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

Forfatterne takke Meng Pan til korrekturlæsning af håndskriftet. Dette arbejde er støttet af Singapore Ministeriet for uddannelse akademisk forskning fond Tier 3 (MOE2012-T3-1-001) til J.Y.; Grundforskningsfond gennem Mechanobiology Institut Singapore til J.Y.; Danmarks Grundforskningsfond, premierministerens kontor, Singapore, under sit NRF Investigatorship program (NRF Investigatorship Award nr. NRF-NRFI2016-03 til J.Y.; den grundlæggende forskningsfonden for Central universiteterne (2017KFYXJJ153) til H. Y.

1. forberedelse af G4 DNA for enkelt-molekyle strækning <ol><li>Forbered 5 &#39;-thiol mærket og 5 &#39;-biotin mærket dsDNA håndtag ved PCR ved hjælp af DDNA polymerase på en lambda phage DNA skabelon ved hjælp af 5 &#39;-thiol og 5 &#39;-biotin primere 14 ( figur 1). Begge dsDNA håndtag har højt indhold af GC (&#62; 60%) for at forhindre DNA smelter når DNA holdes på høje styrker eller under DNA overstretching overgang 15.</l...

<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. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+C-terminus+of+nucleolin+promotes+the+formation+of+the+c-MYC+G-quadruplex+and+inhibits+c-MYC+promoter+activity.">The C-terminus of nucleolin promotes the formation of the c-MYC G-quadruplex and inhibits c-MYC promoter activity.</a> Biochemistry. 49 (45), 9706-9714 (2010).</li><li>Wang, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=telomerase-interacting+protein+that+unfolds+telomere+G-quadruplex+and+promotes+telomere+extension+in+mammalian+cells.">telomerase-interacting protein that unfolds telomere G-quadruplex and promotes telomere extension in mammalian cells.</a> Proc Natl Acad Sci U S A. 109 (50), 20413-20418 (2012).</li><li>Mendoza, O., Bourdoncle, A., Boule, J. B., Brosh, R. M., Mergny, J. L. <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+helicases.">G-quadruplexes and helicases.</a> Nucleic Acids Res. 44 (5), 1989-2006 (2016).</li><li>Schiavone, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PrimPol+Is+Required+for+Replicative+Tolerance+of+G+Quadruplexes+in+Vertebrate+Cells.">PrimPol Is Required for Replicative Tolerance of G Quadruplexes in Vertebrate Cells.</a> Mol Cell. 61 (1), 161-169 (2016).</li><li>Lane, A. N., Chaires, J. B., Gray, R. D., Trent, J. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stability+and+kinetics+of+G-quadruplex+structures.">Stability and kinetics of G-quadruplex structures.</a> Nucleic Acids Res. 36 (17), 5482-5515 (2008).</li><li>Woodside, M. T., Block, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reconstructing+folding+energy+landscapes+by+single-molecule+force+spectroscopy.">Reconstructing folding energy landscapes by single-molecule force spectroscopy.</a> Annu Rev Biophys. 43, 19-39 (2014).</li><li>Neuman, K. C., Nagy, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-molecule+force+spectroscopy:+optical+tweezers,+magnetic+tweezers+and+atomic+force+microscopy.">Single-molecule force spectroscopy: optical tweezers, magnetic tweezers and atomic force microscopy.</a> Nat Methods. 5 (6), 491-505 (2008).</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>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=Dynamics+of+equilibrium+folding+and+unfolding+transitions+of+titin+immunoglobulin+domain+under+constant+forces.">Dynamics of equilibrium folding and unfolding transitions of titin immunoglobulin domain under constant forces.</a> J Am Chem Soc. 137 (10), 3540-3546 (2015).</li><li>You, H., Wu, J., Shao, 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=Stability+and+kinetics+of+c-MYC+promoter+G-quadruplexes+studied+by+single-molecule+manipulation.">Stability and kinetics of c-MYC promoter G-quadruplexes studied by single-molecule manipulation.</a> J Am Chem Soc. 137 (7), 2424-2427 (2015).</li><li>You, H., Lattmann, S., Rhodes, D., 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=RHAU+helicase+stabilizes+G4+in+its+nucleotide-free+state+and+destabilizes+G4+upon+ATP+hydrolysis.">RHAU helicase stabilizes G4 in its nucleotide-free state and destabilizes G4 upon ATP hydrolysis.</a> Nucleic Acids Res. 45 (1), 206-214 (2017).</li><li>You, 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=Dynamics+and+stability+of+polymorphic+human+telomeric+G-quadruplex+under+tension.">Dynamics and stability of polymorphic human telomeric G-quadruplex under tension.</a> Nucleic Acids Res. 42 (13), 8789-8795 (2014).</li><li>Fu, H., Chen, H., Marko, J. 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. J. 82 (6), 3314-3329 (2002).</li><li>Fu, 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=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>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. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Near-field-magnetic-tweezer+manipulation+of+single+DNA+molecules.">Near-field-magnetic-tweezer manipulation of single DNA molecules.</a> Phys Rev E Stat Nonlin Soft Matter Phys. 70 (1 Pt 1), 011905 (2004).</li><li>Le, 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=Disturbance-free+rapid+solution+exchange+for+magnetic+tweezers+single-molecule+studies.">Disturbance-free rapid solution exchange for magnetic tweezers single-molecule studies.</a> Nucleic Acids Res. 43 (17), e113 (2015).</li><li>Neidle, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quadruplex+Nucleic+Acids+as+Novel+Therapeutic+Targets.">Quadruplex Nucleic Acids as Novel Therapeutic Targets.</a> J Med Chem. 59 (13), 5987-6011 (2016).</li><li>Simone, R., Fratta, P., Neidle, S., Parkinson, G. 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. H., Neidle, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+G-quadruplexes+in+gene+promoters:+a+novel+anticancer+strategy?.">Targeting G-quadruplexes in gene promoters: a novel anticancer strategy?.</a> Nat Rev Drug Discov. 10 (4), 261-275 (2011).</li><li>Amato, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toward+the+Development+of+Specific+G-Quadruplex+Binders:+Synthesis,+Biophysical,+and+Biological+Studies+of+New+Hydrazone+Derivatives.">Toward the Development of Specific G-Quadruplex Binders: Synthesis, Biophysical, and Biological Studies of New Hydrazone Derivatives.</a> J Med Chem. 59 (12), 5706-5720 (2016).</li><li>Wells, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-B+DNA+conformations,+mutagenesis+and+disease.">Non-B DNA conformations, mutagenesis and disease.</a> Trends Biochem Sci. 32 (6), 271-278 (2007).</li></ol>

Som beskrevet ovenfor, en platform for at studere den mekaniske stabilitet af G4 DNA og samspillet mellem protein til G4 ved hjælp af er enkelt-molekyle magnetiske pincet rapporteret. Ledsager platformen, er højeffektive protokoller for at finde G4 DNA tether og måling af folde udfoldning dynamik og stabilitet af G4 struktur med nanometer særlig beslutning udviklet. Brændplanet låsning muliggør yderst stabil anti-drift kontrol, hvilket er vigtigt for påvisning af en lille struktur overgang som G4 (trin størrelse...

Fire-strenget DNA eller RNA G4 strukturer spiller en kritisk rolle i mange vigtige biologiske processer1. Mange proteiner er involveret i G4 bindende og regulering, herunder Telomer-bindende proteiner (telomerase, POT1, RPA, TEBPs, TRF2)1,2, transskriptionsfaktorer (nucleolin, PARP1)3, RNA forarbejdning proteiner (hnRNP A1, hnRNP A2)4, helicases (BLM, FANCJ, RHAU, WRN, Dna2, Pif1)5og DNA-replikation relaterede proteiner (Rif1, REV1, PrimPolymerase)6. Protein binding kan stabilisere eller destabilisere G4 strukturer; dermed regulerer de efterfølgende biologiske funktioner. Stabiliteten af G4 blev målt ved termisk smelter ved hjælp af ultraviolet (UV) eller cirkulær dichroism (CD) metoder7. Sådanne forhold er imidlertid ikke fysiologisk relevante og er vanskelige at anvende til at studere virkningerne af bindende proteiner7.
Den hurtige udvikling i enkelt-molekyle manipulation teknologier har aktiveret undersøgelser af folde og udfoldelsen af et biomolekyle, såsom en DNA eller et protein, på en enkelt-molekyle niveau med nanometer opløsning i realtid8. Atomic force mikroskopi (AFM), Optisk pincet og magnetiske pincet er de mest almindeligt anvendte metoder til enkelt-molekyle manipulation. I forhold til AFM og optiske pincet9, tillade magnetiske pincet stabile målinger af folde udfoldning dynamikken i et enkelt molekyle over dage ved hjælp af en anti-drift teknik10,11.
Her, er en enkelt-molekyle manipulation platform ved hjælp af magnetiske pincet til at undersøge regulering af G4 stabilitet af bindende proteiner rapporteret12,13. Dette arbejde skitserer de grundlæggende metoder, herunder prøve og flow-kanal forberedelse, opsætning af magnetiske pincet og kraft kalibrering. Kontrolelementet kraft og protokollerne anti-drift som beskrevet i trin 3 giver mulighed for lang tidsmålinger under forskellige kraft kontrolelementer, som konstant kraft (force klemme) og konstant lastning Vurder (kraft-rampe) og force-jump måling. Kraft kalibrering protokollen beskrevet i trin 4 giver kraft kalibrering af &#60; 1 µm kort bindsler over en bred kraft rækkevidde op til 100 pN, med en relativ fejl inden for 10%. Et eksempel på lovgivning om stabilitet af RNA-Helicase forbundet med AU-rige element (RHAU) helicase (alias DHX36, G4R1), spiller væsentlige roller i løsningen af RNA G4 bruges til at demonstrere applikationer på denne 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

Ikke-kanoniske nukleinsyre sekundærstruktur G-quadruplexes (G4) er involveret i forskellige cellulære processer, såsom DNA-replikation, transskription, RNA forarbejdning og telomere brudforlængelse. Under disse processer, forskellige proteiner binde og løse G4 strukturer til at udføre deres funktion. Funktionen af G4 ofte afhænger af stabiliteten i sine foldede struktur, er det vigtigt at undersøge, hvordan G4-bindende proteiner regulere stabilitet af G4. Dette arbejde præsenterer en metode til at manipulere enkelt G4 molekyler ved hjælp af magnetiske pincet, som giver studier af reguleringen af G4-bindende proteiner på en enkelt G4 molekyle i realtid. I almindelighed, er denne metode velegnet til et bredt anvendelsesområde applikationer i undersøgelser for proteiner/ligander interaktioner og forordninger om forskellige DNA eller RNA sekundære strukturer.

Opsætningen eksperiment for at strække en enkelt G4 molekyle er vist i figur 4. En enkelt-strenget G4 danner sekvens strakte sig mellem to dsDNA håndtag var bundet mellem et coverslip og en Paramagnetiske perle. For at finde en enkelt dsDNA tøjret perle, blev en overstretching analyse udført ved at øge kraft til konstant ladning priser. Tre typer af målinger blev ofte brugt til at studere folde og udfoldelsen af biomolekyler: (i) konstant kraft måling...

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

Enkelt-molekyle Manipulation af G-quadruplexes af magnetiske pincet

En enkelt-molekyle magnetiske pincet platform til at manipulere G-quadruplexes er rapporteret, som giver mulighed for undersøgelse af G4 stabilitet og regulering af forskellige proteiner.

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

Research

JoVE Journal

Biochemistry

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

High Precision FRET på enkeltmolekyliveau for biomolekylstrukturbestemmelse

A protocol on how to perform high-precision interdye distance measurements using F&#246;rster resonance energy transfer (FRET) at the single-molecule ...

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

Ved hjælp af trefarvet enkelt-molekyle FRET at studere sammenhængen af Protein interaktioner

Single-molecule F&#246;rster resonance energy transfer (smFRET) has become a widely used biophysical technique to study the dynamics of biomolecules. For ...

Covalent Immobilization of Proteins for the Single Molecule Force Spectroscopy

Kovalente immobilisering af proteiner for enkelt molekyle kraft spektroskopi

In recent years, atomic force microscopy (AFM) based single molecule force spectroscopy (SMFS) extended our understanding of molecular properties and ...

Proteome-wide Quantification of Labeling Homogeneity at the Single Molecule Level

Proteom-wide kvantificering af mærkning homogenitet på enkelt-molekyle niveau

Cell proteomes are often characterized using electrophoresis assays, where all species of proteins in the cells are non-specifically labeled with a ...

Single-Molecule Tracking Microscopy - A Tool for Determining the Diffusive States of Cytosolic Molecules

Enkelt-molekyle tracking mikroskopi-et værktøj til bestemmelse af Difvøse tilstande af Cytosolic molekyler

Single-molecule localization microscopy probes the position and motions of individual molecules in living cells with tens of nanometer spatial and ...

Conventional BODIPY Conjugates for Live-Cell Super-Resolution Microscopy and Single-Molecule Tracking

Konventionelle BODIPY konjugatter til Live-Cell Super-Resolution mikroskopi og Single-Molecule Tracking

Single molecule localization microscopy (SMLM) techniques overcome the optical diffraction limit of conventional fluorescence microscopy and can resolve ...

Single Cell Analysis Of Transcriptionally Active Alleles By Single Molecule FISH

Enkeltcelleanalyse af transskriptionsaktive alleler efter enkeltmolekyle FISH

Gene transcription is an essential process in cell biology, and allows cells to interpret and respond to internal and external cues. Traditional bulk ...

Single Molecule Fluorescence Energy Transfer Study of Ribosome Protein Synthesis

Enkeltmolekyle fluorescens energioverførsel undersøgelse af ribosom proteinsyntese

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

Enkeltmolekylebilleddannelse af EWS-FLI1-kondensater, der samles på DNA

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

Et optimeret enkeltmolekyle pull-down assay til kvantificering af proteinfosforylering

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