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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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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N., Isaacs, A. M. <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:+Emerging+roles+in+neurodegenerative+diseases+and+the+non-coding+transcriptome.">G-quadruplexes: Emerging roles in neurodegenerative diseases and the non-coding transcriptome.</a> FEBS Lett. 589 (14), 1653-1668 (2015).</li><li>Balasubramanian, S., Hurley, L. 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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

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.

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

The overall goal of this experiment is to directly measure the folding and unfolding of G-quadruplexes, abbreviated G4, and the regulation by binding proteins at the single-molecule level using magnetic tweezers. This message can help answer key questions in G4 field, such as floating dynamics of G4, and the regulation by binding proteins. The main advantage of this technique, is that magnetic tweezers, allow stable measurement, of nanometer sized floating dynamics of a single-molecule over days, by using anti-drift technique.Let's start this with this message, can provide the inside the molecule mechanisms of G4 stability, the helicase activities on G4, it can also use to probe the al-a-do-gees related to G4 binding proteins, or ligans, which may have potential therapeutic applications. In addition, it can also used to probe the outer protein DNA interactions, or protein protein interactions. Begin this procedure with preparation of G4 DNA in the flow channel, as described in the text protocol.G4 DNA was prepared by ligate G4 forming single stranded DNA, two flank single stranded DNA, and a double stranded DNA handle, using T4 DNA ligates. For the magnetic tweezers setup, start the magnetic tweezers program. Here, the magnetic tweezers are controlled by an in-house written lab view program.Next, align the magnet centers before mounting the channel. Use a 10 times objective lens to adjust the X and Y axis of the magnetics, in the optical access of the microscope. Use a computer controlled motorized manipulator to move the magnets along the Z direction, and set the distance between the magnets and coverslip as D equals zero, when the magnetics attach to a coverslip on the microscope.Program the movement of the magnetics through the manipulator to achieve control of the force, including constant force and time varying force. Use a bright light emitting diode light source for back scattered illumination of the bead through the objective. Collect the bead images and a sampling rate of 100 hertz, by a change coupled device camera.For tether formation, gently flow 200 X diluted M280 para magnetic beads in as-say solution into a channel. Incubate the mixture for 10 minutes to allow the beads to bind to biotin labeled DNA molecules immobilized on the SMCC coded surface through the thiol labeled ent. Gently wash away untethered beads using 200 microliters of standard reaction solution.Next, mount the channel onto the microscope stage. Search for the beads on the bottom surface using the 100 times oil immersion objective. Select a reference bead on the surface, and a moving tethered bead.Build the initial image libraries, of both the reference bead and tethered bead and different de-focused plains. Before experiments, use a objective Piezo actuator to obtain images of both reference and tethered beads at different de-focused plains, spaced at 20 to 50 nanometers. Store the images as two separate bead image libraries that are respectively indexed with the de-focused distance.During the experiments, determine the position of the bead in the XY plain, by the bead centroid. Also determine the height change of the tethered bead by comparing the current bead image with those stored in the library. Use the auto correlation function analysis of the power spectrum of the Fourier transform of the bead images.During experiments, use the Pizeo to lock the distance between the objective in a specific reference bead image stored in the library through a low frequency feedback control, so that the stuck bead image, has the best correlation with the specific image stored in the library. Determine whether the tether is a single double stranded DNA molecule, by applying approximately 65 piconewtons of force. A tether is a single double stranded DNA molecule if it undergoes the characteristic DNA overstretching transition.Repeat the process until a single double stranded DNA tether is found. The relationship of force and magnet position, or so called, the force distance curve, was calibrated based on bead fluctuation. Furthermore, the overstretching transition of double stranded DNA that occurred near 65 piconewtons, could also be used to calibrate the force.For force ramp experiments, perform a force increase scan at a loading rate of 0.2 piconewtons per second, followed by a force decrease scan at minus 0.2 piconewtons per second. After each stretching cycle, hold the DNA molecule at one piconewton for 30 seconds, to allow the single stranded DNA to refold to G4.For force-jump experiments, cycle the force between 54 piconewtons for 30 seconds, under which a folded G4 could be unfolded, and 1 piconewton for 60 seconds allowing a folded G4-15T to refold. Finally, analyze the unfolding force, using an in-house written mat lab program.Shown here, is a typical example of a force increase and force decrease curve of a tethered G4 quadruplex. The extension jump in the force increase scan indicated an unfolding transition of G4, at approximately 52 piconewtons. However, in the presence of RHAU, G4 remained folded during the force increase scan for up to 60 piconewtons, indicating G4 force stabilization by RHAU binding.In the presence of RHAU and ATP, the unfolding force distribution of G4, was shifted to a lower force, indicative of an ATP depended destabilization of G4 by RHAU. Representative traces of the bead height during force-jump cycles, shows that the force applied to the molecule was cycled between 54 piconewtons for 30 seconds, under which a folded G4 could be unfolded, and 1 piconewton for 60 seconds, under which it could refold. The average lifetime a folded G4 at 54 piconewtons, was approximately 6.4 seconds, estimated by fitting the lifetime histogram with an exponential decay function.When RHAU helicase was introduced, the extension of tethered DNA remained at folded level throughout the 30 seconds holding time at 54 piconewtons, indicated that the RHAU strongly stabilizes G4 against mechanical unfolding. Upon addition of RHAU and ATP, the extension was at the level of unfolded single stranded DNA, indicating a destabilization of G4.After watching this video, you should have a good understanding of how to measure the unfolding and refolding dynamics of nuclear ACS junctions using this ultra stabled magnetic tweezers. The allotment of this technique paved the way for researchers in field of nucleic acid structure and binding proteins to explore the regression of structure stability and the folding kinetics, as well as the interaction of nucleic acid and binding proteins.

Research

JoVE Journal

Biochemistry

High Precision FRET at Single-molecule Level for Biomolecule Structure Determination

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Using Three-color Single-molecule FRET to Study the Correlation of Protein Interactions

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

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