Name: self-assembly of gamma-modified peptide nucleic acids into complex nanostructures in organic solvent mixtures
Uploaded: 2020-06-26T14:00:00
Description: Watch this Scientific Journal Video about Self-Assembly of Gamma-Modified Peptide Nucleic Acids into Complex Nanostructures in Organic Solvent Mixtures at JoVE.com

这项工作得到了国家科学基金会赠款1739308、NSF CAREER1944130和空军科研办公室赠款F9550-18-1-0199的部分支持。•PNA序列是特鲁科德基因修复公司的图穆尔·斯里瓦斯塔瓦博士的慷慨礼物。我们要感谢埃里克·温弗里博士和里扎尔·哈里阿迪博士就DNA设计工具箱MATLAB代码进行有益的对话。我们还要感谢约瑟夫·苏汉、马拉·沙利文和生物成像中心在收集TEM数据方面提供的援助。

1. [PNA 序列设计]
<ol><li>下载由加州理工学院23 Winfree 实验室开发的 DNA 设计工具箱 22 下载到包含用于设计序列的编程脚本的文件夹中。</li>
	<li>在该序列设计文件夹中，打开与文件扩展名".m"兼容的第四代编程语言，然后使用以下命令将以前下载的"DNAdesign"文件夹添加到路径中： >> 附加路径 DNAdesign</li>
	<li>随后使用以下命令运行以下名为"PNA3naofiber.m"（参见附?...

<ol>
	<li>Fu, T. J., Seeman, N. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+double-crossover+molecules.">DNA double-crossover molecules.</a> Biochemistry. 32 (13), 3211-3220 (1993).</li><li>Winfree, E., Liu, F., Wenzler, L. A., Seeman, N. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design+and+self-assembly+of+two-dimensional+DNA+crystals.">Design and self-assembly of two-dimensional DNA crystals.</a> Nature. 394 (6693), 539-544 (1998).</li><li>Shih, W., Quispe, J., Joyce, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+1.7-kilobase+single-stranded+DNA+that+folds+into+a+nanoscale+octahedron.">A 1.7-kilobase single-stranded DNA that folds into a nanoscale octahedron.</a> Nature. 427 (6975), 618-621 (2004).</li><li>Rothemund, P. W. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Folding+DNA+to+create+nanoscale+shapes+and+patterns.">Folding DNA to create nanoscale shapes and patterns.</a> Nature. 440 (7082), 297-302 (2006).</li><li>He, Y., 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=Hierarchical+self-assembly+of+DNA+into+symmetric+supramolecular+polyhedra.">Hierarchical self-assembly of DNA into symmetric supramolecular polyhedra.</a> Nature. 452 (7184), 198-201 (2008).</li><li>Douglas, S. M., Dietz, H., Liedl, T., H&#246;gberg, B., Graf, F., Shih, W. M. <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+DNA+into+nanoscale+three-dimensional+shapes.">Self-assembly of DNA into nanoscale three-dimensional shapes.</a> Nature. 459 (7245), 414-418 (2009).</li><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>Dietz, H., Douglas, S. M., Shih, W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Folding+DNA+into+twisted+and+curved+nanoscale+shapes.">Folding DNA into twisted and curved nanoscale shapes.</a> Science. 325 (5941), 725-730 (2009).</li><li>Han, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+origami+with+complex+curvatures+in+three-dimensional+space.">DNA origami with complex curvatures in three-dimensional space.</a> Science. 332 (6024), 342-346 (2011).</li><li>Liu, Y., Kumar, S., Taylor, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mix-and-match+nanobiosensor+design:+Logical+and+spa421+tial+programming+of+biosensors+using+self-assembled+DNA+nanostructures.">Mix-and-match nanobiosensor design: Logical and spa421 tial programming of biosensors using self-assembled DNA nanostructures.</a> WIREs Nanomedicne Nanobiotechnology. 10, 1518 (2018).</li><li>Chworos, 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=Building+programmable+jigsaw+puzzles+with+RNA.">Building programmable jigsaw puzzles with RNA.</a> Science. 306 (5704), 2068-2072 (2004).</li><li>Delebecque, C. J., Lindner, A. B., Silver, P. A., Aldaye, F. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organization+of+intracellular+reactions+with+rationally+designed+RNA+assemblies.">Organization of intracellular reactions with rationally designed RNA assemblies.</a> Science. 333 (6041), 470-474 (2011).</li><li>Wang, X., Lim, H. J., Son, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+denaturation+and+renaturation+of+DNA+for+DNA+hybridization.">Characterization of denaturation and renaturation of DNA for DNA hybridization.</a> Environmental health and toxicology. 29, 2014007 (2014).</li><li>Bonner, G., Klibanov, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+stability+of+DNA+in+nonaqueous+solvents.">Structural stability of DNA in nonaqueous solvents.</a> Biotechnology and Bioengineering. 68, 339 (2000).</li><li>Kumar, S., Pearse, A., Liu, Y., Taylor, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modular+self-assembly+of+gamma-modified+peptide+nucleic+acids+in+organic+solvent+mixtures.">Modular self-assembly of gamma-modified peptide nucleic acids in organic solvent mixtures.</a> Nature Communications. 11 (1), 1-10 (2020).</li><li>Barluenga, S., Winssinger, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PNA+as+a+biosupramolecular+tag+for+programmable+assemblies+and+reactions.">PNA as a biosupramolecular tag for programmable assemblies and reactions.</a> Accounts of chemical research. 48, 1319-1331 (2015).</li><li>Berger, O., Gazit, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+self-assembly+using+peptide+nucleic+acids.">Molecular self-assembly using peptide nucleic acids.</a> Peptide Science. 108, 22930 (2017).</li><li>Rothemund, P. W., 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+and+characterization+of+programmable+DNA+nanotubes.">Design and characterization of programmable DNA nanotubes.</a> Journal of American Chemical Society. 126, 16344-16352 (2004).</li><li>Yin, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Programming+DNA+tube+circumferences.">Programming DNA tube circumferences.</a> Science. 321, 824-826 (2008).</li><li>Yang, Y., 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+DNA+rings+from+scaffold-free+DNA+tiles.">Self-assembly of DNA rings from scaffold-free DNA tiles.</a> Nano Letters. 13, 1862-1866 (2013).</li><li>Sahu, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis+and+characterization+of+conformationally+preorganized,+(R)-diethylene+glycol-containing+-peptide+nucleic+acids+with+superior+hybridization+properties+and+water+solubility.">Synthesis and characterization of conformationally preorganized, (R)-diethylene glycol-containing -peptide nucleic acids with superior hybridization properties and water solubility.</a> Journal of Organic Chemisty. 76, 5614-5627 (2011).</li><li>Dirks, R. M., Lin, M., Winfree, E., Pierce, N. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Paradigms+for+computational+nucleic+acid+design.">Paradigms for computational nucleic acid design.</a> Nucleic Acids Research. 32 (4), 1392-1403 (2004).</li><li>Sen, A., Nielsen, P. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+the+stability+of+peptide+nucleic+acid+duplexes+in+the+presence+of+organic+solvents.">On the stability of peptide nucleic acid duplexes in the presence of organic solvents.</a> Nucleic Acids Research. 35, 3367-3374 (2007).</li><li>Yang, Z., Williams, D., Russell, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis+of+protein-containing+polymers+in+organic+solvents.">Synthesis of protein-containing polymers in organic solvents.</a> Biotechnology and Bioengineering. 45, 10-17 (1995).</li><li>Coin, I., Beyermann, M., Bienert, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Solid-phase+peptide+synthesis:+from+standard+procedures+to+the+synthesis+of+difficult+sequences.">Solid-phase peptide synthesis: from standard procedures to the synthesis of difficult sequences.</a> Nature Protocols. 2, 3247 (2007).</li></ol>

本文重点介绍对有机溶剂混合物的现有核酸纳米技术方案的适应和改进。此处介绍的方法侧重于在选择极性有机溶剂的固定实验空间内进行修改和故障排除。其他已建立的核酸纳米技术方案尚未在此空间内加以调整，目前尚有未探索的潜力。这可以通过整合在其他领域，如聚合物和肽合成，通常执行在类似的有机溶剂25，26的潜在应用。此外，我们在此?...

成功建造许多复杂的纳米结构1、2、3、 4、5、6、7、8、9、10、11、12在水性或实质水合介质中使用 天然存在的核酸，如DNA1、2、3、4、5、6、7、8、9、10和RNA 11、12等，在以往的作品中已经得到证明。然而，自然产生的核酸会经历双面的脂肪构象变化，或在有机溶剂混合物13，14中降低热稳定性。
此前，我们的实验室已经报告了一种利用伽马位置改性合成核酸模拟伽马-肽核酸（+PNA）15（图1A）构建3螺旋纳米纤维的方法。在16、17领域讨论了这种开发需求以及合成核酸模拟PNA的潜在应用。我们已经表明，通过适应为DNA纳米结构18，19，20提出的单链瓷砖（SST）策略，9个顺序明显的αPNA寡聚物可以设计为在选择极性螺旋有机溶剂混合物（如DMSO和DMF）中形成3螺旋纳米纤维。[PNA寡聚物是商业订购的，根据Sahu等人发布的方法，在每个12基寡聚物的基础上，在每个12基寡聚体上，每个12基寡聚体沿3个γ位置（1个、4个和8个基位）对（R）-二乙二醇（微型PEG）进行了修改。21这些伽玛修饰导致与 +PNA 相对于未修改 PNA 的较高结合亲和力和热稳定性相关的级级预组织。
本文是本文对所报道工作的改编，本文对溶剂溶液和DNA替代对基于αPNA的纳米结构15的形成的影响进行了调查。本文的目的是提供设计的详细描述，以及为αPNA纳米纤维的自组装和特性而开发的溶剂适应方法的详细方案。因此，我们首先介绍了模块化SST策略，即使用合成核酸模拟PNA的纳米结构设计的通用平台。
据报道，与每10.5个碱基相比，PNA双工的利形间距每回合18个碱基（图1B）。因此，将演示的 +PNA SST 的域长度设置为 6 个基座，以适应三分之一的完全旋转或 120° 的旋转，从而实现三个三角形排列的螺旋之间的交互。此外，与以前的SST图案不同，每个SST只包含2个域，有效地创造了一个一维功能区状的结构，该结构包装形成一个三螺旋束（图1C）。每个 12 基 +PNA 寡聚物在 1、4 和 8 位置进行伽玛改度，以确保迷你 PEG 组在整个 SST 主题中均匀分布。此外，在图案中，有两种类型的寡聚物："连续"股存在于单个螺旋和螺旋跨度"交叉"链（图1D）。此外，寡聚物P8和P6分别标有荧光Cy3（绿星）和生物素（黄色椭圆形）（图1D），以便使用荧光显微镜检测结构形成。SST 图案总共由 9 个总寡聚物组成，通过每个域的编程互补，使 3 螺旋纳米纤维与相邻寡聚体上的相应域形成三螺旋纳米纤维（图 1E）。

<table>
	<tbody>
		<tr>
			<td>&#947;PNA strands/oligomers</td>
			<td>Trucode Gene Repair Inc.</td>
			<td></td>
			<td>Section 2.1</td>
		</tr>
		<tr>
			<td>UV-Vis Spectrophotometer</td>
			<td>Agilent</td>
			<td>Varian Cary 300</td>
			<td>Section 3.1.2</td>
		</tr>
		<tr>
			<td>Quartz cuvettes</td>
			<td>Starna</td>
			<td>29-Q-10</td>
			<td>Section 3.1.1</td>
		</tr>
		<tr>
			<td>Thermal cycler</td>
			<td>Bio Rad</td>
			<td>C1000 touch</td>
			<td>Section 4.1</td>
		</tr>
		<tr>
			<td>0.2 mL PCR tubes</td>
			<td>VWR</td>
			<td>53509-304</td>
			<td>Section 4.5</td>
		</tr>
		<tr>
			<td>Anhydrous DMF</td>
			<td>VWR</td>
			<td>EM-DX1727-6</td>
			<td>Section 4.6</td>
		</tr>
		<tr>
			<td>Anhydrous DMSO</td>
			<td>VWR</td>
			<td>EM-MX1457-6</td>
			<td>Section 4.6</td>
		</tr>
		<tr>
			<td>Anhydrous 1,4-Dioxane</td>
			<td>Fisher Scientific</td>
			<td>AC615121000</td>
			<td>Section 4.6</td>
		</tr>
		<tr>
			<td>10X Phosphate Buffered Saline (PBS)</td>
			<td>VWR</td>
			<td>75800-994</td>
			<td>Section 3.1.1</td>
		</tr>
		<tr>
			<td>Microscope slides</td>
			<td>VWR</td>
			<td>89085-399</td>
			<td>Section 5.2</td>
		</tr>
		<tr>
			<td>Glass cover slips</td>
			<td>VWR</td>
			<td>48382-126</td>
			<td>Section 5.2</td>
		</tr>
		<tr>
			<td>2% Collodion in Amyl Acetate</td>
			<td>Sigma-Aldrich</td>
			<td>9817</td>
			<td>Section 5.2</td>
		</tr>
		<tr>
			<td>Isoamyl Acetate</td>
			<td>VWR</td>
			<td>200001-180</td>
			<td>Section 5.2</td>
		</tr>
		<tr>
			<td>Biotinylated Bovine Serum Albumin (Biotin-BSA)</td>
			<td>Sigma-Aldrich</td>
			<td>A8549</td>
			<td>Section 5.3</td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin (BSA)</td>
			<td>Sigma-Aldrich</td>
			<td>A2153</td>
			<td>Section 5.4</td>
		</tr>
		<tr>
			<td>Streptavidin</td>
			<td>Sigma-Aldrich</td>
			<td>189730</td>
			<td>Section 5.5</td>
		</tr>
		<tr>
			<td>Trolox</td>
			<td>Sigma-Aldrich</td>
			<td>238813</td>
			<td>Section 5.7</td>
		</tr>
		<tr>
			<td>Total Internal Reflection Fluorescence microscope</td>
			<td>Nikon</td>
			<td>Nikon Ti2-E</td>
			<td>Section 5.8</td>
		</tr>
		<tr>
			<td>Transmission Electron Microscope</td>
			<td>Joel</td>
			<td>JEM 1011</td>
			<td>Section 6.6</td>
		</tr>
		<tr>
			<td>Tweezers</td>
			<td>Dumont</td>
			<td>0203-N5AC-PO</td>
			<td>Section 6.3</td>
		</tr>
		<tr>
			<td>Uranyl Acetate</td>
			<td>Electron Microscopy Sciences</td>
			<td>22400</td>
			<td>Section 6.1</td>
		</tr>
		<tr>
			<td>Formvar, 300 mesh, Copper grids</td>
			<td>Ted Pella Inc.</td>
			<td>1701-F</td>
			<td>Section 6.2</td>
		</tr>
		<tr>
			<td>Formvar-Silicon monoxide Type A, 300 mesh, Copper grids</td>
			<td>Ted Pella Inc.</td>
			<td>1829</td>
			<td>Section 6.2</td>
		</tr>
		<tr>
			<td>DNA oligomers/strands</td>
			<td>IDT</td>
			<td></td>
			<td>Section 7.1</td>
		</tr>
		<tr>
			<td>Sodium Dodecyl Sulphate (SDS)</td>
			<td>VWR</td>
			<td>97064-860</td>
			<td>Section 8.1</td>
		</tr>
	</tbody>
</table>

self-assembly of gamma-modified peptide nucleic acids into complex nanostructures in organic solvent mixtures

DNA 和 RNA 纳米技术中的当前策略使各种核酸纳米结构在水性或基本水合介质中自组装。在这篇文章中，我们描述了详细的协议，通过独特的可处理、单链、伽马改性肽核酸 （+PNA） 瓷砖的自组装，在有机溶剂混合物中构建纳米纤维结构。每个单链瓦 （SST） 是一个 12 基 +PNA 寡聚物，由两个串联的模块化域组成，每个模块有 6 个基。每个域都可以绑定到相邻链上的相互互补域，使用编程的互补性来形成纳米纤维，可以长到微米的长度。SST 图案由 9 个总寡聚物组成，能够形成 3 螺旋纳米纤维。与形成直径单分散结构的DNA纳米结构相比，这些αPNA系统形成纳米纤维，在有机溶剂混合物自组装过程中沿宽度捆绑。因此，此处描述的自组装协议还包括传统的表面活性剂，硫酸钠 （SDS），以减少捆绑效应。

上述各节中讨论的协议描述了DNA纳米纤维的经过调整的SST图案的设计，用于使用多种不同αPNA寡聚物的自组装纳米纤维结构的强健生成。本节介绍对从成功娱乐所述协议中获得的数据的解释。
按照第5节所述的TIRF对75%DMSO中退火的αPNA寡聚物样本的TIRF成像方案，H2O （v/v） 最容易提供在微观观察下组织良好的结构的证据，如图4A所示。75% DMF： H2...

Watch this Scientific Journal Video about Self-Assembly of Gamma-Modified Peptide Nucleic Acids into Complex Nanostructures in Organic Solvent Mixtures at JoVE.com

Self-Assembly of Gamma-Modified Peptide Nucleic Acids into Complex Nanostructures in Organic Solvent Mixtures

本文为有机溶剂混合物中伽马改性肽核酸寡聚物的纳米结构设计和自组装提供了方案。

将伽马改性肽核酸自组装成有机溶剂混合物中复杂的纳米结构

Current strategies in DNA and RNA nanotechnology enable the self-assembly of a variety of nucleic acid nanostructures in aqueous or substantially hydrated ...

这种新颖的协议描述了使用多种不同序列的合成核酸模仿伽马PNA形成纳米结构在特定的有机溶剂混合物。此处描述的测定表明，需要将现有的核酸纳米技术协议调整为有机溶剂，而有机溶剂很少或没有发布。由于纳米材料PNA具有聚合的倾向，新接触这项技术的从业者可能会发现，很难将为水富环境开发的现有协议适应有机溶剂丰富的环境。首先准备伽马PNA库存。从一家商业制造商那里获得 HPLC 级纯化伽马 PNA 链后，将去化水中的每股重新暂停至 300 微摩尔浓度。将伽玛 PNA 储存在零下 20 摄氏度，长达数月。当准备执行自组装时，在 75%DMSO、75%DMF 和 40%1、4-二恶烷中准备退火批次样品。首先，从主库存中加入0.67微升，加入除离子水，最终为10微升，为每个寡聚物准备20微摩尔子。将20微摩尔子库存中每个寡聚物的微升添加到200微升PCR管中，9个寡聚物的总体积为9微升。添加 30 微升无水 DMSO 或 DMF，再加一微升去维水，最终体积为 40 微升。要准备40%1，4二氧烷批次，加入16微升1，4-二氧烷，并带体积到40微升与去电化水。在 90 至 20 摄氏度的热循环器冷却中，将样品退去 22.5 小时。通常，两个寡聚物和三个寡聚伽马 PNA 子集的熔化温度在 40 至 70 摄氏度的范围内，用于不同的溶剂条件。根据手稿指示对热循环器进行编程。退火完成后，样品可以在4摄氏度下储存12至24小时，然后进行表征。通过将大约五毫升的水填充到包装盒中，从空移液器提示盒中制作一个湿度室。用乙酰醋酸酯溶剂准备20倍的商用2%的醋酸胺醇稀释，以产生硝基纤维素溶液。使用钳子将盖玻片浸入硝基纤维素溶液中，使其风干，然后用显微镜幻灯片、两个双面胶带和硝基纤维素涂层盖玻片制作流动室。通过称量一毫克生物素 BSA 并在 PBS 中溶解它，准备生物素 BSA 溶液。将生物素 BSA 的 15 微升移液器以一定角度进入流通道，并在加湿室的室温下孵育两到四分钟。将洗涤缓冲液的15微升（由1毫升PBS中的1毫克BSA组成）吹入通道，洗去多余的生物素BSA，然后通过在加湿室中孵化流通道2至4分钟来钝化表面。测量0.5毫克的链球菌素和1毫克的BSA，然后溶解在PBS的一毫升。将链球菌溶液的15微升移液放入流道中，放入加湿室2至4分钟，然后通过流动15微升的洗涤缓冲液清洗通道。接下来，在加湿室中流15微升的退火批次伽马PNA寡聚物，然后以与纳米结构相同的溶剂成分流15微升15微升，清洗未绑定的纳米结构。将流量通道传输到滑架，使用配备 TIRF 成像、60 倍油浸夹目标和 1.5 倍放大镜的荧光显微镜对它进行成像。在监控 Cy3 通道时，以 60 倍或 90 倍的放大率扫描流量通道。根据手稿方向准备 20 和 6% SDS 库存，然后将伽马 PNA 寡聚物退火，将每 20 微摩尔寡聚物中的一微升添加到 200 微升 PCR 管中。在 PCR 管中加入 30 微升无水 DMSO 和一个 6%或 20%SDS 微升，最终体积为 40 微升，最终 SDS 浓度将分别达到 5.25 或 17.5 毫摩尔。如前面所述，将热循环器中的伽马 PNA 退火。使用 TIRF 或 TEM 协议在存在 SDS 的情况下描述伽马 PNA 纳米结构。75% DMSO 中退火的伽马 PNA 寡聚物的 TIRF 成像显示结构良好，而 75% DMF 的退火则产生了尖刺状或针状纳米结构。40%1，4-二氧烷条件产生了丝丝纳米结构的稀疏装饰。此外，在 75% DMSO 中形成的伽马 PNA 纳米管的样本表明，在 TEM 成像过程中，纳米纤维以高放大倍率或纳米分辨率捆绑在一起。对纳米结构宽度的定量分析表明，中位宽度为16.3纳米，最大值超过80纳米。取代连续伽马PNA的异质DNA寡聚物形成直丝结构，而交叉伽马PNA的替代则产生硬质结构。被连续DNA寡聚物取代的纳米结构的中位宽度约为19纳米。在高 SDS 浓度下，伽马 PNA 纳米纤维在使用 TIRF 成像时，也采用高度网络化形态，而与其关键的 micelle 浓度相比。TEM 成像表明，5.25 毫摩尔 SDS 中的伽玛 PNA 组件在结构捆绑方面具有最大的缩减。在尝试此协议时，重要的是要记住保持上述溶剂组合物，因为纳米结构有聚合的倾向。这些方法鼓励从业者探索其他未探索的不同溶剂组合物、表面活性剂和其他PNA DNA纳米结构的途径。

Research

JoVE Journal

Bioengineering

Fabrication of Electrochemical-DNA Biosensors for the Reagentless Detection of Nucleic Acids, Proteins and Small Molecules

无试剂检测核酸，蛋白质和小分子的电化学DNA生物传感器的制备

As medicine is currently practiced, doctors send specimens to a central laboratory for testing and thus must wait hours or days to 
receive the results. ...

科研

生物工程

Directed Cellular Self-Assembly to Fabricate Cell-Derived Tissue Rings for Biomechanical Analysis and Tissue Engineering

定向细胞自我组装制造，为生物力学分析与组织工程细胞衍生的组织环

Each year, hundreds of thousands of patients undergo coronary artery bypass surgery in the United States.1 Approximately one third of these patients do ...

Solid-phase Submonomer Synthesis of Peptoid Polymers and their Self-Assembly into Highly-Ordered Nanosheets

固相Submonomer Peptoid聚合物及其自组装成高度有序的纳米片的合成

Peptoids are a novel class of biomimetic, non-natural, sequence-specific heteropolymers that resist proteolysis, exhibit potent biological activity, and ...

On-chip Isotachophoresis for Separation of Ions and Purification of Nucleic Acids

对芯片等速电泳离子的分离和纯化核酸

Electrokinetic techniques are a staple of microscale applications because of their unique ability to perform a variety of fluidic and electrophoretic ...

Continuously-stirred Anaerobic Digester to Convert Organic Wastes into Biogas: System Setup and Basic Operation

连续搅拌厌氧消化有机废物转化为沼气系统的安装和基本操作

Anaerobic digestion (AD) is a bioprocess that is commonly used to convert complex organic wastes into a useful biogas with methane as the energy carrier ...

An Injectable and Drug-loaded Supramolecular Hydrogel for Local Catheter Injection into the Pig Heart

可注射药物加载超分子凝胶局部导管注入猪心

Regeneration of lost myocardium is an important goal for future therapies because of the increasing occurrence of chronic ischemic heart failure and the ...

Biomembrane Fabrication by the Solvent-assisted Lipid Bilayer (SALB) Method

Biomembrane Fabrication by the Solvent-assisted Lipid Bilayer SALB Method

生物膜制作的溶剂辅助脂质双分子层（SALB）方法

In order to mimic cell membranes, the supported lipid bilayer (SLB) is an attractive platform which enables in vitro investigation of membrane-related ...

Polyelectrolyte Complex for Heparin Binding Domain Osteogenic Growth Factor Delivery

聚电解质复合物肝素结合域成骨生长因子交货

During reconstructive bone surgeries, supraphysiological amounts of growth factors are empirically loaded onto scaffolds to promote successful bone ...

Wet Chemistry and Peptide Immobilization on Polytetrafluoroethylene for Improved Cell-adhesion

湿化学和肽固定在聚四氟乙烯为改善细胞粘附

Endowing materials surface with cell-adhesive properties is a common strategy in biomaterial research and tissue engineering. This is particularly ...

Fabricating Degradable Thermoresponsive Hydrogels on Multiple Length Scales via Reactive Extrusion, Microfluidics, Self-assembly, and Electrospinning

通过反应挤出、微流体、自组装和电纺丝制备多长尺度可降解 Thermoresponsive 水凝胶

While various smart materials have been explored for a variety of biomedical applications (e.g., drug delivery, tissue engineering, bioimaging, etc.), ...