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

この研究は、国立研究財団の Korea(NRF) 省の科学と ICT (MSIT) (2015K1A4A3047345) Nano· によって資金を供給を通じてグローバル研究開発センター プログラムによって部分的に支持されました。材料技術開発プログラムを通じて、国立研究財団の韓国 (NRF) 科学省と ICT (MSIT) (2012M3A7A9671610) によって資金を供給します。ソウル大学校工学研究所は、この作品の研究施設を提供しました。著者は、フレットの分析のための蛍光分光法に関するユン ・ テヨン (生物科学, ソウル国立大学) への感謝をご了承ください。

1. 6 DNA アコーディオン ラック caDNAno14で 6 のデザイン
<ol><li>ダウンロードし、インストール caDNAno 2.0 ソフトウェア14 DNA アコーディオン ラックを設計する (caDNAno 2.5 も https://github.com/cadnano/cadnano2.5 で利用可能です)。CaDNAno14を開き、正方格子で新しいパーツを追加する正方形ツールをクリックします。</li>
	<li>アコーディオン?...

<ol>
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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. G., Praetorius, F., Dietz, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Facile+and+Scalable+Preparation+of+Pure+and+Dense+DNA+Origami+Solutions.">Facile and Scalable Preparation of Pure and Dense DNA Origami Solutions.</a> Angewandte Chemie International Edition. 53 (47), 12735-12740 (2014).</li><li>Wei, B., Vhudzijena, M. K., Robaszewski, J., Yin, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Self-assembly+of+Complex+Two-dimensional+Shapes+from+Single-stranded+DNA+Tiles.">Self-assembly of Complex Two-dimensional Shapes from Single-stranded DNA Tiles.</a> Journal of Visualized Experiments. (99), e52486 (2015).</li><li>Clegg, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+resonance+energy+transfer+and+nucleic+acids.">Fluorescence resonance energy transfer and nucleic acids.</a> Methods in enzymology. 211, 353-388 (1992).</li><li>Kopperger, 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+self-assembled+nanoscale+robotic+arm+controlled+by+electric+fields.">A self-assembled nanoscale robotic arm controlled by electric fields.</a> Science. 359 (6373), 296-301 (2018).</li><li>Lauback, 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=Real-time+magnetic+actuation+of+DNA+nanodevices+via+modular+integration+with+stiff+micro-levers.">Real-time magnetic actuation of DNA nanodevices via modular integration with stiff micro-levers.</a> Nature Communications. 9 (1), 1446 (2018).</li></ol>

このプロトコルは、設計、シミュレーション、合成、および基本的な 2D DNA アコーディオン ラックの解析から全体のプロセスを紹介します。変更された設計とシミュレーション ルールは記載柔軟性14,15のクロス オーバーで、DNA アコーディオン ラックはヌクレオチドという点で標準的な DNA 折り紙の設計ルールが異なるためにをされています。この...

DNA ナノ構造や DNA ナノマシン1,2,3,4,5に基づく機械システムが一意にナノメータ単位で 2 D および 3 D の複雑なナノスケール モーションを出すのでオングストローム分解能、様々 な生体分子の刺激2,3,6によると。これらの構造の機能性材料を取り付けると自分の位置を制御する、これらの構造は、様々 な分野に適用できます。たとえば、DNA ナノマシンは分子原子炉7、薬配信8、および nanoplasmonic システム9,10の提案されている.
以前、紹介した再構成の DNA アコーディオン ラックは、要素11 (図 1 a) の 2D または 3D のナノスケール ネットワークを操作することができます。異なり、いくつかの要素を制御するだけ他の DNA ナノマシン、プラットフォームはまとめて、さまざまな段階に周期的に配列した 2D または 3D 要素を操作できます。プログラム可能な生物・化学反応ネットワークまたは分子コンピューティング システム ビルドできることを私達のシステムから制御可能な要素の数を増やすことで期待しています。DNA アコーディオン ラックは、複数の DNA 梁のネットワークは単一座礁させた DNA (図 1 b) の接合に接続されて、構造です。DNA 梁によって生成されたアコーディオンのラックは、梁の粘着部分に交配させるし、ロック (ロック状態) のブリッジ部分の長さによると梁の間の角度を変更する DNA ロックによって再構成されます。さらに、鎖の足掛かりに基づく変位12,13を介して DNA ロックをデタッチによって自由国の形成の後新しいロックを加えることによって多段の再構成を示します。
このプロトコルで再構成可能な DNA アコーディオン ラックの全体設計・合成プロセスをについて説明します。プロトコルには、設計、シミュレーション、ぬれた実験室実験および 6 によって 6 メッシュの DNA アコーディオン ラックとこれらの再構成の合成の分析が含まれています。以前研究11の基本的なモデルのプロトコルで覆われた構造で、65 65 nm nm の 14 の梁から成る。設計とシミュレーションの面でのアコーディオンのラック構造設計は従来 DNA 折り紙14,15 (すなわち、ぎっしり詰まった) によって違います。したがって、設計ルールと分子シミュレーションを従来の方法から変更されています。示すために、caDNAno14の変更されたアプローチと追加のスクリプト oxDNA16,17を使用してアコーディオンのラックのシミュレーションを用いた設計法を示す.最後に、構成されたアコーディオン ラック構造の分析 TEM およびフレットの両方のプロトコルを説明します。

<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 ナノ構造に基づく機械システムまたは DNA ナノマシン オングストローム分解能をナノメータ単位で 2 D および 3 D の複雑なナノスケール運動を作り出す、ドラッグデリバリー、分子の原子炉などナノテクノロジーの様々 な分野で大きな可能性を示してください。・ nanoplasmonic システム。DNA の入力への応答の複数の段階で、要素の 2D または 3D のナノスケール ネットワークを操作できますまとめて、再構成可能な DNA アコーディオン ラックが説明されています。プラットフォームには、再構成の複数の段階のネットワーク規模にいくつかの要素から DNA ナノマシンの制御できる要素の数を増やす可能性があります。
このプロトコルで再構成可能な 6 によって 6 メッシュの DNA アコーディオン ラック全体の実験プロセスをについて説明します。プロトコルには、構造と合成と再構成のウェット研究室の実験の設計ルールとシミュレーションの手順が含まれています。さらに、TEM (透過型電子顕微鏡) とフレット (蛍光共鳴エネルギー移動) を使用して構造の解析は、プロトコルに含まれます。このプロトコルで覆われている新規の設計とシミュレーションの方法は、さらにアプリケーションの DNA アコーディオン ラックを使用する研究者を支援します。

OxDNA16,17から 6 によって設計された 6 DNA のアコーディオン ラックをシミュレートし、結果を図 6に示します。シミュレーション結果から構造の歪みのない意図されていた構造が形成されることが確認されました。
図 7の TEM イメージ 2?...

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

設計、シミュレーション、ぬれた実験室実験および 6 によって 6 メッシュの再構成可能な DNA アコーディオン ラックの解析のための詳しいプロトコルについて述べる。

再構成可能な DNA アコーディオン ラックの設計と合成

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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のための周期的圧力のバイオリアクターの設計生体外で</em大動脈心臓弁の>研究

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Synthesis of Keratin-based Nanofiber for Biomedical Engineering

医工学のためのケラチンベースのナノファイバーの合成

Electrospinning, due to its versatility and potential for applications in various fields, is being frequently used to fabricate nanofibers. Production of ...

A Protocol for Bioinspired Design: A Ground Sampler Based on Sea Urchin Jaws

バイオインスパイアードデザインのためのプロトコル：グランドサンプラーウニジョーズに基づいて、

Bioinspired design is an emerging field that takes inspiration from nature to develop high-performance materials and devices. The sea urchin mouthpiece, ...

Automated Robotic Liquid Handling Assembly of Modular DNA Devices

モジュラー DNA デバイスの液体ハンドリング アセンブリを自動化

Recent advances in modular DNA assembly techniques have enabled synthetic biologists to test significantly more of the available &#34;design space&#34; ...

Reconfigurable Microfluidic Channel with Pin-discretized Sidewalls

ピン分離側壁を持つ再構成可能なマイクロ流路

Microfluidic components need to have various shapes to realize different key microfluidic functions such as mixing, separation, particle trapping, or ...

In Vesiculo Synthesis of Peptide Membrane Precursors for Autonomous Vesicle Growth

自律性小胞成長のためのペプチド膜前駆体のベシキュロ合成

Compartmentalization of biochemical reactions is a central aspect of synthetic cells. For this purpose, peptide-based reaction compartments serve as an ...

Design of an Open-Source, Low-Cost Bioink and Food Melt Extrusion 3D Printer

オープンソース、低コストのバイオインク、食品溶融物押出3Dプリンタの設計

Three-dimensional (3D) printing is an increasingly popular manufacturing technique that allows highly complex objects to be fabricated with no retooling ...

再構成可能な DNA アコーディオン ラックの設計と合成

再構成可能な DNA アコーディオンラックの設計と合成