这项研究由科学和信息和通信技术部 (MSIT) (2015K1A4A3047345) 资助的韩国国家研究基金会 (NRF) 在全球研究发展中心的项目中得到部分支持, Nano·通过由科学和信息和通信技术部 (MSIT) 资助的韩国国家研究基金会 (NRF) 提供的材料技术发展方案 (2012M3A7A9671610)。首尔国立大学工程研究所为这项工作提供了研究设施。作者感谢泰青年尹 (生物科学, 首尔国立大学) 关于荧光光谱的烦恼分析。

1. 设计6由6个 DNA 手风琴架与 caDNAno14
<ol><li>下载并安装 caDNAno 2.0 软件14设计一个 DNA 手风琴架 (caDNAno 2.5 也可在 https://github.com/cadnano/cadnano2.5)。打开 caDNAno14并单击正方形工具以添加一个正方形格子的新零件。</li>
	<li>将手风琴架的每束光束编号, 并在 caDNAno14的左格子面板上绘制 (图 2)。</li>
	<li>单击铅笔工具并在 caDNAno14的右侧编辑面板上绘制每个光束。每32的 bp, 这是为相邻梁之间的连接断裂光束。将短钉交叉处与接头位置相同。使用插入工具和铅笔工具在 caDNAno14 , 让关节有额外的单链分频器。</li>
	<li>单击铅笔工具并连接接头。每束有七个关节。</li>
	<li>使用先前报告的脚手架路由算法11, 生成脚手架分频器将支架合并到单个循环中。不要让脚手架和短链之间的最小绑定域小于 8 bp (图 3)。</li>
	<li>将未在组件中使用的脚手架放置在折叠机架的另一侧的顶点上, 如图 3所示。</li>
	<li>单击 "中断" 工具。折断短链是圆形或长于 60 bp。</li>
	<li>设计 DNA 锁链。<ol>
<li>单击 "中断" 工具。打破 8 bp 的主要 dna 区域, 使一个粘性部分和删除 8 bp 的主要 dna 区域。6 6 个手风琴架上有18个粘性零件 (图 1)。</li>
		<li>将在锁链两端的粘滞部分反向互补的序列, 并由桥接区域连接, 由所需长度的聚 T 链组成 (图 1B)。</li>
		<li>对于重构, 添加 8 bp 的立足点序列在 DNA 锁的末端为绞线位移。所用的立足点序列在表 2中。</li>
		<li>准备聚一股, 这是反向互补的桥接区域。</li>
		<li>设计的链, 是反向互补的 DNA 锁的重新配置实验。</li>
	</ol>
</li>
	<li>单击序列工具, 然后单击 "脚手架 DNA"。选择脚手架作为标准 M13mp18. 单击导出工具并以 csv 格式保存序列 (表 1)。</li>
</ol>2. 用 oxDNA 模拟结构
<ol><li>下载并安装 oxDNA16,17。最新的源代码可在 https://sourceforge.net/projects/oxdna/files/上使用。</li>
	<li>使用 python 脚本 "cadnano_interface" (oxDNA16、17包中提供), 从 caDNAno14文件中启动配置文件。其用法如下: "python cadnano_interface cadnano_file. json 平方 '。此时将生成拓扑文件和配置文件。 注: 拓扑文件包括结构中有多少股和核苷酸, 以及核苷酸之间骨干-骨干键之间的信息。配置文件包含一般信息, 如 timestep、能量和框大小。研究了核苷酸的位置向量、主干基矢量、正常矢量、速度和角速度等方向信息 (图 4)。</li>
	<li>从 caDNAno14更改拓扑和配置文件中的信息, 使其反映手风琴机架的真实结构信息。当 caDNAno14中的拓扑和配置文件可视化时, 所有光束都是平行排列的。然而, 手风琴架是一个格子结构, 所以粘结核苷酸之间的距离是很远的模拟 (图 5)。<ol>
<li>旋转并移动每个光束到所需的晶格结构。配置文件左侧的九列是位置向量、主干基向量和正则向量 (图 4)。要旋转光束, 请使用旋转变换旋转所有位置、主干基和正常矢量。然后通过改变位置向量来移动一个光束, 以找到它, 如图 5所示。</li>
		<li>使用 oxDNA 包中提供的脚本放松结构 (请参见 $oxDNA 中的示例/示例/RELAX_INITIAL_CONFIGURATION 以了解更多信息)。</li>
	</ol>
</li>
	<li>使用轻松配置文件运行1000万步骤的分子动力学模拟。其用法如下: './oxDNA < 输入 > ' 每5000或10000个步骤保存数据。</li>
	<li>可视 化 注意: 这些结构是使用 cogli 可视化的。<ol>
<li>下载并安装最新版本的 cogli (https://sourceforge.net/projects/cogli1/)。</li>
		<li>使用 oxDNA 模拟中的拓扑和配置文件运行 cogli。其用法如下: "./cogli1 < 拓扑文件 > < 配置文件 >"。</li>
		<li>按b把盒子藏起来。</li>
	</ol>
</li>
</ol>3. 结构的综合
注: 合成方法适用于以前的15、18号议定书。
<ol><li>从寡核苷酸提供商那里购买设计的 DNA 主食。</li>
	<li>使用无核酸酶的水, 将这些 DNA 主食的浓度调整为100微米。<ol>
<li>将构成 "自由状态" 结构的每个 DNA 链放入一个管中, 并将每个链的浓度调整为2微米。</li>
		<li>池 DNA 锁链的长度和数量的锁定点进入管和调整浓度为2微米的每一个链。使用18、9和4个锁定站点。添加聚一股, 这是互补的桥区, 在同一浓度。</li>
		<li>池链, 是反向互补的 DNA 锁链长度进入管和调整浓度为2微米的每一个链。</li>
	</ol>
</li>
	<li>准备氯化镁2溶液 300 nM 混合 70 ul 的核酸酶水和 30 ul 的1微米氯化镁2解决方案。通过混合 95 ul 的核酸酶水和 5 ul 的100x 三 edta 溶液, 制备5x 三 edta 溶液。</li>
	<li>添加 2 ul 的主食 dna, 1.1 ul 的氯化镁2溶液, 2 ul 的三 EDTA 溶液, 7.6 ul 的核酸酶水和 7.3 ul 的脚手架 DNA 的浓度是 110 nM, 使 20 ul 的混合股票。将支架 dna 的最终浓度设置为 40 nm, 将 dna 短至 200 nm, 氯化镁2至16毫米, 三 EDTA 溶液为0.5x。</li>
	<li>迅速加热混合股票溶液在一个热循环仪到80°c 和冷却到60°c 以每°c 4 分钟的速度和冷却从60°c 到4°c 以每°c 的速度40分钟。</li>
</ol>4. 结构的净化
注: 在分析前, 所有结构的样品都经过纯化。在这一节中, 我们描述了 PEG 纯化的协议, 这是根据以前的文献19。样品也可以通过凝胶电泳纯化, 如前文15、18所述。
<ol><li>准备5米氯化钠和100x 三乙 EDTA。</li>
	<li>通过混合 150 ul 的 PEG 8000, 500 ul 100x 三 EDTA 和 101 ul 的5米氯化钠和249核酸酶水的 ul, 准备沉淀缓冲。</li>
	<li>通过混合 5.5 ul 300 nM 氯化镁2溶液从 3.3, 10 ul 5x 三 EDTA 溶液从3.3 和 84.5 ul 的核酸酶水, 准备目标缓冲区。</li>
	<li>混合 20 ul 的合成结构从3和 20 ul 的沉淀缓冲从4.2 节。然后旋转的混合股票在 16000 x g 在4摄氏度。取出上清液, 从4.3 节中溶解靶缓冲器中的颗粒。</li>
</ol>5. 将手风琴架从 "自由状态" 重新配置为 "锁定状态"
<ol><li>对构型实验无 DNA 锁的结构进行综合。</li>
	<li>从3节准备 DNA 锁链。</li>
	<li>将所需长度的 DNA 锁链的 2 ul 添加到合成结构的 20 ul 中。DNA 锁链的浓度比结构高五倍。</li>
	<li>将样本孵化0、10、25、50或100分钟, 以查看重新配置的速度。<ol>
<li>为100分钟孵化, 孵化样品在50°c 30 分钟, 慢慢降温到25°c 的速率0.33 °c/分钟。</li>
		<li>为50分钟孵化, 孵化样品在50°c 15 分钟, 慢慢降温到25°c 的速率0.66 °c/分钟。</li>
		<li>为25分钟孵化, 孵化样品在50°c 7.5 分钟, 慢慢降温到25°c 的速率1.32 °c/分钟。</li>
		<li>为10分钟孵化, 孵化样品在50°c 3 分钟, 慢慢降温到25°c 的速率3.3 °c/分钟。</li>
		<li>在0分钟的孵化, 储存样本在4摄氏度右后 DNA 锁链的添加。</li>
	</ol>
</li>
	<li>在附加步骤后, 迅速冷却到4摄氏度的样品, 以防止不必要的变性。</li>
</ol>6. 将手风琴架从 "锁定状态" 重新配置为 "自由状态"
<ol><li>用所需长度的 DNA 锁合成结构实验。</li>
	<li>准备反向互补链从3节。</li>
	<li>添加 2 ul 的股, 是反向互补的锁链的期望长度为 20 ul 的合成结构。DNA 锁链的浓度比结构高五倍。</li>
	<li>孵化样本 0, 12, 60, 120, 240 分钟, 看看如何快速重构发生。<ol>
<li>为 12, 60, 120, 240 分钟孵化, 迅速加热样品到40°c 和慢慢地冷却下来到20°c 为相应的时间。在分离步骤后, 快速冷却样品到4°c, 以防止不必要的变性。</li>
		<li>在0分钟的孵化, 存储样品在4摄氏度右后, 反向互补链的补充。</li>
	</ol>
</li>
</ol>7. TEM 成像
注: TEM 成像协议是根据以前的文献18,20改编的。
<ol><li>通过混合 87.5 ul 的无核酸酶水和 12.5 ul 10 米氢氧化钠溶液, 制备1.25 米氢氧化钠溶液。</li>
	<li>添加 1 ul 1.25 米氢氧化钠溶液, 50 ul 的2% 甲酸铀溶液。</li>
	<li>涡流解决方案3分钟, 离心机以最大速度3分钟。将纯化样品的 3 ul 存入辉光放电 TEM 网格上3分钟, 用滤纸迅速冲洗出来。</li>
	<li>将制备的甲酸铀溶液的 7 ul 存入30秒, 用滤纸迅速冲洗。</li>
	<li>用透射电镜测量手风琴结构的长度和角度。</li>
</ol>8. 烦恼分析
<ol><li>使用阿托550和阿托647N 染料, Förster 距离为 6.5 nm。用荧光标记的链替换表 1中的主食58和主食117。然后用3节中描述的方法合成荧光标记的链结构。</li>
	<li>测量纯化样品的浓度。</li>
	<li>将样品正常化为 10 nM, 并将 50 ul 加载到384微型井。</li>
	<li>激发样品与捐赠者和受体染料在550毫微米和测量荧光光谱从570毫微米到800毫微米与荧光计。</li>
	<li>用同样的方法测量供体样本的荧光光谱。</li>
	<li>激发样品的染料在650毫微米和荧光光谱和测量从670毫微米到800毫微米。这是测量受体的浓度。</li>
	<li>通过重复相同的实验, 分别合成纯化三个样品, 获得标准偏差。</li>
	<li>计算的烦恼效率与比率的方法, 如所描述的等式低于21。 <img alt="Equation" src="/files/ftp_upload/58364/58364eq1.jpg"> DA550: 受体的峰值荧光强度的样本与捐助者和受体在 550 nm 激发。 D550: 在 550 nm 激发下, 仅供方样品的受体发射范围内的荧光强度。 DA650: 受体的峰值荧光强度的样本与捐助者和受体在 650 nm 激发。</li>
</ol>

<ol>
	<li>Andersen, E. 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=Self-assembly+of+a+nanoscale+DNA+box+with+a+controllable+lid.">Self-assembly of a nanoscale DNA box with a controllable lid.</a> Nature. 459 (7243), 73-76 (2009).</li><li>Cha, T. -. G., 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=Design+principles+of+DNA+enzyme+based+walkers:+Translocation+kinetics+and+photo-regulation.">Design principles of DNA enzyme based walkers: Translocation kinetics and photo-regulation.</a> Journal of the American Chemical Society. 137 (29), 9429-9437 (2015).</li><li>Gerling, T., Wagenbauer, K. F., Neuner, A. M., 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=Dynamic+DNA+devices+and+assemblies+formed+by+shape-complementary,+non-base+pairing+3D+components.">Dynamic DNA devices and assemblies formed by shape-complementary, non-base pairing 3D components.</a> Science. 347 (6229), 1446-1452 (2015).</li><li>Pinheiro, A. V., Han, D., Shih, W. M., 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=Challenges+and+opportunities+for+structural+DNA+nanotechnology.">Challenges and opportunities for structural DNA nanotechnology.</a> Nature nanotechnology. 6 (12), 763-772 (2011).</li><li>Li, 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=Exploring+the+speed+limit+of+toehold+exchange+with+a+cartwheeling+DNA+acrobat.">Exploring the speed limit of toehold exchange with a cartwheeling DNA acrobat.</a> Nature Nanotechnology. 1, (2018).</li><li>Krishnan, Y., Simmel, F. 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. 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>

作者没有什么可透露的

该协议介绍了从设计、仿真、合成和分析基本的 2D DNA 手风琴机架的整个过程。修改后的设计和仿真规则被描述, 因为设计规则不同于标准的 dna 折纸, 因为 dna 手风琴架有额外的核苷酸在分频器为灵活性14,15。由此, 我们期望该协议可以加速各种研究使用 DNA 手风琴架。此外, 所描述的协议也可以应用于其他研究使用 dna 纳米结构, 而不是标准的 dna 折纸。</...

基于 dna 纳米结构或 dna nanomachines 的机械系统,1,2,3,4,5是独一无二的, 因为它们在纳米的2D 和3D 中产生复杂的纳米尺度运动, 以ångström 决议, 根据各种生物分子刺激2,3,6。通过在这些结构上附加功能材料并控制它们的位置, 这些结构可应用于各个领域。例如, 为分子反应器7, 药物交付8和 nanoplasmonic 系统9,10提出了 DNA nanomachines。
以前, 我们介绍了可重构的 DNA 手风琴架, 它可以操纵2D 或3D 元素11 (图 1A) 的纳米尺度网络。与其他仅控制少数元素的 DNA nanomachines 不同, 该平台可以将周期性地排列2D 或3D 元素的过程集体地操作到不同的阶段。我们预计, 可编程的化学和生物反应网络或分子计算系统可以建立从我们的系统, 通过增加数量的可控元素。dna 手风琴架是一种结构, 其中多个 dna 束网络连接到由单链 dna 组成的关节 (图 1B)。由 dna 束产生的手风琴架由 dna 锁重新配置, 杂交在光束的粘性部分, 根据锁 (锁定状态) 的桥接部分的长度改变光束之间的夹角。此外, 通过通过基于立足点的绞线12、13分离 DNA 锁, 在自由状态形成后添加新锁, 可以证明多步重构。
在本协议中, 我们描述了可重构 DNA 手风琴架的整个设计和合成过程。该协议包括设计, 模拟, 湿实验室实验, 并分析了 6 6 网格的 DNA 手风琴架的合成和重新配置这些。该协议所涵盖的结构是前一项研究的基本模型,11是 65 nm, 大小为65纳米, 由14束组成。在设计和仿真方面, 手风琴架的结构设计不同于传统的 DNA 折纸14、15 (即紧密包装)。因此, 采用传统的方法对设计规则和分子模拟进行了改进。为了演示, 我们用 caDNAno14的改进方法和使用 oxDNA16、17和附加脚本的手风琴机架的仿真来展示设计技术。最后, 介绍了该方法的基本原理和对所配置的手风琴架结构分析的烦恼。

<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

基于 dna 纳米结构的机械系统或 dna nanomachines, 在纳米到ångström 分辨率的2D 和3D 中产生复杂的纳米尺度运动, 在纳米技术的各个领域, 如分子反应器、药物输送, 都显示出巨大的潜力,和 nanoplasmonic 系统。描述了可重构的 dna 手风琴机架, 它可以集体操纵2D 或3D 纳米元素网络, 在多个阶段响应 dna 输入。该平台有可能增加 DNA nanomachines 可以从几个元素控制到网络规模的元素数量, 并可重新配置多个阶段。
在本协议中, 我们描述了 6 6 网格可重构 DNA 手风琴架的整个实验过程。该协议包括结构的设计规则和仿真程序, 以及用于合成和重构的湿实验室实验。此外, 该协议还包括了透射电镜 (透射电子显微镜) 和焦虑 (荧光共振能量传递) 的结构分析。本协议所涵盖的新颖设计和仿真方法将帮助研究人员使用 DNA 手风琴机架进行进一步的应用。

设计的6由 6 DNA 手风琴机架模拟从 oxDNA16,17和结果显示在图 6。仿真结果表明, 该结构在不变形的情况下形成了拟合结构。
图 7中的 TEM 图像是配置结构的图像, 其锁定长度为2、8、13和 20 bp。在图像上, 当锁的长度变长时, 结构的角度 (<strong c...

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

我们描述了设计, 模拟, 湿实验室实验的详细协议, 和分析的可重构的 DNA 手风琴架 6 6 网格。

可重构 DNA 手风琴架的设计与合成

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 ...

design-and-synthesis-of-a-reconfigurable-dna-accordion-rack

Research

JoVE Journal

Bioengineering

7.0K 次观看.  Seoul National University. 我们描述了设计, 模拟, 湿实验室实验的详细协议, 和分析的可重构的 DNA 手风琴架 6 6 网格。

视频: Design and Synthesis of a Reconfigurable DNA Accordion Rack

Gold Nanostar Synthesis with a Silver Seed Mediated Growth Method

The physical, chemical and optical properties of nano-scale colloids depend on their material composition, size and shape 1-5. There is a great interest in using nano-colloids for photo-thermal ablation, drug delivery and many other biomedical applications 6. Gold is particularly used because of its low toxicity 7-9. A property of metal nano-colloids is that they can have a strong surface plasmon resonance 10. The peak of the surface plasmon resonance mode depends on the structure and composition of the metal nano-colloids. Since the surface plasmon resonance mode is stimulated with light there is a need to have the peak absorbance in the near infrared where biological tissue transmissivity is maximal 11, 12.
We present a method to synthesize star shaped colloidal gold, also known as star shaped nanoparticles 13-15 or nanostars 16. This method is based on a solution containing silver seeds that are used as the nucleating agent for anisotropic growth of gold colloids 17-22. Scanning electron microscopy (SEM) analysis of the resulting gold colloid showed that 70 % of the nanostructures were nanostars. The other 30 % of the particles were amorphous clusters of decahedra and rhomboids. The absorbance peak of the nanostars was detected to be in the near infrared (840 nm). Thus, our method produces gold nanostars suitable for biomedical applications, particularly for photo-thermal ablation.

We synthesized star shaped gold nanostars using a silver seed mediated growth method. The diameter of the nanostars ranges from 200 to 300 nm and the number of tips vary from 7 to 10. The nanoparticles have a broad surface plasmon resonance mode centered in the near infrared.

黄金Nanostar合成银种子介导的生长方法

The physical, chemical and optical properties of nano-scale colloids depend on their material composition, size and shape 1-5. There is a great interest ...

科研

生物工程

Design of a Biaxial Mechanical Loading Bioreactor for Tissue Engineering

We designed a loading device that is capable of applying uniaxial or biaxial mechanical strain to a tissue engineered biocomposites fabricated for transplantation. While the device primarily functions as a bioreactor that mimics the native mechanical strains, it is also outfitted with a load cell for providing force feedback or mechanical testing of the constructs. The device subjects engineered cartilage constructs to biaxial mechanical loading with great precision of loading dose (amplitude and frequency) and is compact enough to fit inside a standard tissue culture incubator. It loads samples directly in a tissue culture plate, and multiple plate sizes are compatible with the system. The device has been designed using components manufactured for precision-guided laser applications. Bi-axial loading is accomplished by two orthogonal stages. The stages have a 50 mm travel range and are driven independently by stepper motor actuators, controlled by a closed-loop stepper motor driver that features micro-stepping capabilities, enabling step sizes of less than 50 nm. A polysulfone loading platen is coupled to the bi-axial moving platform. Movements of the stages are controlled by Thor-labs Advanced Positioning Technology (APT) software. The stepper motor driver is used with the software to adjust load parameters of frequency and amplitude of both shear and compression independently and simultaneously. Positional feedback is provided by linear optical encoders that have a bidirectional repeatability of 0.1 &mu;m and a resolution of 20 nm, translating to a positional accuracy of less than 3 &mu;m over the full 50 mm of travel. These encoders provide the necessary position feedback to the drive electronics to ensure true nanopositioning capabilities. In order to provide the force feedback to detect contact and evaluate loading responses, a precision miniature load cell is positioned between the loading platen and the moving platform. The load cell has high accuracies of 0.15% to 0.25% full scale.

We designed a novel mechanical loading bioreactor that can apply uniaxial or biaxial mechanical strain to a cartilage biocomposite prior to transplantation into an articular cartilage defect.

组织工程生物反应器的双轴机械装载设计

We  designed a loading device that is capable of applying uniaxial or biaxial  mechanical strain to a tissue engineered biocomposites fabricated for  ...

Millifluidics for Chemical Synthesis and Time-resolved Mechanistic Studies

Procedures utilizing millifluidic devices for chemical synthesis and time-resolved mechanistic studies are described by taking three examples. In the first, synthesis of ultra-small copper nanoclusters is described. The second example provides their utility for investigating time resolved kinetics of chemical reactions by analyzing gold nanoparticle formation using in situ X-ray absorption spectroscopy. The final example demonstrates continuous flow catalysis of reactions inside millifluidic channel coated with nanostructured catalyst.

Millifluidic devices are utilized for controlled synthesis of nanomaterials, time-resolved analysis of reaction mechanisms and continuous flow catalysis.

Millifluidics的化学合成和时间分辨机理研究

Procedures utilizing millifluidic devices for chemical synthesis and time-resolved mechanistic studies are described by taking three examples. In the ...

Synthesis of an Intein-mediated Artificial Protein Hydrogel

We present the synthesis of a highly stable protein hydrogel mediated by a split-intein-catalyzed protein trans-splicing reaction. The building blocks of this hydrogel are two protein block-copolymers each containing a subunit of a trimeric protein that serves as a crosslinker and one half of a split intein. A highly hydrophilic random coil is inserted into one of the block-copolymers for water retention. Mixing of the two protein block copolymers triggers an intein trans-splicing reaction, yielding a polypeptide unit with crosslinkers at either end that rapidly self-assembles into a hydrogel. This hydrogel is very stable under both acidic and basic conditions, at temperatures up to 50 &#176;C, and in organic solvents. The hydrogel rapidly reforms after shear-induced rupture. Incorporation of a &#34;docking station peptide&#34; into the hydrogel building block enables convenient incorporation of &#34;docking protein&#34;-tagged target proteins. The hydrogel is compatible with tissue culture growth media, supports the diffusion of 20 kDa molecules, and enables the immobilization of bioactive globular proteins. The application of the intein-mediated protein hydrogel as an organic-solvent-compatible biocatalyst was demonstrated by encapsulating the horseradish peroxidase enzyme and corroborating its activity.

We present the synthesis of a split-intein-mediated protein hydrogel. The building blocks of this hydrogel are two protein copolymers each containing a subunit of a trimeric protein that serves as a crosslinker and one half of a split intein. Mixing of the two protein copolymers triggers an intein trans-splicing reaction, yielding a polypeptide unit that self-assembles into a hydrogel. This hydrogel is highly pH- and temperature-stable, compatible with organic solvents, and easily incorporates functional globular proteins.

内含肽介导的人工蛋白水凝胶的合成

We present the synthesis of a highly stable protein hydrogel mediated by a split-intein-catalyzed protein trans-splicing reaction. The building blocks of ...

Synthesis of Keratin-based Nanofiber for Biomedical Engineering

Electrospinning, due to its versatility and potential for applications in various fields, is being frequently used to fabricate nanofibers. Production of these porous nanofibers is of great interest due to their unique physiochemical properties. Here we elaborate on the fabrication of keratin containing poly (&#949;-caprolactone) (PCL) nanofibers (i.e., PCL/keratin composite fiber). Water soluble keratin was first extracted from human hair and mixed with PCL in different ratios. The blended solution of PCL/keratin was transformed into nanofibrous membranes using a laboratory designed electrospinning set up. Fiber morphology and mechanical properties of the obtained nanofiber were observed and measured using scanning electron microscopy and tensile tester. Furthermore, degradability and chemical properties of the nanofiber were studied by FTIR. SEM images showed uniform surface morphology for PCL/keratin fibers of different compositions. These PCL/keratin fibers also showed excellent mechanical properties such as Young&#39;s modulus and failure point. Fibroblast cells were able to attach and proliferate thus proving good cell viability. Based on the characteristics discussed above, we can strongly argue that the blended nanofibers of natural and synthetic polymers can represent an excellent development of composite materials that can be used for different biomedical applications.

Electrospun nanofibers have a high surface area to weight ratio, excellent mechanical integrity, and support cell growth and proliferation. These nanofibers have a wide range of biomedical applications. Here we fabricate keratin/ PCL nanofibers, using the electrospinning technique, and characterize the fibers for possible applications in tissue engineering.

基于角蛋白纳米纤维生物医学工程合成

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

Automated Robotic Liquid Handling Assembly of Modular DNA Devices

Recent advances in modular DNA assembly techniques have enabled synthetic biologists to test significantly more of the available &#34;design space&#34; represented by &#34;devices&#34; created as combinations of individual genetic components. However, manual assembly of such large numbers of devices is time-intensive, error-prone, and costly. The increasing sophistication and scale of synthetic biology research necessitates an efficient, reproducible way to accommodate large-scale, complex, and high throughput device construction.
Here, a DNA assembly protocol using the Type-IIS restriction endonuclease based Modular Cloning (MoClo) technique is automated on two liquid-handling robotic platforms. Automated liquid-handling robots require careful, often times tedious optimization of pipetting parameters for liquids of different viscosities (e.g. enzymes, DNA, water, buffers), as well as explicit programming to ensure correct aspiration and dispensing of DNA parts and reagents. This makes manual script writing for complex assemblies just as problematic as manual DNA assembly, and necessitates a software tool that can automate script generation. To this end, we have developed a web-based software tool, http://mocloassembly.com, for generating combinatorial DNA device libraries from basic DNA parts uploaded as Genbank files. We provide access to the tool, and an export file from our liquid handler software which includes optimized liquid classes, labware parameters, and deck layout. All DNA parts used are available through Addgene, and their digital maps can be accessed via the Boston University BDC ICE Registry. Together, these elements provide a foundation for other organizations to automate modular cloning experiments and similar protocols.
The automated DNA assembly workflow presented here enables the repeatable, automated, high-throughput production of DNA devices, and reduces the risk of human error arising from repetitive manual pipetting. Sequencing data show the automated DNA assembly reactions generated from this workflow are ~95% correct and require as little as 4% as much hands-on time, compared to manual reaction preparation.

Here, an automated workflow to perform modular DNA &#34;device&#34; assembly using a modular cloning DNA assembly method on liquid-handling robots is presented. The protocol uses an intuitive software tool for generating liquid handler picklists for combinatorial DNA device library generation, which we demonstrate using two liquid handling platforms.

模块化 DNA 设备的自动机器人液体处理组件

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

Microfluidic components need to have various shapes to realize different key microfluidic functions such as mixing, separation, particle trapping, or reactions. A microfluidic channel that deforms even after fabrication while retaining the channel shape enables high spatiotemporal reconfigurability. This reconfigurability is required in such key microfluidic functions that are difficult to achieve in existing "reconfigurable" or "integrated" microfluidic systems. We describe a method for the fabrication of a microfluidic channel with a deformable sidewall consisting of a laterally aligned array of the ends of rectangular pins. Actuating the pins in their longitudinal directions changes the pins' end positions, and thus, the shape of discretized channel sidewalls.Pin gaps can cause unwanted leakage or adhesion to adjacent pins caused by meniscus forces. To close the pin gaps, we have introduced hydrocarbon-fluoropolymer suspension-based gap filler accompanied by an elastomeric barrier. This reconfigurable microfluidic device can generate strong temporal in-channel displacement flow, or can stop the flow in any region of the channel. This feature will facilitate, on demand, the handling of cells, viscous liquids, gas bubbles, and non-fluids, even if their existence or behavior is unknown at the time of fabrication.

A microfluidic channel with deformable sidewalls offers flow control, particle handling, channel dimension customization and other reconfigurations while in use. We describe a method for fabricating a microfluidic channel with sidewalls made of an array of pins that allows their shape to change.

基于 Pin 离散壁的可重构微流控通道

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

Compartmentalization of biochemical reactions is a central aspect of synthetic cells. For this purpose, peptide-based reaction compartments serve as an attractive alternative to liposomes or fatty acid-based vesicles. Externally or within the vesicles, peptides can be easily expressed and simplify the synthesis of membrane precursors. Provided here is a protocol for the creation of vesicles with diameters of ~200 nm based on the amphiphilic elastin-like polypeptides (ELP) utilizing dehydration-rehydration from glass beads. Also presented are protocols for bacterial ELP expression and purification via inverse temperature cycling, as well as their covalent functionalization with fluorescent dyes. Furthermore, this report describes a protocol to enable the transcription of RNA aptamer dBroccoli inside ELP vesicles as a less complex example for a biochemical reaction. Finally, a protocol is provided, which allows in vesiculo expression of fluorescent proteins and the membrane peptide, whereas synthesis of the latter results in vesicle growth.

Presented here are protocols for the creation of peptide-based small unilamellar vesicles capable of growth. To facilitate in vesiculo production of the membrane peptide, these vesicles are equipped with a transcription-translation system and the peptide-encoding plasmid.

在肽膜前体的维西图洛合成中,用于自主囊泡生长

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

Three-dimensional (3D) printing is an increasingly popular manufacturing technique that allows highly complex objects to be fabricated with no retooling costs. This increasing popularity is partly driven by falling barriers to entry such as system set-up costs and ease of operation. The following protocol presents the design and construction of an Additive Manufacturing Melt Extrusion (ADDME) 3D printer for the fabrication of custom parts and components. ADDME has been designed with a combination of 3D-printed, laser-cut, and online-sourced components. The protocol is arranged into easy-to-follow sections, with detailed diagrams and parts lists under the headings of framing, y-axis and bed, x-axis, extrusion, electronics, and software. The performance of ADDME is evaluated through extrusion testing and 3D printing of complex objects using viscous cream, chocolate, and Pluronic F-127 (a model for bioinks). The results indicate that ADDME is a capable platform for the fabrication of materials and constructs for use in a wide range of industries. The combination of detailed diagrams and video content facilitates access to low-cost, easy-to-operate equipment for individuals interested in 3D printing of complex objects from a wide range of materials.

The aim of this work is to design and construct a reservoir-based melt extrusion three-dimensional printer made from open-source and low-cost components for applications in the biomedical and food printing industries.

设计开源、低成本生物墨水和食品熔炼挤出3D打印机

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

Design of a Biocompatible Drug-Eluting Tracheal Stent in Mice with Laryngotracheal Stenosis

Laryngotracheal stenosis (LTS) is a pathologic narrowing of the subglottis and trachea leading to extrathoracic obstruction and significant shortness of breath. LTS results from mucosal injury from a foreign body in the trachea, leading to tissue damage and a local inflammatory response that goes awry, leading to the deposition of pathologic scar tissue. Treatment for LTS is surgical due to the lack of effective medical therapies. The purpose of this method is to construct a biocompatible stent that can be miniaturized to place into mice with LTS. We demonstrated that a PLLA-PCL (70% poly-L-lactide and 30% polycaprolactone) construct had optimal biomechanical strength, was biocompatible, practicable for an in vivo placement stent, and capable of eluting drug. This method provides a drug delivery system for testing various immunomodulatory agents to locally inhibit inflammation and reduce airway fibrosis. Manufacturing the stents takes 28&#8722;30 h and can be reproduced easily, allowing for experiments with large cohorts. Here we incorporated the drug rapamycin within the stent to test its effectiveness in reducing fibrosis and collagen deposition. Results revealed that PLLA-PCL tents showed reliable rapamycin release, were mechanically stable in physiological conditions, and were biocompatible, inducing little inflammatory response in the trachea. Further, the rapamycin-eluting PLLA-PCL stents reduced scar formation in the trachea in vivo.

Laryngotracheal stenosis results from pathologic scar deposition that critically narrows the tracheal airway and lacks effective medical therapies. Using a PLLA-PCL (70% poly-L-lactide and 30% polycaprolactone) stent as a local drug delivery system, potential therapies aimed at decreasing scar proliferation in the trachea can be studied.

与拉林戈切尔·斯泰斯·斯泰斯特的小鼠中生物相容性药物-脱酸支架的设计

Laryngotracheal stenosis (LTS) is a pathologic narrowing of the subglottis and trachea leading to extrathoracic obstruction and significant shortness of ...