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.

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