biotinylation+of+bioactive+small+molecules.">Synthetic strategies for the biotinylation of bioactive small molecules.</a> ChemMedChem. 8 (2), 190-203 (2013).</li><li>Rodgers, J. T., Patel, P., Hennes, J. L., Bolognia, S. L., Mascotti, D. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+biotin-labeled+nucleic+acids+for+protein+purification+and+agarose-based+chemiluminescent+electromobility+shift+assays.">Use of biotin-labeled nucleic acids for protein purification and agarose-based chemiluminescent electromobility shift assays.</a> Analytical Biochemistry. 277 (2), 254-259 (2000).</li><li>Panda, A. C., Martindale, J. L., Gorospe, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Affinity+Pulldown+of+Biotinylated+RNA+for+Detection+of+Protein-RNA+Complexes.">Affinity Pulldown of Biotinylated RNA for Detection of Protein-RNA Complexes.</a> Bio-Protocol. 6 (24), (2016).</li><li>Bednarek, S., 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=mRNAs+biotinylated+within+the+5'+cap+and+protected+against+decapping:+new+tools+to+capture+RNA+-+protein+complexes.">mRNAs biotinylated within the 5' cap and protected against decapping: new tools to capture RNA - protein complexes.</a> Philosophical Transactions Of the Royal Society B-Biological Sciences. 373 (1762), (2018).</li><li>Souquet, 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=MEIOB+Targets+Single-Strand+DNA+and+Is+Necessary+for+Meiotic+Recombination.">MEIOB Targets Single-Strand DNA and Is Necessary for Meiotic Recombination.</a> Plos Genetics. 9 (9), (2013).</li><li>Luo, 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=MEIOB+exhibits+single-stranded+DNA-binding+and+exonuclease+activities+and+is+essential+for+meiotic+recombination.">MEIOB exhibits single-stranded DNA-binding and exonuclease activities and is essential for meiotic recombination.</a> Nature Communications. 4, 2788 (2013).</li><li>Xu, Y., Greenberg, R. A., Schonbrunn, E., Wang, 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=Meiosis-specific+proteins+MEIOB+and+SPATA22+cooperatively+associate+with+the+single-stranded+DNA-binding+replication+protein+A+complex+and+DNA+double-strand+breaks.">Meiosis-specific proteins MEIOB and SPATA22 cooperatively associate with the single-stranded DNA-binding replication protein A complex and DNA double-strand breaks.</a> Biology of Reproduction. 96 (5), 1096-1104 (2017).</li></ol>

Investigating protein-nucleic acid interactions is critical to our understanding of molecular mechanisms underlying diverse biological processes. For example, MEIOB is a testis-specific protein essential for meiosis and fertility in mammals25,26,27. MEIOB contains an OB domain that binds to single-stranded DNA and exhibits 3&#39;&#160;to 5&#39;&#160;exonuclease activity26, which directly relates to its ph...

Protein-nucleic acid interactions are involved in many essential cellular processes such as DNA repair, replication, transcription, RNA processing, and translation. Protein interactions with specific DNA sequences within the chromatin are required for the tight control of gene expression at the transcriptional level1. Precise posttranscriptional regulation of numerous coding and noncoding RNAs necessitates extensive and complicated interactions between any protein and RNA2. These layers of gene expression regulatory mechanism comprise a cascade of dynamic intermolecular events, which are coordinated by interactions of transcription/epigenetic factors or RNA-binding proteins with their nucleic acid targets, as well as protein-protein interactions. To dissect whether proteins in vivo are directly or indirectly associated with nucleic acids and how such associations occur and culminate, in vitro biochemical assays are conducted to examine the binding affinity or enzymatic activity of proteins of interest on designed substrates of DNA and/or RNA.
Many techniques have been developed to detect and characterize nucleic acid-protein complexes, including the electrophoretic mobility shift assay (EMSA), also termed gel retardation assay or band shift assay3,4,5. EMSA is a versatile and sensitive biochemical method that is widely used for studying the direct binding of proteins with nucleic acids. EMSA relies on gel electrophoretic shift in bands, which are routinely visualized using chemiluminescence to detect biotin labels6,7, the fluorescence of fluorophore labels8,9, or by autoradiography of radioactive 32P labels10,11. Other purposes of biochemical studies are the identification and characterization of nucleic acid-processing activity of proteins, such as &#160;nuclease-based reactions to assess the cleavage products from nucleic acid substrates12,13,14 and DNA/RNA structure-unwinding assays to evaluate helicase activities15,16,17.
In such enzymatic activity assays, the radioisotope-labeled or fluorophore-labeled nucleic acids are often used as substrates due to their high sensitivity. Analysis of radiographs of enzymatic reactions involving 32P labeled radiotracers has been found to be sensitive and reproducible18. Yet, in an increasing number of laboratories in the world, the usage of radioisotopes is restricted or even prohibited due to the health risks associated with potential exposure. In addition to biosafety concerns, other drawbacks are the required necessary equipment to conduct work with radioisotopes, required radioactivity license, short half-life of 32P (about 14 days), and gradual deterioration of the probe quality due to radiolysis. Thus, alternative non-isotopic methods have been developed (i.e., labeling the probe with fluorophores enables detection by fluorescent imaging19). However, a high-resolution imaging system is needed when using fluorescently labeled probes. Biotin, a commonly used label, readily binds to biological macromolecules such as proteins and nucleic acids. Biotin-streptavidin system operates efficiently and improves detection sensitivity without increasing non-specific background20,21. Besides EMSA, biotin is widely used for protein purification and RNA pull-down, among others22,23,24.
This protocol successfully uses biotin-labeled nucleic acids as substrates for in vitro biochemical assays that include EMSA, in addition to enzymatic reactions in which biotin has not been commonly used. The MEIOB OB domain is constructed and two mutants (truncation and point mutation) are expressed as GST fusion proteins25,26,27, as well as mouse MOV10 recombinant FLAG fusion protein16. This report highlights the effectiveness of this combined protocol for protein purification and biotin-labeled assays for miscellaneous experimental purposes.

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			<td height="21" style="height:21px;width:351px;">Equipment</td>
			<td style="width:163px;"></td>
			<td style="width:104px;"></td>
			<td style="width:176px;"></td>
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			<td height="21" style="height:21px;width:351px;">Centrifuge</td>
			<td style="width:163px;">Eppendorf, Germany</td>
			<td style="width:104px;">5242R</td>
			<td style="width:176px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Chemiluminescent Imaging System</td>
			<td>Tanon, China</td>
			<td>5200</td>
			<td style="width:176px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Digital sonifer</td>
			<td>Branson, USA</td>
			<td>BBV12081048A</td>
			<td style="width:176px;">450 Watts; 50/60 HZ</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Semi-dry electrophoretic blotter</td>
			<td style="width:163px;">Hoefer, USA</td>
			<td style="width:104px;">TE77XP</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">Tube Revolver&#160;</td>
			<td style="width:163px;">Crystal, USA</td>
			<td style="width:104px;">3406051</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">UV-light cross-linker</td>
			<td>UVP, USA</td>
			<td style="width:104px;">CL-1000</td>
			<td></td>
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			<td height="21" style="height:21px;width:351px;">Materials</td>
			<td></td>
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			<td height="21" style="height:21px;">Amicon Ultra-4 Centrifugal Filter&#160;</td>
			<td>Milipore, USA</td>
			<td>UFC801096</td>
			<td>4 ml/10 K</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">Nylon membrane</td>
			<td>Thermo Scientific, USA</td>
			<td style="width:104px;">TG263940A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">TC-treated Culture Dish</td>
			<td>Corning, USA</td>
			<td style="width:104px;">430167</td>
			<td style="width:176px;">100 mm&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">TC-treated Culture Dish</td>
			<td>Corning, USA</td>
			<td style="width:104px;">430597</td>
			<td style="width:176px;">150 mm&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">Microtubes tubes</td>
			<td style="width:163px;">AXYGEN, USA</td>
			<td style="width:104px;">MCT-150-C</td>
			<td style="width:176px;">1.5 mL&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">Tubes</td>
			<td style="width:163px;">Corning, USA</td>
			<td style="width:104px;">430791</td>
			<td style="width:176px;">15 mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">Reagents&#160;</td>
			<td style="width:163px;"></td>
			<td style="width:104px;"></td>
			<td style="width:176px;"></td>
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			<td height="21" style="height:21px;">Ampicillin</td>
			<td>SunShine Bio, China</td>
			<td>8h288h28</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">Anti-FLAG M2 magnetic beads</td>
			<td style="width:163px;">Sigma, USA</td>
			<td style="width:104px;">M8823</td>
			<td></td>
		</tr>
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			<td height="21" style="height:22px;">ATP</td>
			<td>Thermo Scientific, USA</td>
			<td>591136</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BCIP/NBT Alkaline Phosphatase Color Development Kit</td>
			<td>Beyotime, China</td>
			<td>C3206</td>
			<td></td>
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			<td height="24" style="height:24px;">CelLyticTM M Cell Lysis Reagent&#160;</td>
			<td>Sigma, USA</td>
			<td>107M4071V</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ClonExpress II one step cloning kit&#160;</td>
			<td>Vazyme, China</td>
			<td>C112</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Chemiluminescent Nucleic Acid Detection Kit</td>
			<td>Thermo Scientific, USA</td>
			<td>T1269950</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">dNTP</td>
			<td>Sigma-Aldrich, USA</td>
			<td>DNTP100-1KT</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">DMEM</td>
			<td style="width:163px;">Gibco, USA</td>
			<td style="width:104px;">10569044</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">DPBS buffer</td>
			<td style="width:163px;">Gibco, USA</td>
			<td style="width:104px;">14190-136</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">EDTA</td>
			<td>Invitrogen, USA</td>
			<td style="width:104px;">AM9260G</td>
			<td>0.5 M</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">EDTA free protease inhibitor cocktail</td>
			<td style="width:163px;">Roche, USA</td>
			<td style="width:104px;">04693132001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">EndoFree Maxi Plasmid Kit&#160;</td>
			<td>Vazyme, China</td>
			<td>&#160; 
			DC202</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FastPure Gel DNA Extraction Mini Kit</td>
			<td>Vazyme, China</td>
			<td>DC301-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">FBS</td>
			<td style="width:163px;">Gibco, USA</td>
			<td style="width:104px;">10437028</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">FLAG peptide</td>
			<td style="width:163px;">Sigma, USA</td>
			<td style="width:104px;">F4799</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">Glycerol</td>
			<td style="width:163px;">Sigma, USA</td>
			<td style="width:104px;">SHBK3676</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">GST Bulk Kit</td>
			<td style="width:163px;">GE Healthcare, USA</td>
			<td style="width:104px;">27-4570-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HEPES buffer</td>
			<td>Sigma, USA</td>
			<td>SLBZ2837</td>
			<td>1 M&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">IPTG</td>
			<td>Thermo Scientific, USA</td>
			<td>34060</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">KoAc</td>
			<td style="width:163px;">Sangon Biotech, China</td>
			<td style="width:104px;">127-08-02</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Lipofectamin 3000 Transfection Reagent</td>
			<td>Thermo Scientific, USA</td>
			<td style="width:104px;">L3000001</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:351px;">MgCl2</td>
			<td>Invitrogen, USA</td>
			<td style="width:104px;">AM9530G</td>
			<td>1 M</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NaCl</td>
			<td>Invitrogen, USA</td>
			<td>AM9759 
			&#160;</td>
			<td>5 M&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">NP-40</td>
			<td style="width:163px;">Amresco, USA</td>
			<td style="width:104px;">M158-500ML</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Opti-MEM medium</td>
			<td>Gibco, USA</td>
			<td style="width:104px;">31985062</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PBS</td>
			<td>Gibco, USA</td>
			<td>10010023</td>
			<td>PH 7.4</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">RNase Inhibitor</td>
			<td style="width:163px;">Promega, USA</td>
			<td style="width:104px;">N251B</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">Streptavidin alkaline phosphatase</td>
			<td style="width:163px;">Promega, USA</td>
			<td style="width:104px;">V5591</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TBE</td>
			<td style="width:163px;">Invitrogen, USA</td>
			<td style="width:104px;">15581044</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tris-HCI Buffer&#160;</td>
			<td>Invitrogen, USA</td>
			<td>15567027</td>
			<td>1 M, PH 7.4</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tris-HCI Buffer&#160;</td>
			<td>Invitrogen, USA</td>
			<td>15568025</td>
			<td>1 M, PH 8.0</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">Tween-20</td>
			<td style="width:163px;">Sangon Biotech, China</td>
			<td style="width:104px;">A600560</td>
			<td></td>
		</tr>
	</tbody>
</table>

in vitro biochemical assays using biotin labels to study protein-nucleic acid interactions

Protein-nucleic acid interactions play important roles in biological processes such as transcription, recombination, and RNA metabolism. Experimental methods to study protein-nucleic acid interactions require the use of fluorescent tags, radioactive isotopes, or other labels to detect and analyze specific target molecules. Biotin, a non-radioactive nucleic acid label, is commonly used in electrophoretic mobility shift assays (EMSA) but has not been regularly employed to monitor protein activity during nucleic acid processes. This protocol illustrates the utility of biotin labeling during in vitro enzymatic reactions, demonstrating that this label works well with a range of different biochemical assays. Specifically, in alignment with previous findings using radioisotope 32P-labeled substrates, it is confirmed via biotin-labeled EMSA that MEIOB (a protein specifically involved in the meiotic recombination) is a DNA-binding protein, that MOV10 (an RNA helicase) resolves biotin-labeled RNA duplex structures, and that MEIOB cleaves biotin-labeled single-stranded DNA. This study demonstrates that biotin is capable of substituting 32P in various nucleic acid-related biochemical assays in vitro.&#160;

The protein structure of MEIOB and the expression constructs used in this study are illustrated in Figure 1A. OB folds in MEIOB are compact barrel-like structures that can recognize and interact with single-stranded nucleic acids. One of the OB domains (aa 136-307, construct A) binds single stranded DNA (ssDNA), the truncated protein (aa 136-178 truncations, construct C) and the point mutant form (R235A mutation, construct E) of MEIOB do not have DNA-binding activity<sup cla...

Watch this Scientific Journal Video about In Vitro Biochemical Assays using Biotin Labels to Study Protein-Nucleic Acid Interactions at JoVE.com

In Vitro Biochemical Assays using Biotin Labels to Study Protein-Nucleic Acid Interactions

Presented here are protocols for in vitro biochemical assays using biotin labels that may be widely&#160;applicable for studying protein-nucleic acid interactions.

Protein-nucleic acid interactions play important roles in biological processes such as transcription, recombination, and RNA metabolism. Experimental ...

This protocol offers a biotin-labeled platform for the study of protein-nucleic acid interactions that proves to be robust, reliable, efficient, and affordable. The same batch of the biotin-labeled nucleic acids can be used over a long period of time, maintaining reproducibility of experiments. Finally, all biotin-labeled assays described here can be performed within a day and do not require special equipment.Demonstrating the procedure will be Lina Yu, a postgraduate from our laboratory. Prepare MEIOB and MOV10 constructs, and transform them into the appropriate bacteria. To extract MEIOB, pellet the cells via centrifugation and resuspend them in 20 milliliters of ice-cold Dulbecco's phosphate buffered saline, or DPBS, buffer.Sonicate the bacterial suspension on ice. Then, centrifuge the lysate at 12, 000 times g and four degrees Celsius for 15 minutes, and transfer the supernatant to a fresh tube. Pre-wash glutathione-Sepharose beads, and incubate them with the lysate at four degrees Celsius for two hours.Centrifuge the mixture at 750 times g and four degrees Celsius for one minute to pellet the beads. Rinse the beads with 10 milliliters of ice-cold PBS eight times. Then, add one milliliter of elution buffer, incubate the beads at four degrees Celsius for 10 minutes, and elute the beads via centrifugation.Repeat the elution six times, and pool together the six fractions. Transfer the eluted proteins into a centrifugal filter, and concentrate by centrifugation at 7, 500 times g for a final volume of 100 to 200 microliters. To extract MOV10, express the protein in HEK293T cells and pellet the cells according to the manuscript directions.Resuspend the pellet in three milliliters of cell lysis buffer with complete EDTA-free protease inhibitor cocktail, and incubate for 30 minutes on ice. Then, centrifuge the lysate at four degrees Celsius. Prepare anti-FLAG magnetic beads by washing them twice with K150 buffer.After the wash, resuspend the beads in one milliliter of ice-cold K150 buffer and incubate them for two minutes at four degrees Celsius with gentle rotation. Collect the beads on a magnet, and remove the supernatant. Add the beads to the cell lysate, and incubate at four degrees Celsius for two hours.Wash the beads with K150 buffer according to the manuscript directions, and resuspend the beads in 300 microliters of FLAG elution buffer. Place the beads on a magnet, collect the supernatant with the MOV10 proteins, and determine the protein concentration. Prepare the RNA oligonucleotides by diluting each oligo to 20 micromolar and annealing them to form RNA duplexes according to the manuscript directions.For the MEIOB electrophoretic mobility shift assay, or EMSA, mix the reagents according to the manuscript directions. Add water for a final reaction volume of 20 microliters. Incubate the reaction at room temperature for 30 minutes, and then add 5x stop buffer.For the MOV10 helicase activity assay, mix the reagents according to the manuscript directions and add water for a final reaction volume of 20 microliters. Incubate the reaction at 37 degrees Celsius for 10 minutes, 30 minutes, and 60 minutes, and then add 5x stop buffer to stop the reaction. Then, prepare a 20%native polyacrylamide gel, and load 20 to 25 microliters of each sample into each well.Run the gel at 100 volts on an ice bath until the bromophenol blue marker has migrated to the bottom quarter of the gel. Acrylamide is harmful and toxic. It is important to handle it with appropriate personal protective equipment.Disassemble the gel plates, and trim the gel by removing loading wells and unused lanes. Place the gel in 0.5x TBE buffer. Cut filter paper and nylon membrane to the size of the gel, pre-wet the paper and membrane, and assemble the stack for transfer.Transfer the samples from the gel to the membrane in a semi-dry electrophoretic apparatus at 90 milliamperes for 20 minutes. Then, crosslink the samples by irradiating the membrane at 120 millijoules per centimeter squared for 45 to 60 seconds. For chemiluminescence detection, begin by adding 20 milliliters of blocking buffer to the membrane and incubating it for 15 to 30 minutes while gently shaking.Carefully remove the blocking buffer, and replace it with conjugate/blocking buffer. Incubate the membrane again for 15 minutes. Wash the membrane four times while shaking for five minutes per wash.Then, add 30 milliliters of substrate equilibration buffer to the membrane, and incubate it for five minutes while shaking at 20 to 25 rpm. Cover the entire surface of the membrane with substrate working solution, and incubate for five minutes. After the incubation, scan the membrane in a chemiluminescent imaging system for one to three seconds.Purification of the MEIOB proteins A, C, and E can be verified with Coomassie blue staining and western blot analysis. The red arrows indicate the positions of the purified MEIOB proteins. Interactions of MEIOB and single-stranded DNA can be visualized using EMSA.Concentration-dependent binding and cleavage are observed. As expected, only the wild-type MEIOB-A interacts with the DNA while the mutant MEIOB-E and MEIOB-C do not. Interactions of MEIOB and single-stranded RNA can also be observed.Wild-type MEIOB shows concentration-dependent binding and exonuclease activity on single-stranded RNA. However, quantitative comparison shows that MEIOB binds DNA with higher efficiency than RNA. Purification of MOV10 was verified with Coomassie blue staining, and the protein's helicase activity was measured on an RNA duplex with a five-prime overhang.As reaction time increases, MOV10 progressively unwinds the double-stranded RNA. To reduce the cost of the assay, the chemiluminescence detection can be performed with either a two-fold dilution of the detection kit reagents or with self-made reagents. The protocol described here successfully used biotin-labeled nucleic acids as substrates for in vitro biochemical assays such as EMSA and for enzymatic reactions in which biotin has not been commonly used.

in-vitro-biochemical-assays-using-biotin-labels-to-study-protein

Research

JoVE Journal

Genetics

In Vitro Transcription Assays and Their Application in Drug Discovery

In vitro transcription assays have been developed and widely used for many years to study the molecular mechanisms involved in transcription. This process requires multi-subunit DNA-dependent RNA polymerase (RNAP) and a series of transcription factors that act to modulate the activity of RNAP during gene expression. Sequencing gel electrophoresis of radiolabeled transcripts is used to provide detailed mechanistic information on how transcription proceeds and what parameters can affect it. In this paper we describe the protocol to study how the essential elongation factor NusA regulates transcriptional pausing, as well as a method to identify an antibacterial agent targeting transcription initiation through inhibition of RNAP holoenzyme formation. These methods can be used a as platform for the development of additional approaches to explore the mechanism of action of the transcription factors which still remain unclear, as well as new antibacterial agents targeting transcription which is an underutilized drug target in antibiotic research and development.

In Vitro Transcription Assays and Their Application in Drug Discovery

In this manuscript, we describe a protocol to functionally examine transcription and the inhibitory activity of antibacterial agents targeting bacterial transcription.

In vitro transcription assays have been developed and widely used for many years to study the molecular mechanisms involved in transcription. This process ...

Genetic Manipulation of the Plant Pathogen Ustilago maydis to Study Fungal Biology and Plant Microbe Interactions

Gene deletion plays an important role in the analysis of gene function. One of the most efficient methods to disrupt genes in a targeted manner is the replacement of the entire gene with a selectable marker via homologous recombination. During homologous recombination, exchange of DNA takes place between sequences with high similarity. Therefore, linear genomic sequences flanking a target gene can be used to specifically direct a selectable marker to the desired integration site. Blunt ends of the deletion construct activate the cell&#39;s DNA repair systems and thereby promote integration of the construct either via homologous recombination or by non-homologous-end-joining. In organisms with efficient homologous recombination, the rate of successful gene deletion can reach more than 50% making this strategy a valuable gene disruption system. The smut fungus Ustilago maydis is a eukaryotic model microorganism showing such efficient homologous recombination. Out of its about 6,900 genes, many have been functionally characterized with the help of deletion mutants, and repeated failure of gene replacement attempts points at essential function of the gene. Subsequent characterization of the gene function by tagging with fluorescent markers or mutations of predicted domains also relies on DNA exchange via homologous recombination. Here, we present the U. maydis strain generation strategy in detail using the simplest example, the gene deletion.

Genetic Manipulation of the Plant Pathogen Ustilago maydis to Study Fungal Biology and Plant Microbe Interactions

We describe a robust gene replacement strategy to genetically manipulate the smut fungus Ustilago maydis. This protocol explains how to generate deletion mutants to investigate infection phenotypes. It can be extended to modify genes in any desired way, e.g., by adding a sequence encoding a fluorescent protein tag.

Gene deletion plays an important role in the analysis of gene function. One of the most efficient methods to disrupt genes in a targeted manner is the ...

Conjugative Mating Assays for Sequence-specific Analysis of Transfer Proteins Involved in Bacterial Conjugation

The transfer of genetic material by bacterial conjugation is a process that takes place via complexes formed by specific transfer proteins. In Escherichia coli, these transfer proteins make up a DNA transfer machinery known as the mating pair formation, or DNA transfer complex, which facilitates conjugative plasmid transfer. The objective of this paper is to provide a method that can be used to determine the role of a specific transfer protein that is involved in conjugation using a series of deletions and/or point mutations in combination with mating assays. The target gene is knocked out on the conjugative plasmid and is then provided in trans through the use of a small recovery plasmid harboring the target gene. Mutations affecting the target gene on the recovery plasmid can reveal information about functional aspects of the target protein that result in the alteration of mating efficiency of donor cells harboring the mutated gene. Alterations in mating efficiency provide insight into the role and importance of the particular transfer protein, or a region therein, in facilitating conjugative DNA transfer. Coupling this mating assay with detailed three-dimensional structural studies will provide a comprehensive understanding of the function of the conjugative transfer protein as well as provide a means for identifying and characterizing regions of protein-protein interaction.

Here, we present a protocol to knockout a gene of interest involved in plasmid conjugation and subsequently analyze the impact of its absence using mating assays. The function of the gene is further explored to a specific region of its sequence using deletion or point mutations.

The transfer of genetic material by bacterial conjugation is a process that takes place via complexes formed by specific transfer proteins. In Escherichia ...

The Nematode Caenorhabditis Elegans - A Versatile In Vivo Model to Study Host-microbe Interactions

We demonstrate a method using Caenorhabditis elegans as a model host to study microbial interaction. Microbes are introduced via the diet making the intestine the primary location for disease. The nematode intestine structurally and functionally mimics mammalian intestines and is transparent making it amenable to microscopic study of colonization. Here we show that pathogens can cause disease and death. We are able to identify microbial mutants that show altered virulence. Its conserved innate response to biotic stresses makes C. elegans an excellent system to probe facets of host innate immune interactions. We show that hosts with mutations in the dual oxidase gene cannot produce reactive oxygen species and are unable to resist microbial insult. We further demonstrate the versatility of the presented survival assay by showing that it can be used to study the effects of inhibitors of microbial growth. This assay may also be used to discover fungal virulence factors as targets for the development of novel antifungal agents, as well as provide an opportunity to further uncover host-microbe interactions. The design of this assay lends itself well to high throughput whole-genome screens, while the ability to cryo-preserve worms for future use makes it a cost-effective and attractive whole animal model to study.

The Nematode Caenorhabditis Elegans - A Versatile In Vivo Model to Study Host-microbe Interactions

Here, we present the nematode Caenorhabditis elegans as a versatile host model to study microbial interaction.

We demonstrate a method using Caenorhabditis elegans as a model host to study microbial interaction. Microbes are introduced via the diet making the ...

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

Cells should respond properly to temporally changing environments, which are influenced by various factors from surrounding cells. The Notch signaling pathway is one of such essential molecular machinery for cell-to-cell communications, which plays key roles in normal development of embryos. This pathway involves a cell-to-cell transfer of oscillatory information with ultradian rhythms, but despite the progress in molecular biology techniques, it has been challenging to elucidate the impact of multicellular interactions on oscillatory gene dynamics. Here, we present a protocol that permits optogenetic control and live monitoring of gene expression patterns in a precise temporal manner. This method successfully revealed that intracellular and intercellular periodic inputs of Notch signaling entrain intrinsic oscillations by frequency tuning and phase shifting at the single-cell resolution. This approach is applicable to the analysis of the dynamic features of various signaling pathways, providing a unique platform to test a functional significance of dynamic gene expression programs in multicellular systems.

Here, we present a protocol to analyze cell-to-cell transfer of oscillatory information by optogenetic control and live monitoring of gene expression. This approach provides a unique platform to test a functional significance of dynamic gene expression programs in multicellular systems.

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

Biotin-based Pulldown Assay to Validate mRNA Targets of Cellular miRNAs

MicroRNAs (miRNAs) are a class of small noncoding RNAs that post-transcriptionally regulate cellular gene expression. MiRNAs bind to the 3&#39; untranslated region (UTR) of target mRNA to inhibit protein translation or in some instances cause mRNA degradation. The binding of the miRNA to the 3&#39; UTR of the target mRNA is mediated by a 2&#8211;8 nucleotide seed sequence at the 5&#39; end of miRNA. While the role of miRNAs as cellular regulatory molecules is well established, identification of the target mRNAs with functional relevance remains a challenge. Bioinformatic tools have been employed to predict sequences within the 3&#39; UTR of mRNAs as potential targets for miRNA binding. These tools have also been utilized to determine the evolutionary conservation of such sequences among related species in an attempt to predict functional role. However, these computational methods often generate false positive results and are limited to predicting canonical interaction between miRNA and mRNA. Therefore, experimental procedures that measure direct binding of miRNA to its mRNA target are necessary to establish functional interaction. In this report, we describe a sensitive method for validating direct interaction between the cellular miRNA miR-125b and the 3&#39; UTR of PARP-1 mRNA. We elaborate a protocol in which synthetic biotinylated-miRNA mimics were transfected into mammalian cells and the miRNA-mRNA complex in the cellular lysate was pulled down with streptavidin-coated magnetic beads. Finally, the target mRNA in the pulled-down nucleic acid complex was quantified using a qPCR-based strategy.

This report describes a fast and reliable method for validating mRNA targets of cellular miRNAs. The method uses synthetic biotinylated Locked Nucleic Acid (LNA)-based miRNA mimics to capture target mRNA. Subsequently, streptavidin-coated magnetic beads are employed to pulldown the target mRNA for quantification by qPCR polymerase chain reaction.

MicroRNAs (miRNAs) are a class of small noncoding RNAs that post-transcriptionally regulate cellular gene expression. MiRNAs bind to the 3&#39; ...

High-throughput Identification of Gene Regulatory Sequences Using Next-generation Sequencing of Circular Chromosome Conformation Capture (4C-seq)

The identification of regulatory elements for a given target gene poses a significant technical challenge owing to the variability in the positioning and effect sizes of regulatory elements to a target gene. Some progress has been made with the bioinformatic prediction of the existence and function of proximal epigenetic modifications associated with activated gene expression using conserved transcription factor binding sites. Chromatin conformation capture studies have revolutionized our ability to discover physical chromatin contacts between sequences and even within an entire genome. Circular chromatin conformation capture coupled with next-generation sequencing (4C-seq), in particular, is designed to discover all possible physical chromatin interactions for a given sequence of interest (viewpoint), such as a target gene or a regulatory enhancer. Current 4C-seq strategies directly sequence from within the viewpoint but require numerous and diverse viewpoints to be simultaneously sequenced to avoid the technical challenges of uniform base calling (imaging) with next generation sequencing platforms. This volume of experiments may not be practical for many laboratories. Here, we report a modified approach to the 4C-seq protocol that incorporates both an additional restriction enzyme digest and qPCR-based amplification steps that are designed to facilitate a greater capture of diverse sequence reads and mitigate the potential for PCR bias, respectively. Our modified 4C method is amenable to the standard molecular biology lab for assessing chromatin architecture.

High-throughput Identification of Gene Regulatory Sequences Using Next-generation Sequencing of Circular Chromosome Conformation Capture 4C-seq

The identification of physical interactions between genes and regulatory elements is challenging but has been facilitated by chromosome conformation capture methods. This modification to the 4C-seq protocol mitigates PCR bias by minimizing over-amplification of PCR templates and maximizes the mappability of reads by incorporating an addition restriction enzyme digest step.

The identification of regulatory elements for a given target gene poses a significant technical challenge owing to the variability in the positioning and ...

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

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

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

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

In Vivo Functional Study of Disease-associated Rare Human Variants Using Drosophila

Advances in sequencing technology have made whole-genome and whole-exome datasets more accessible for both clinical diagnosis and cutting-edge human genetics research. Although a number of in silico algorithms have been developed to predict the pathogenicity of variants identified in these datasets, functional studies are critical to determining how specific genomic variants affect protein function, especially for missense variants. In the Undiagnosed Diseases Network (UDN) and other rare disease research consortia, model organisms (MO) including Drosophila, C. elegans, zebrafish, and mice are actively used to assess the function of putative human disease-causing variants. This protocol describes a method for the functional assessment of rare human variants used in the Model Organisms Screening Center Drosophila Core of the UDN. The workflow begins with gathering human and MO information from multiple public databases, using the MARRVEL web resource to assess whether the variant is likely to contribute to a patient&#39;s condition as well as design effective experiments based on available knowledge and resources. Next, genetic tools (e.g., T2A-GAL4 and UAS-human cDNA lines) are generated to assess the functions of variants of interest in Drosophila. Upon development of these reagents, two-pronged functional assays based on rescue and overexpression experiments can be performed to assess variant function. In the rescue branch, the endogenous fly genes are &#34;humanized&#34;&#160;by replacing the orthologous Drosophila gene with reference or variant human transgenes. In the overexpression branch, the reference and variant human proteins are exogenously driven in a variety of tissues. In both cases, any scorable phenotype (e.g., lethality, eye morphology, electrophysiology) can be used as a read-out, irrespective of the disease of interest. Differences observed between reference and variant alleles suggest a variant-specific effect, and thus likely pathogenicity. This protocol allows rapid, in vivo assessments of putative human disease-causing variants of genes with known and unknown functions.

In Vivo Functional Study of Disease-associated Rare Human Variants Using Drosophila

The goal of this protocol is to outline the design and performance of in vivo experiments in Drosophila melanogaster to assess the functional consequences of rare gene variants associated with human diseases.

Advances in sequencing technology have made whole-genome and whole-exome datasets more accessible for both clinical diagnosis and cutting-edge human ...

Transcriptome-Wide Profiling of Protein-RNA Interactions by Cross-Linking and Immunoprecipitation Mediated by FLAG-Biotin Tandem Purification

RNA and RNA-binding proteins (RBPs) control multiple biological processes. The spatial and temporal arrangement of RNAs and RBPs underlies the delicate regulation of these processes. A strategy called CLIP-seq (cross-linking and immunoprecipitation) has been developed to capture endogenous protein-RNA interactions with UV cross-linking followed by immunoprecipitation. Despite the wide use of conventional CLIP-seq method in RBP study, the CLIP method is limited by the availability of high-quality antibodies, potential contaminants from the copurified RBPs, requirement of isotope manipulation, and potential loss of information during a tedious experimental procedure. Here we describe a modified CLIP-seq method called FbioCLIP-seq using the FLAG-biotin tag tandem purification. Through tandem purification and stringent wash conditions, almost all the interacting RNA-binding proteins are removed. Thus, the RNAs interacting indirectly mediated by these copurified RBPs are also reduced. Our FbioCLIP-seq method allows efficient detection of direct protein-bound RNAs without SDS-PAGE and membrane transfer procedures in an isotope-free and protein-specific antibody-free manner.

Here we present a modified CLIP-seq protocol called FbioCLIP-seq with FLAG-biotin tandem purification to determine the RNA targets of RNA-binding proteins (RBPs) in mammalian cells.

RNA and RNA-binding proteins (RBPs) control multiple biological processes. The spatial and temporal arrangement of RNAs and RBPs underlies the delicate ...