Name: design and synthesis of a reconfigurable dna accordion rack
Uploaded: 2018-08-15T15:00:00
Description: Watch this Scientific Journal Video about Design and Synthesis of a Reconfigurable DNA Accordion Rack at JoVE.com

Denne forskningen ble delvis støttet av Global Development Center forskningsprogrammet gjennom National Research Foundation av Korea(NRF) finansiert av departementet for vitenskap og IKT (MSIT) (2015K1A4A3047345) og Nano· Materielle Technology Development Program gjennom National Research Foundation av Korea (NRF) finansiert av departementet for vitenskap og IKT (MSIT) (2012M3A7A9671610). Institute of Engineering Research ved Seoul National University gitt forskningsanlegg for dette arbeidet. Forfatterne bekrefter takknemlighet mot Tae-unge Yoon (biologi, Seoul National University) om fluorescens spektroskopi for bånd analyse.

1. prosjektert av 6 av 6 DNA trekkspill Rack med caDNAno14
<ol><li>Laste ned og installere caDNAno 2.0 programvare14 for å designe en DNA trekkspill rack (caDNAno 2.5 er også tilgjengelig på https://github.com/cadnano/cadnano2.5). Åpne caDNAno14 og klikk Square verktøyet for å legge til en ny del med en firkantet gitter.</li>
	<li>Nummerere hver stråle av trekkspill stativet og trekke på venstre gitter panelet caDNAno<sup c...

<ol>
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C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nucleic+acid+based+molecular+devices.">Nucleic acid based molecular devices.</a> Angewandte Chemie International Edition. 50 (14), 3124-3156 (2011).</li><li>Liu, 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=A+DNA+tweezer-actuated+enzyme+nanoreactor.">A DNA tweezer-actuated enzyme nanoreactor.</a> Nature communications. 4, 2127 (2013).</li><li>Douglas, S. M., Bachelet, I., Church, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+logic-gated+nanorobot+for+targeted+transport+of+molecular+payloads.">A logic-gated nanorobot for targeted transport of molecular payloads.</a> Science. 335 (6070), 831-834 (2012).</li><li>Kuzyk, 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=Reconfigurable+3D+plasmonic+metamolecules.">Reconfigurable 3D plasmonic metamolecules.</a> Nature Materials. 13 (9), 862-866 (2014).</li><li>Zhou, C., Duan, X., Liu, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+plasmonic+nanorod+that+walks+on+DNA+origami.">A plasmonic nanorod that walks on DNA origami.</a> Nature communications. 6, 8102 (2015).</li><li>Choi, Y., Choi, H., Lee, A. C., Lee, H., Kwon, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Reconfigurable+DNA+Accordion+Rack.">A Reconfigurable DNA Accordion Rack.</a> Angewandte Chemie International Edition. 57 (11), 2811-2815 (2018).</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=Understanding+the+Mechanical+Properties+of+DNA+Origami+Tiles+and+Controlling+the+Kinetics+of+their+Folding+and+Unfolding+Reconfiguration.">Understanding the Mechanical Properties of DNA Origami Tiles and Controlling the Kinetics of their Folding and Unfolding Reconfiguration.</a> Journal of the American Chemical Society. 136 (19), 6995-7005 (2014).</li><li>Han, D., Pal, S., Liu, Y., Yan, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Folding+and+cutting+DNA+into+reconfigurable+topological+nanostructures.">Folding and cutting DNA into reconfigurable topological nanostructures.</a> Nature Nanotechnology. 5 (10), 712-717 (2010).</li><li>Douglas, 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=Rapid+prototyping+of+3D+DNA-origami+shapes+with+caDNAno.">Rapid prototyping of 3D DNA-origami shapes with caDNAno.</a> Nucleic Acids Research. 37 (15), 5001-5006 (2009).</li><li>Castro, C. E., 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=A+primer+to+scaffolded+DNA+origami.">A primer to scaffolded DNA origami.</a> Nature methods. 8 (3), 221-229 (2011).</li><li>Ouldridge, T. E., Louis, A. A., Doye, J. P. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+Nanotweezers+Studied+with+a+Coarse-Grained+Model+of+DNA.">DNA Nanotweezers Studied with a Coarse-Grained Model of DNA.</a> Physical Review Letters. 104 (17), 178101 (2010).</li><li>Snodin, B. E. K., 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=Direct+Simulation+of+the+Self-Assembly+of+a+Small+DNA+Origami.">Direct Simulation of the Self-Assembly of a Small DNA Origami.</a> ACS Nano. 10 (2), 1724-1737 (2016).</li><li>Amir, Y., Abu-Horowitz, A., Bachelet, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Folding+and+Characterization+of+a+Bio-responsive+Robot+from+DNA+Origami.">Folding and Characterization of a Bio-responsive Robot from DNA Origami.</a> Journal of Visualized Experiments. (106), e51272 (2015).</li><li>Stahl, E., Martin, T. 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Denne protokollen lanserer hele prosessen fra design, simulering, syntese og analyse av grunnleggende 2D DNA trekkspill stativet. Modifisert design og simulering regler har blitt beskrevet fordi design regelen er forskjellig fra standard DNA origami, i at DNA trekkspill stativet har flere nukleotider på overganger for fleksibilitet14,15. Fra dette forventer vi at protokollen kan akselerere ulike undersøkelser med DNA trekkspill stativer. I tillegg kan beskrives...

Mekaniske systemer basert på DNA nanostrukturer eller DNA nanomachines1,2,3,4,5 er unike fordi de produserer komplekse nanoskala bevegelse i 2D og 3D i nanometer til Ångström oppløsning, ifølge ulike biomolecular stimuli2,3,6. Feste funksjonelle materialer på disse strukturene og kontrollere sine posisjoner, kan disse strukturene brukes til ulike områder. For eksempel er DNA nanomachines foreslått for molekylær reaktoren7, stoffet levering8og nanoplasmonic systemer9,10.
Tidligere har innført vi det rekonfigurerbare DNA trekkspill racket, som kan manipulere et 2D eller 3D nanoskala nettverk av elementer11 (figur 1A). I motsetning til andre DNA nanomachines som kontrollerer bare noen elementer, kan plattformen kollektivt manipulere regelmessig plassert 2D eller 3D-elementer i ulike stadier. Vi forventer at en programmerbar kjemiske og biologiske reaksjon nettverk eller en molekylær datasystem kan bygges fra våre system, ved å øke antall kontrollerbar elementer. DNA trekkspill stativet er en struktur som nettverk av flere DNA bjelker er koblet til leddene består av enkelt-strandet DNA (figur 1B). Trekkspill stativet generert av DNA bjelker konfigureres av DNA-låser, som hybridize til klissete del av bjelker og endre vinkelen mellom bjelker ifølge den bygge bro delen av låser (låst). I tillegg er flere trinn rekonfigurering demonstrert ved å legge til nye låser etter dannelsen av free state ved frakobling DNA låser gjennom toehold-baserte strand forskyvning12,13.
I denne protokollen beskriver vi hele design og syntese prosessen med rekonfigurerbare DNA trekkspill stativet. Protokollen inneholder design, simulering, våt-lab eksperimenter og analyse for syntese av DNA trekkspill rack med 6 av 6 masker og en ny konfigurering av disse. Strukturen i protokollen er grunnmodellen til den tidligere forskning11 og er 65 nm ved 65 nm i størrelse, bestående av 14 bjelker. Når det gjelder design og simulering er den strukturelle utformingen av trekkspill stativet forskjellig fra konvensjonelle DNA origami14,15 (dvs. tett pakket). Derfor er design regelen og molekylære simulering endret fra tradisjonelle metoder. For å demonstrere, viser vi design teknikken bruker den endrede tilnærmingen caDNAno14 og simulering av trekkspill stativet oxDNA16,17 med skript. Til slutt, begge protokollene TEM og bånd for analyse av konfigurerte trekkspill rack strukturer er beskrevet.

<table>
	<tbody>
		<tr>
			<td>M13mp18 Single-stranded DNA</td>
			<td>NEB</td>
			<td>N4040s</td>
			<td></td>
		</tr>
		<tr>
			<td>1M MgCl2 Solution</td>
			<td>Biosesang</td>
			<td>M2001</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris-EDTA buffer</td>
			<td>Biosesang</td>
			<td>T2142</td>
			<td></td>
		</tr>
		<tr>
			<td>Nuclease-Free Water</td>
			<td>Qiagen</td>
			<td>129114</td>
			<td></td>
		</tr>
		<tr>
			<td>5M Sodium Chloride solution</td>
			<td>Biosesang</td>
			<td>s2007</td>
			<td></td>
		</tr>
		<tr>
			<td>PEG 8000</td>
			<td>Sigma Aldrich</td>
			<td>1546605</td>
			<td></td>
		</tr>
		<tr>
			<td>10N NaOH</td>
			<td>Biosesang</td>
			<td>S2038</td>
			<td></td>
		</tr>
		<tr>
			<td>Uranyl formate</td>
			<td>Thomas Science</td>
			<td>C993L42</td>
			<td></td>
		</tr>
		<tr>
			<td>Thermal cycler C1000</td>
			<td>Biorad</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Nanodropic 2000</td>
			<td>Thermo Fisher Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>TEM (LIBRA 120)&#160;&#160;</td>
			<td>Carl Zeiss</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorometer Enspire 2300</td>
			<td>Perkin-Elmer</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Labogene</td>
			<td>LZ-1580</td>
			<td></td>
		</tr>
	</tbody>
</table>

design and synthesis of a reconfigurable dna accordion rack

DNA nanostructure-baserte mekaniske systemer eller DNA nanomachines, som produsere komplekse nanoskala bevegelse i 2D og 3D i nanometer ångström oppløsning, viser stort potensial i ulike nanoteknologi som de molekylære reaktorene, narkotika-leveranser, og nanoplasmonic systemer. Det rekonfigurerbare DNA trekkspill racket, som kan kollektivt manipulere et 2D eller 3D nanoskala nettverk av elementer i flere etapper svar på DNA innganger, er beskrevet. Plattformen har potensial til å øke antall elementer som DNA nanomachines kan styre fra noen elementer til en nettverk skala med flere etapper for konfigurering.
I denne protokollen beskriver vi hele eksperimentelle prosessen med rekonfigurerbare DNA trekkspill rack med 6 av 6 masker. Protokollen inneholder en design regelen og simulering prosedyre av strukturer og en våt-lab eksperiment for syntese og konfigurering. I tillegg er analyse av strukturen TEM (overføring elektronmikroskop) og bånd (fluorescens resonans energioverføring) inkludert i protokollen. De nye design og simulering metodene dekket i denne protokollen vil assistere forskere bruke DNA trekkspill stativet for videre søknader.

Designet 6 av 6 DNA trekkspill stativet er simulert oxDNA16,17 , og resultatene vises i figur 6. Fra simulering resultatet, ble det bekreftet at den tiltenkte strukturen dannes uten forvrengning av strukturen.
TEM bildene i figur 7 er bilder av konfigurerte strukturer med en lås lengde på 2, 8, 13 og 20 bp. P?...

Watch this Scientific Journal Video about Design and Synthesis of a Reconfigurable DNA Accordion Rack at JoVE.com

Design and Synthesis of a Reconfigurable DNA Accordion Rack

Vi beskriver detaljert protokollen for design, simulering, våt-lab eksperimenter og analyse for en rekonfigurerbare DNA trekkspill rack med 6 av 6 masker.

Design og syntese av en rekonfigurerbare DNA trekkspill Rack

DNA nanostructure-based mechanical systems or DNA nanomachines, which produce complex nanoscale motion in 2D and 3D in the nanometer to &#229;ngstr&#246;m ...

This method can help answer key questions in the nano molecules field, such as DNA nano technology, nano molecular circuits, and nano medicine. The main advantage of this technique is that it dramatically increases the category of controlling periodically irate elements in both two and three dimensions. To begin, download and install Cadnano software to design a DNA accordion rack.Open Cadnano and click the square tool to add a new part with a square lattice. Click the pencil tool and draw each beam on the right edit panel of Cadnano. Break beams every 32 base pairs for joints between adjacent beams.Place staple crossovers in the same position as the joints. Use the insert tool and the pencil tool in Cadnano to let the joints have additional single stranded crossovers. Now, click the break tool.Break the strands where staple strands are circular or longer than 60 base pairs. To design the DNA lockstrands, click the break tool. Break eight base pairs of a staple DNA region to make a sticky part and delete eight base pairs of a staple DNA region.There are 18 sticky parts in the six by six accordion rack. Place sequences that are reverse complimentary to the sticky parts at both ends of lockstrands and connect them by a bridging region, which consists of poly T strands of the desired length. For the reconfiguration, add eight base pairs of toehold sequences at the end of the DNA locks for strand displacement.Design strands that are reverse complimentary to the DNA locks for the reconfiguration experiment. Generate a topology file and configuration file using OxDNA as described in the text protocol. The topology file includes how many strands and nucleotides are in the structure and information regarding backbone-backbone bonds between nucleotides.The configuration file includes general information, such as time step, box size, and energy. Orientation information, such as position vector, backbone-base vector, normal vector, velocity and angular velocity of nucleotides is also included. Change the information in the topology and configuration file from Cadnano to make them reflect the real structural information of the accordion rack.Then, run the molecular dynamics program as described in the text protocol. To visualize the structures, run the Cogli program with the topology and configuration files from the OxDNA simulation. Hide the box by pressing B.Purchase the design DNA staples and prepare them as described in the text protocol.Combine staple DNA, magnesium chloride solution, tris EDTA solution, nuclease free water, and scaffold DNA to make 20 micro liters of mixed stock with the final concentrations listed in the text protocol. Now, rapidly heat the mixed stock solution in a thermal cycler to 80 degrees Celsius. Cool the solution to 60 degrees Celsius, at a rate of four minutes per degree Celsius.Then, cool from 60 degrees Celsius to four degrees Celsius at a rate of 40 minutes per degree Celsius. To purify the structure, mix 20 micro liters of the synthesized structure in 20 micro liters of precipitation buffer. Then, spin the mixed stock at 16, 000 times G and four degrees celsius.Remove the super natant and dissolve pellet in the target buffer. To reconfigure the accordion rack from a free state to a locked state, first add two micro liters of DNA lockstrands of the desired length into 20 micro liters of the synthesized structure. Incubate the sample for 100 minutes at 50 degrees Celsius for 30 minutes and slowly cool down to 25 degrees Celsius at a rate of 0.33 degrees Celsius per minute.To reconfigure the accordion rack from a locked state to a free state, add two micro liters of strands that are reverse complimentary to the locked strands of the desired length into 20 microliters of synthesized structure. Incubate the sample for 12, 60, 120, and 240 minutes by rapidly heating the sample to 40 degrees Celsius and slowly cooling down to 20 degrees Celsius for the time corresponding to each. Right after the detaching step, rapidly cool down the sample to four degrees Celsius to prevent unwanted denaturation.To perform FRET analysis, synthesize the structure as before, expect with fluorescently labeled strands. Measure the concentration of the purified sample. After normalizing the sample to 10 nano molar, load 50 micro liters to a well in a 384 well micro plate.Measure the fluorescence of the sample with donor and acceptor dyes, as well as the donor only sample by exciting at 550 nano meters and measuring the fluorescent spectrum from 570 nano meters to 800 nano meters with a fluorometer. To measure the concentration of the acceptor, excite the dyes of the sample at 650 nano meters and measure the fluorescent spectrum from 670 nano meters to 800 nanometers. Then, calculate the FRET efficiency as described in the text protocol.A simulation result from OxDNA is visualized here by Cogli. The accordion rack is shown with DNA locks, of which the lengths are two, eight, 13, and 20 base pairs. The angle of the accordion rack, comprised of six by six meshes, was controlled by the length of the DNA locks.By adding DNA locks with lengths of two, eight, 13, and 20 base pairs to the 18 locking sights of the accordion rack, the angle is configured as seen in the representative transmission electron microscopy, or TEM images. The dye pair is attached to the structure for FRET efficiency analysis. Shown here are representative TEM images of configured six by six DNA accordion racks with different DNA lock lengths.After its development, this technique paved the way for researchers in the field of DNA nano technology to explore interactions of different molecules in a collectively controlled system.

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