Name: design and synthesis of a reconfigurable dna accordion rack
Uploaded: 2018-08-15T15:00:00.000+00:00
Duration: 7 min 44 s
Description: Watch this Scientific Journal Video about Design and Synthesis of a Reconfigurable DNA Accordion Rack at JoVE.com

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>

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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=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 MgCl&lt;sub&gt;2&lt;/sub&gt; 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)&amp;nbsp;&amp;nbsp;</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

Folding and Characterization of a Bio-responsive Robot from DNA Origami

The DNA nanorobot is a hollow hexagonal nanometric device, designed to open in response to specific stimuli and present cargo sequestered inside. Both stimuli and cargo can be tailored according to specific needs. Here we describe the DNA nanorobot fabrication protocol, with the use of the DNA origami technique. The procedure initiates by mixing short single-strand DNA staples into a stock mixture which is then added to a long, circular, single-strand DNA scaffold in presence of a folding buffer. A standard thermo cycler is programmed to gradually lower the mixing reaction temperature to facilitate the staples-to-scaffold annealing, which is the guiding force behind the folding of the nanorobot. Once the 60 hr folding reaction is complete, excess staples are discarded using a centrifugal filter, followed by visualization via agarose-gel electrophoresis (AGE). Finally, successful fabrication of the nanorobot is verified by transmission electron microscopy (TEM), with the use of uranyl-formate as negative stain.

DNA origami is a powerful method for fabricating precise nanoscale objects by programming the self-assembly of DNA molecules. Here we describe a protocol for the folding of a bio-responsive robot from DNA origami, its purification and negative staining for transmission electron microscopic imaging (TEM).

折叠的DNA折纸一个生物反应机器人与表征

The DNA nanorobot is a hollow hexagonal nanometric device, designed to open in response to specific stimuli and present cargo sequestered inside. Both ...

科研

化学

Self-assembly of Complex Two-dimensional Shapes from Single-stranded DNA Tiles

Current methods in DNA nano-architecture have successfully engineered a variety of 2D and 3D structures using principles of self-assembly. In this article, we describe detailed protocols on how to fabricate sophisticated 2D shapes through the self-assembly of uniquely addressable single-stranded DNA tiles which act as molecular pixels on a molecular canvas. Each single-stranded tile (SST) is a 42-nucleotide DNA strand composed of four concatenated modular domains which bind to four neighbors during self-assembly. The molecular canvas is a rectangle structure self-assembled from SSTs. A prescribed complex 2D shape is formed by selecting the constituent molecular pixels (SSTs) from a 310-pixel molecular canvas and then subjecting the corresponding strands to one-pot annealing. Due to the modular nature of the SST approach we demonstrate the scalability, versatility and robustness of this method. Compared with alternative methods, the SST method enables a wider selection of information polymers and sequences through the use of de novo designed and synthesized short DNA strands.

DNA tiling is an effective approach to make programmable nanostructures. We describe the protocols to construct complex two-dimensional shapes by the self-assembly of single-stranded DNA tiles.

复杂的二维形状的从单链DNA的瓷砖自组装

Current methods in DNA nano-architecture have successfully engineered a variety of 2D and 3D structures using principles of self-assembly. In this ...

Designing a Bio-responsive Robot from DNA Origami

Nucleic acids are astonishingly versatile. In addition to their natural role as storage medium for biological information1, they can be utilized in parallel computing2,3 , recognize and bind molecular or cellular targets4,5 , catalyze chemical reactions6,7 , and generate calculated responses in a biological system8,9. Importantly, nucleic acids can be programmed to self-assemble into 2D and 3D structures10-12, enabling the integration of all these remarkable features in a single robot linking the sensing of biological cues to a preset response in order to exert a desired effect.
Creating shapes from nucleic acids was first proposed by Seeman13, and several variations on this theme have since been realized using various techniques11,12,14,15 . However, the most significant is perhaps the one proposed by Rothemund, termed scaffolded DNA origami16. In this technique, the folding of a long (&gt;7,000 bases) single-stranded DNA 'scaffold' is directed to a desired shape by hundreds of short complementary strands termed 'staples'. Folding is carried out by temperature annealing ramp. This technique was successfully demonstrated in the creation of a diverse array of 2D shapes with remarkable precision and robustness. DNA origami was later extended to 3D as well17,18 .
 
The current paper will focus on the caDNAno 2.0 software19 developed by Douglas and colleagues. caDNAno is a robust, user-friendly CAD tool enabling the design of 2D and 3D DNA origami shapes with versatile features. The design process relies on a systematic and accurate abstraction scheme for DNA structures, making it relatively straightforward and efficient.
In this paper we demonstrate the design of a DNA origami nanorobot that has been recently described20. This robot is 'robotic' in the sense that it links sensing to actuation, in order to perform a task. We explain how various sensing schemes can be integrated into the structure, and how this can be relayed to a desired effect. Finally we use Cando21 to simulate the mechanical properties of the designed shape. The concept we discuss can be adapted to multiple tasks and settings.

DNA origami is a powerful method for fabricating precise nanoscale objects by programming the self-assembly of DNA molecules. Here, we describe how DNA origami can be utilized to design a robotic robot capable of sensing biological cues and responding by shape shifting, subsequently relayed to a desired effect.

DNA折纸设计生物反应灵敏的机器人

Nucleic acids are astonishingly versatile. In addition to their natural role as storage medium for biological information1, they can be utilized in ...

生物工程

DNA Curtains Shed Light on Complex Molecular Systems During Homologous Recombination

Homologous recombination (HR) is important for the repair of double-stranded DNA breaks (DSBs) and stalled replication forks in all organisms. Defects in HR are closely associated with a loss of genome integrity and oncogenic transformation in human cells. HR involves coordinated actions of a complex set of proteins, many of which remain poorly understood. The key aspect of the research described here is a technology called "DNA curtains", a technique which allows for the assembly of aligned DNA molecules on the surface of a microfluidic sample chamber. They can then be visualized by total internal reflection fluorescence microscopy (TIRFM). DNA curtains was pioneered by our laboratory and allows for direct access to spatiotemporal information at millisecond time scales and nanometer scale resolution, which cannot be easily revealed through other methodologies. A major advantage of DNA curtains is that it simplifies the collection of statistically relevant data from single molecule experiments. This research continues to yield new insights into how cells regulate and preserve genome integrity.

DNA curtains present a novel method for visualizing hundreds or even thousands of DNA-binding proteins in real-time as they interact with DNA molecules aligned on the surface of a microfluidic sample chamber.

Homologous recombination (HR) is important for the repair of double-stranded DNA breaks (DSBs) and stalled replication forks in all organisms. Defects in ...

遗传学

Stable DNA Motifs, 1D and 2D Nanostructures Constructed from Small Circular DNA Molecules

This article presents a detailed protocol for synthesis of small circular DNA molecules, annealing of circular DNA motifs, and construction of 1D and 2D DNA nanostructures. Over decades, the rapid development of DNA nanotechnology is attributed to the use of linear DNAs as the source materials. For example, the DAO (double crossover, antiparallel, odd half-turns) tile is well-known as a building block for construction of 2D DNA lattices; the core structure of DAO is made from two linear single-stranded (ss) oligonucleotides, like two ropes making a right hand granny&#160;knot. Herein, a new type of DNA tiles called cDAO (coupled DAO) are built using a small circular ss-DNA of c64nt or c84nt (circular 64 or 84 nucleotides) as the scaffold strand and several linear ss-DNAs as the staple strands. Perfect 1D and 2D nanostructures are assembled from cDAO tiles: infinite nanowires, nanospirals, nanotubes, nanoribbons; and finite nano-rectangles. Detailed protocols are described: 1) preparation by T4 ligase and purification by denaturing PAGE (polyacrylamide gel electrophoresis) of small circular oligonucleotides, 2) annealing of stable circular tiles, followed by native PAGE analysis, 3) assembling of infinite 1D nanowires, nanorings, nanospirals, infinite 2D lattices of nanotubes and nanoribbons, and finite 2D nano-rectangles, followed by AFM (Atomic Force Microscopy) imaging. The method is simple, robust, and affordable for most labs.

This article presents a detailed protocol for T4 ligation and denaturing PAGE purification of small circular DNA molecules, annealing and native PAGE analysis of circular tiles, assembling and AFM imaging of 1D and 2D DNA nanostructures, as well as agarose gel electrophoresis and centrifugation purification of finite DNA nanostructures.

由小圆形 DNA 分子构建的稳定 DNA 分子、一维和二维纳米结构

This article presents a detailed protocol for synthesis of small circular DNA molecules, annealing of circular DNA motifs, and construction of 1D and 2D ...

DNA Origami-Mediated Substrate Nanopatterning of Inorganic Structures for Sensing Applications

Structural DNA nanotechnology provides a viable route for building from the bottom-up using DNA as construction material. The most common DNA nanofabrication technique is called DNA origami, and it allows high-throughput synthesis of accurate and highly versatile structures with nanometer-level precision. Here, it is shown how the spatial information of DNA origami can be transferred to metallic nanostructures by combining the bottom-up DNA origami with the conventionally used top-down lithography approaches. This allows fabrication of billions of tiny nanostructures in one step onto selected substrates. The method is demonstrated using bowtie DNA origami to create metallic bowtie-shaped antenna structures on silicon nitride or sapphire substrates. The method relies on the selective growth of a silicon oxide layer on top of the origami deposition substrate, thus resulting in a patterning mask for following lithographic steps. These nanostructure-equipped surfaces can be further used as molecular sensors (e.g., surface-enhanced Raman spectroscopy (SERS)) and in various other optical applications at the visible wavelength range owing to the small feature sizes (sub-10 nm). The technique can be extended to other materials through methodological modifications; therefore, the resulting optically active surfaces may find use in development of metamaterials and metasurfaces.

Here, we describe a protocol to create discrete and accurate inorganic nanostructures on substrates using DNA origami shapes as guiding templates. The method is demonstrated by creating plasmonic gold bowtie-shaped antennas on a transparent substrate (sapphire).

脱氧核糖核酸介导基板无机结构纳米图案用于传感应用

Structural DNA nanotechnology provides a viable route for building from the bottom-up using DNA as construction material. The most common DNA ...

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; ...

Simple, Affordable, and Modular Patterning of Cells using DNA

The relative positioning of cells is a key feature of the microenvironment that organizes cell-cell interactions. To study the interactions between cells of the same or different type, micropatterning techniques have proved useful. DNA Programmed Assembly of Cells (DPAC) is a micropatterning technique that targets the adhesion of cells to a substrate or other cells using DNA hybridization. The most basic operations in DPAC begin with decorating cell membranes with lipid-modified oligonucleotides, then flowing them over a substrate that has been patterned with complementary DNA sequences. Cells adhere selectively to the substrate only where they find a complementary DNA sequence. Non-adherent cells are washed away, revealing a pattern of adherent cells. Additional operations include further rounds of cell-substrate or cell-cell adhesion, as well as transferring the patterns formed by DPAC to an embedding hydrogel for long-term culture. Previously, methods for patterning oligonucleotides on surfaces and decorating cells with DNA sequences required specialized equipment and custom DNA synthesis, respectively. We report an updated version of the protocol, utilizing an inexpensive benchtop photolithography setup and commercially available cholesterol modified oligonucleotides (CMOs) deployed using a modular format. CMO-labeled cells adhere with high efficiency to DNA-patterned substrates. This approach can be used to pattern multiple cell types at once with high precision and to create arrays of microtissues embedded within an extracellular matrix. Advantages of this method include its high resolution, ability to embed cells into a three-dimensional microenvironment without disrupting the micropattern, and flexibility in patterning any cell type.

Here we present a protocol to micropattern cells at single-cell resolution using DNA-programmed adhesion. This protocol uses a benchtop photolithography platform to create patterns of DNA oligonucleotides on a glass slide and then labels cell membranes with commercially available complementary oligonucleotides. Hybridization of the oligos results in programmed cell adhesion.

使用DNA对细胞进行简单、经济、模块化的图案化

The relative positioning of cells is a key feature of the microenvironment that organizes cell-cell interactions. To study the interactions between cells ...

DNA-Tethered RNA Polymerase for Programmable In vitro Transcription and Molecular Computation

DNA nanotechnology enables programmable self-assembly of nucleic acids into user-prescribed shapes and dynamics for diverse applications. This work demonstrates that concepts from DNA nanotechnology can be used to program the enzymatic activity of the phage-derived T7 RNA polymerase (RNAP) and build scalable synthetic gene regulatory networks. First, an oligonucleotide-tethered T7 RNAP is engineered via expression of an N-terminally SNAP-tagged RNAP and subsequent chemical coupling of the SNAP-tag with a benzylguanine (BG)-modified oligonucleotide. Next, nucleic-acid strand displacement is used to program polymerase transcription on-demand. In addition, auxiliary nucleic acid assemblies can be used as &#34;artificial transcription factors&#34; to regulate the interactions between the DNA-programmed T7 RNAP with its DNA templates. This in vitro transcription regulatory mechanism can implement a variety of circuit behaviors such as digital logic, feedback, cascading, and multiplexing. The composability of this gene regulatory architecture facilitates design abstraction, standardization, and scaling. These features will enable the rapid prototyping of in vitro&#160;genetic devices for applications such as bio-sensing, disease detection, and data storage.

We describe the engineering of a novel DNA-tethered T7 RNA polymerase to regulate in vitro transcription reactions. We discuss the steps for protein synthesis and characterization, validate proof-of-concept transcriptional regulation, and discuss its applications in molecular computing, diagnostics, and molecular information processing.

DNA系留RNA聚合酶用于可编程体外转录和分子计算

DNA nanotechnology enables programmable self-assembly of nucleic acids into user-prescribed shapes and dynamics for diverse applications. This work ...

Plasmid-derived DNA Strand Displacement Gates for Implementing Chemical Reaction Networks

DNA nanotechnology requires large amounts of highly pure DNA as an engineering material. Plasmid DNA could meet this need since it is replicated with high fidelity, is readily amplified through bacterial culture and can be stored indefinitely in the form of bacterial glycerol stocks. However, the double-stranded nature of plasmid DNA has so far hindered its efficient use for construction of DNA nanostructures or devices that typically contain single-stranded or branched domains. In recent work, it was found that nicked double stranded DNA (ndsDNA) strand displacement gates could be sourced from plasmid DNA. The following is a protocol that details how these ndsDNA gates can be efficiently encoded in plasmids and can be derived from the plasmids through a small number of enzymatic processing steps. Also given is a protocol for testing ndsDNA gates using fluorescence kinetics measurements. NdsDNA gates can be used to implement arbitrary chemical reaction networks (CRNs) and thus provide a pathway towards the use of the CRN formalism as a prescriptive molecular programming language. To demonstrate this technology, a multi-step reaction cascade with catalytic kinetics is constructed. Further it is shown that plasmid-derived components perform better than identical components assembled from synthetic DNA.

This protocol describes a method for deriving DNA strand displacement gates from plasmids and testing them using fluorescence kinetics measurements. Gates can be modularly composed into multi-component systems to approximate the behavior of formal chemical reaction networks (CRN), demonstrating a new use for CRNs as a molecular programming language.

质粒来源的DNA链置换盖茨实施化学反应网络

DNA nanotechnology requires large amounts of highly pure DNA as an engineering material. Plasmid DNA could meet this need since it is replicated with high ...