Name: single-molecule manipulation of g-quadruplexes by magnetic tweezers
Uploaded: 2017-09-19T14:00:00
Description: Watch this Scientific Journal Video about Single-molecule Manipulation of G-quadruplexes by Magnetic Tweezers at JoVE.com

The authors thank Meng Pan for proofreading the manuscript. This work is supported by Singapore Ministry of Education Academic Research Fund Tier 3 (MOE2012-T3-1-001) to J.Y.; the National Research Foundation through the Mechanobiology Institute Singapore to J.Y.; the National Research Foundation, Prime Minister&#39;s Office, Singapore, under its NRF Investigatorship Programme (NRF Investigatorship Award No. NRF-NRFI2016-03 to J.Y.; the Fundamental Research Fund for the Central Universities (2017KFYXJJ153) to H. Y.

1. Preparation of G4 DNA for Single-molecule Stretching
<ol>
	<li>Prepare 5&#39;-thiol labeled and 5&#39;-biotin labeled dsDNA handles by PCR using DDNA polymerase on a lambda phage DNA template using 5&#39;-thiol and 5&#39;-biotin primers14 (Figure 1). Both dsDNA handles have high GC content (&#62; 60%) to prevent DNA melting when DNA is held at high forces or during DNA overstretching transition15.</li>
	<li>Purify PCR products using a comm...

<ol>
	<li>Rhodes, D., Lipps, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=G-quadruplexes+and+their+regulatory+roles+in+biology.">G-quadruplexes and their regulatory roles in biology.</a> Nucleic Acids Res. 43 (18), 8627-8637 (2015).</li><li>Brazda, V., Haronikova, L., Liao, J. C., Fojta, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+and+RNA+quadruplex-binding+proteins.">DNA and RNA quadruplex-binding proteins.</a> Int J Mol Sci. 15 (10), 17493-17517 (2014).</li><li>Gonzalez, V., Hurley, L. 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F., Yan, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two+distinct+overstretched+DNA+states.">Two distinct overstretched DNA states.</a> Nucleic Acids Res. 38 (16), 5594-5600 (2010).</li><li>Gosse, C., Croquette, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Magnetic+tweezers:+micromanipulation+and+force+measurement+at+the+molecular+level.">Magnetic tweezers: micromanipulation and force measurement at the molecular level.</a> Biophys. 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S., Yan, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two+distinct+overstretched+DNA+structures+revealed+by+single-molecule+thermodynamics+measurements.">Two distinct overstretched DNA structures revealed by single-molecule thermodynamics measurements.</a> Proc Natl Acad Sci U S A. 109 (21), 8103-8108 (2012).</li><li>Zhang, X., 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=Revealing+the+competition+between+peeled+ssDNA,+melting+bubbles,+and+S-DNA+during+DNA+overstretching+by+single-molecule+calorimetry.">Revealing the competition between peeled ssDNA, melting bubbles, and S-DNA during DNA overstretching by single-molecule calorimetry.</a> Proc Natl Acad Sci U S A. 110 (10), 3865-3870 (2013).</li><li>Chen, 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=Improved+High-Force+Magnetic+Tweezers+for+Stretching+and+Refolding+of+Proteins+and+Short+DNA.">Improved High-Force Magnetic Tweezers for Stretching and Refolding of Proteins and Short DNA.</a> Biophys. J. 100 (2), 517-523 (2011).</li><li>Fu, H. X., 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=Transition+dynamics+and+selection+of+the+distinct+S-DNA+and+strand+unpeeling+modes+of+double+helix+overstretching.">Transition dynamics and selection of the distinct S-DNA and strand unpeeling modes of double helix overstretching.</a> Nucleic Acids Res. 39 (8), 3473-3481 (2011).</li><li>Zhang, X., Chen, H., Fu, H., Doyle, P. S., Yan, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two+distinct+overstretched+DNA+structures+revealed+by+single-molecule+thermodynamics+measurements.">Two distinct overstretched DNA structures revealed by single-molecule thermodynamics measurements.</a> Proc. Natl. Acad. Sci. U.S.A. 109 (21), 8103-8108 (2012).</li><li>Zhang, X., 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=Revealing+the+competition+between+peeled+ssDNA,+melting+bubbles,+and+S-DNA+during+DNA+overstretching+by+single-molecule+calorimetry.">Revealing the competition between peeled ssDNA, melting bubbles, and S-DNA during DNA overstretching by single-molecule calorimetry.</a> Proc. Natl. Acad. Sci. U.S.A. 110 (10), 3865-3870 (2013).</li><li>Vaughn, J. P., 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+DEXH+protein+product+of+the+DHX36+gene+is+the+major+source+of+tetramolecular+quadruplex+G4-DNA+resolving+activity+in+HeLa+cell+lysates.">The DEXH protein product of the DHX36 gene is the major source of tetramolecular quadruplex G4-DNA resolving activity in HeLa cell lysates.</a> J Biol Chem. 280 (46), 38117-38120 (2005).</li><li>Giri, 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=G4+resolvase+1+tightly+binds+and+unwinds+unimolecular+G4-DNA.">G4 resolvase 1 tightly binds and unwinds unimolecular G4-DNA.</a> Nucleic Acids Res. 39 (16), 7161-7178 (2011).</li><li>De Vlaminck, I., Dekker, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+advances+in+magnetic+tweezers.">Recent advances in magnetic tweezers.</a> Annu Rev Biophys. 41, 453-472 (2012).</li><li>Yan, J., Skoko, D., Marko, J. 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As described above, a platform for studying the mechanical stability of G4 DNA and the interactions of protein to G4 using single-molecule magnetic tweezers is reported. Accompanying the platform, highly efficient protocols of finding G4 DNA tether, and measurement of the folding-unfolding dynamics and stability of the G4 structure with nanometers special resolution are developed. The focal plane locking enables highly stable anti-drift control, which is important for detecting a small structure transition such as G4 (st...

Four-stranded DNA or RNA G4 structures play critical roles in many important biological processes1. Many proteins are involved in G4 binding and regulation, including telomere binding proteins (telomerase, POT1, RPA, TEBPs, TRF2)1,2, transcription factors (nucleolin, PARP1)3, RNA processing proteins (hnRNP A1, hnRNP A2)4, helicases (BLM, FANCJ, RHAU, WRN, Dna2, Pif1)5, and DNA replication related proteins (Rif1, REV1, PrimPolymerase)6. Protein binding can stabilize or destabilize G4 structures; thus regulating the subsequent biological functions. The stability of G4 was measured by thermal melting using ultraviolet (UV) or circular dichroism (CD) methods7. However, such conditions are not physiological relevant and are difficult to apply to studying the effects of binding proteins7.
The rapid development in single-molecule manipulation technologies has enabled studies of folding and unfolding of a biomolecule, such as a DNA or a protein, at a single-molecule level with nanometer resolution in real time8. Atomic force microscopy (AFM), optical tweezers, and magnetic tweezers are the most commonly used single-molecule manipulation methods. Compared to AFM and optical tweezers9, magnetic tweezers allow stable measurements of folding-unfolding dynamics of a single molecule over days by using an anti-drift technique10,11.
Here, a single-molecule manipulation platform using magnetic tweezers to study the regulation of G4 stability by binding proteins is reported12,13. This work outlines the basic approaches, including sample and flow channel preparation, the setup of magnetic tweezers, and the force calibration. The force control and the anti-drift protocols as described in step 3 allow for long time measurements under various force controls, such as constant force (force clamp) and constant loading rate (force-ramp), and force-jump measurement. The force calibration protocol described in step 4 enables force calibration of &#60; 1 &#181;m short tethers over a wide force range up to 100 pN, with a relative error within 10%. An example of regulation of the stability of the RNA Helicase associated with AU-rich element (RHAU) helicase (alias DHX36, G4R1) that plays essential roles in resolving RNA G4 is used to demonstrate the applications of this platform13.

<table>
	<tbody>
		<tr>
			<td>DNA PCR primers</td>
			<td>IDT</td>
			<td></td>
			<td>DNA preparations</td>
		</tr>
		<tr>
			<td>DNA PCR chemicals</td>
			<td>NEB</td>
			<td></td>
			<td>DNA preparations</td>
		</tr>
		<tr>
			<td>restriction enzyme BstXI</td>
			<td>NEB</td>
			<td>R0113S</td>
			<td>DNA preparations</td>
		</tr>
		<tr>
			<td>coverslips (#1.5, 22*32 mm, and 20*20 mm)</td>
			<td>BMH.BIOMEDIA</td>
			<td>72204</td>
			<td>flow channel preparation</td>
		</tr>
		<tr>
			<td>Decon90</td>
			<td>Decon Laboratories Limited</td>
			<td></td>
			<td>flow channel preparation</td>
		</tr>
		<tr>
			<td>APTES</td>
			<td>Sigma</td>
			<td>440140-500ML</td>
			<td>flow channel preparation</td>
		</tr>
		<tr>
			<td>Sulfo-SMCC</td>
			<td>ThermoFisher Scientific</td>
			<td>22322</td>
			<td>flow channel preparation</td>
		</tr>
		<tr>
			<td>M-280, paramganetic beads,streptavidin</td>
			<td>ThermoFisher Scientific</td>
			<td>11205D</td>
			<td>flow channel preparation</td>
		</tr>
		<tr>
			<td>Polybead Amino Microspheres 3.00 &#956;m</td>
			<td>Polysciences, Inc</td>
			<td>17145-5</td>
			<td>flow channel preparation</td>
		</tr>
		<tr>
			<td>2-Mercaptoethanol</td>
			<td>Sigma</td>
			<td>M6250-250ML</td>
			<td>flow channel preparation</td>
		</tr>
		<tr>
			<td>Olympus Microscopes IX71</td>
			<td>Olympus</td>
			<td>IX71</td>
			<td>Magnetic tweezers setup</td>
		</tr>
		<tr>
			<td>Piezo-Z Stages P-721</td>
			<td>Physik Instrumente</td>
			<td>P-721</td>
			<td>Magnetic tweezers setup</td>
		</tr>
		<tr>
			<td>Olympus Objective lense MPLAPON-Oil 100X</td>
			<td>Olympus</td>
			<td>MPLAPON-Oil 100X</td>
			<td>Magnetic tweezers setup</td>
		</tr>
		<tr>
			<td>CCD/CMOS camera</td>
			<td>AVT</td>
			<td>Pike F-032B</td>
			<td>Magnetic tweezers setup</td>
		</tr>
		<tr>
			<td>Translation linear stage</td>
			<td>Physik Instrumente</td>
			<td>MoCo DC</td>
			<td>Magnetic tweezers setup</td>
		</tr>
		<tr>
			<td>LED</td>
			<td>Thorlabs</td>
			<td>MCWHL</td>
			<td>Magnetic tweezers setup</td>
		</tr>
		<tr>
			<td>Cubic Magnets</td>
			<td>Supermagnete</td>
			<td></td>
			<td>Magnetic tweezers setup</td>
		</tr>
		<tr>
			<td>Labview</td>
			<td>National Instruments</td>
			<td></td>
			<td>Magnetic tweezers setup</td>
		</tr>
		<tr>
			<td>OriginPro/Matlab</td>
			<td>OriginLab/MathWorks</td>
			<td></td>
			<td>Data analysis</td>
		</tr>
	</tbody>
</table>

single-molecule manipulation of g-quadruplexes by magnetic tweezers

Non-canonical nucleic acid secondary structure G-quadruplexes (G4) are involved in diverse cellular processes, such as DNA replication, transcription, RNA processing, and telomere elongation. During these processes, various proteins bind and resolve G4 structures to perform their function. As the function of G4 often depends on the stability of its folded structure, it is important to investigate how G4 binding proteins regulate the stability of G4. This work presents a method to manipulate single G4 molecules using magnetic tweezers, which enables studies of the regulation of G4 binding proteins on a single G4 molecule in real time. In general, this method is suitable for a wide scope of applications in studies for proteins/ligands interactions and regulations on various DNA or RNA secondary structures.

The experiment setup for stretching a single G4 molecule is shown in Figure 4. A single-stranded G4 forming sequence spanned between two dsDNA handles was tethered between a coverslip and a paramagnetic bead. To find a single dsDNA tethered bead, an overstretching assay was performed by increasing the force at constant loading rates. Three types of measurements were often used for studying the folding and unfolding of biomolecules: (i) constant force measurem...

Watch this Scientific Journal Video about Single-molecule Manipulation of G-quadruplexes by Magnetic Tweezers at JoVE.com

Single-molecule Manipulation of G-quadruplexes by Magnetic Tweezers (Video) | JoVE

A single-molecule magnetic tweezers platform to manipulate G-quadruplexes is reported, which allows for the study of G4 stability and regulation by various proteins.

Single-molecule Manipulation of G-quadruplexes by Magnetic Tweezers

Non-canonical nucleic acid secondary structure G-quadruplexes (G4) are involved in diverse cellular processes, such as DNA replication, transcription, RNA ...

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Single Molecule Fluorescence Energy Transfer Study of Ribosome Protein Synthesis

Single Molecule Fluorescence Energy Transfer Study of Ribosome Protein Synthesis (Video) | JoVE

The ribosome is a large ribonucleoprotein complex that assembles proteins processively along mRNA templates. The diameter of the ribosome is approximately ...

Single-Molecule Imaging of EWS-FLI1 Condensates Assembling on DNA

Single-Molecule Imaging of EWS-FLI1 Condensates Assembling on DNA (Video) | JoVE

The fusion genes resulting from chromosomal translocation have been found in many solid tumors or leukemia. EWS-FLI1, which belongs to the FUS/EWS/TAF15 ...

An Optimized Single-Molecule Pull-Down Assay for Quantification of Protein Phosphorylation

An Optimized Single-Molecule Pull-Down Assay for Quantification of Protein Phosphorylation (Video) | JoVE

Phosphorylation is a necessary posttranslational modification that regulates protein function and directs cell signaling outcomes. Current methods to ...