Name: dna nanotubes as a versatile tool to study semiflexible polymers
Uploaded: 2017-10-25T14:00:00
Description: Watch this Scientific Journal Video about DNA Nanotubes as a Versatile Tool to Study Semiflexible Polymers at JoVE.com

我们承认 DFG (1116/17-1) 和莱比锡自然科学学校 "BuildMoNa" (GSC 185) 的资助。这项工作得到了通过弗劳恩霍夫吸引项目 601 683。骅承认欧洲社会基金 (ESF-100077106) 提供的资金。

1. 准备 n 高温超导 注: 在这里, n 表示在某一大小的螺旋管的形成中所涉及的不同单 DNA 链的数量。对于 n = 8, 八不同的单个 DNA 链组成一个单位环. 
<ol> <li> 从合适的 dna 合成服务中购买 dna 序列 (HPLC 纯度级或更高), 或执行高质量的合成和纯化 (在 表 1 中给出的模范序列). </li>
	<li> 在纯化水中重冻干寡核苷酸, 并按照该公司相应手册中的...

要获得适当形成的网络, 组装 DNA 纳米管是一个关键的步骤。合成过程中的误差对管质量产生负面影响;因此, 建议采用 HPLC 法或更严格的工艺来纯化寡核苷酸。由于离散而非聚合的 DNA 纳米管的形成, 以及它们的长度分布, 取决于集合中的n组成的寡核苷酸的摩尔化学计量, 因此必须测量浓度被购买的子线, 因为被给的价值可能从实际集中极大地变化。例如, 可以通过紫外可见光谱在 260 nm 的吸收...

由于其独特的力学性能使复杂的行为, 半聚合物是基本的生活物质的基石。与柔性聚合物相比, 半聚合物采用了一种伸展的结构, 由于其非的骨架刚度, 同时仍受强热波动的影响1。因此, 纯粹的随机模型不能应用于他们的行为, 就像极端的完全灵活或刚性的聚合物。so-called 蠕虫样链模型2,3,4被开发为通过lp来量化此刚度, 它是沿灯丝4的切线相关的衰减常数。如果lp与灯丝的轮廓长度 (lc) 相媲美, 则该聚合物被视为半1。类似于帐篷的两极, 他们在网络或捆绑的安排稳定整个集体系统在低体积分数, 导致异常粘弹性属性5,6,7, 8,9。这些结构在大型网格大小10中提供了高弹性, 保持了机械完整性, 同时还促进了扩散和活动的传输过程。该特性特别适用于生物系统, 如骨架或胞外基质, 但它也广泛应用于食品工程1,11,12。
为了能有发展仿生材料或新型水凝胶的工具, 对这些结构的物理性质进行全面的检验, 对其意义深远。在半聚合物方面, 这意味着系统地确定由 single-filament 属性 (如lp和描述性理论框架的开发) 产生的网络的集体属性。在开创性研究中, 细胞生物肌动蛋白被建立为半聚合物的模型系统, 并仍被广泛认为是金标准5,13,14,15,16,17. 然而, 这一系统的详尽研究是有限的, 因为它们与这种蛋白质的固有特性息息相关。各种理论方法的目的是建立一个描述的非平凡的机械行为在 single-filament 水平, 并导致明显不同的比例预测的依赖线性弹性高原剪切模量, G 0 (即网络的 "弹性"), 就集中度 (c) 和lp6,7,13,14, 15,18,19,20,21,22,23。虽然在 actin-based 或其他模型系统的实验中可以方便地进行浓度缩放, 而理论预测已得到严格验证13,16,24,25, 相对于lp的缩放在实验上仍然不可访问。但是, 这是一个主要的限制, 因为lp也是一个独立变量, 它是半聚合物的定义数量。
这种中央, 自然的限制强加由固定的lp的肌动蛋白或其他生物衍生聚合物, 如胶原蛋白, 最近已经解决了使用型 DNA 管, 这是可调谐的机械性能9,26,27,28. 管结构的细微变化 (例如,在单位环内的不同数量的组成 DNA 链) 产生独特的值为lp, 可以通过荧光显微镜进行评估,通过分析一个波动管或评估几个粘附管的弯曲配置, 如前面所描述的9,28。这些分析显示, 不同管种群的lp值在多个数量级上变化, 并且不同的评估技术产生一致的结果9,28。
令人惊讶的是, 线性弹性高原切变模量的整体比例G0关于浓度和lp已报告与所有以前的理论方法不一致9, 特别是在lp上演示了比预测依赖强得多的依赖性。这些发现强调了一个新的模型系统的价值, 研究半聚合物的中心性质。使用n-螺旋 DNA 管极大地拓宽了这些调查的范围。lp不仅可以在不改变基本材料的情况下自由变化, 而且 DNA 的固有可编程性可以使系统地检查其他元素, 如通道或动态切换过程。此外, 这些管子可溶于水, 与大多数蛋白质相比, 在适当的 pH 值和离子条件下稳定数周, 没有可检测的降解9。
为了组装这些管子, 我们使用了一组离散的 DNA 寡核苷酸, 每个都包含两个域, 共享互补的碱基序列到两个相邻的链 (由于特定的序列, 单链不能形成结构, 如发夹)。互补序列杂交的循环方式, 形成封闭的, half-overlapping 环的n互联的双段 (图 1A和 B)。这些环形成在一个离散的直径 (图 1C), 其 half-overlapping 配置暴露了轴向粘性两端互补的另一环的粘性两端。这种选择性添加匹配的寡核苷酸触发了环的堆叠, 导致有效聚合的丝状 DNA 螺旋管的大小n (nHT)。他们的外形长度通常测量几微米长度, 并且他们的长度分布是可比较的肌动蛋白花丝9,26,27,28。它已经证明了类似的 DNA 纳米管, 他们确实展示聚合动力学类似的肌动蛋白丝和微管内<su></su>p 类 = "xref" > 29。根据组成基本环结构的单个 DNA 链的数字n , nHT 体系结构以及其周长和直径可控变化。使用更多的 DNA 链增加环/管的周长, 相应的结构变化将机械性能转移到更高的lp值 (图 1C), 对应于更高的刚性。在介观尺度上, 这些较大的lp值转换为更高的刚度 (图 1D和E) 的弯曲构象。

<table border="0" cellpadding="0" cellspacing="0" width="667">
	<tbody>
		<tr height="22">
			<td height="22" style="height:22px;width:169px;">AFM cantilever ACTA</td>
			<td style="width:196px;">AppNano</td>
			<td style="width:123px;"></td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:169px;">AFM - NanoWizard 3</td>
			<td style="width:196px;">JPK Instruments</td>
			<td style="width:123px;"></td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:169px;">CCD camera</td>
			<td style="width:196px;">Andor</td>
			<td style="width:123px;">iXon DV887</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:169px;">DMSO</td>
			<td style="width:196px;">Sigma-Aldrich</td>
			<td style="width:123px;">D2650</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:169px;">DNA oligonucleotides</td>
			<td style="width:196px;">Biomers.net</td>
			<td style="width:123px;"></td>
			<td style="width:180px;">For sequences see Table 1</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:169px;">DNA Cy3-labeled oligonucleotides</td>
			<td style="width:196px;">Biomers.net</td>
			<td style="width:123px;"></td>
			<td style="width:180px;">For sequence see Table 1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:169px;">EDTA</td>
			<td style="width:196px;">Sigma-Aldrich</td>
			<td style="width:123px;">E-9884</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:169px;">Epi-fluorescence micro-scope</td>
			<td style="width:196px;">Leica</td>
			<td style="width:123px;">DM-IRB</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:169px;">MgCl2</td>
			<td style="width:196px;">Sigma-Aldrich</td>
			<td style="width:123px;">M-8266</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:169px;">Mica &#34;V1&#34;, 12 mm round</td>
			<td style="width:196px;">Plano GmbH</td>
			<td style="width:123px;">50-12</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:169px;">MicroAmp&#174; Fast Optical 96-Well Reaction Plate</td>
			<td style="width:196px;">Thermo Fisher Scientific Inc.</td>
			<td style="width:123px;">4346907</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:169px;">MicroAmp&#174; Optical Adhesive Film</td>
			<td style="width:196px;">Thermo Fisher Scientific Inc.</td>
			<td style="width:123px;">4306311</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:169px;">NanoDrop 1000 Spectrophotometer</td>
			<td style="width:196px;">Thermo Fisher Scientific Inc.</td>
			<td style="width:123px;"></td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:169px;">100x objective</td>
			<td style="width:196px;">Leica5</td>
			<td style="width:123px;">506168</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:169px;">Purified water</td>
			<td style="width:196px;">Merk Millipore - Milli-Q &#38; Elix</td>
			<td style="width:123px;"></td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:169px;">Sapphire PCR tubes</td>
			<td style="width:196px;">Greiner Bio-One</td>
			<td style="width:123px;">683271</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:169px;">TProfessional Standard PCR Thermocycler</td>
			<td style="width:196px;">Core Life Sciences Inc.</td>
			<td style="width:123px;">070- Standard</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="21">
			<td height="42" style="height:42px;width:169px;">7900HT Fast Real-Time PCR System</td>
			<td style="width:196px;">Applied Biosystems</td>
			<td style="width:123px;">4351405</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:169px;">Rheometer</td>
			<td style="width:196px;">TA Instruments</td>
			<td style="width:123px;">ARES</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:169px;">SYBR&#174; Green I nucleic acid gel stain</td>
			<td style="width:196px;">Thermo Fisher Scientific Inc.</td>
			<td style="width:123px;">S7567</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:169px;">Tris</td>
			<td style="width:196px;">Sigma-Aldrich</td>
			<td style="width:123px;">T4661</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:169px;">Triton X-100</td>
			<td style="width:196px;">Sigma-Aldrich Co.</td>
			<td style="width:123px;">X-100</td>
			<td style="width:180px;">Suppresses evaporation of sample at air-water interface</td>
		</tr>
	</tbody>
</table>

dna nanotubes as a versatile tool to study semiflexible polymers

复杂的, 聚合物的软物质的机械性能, 如细胞或生物网络, 可以理解在既不经典框架的柔性聚合物也不是刚性棒。由于其非的主干刚度 (通过持久长度 (lp) 进行量化), 底层细丝仍会伸展, 但它们也会受到强烈的热波动的影响。它们的有限弯曲刚度导致了独特的、非平凡的体积网络集体力学, 使得在低体积分数下形成稳定的支架, 同时提供大的网格尺寸。这一基本原理在自然界中很普遍 (例如,在细胞或组织中的), 使高分子含量最小化, 从而促进扩散或主动运输。由于其生物学意义和潜在的技术应用在生物相容的水凝胶, 半聚合物已受到相当多的研究。然而, 可理解的调查仍然具有挑战性, 因为他们依赖天然的聚合物, 如肌动蛋白丝, 这是不能自由调谐。尽管有这些限制, 由于缺乏合成, 机械可调谐, 和半聚合物, 肌动蛋白花丝被建立作为共同的模型系统。一个主要的限制是, 不能自由调整中心数量lp来研究其对宏观体积结构的影响。通过采用结构化可编程的 DNA 纳米管, 实现了对灯丝刚度的控制改变, 从而解决了这一缺陷。它们是通过型的设计形成的, 在这里, 一组离散的部分互补的链杂交在一个具有离散圆周的环形结构中。这些环的特点是粘性的两端, 使有效的聚合成丝数微米的长度, 并显示类似的聚合动力学作为天然的生物聚合物。由于他们的可编程的力学, 这些管是多才多艺的, 新颖的工具, 研究的影响, lp对分子和散装规模。与肌动蛋白丝相比, 它们在几周内保持稳定, 没有明显的变性, 它们的处理也比较简单。

通过温度斜坡 (图 2) 进行 DNA 纳米管的组装是形成这些人造半聚合物的一种非常可靠的方法。这些聚合物与自然发生的对应物有相似的特征, 例如肌动蛋白丝, 但提供一个更加宽广的实验框架, 因为他们的机械物产可以被控改变9,27.与生物聚合物一样, 它们可以在网络中进行排列 (图 3), ?...

Watch this Scientific Journal Video about DNA Nanotubes as a Versatile Tool to Study Semiflexible Polymers at JoVE.com

DNA Nanotubes as a Versatile Tool to Study Semiflexible Polymers

半聚合物显示独特的机械性能, 广泛应用于生活系统。然而, 由于聚合物刚性等性质无法进入, 对生物聚合物的系统研究是有限的。这篇手稿描述了如何利用可编程的 DNA 纳米管规避这一限制, 从而对纤维刚性的影响进行实验研究。

DNA 纳米管作为研究半聚合物的万能工具

Mechanical properties of complex, polymer-based soft matter, such as cells or biopolymer networks, can be understood in neither the classical frame of ...

The overall goal of this new DNA-based model system is to understand the fundamental properties of a class of semiflexible polymers, which also describes the behavior of a vital class of biopolymers. This new method allows us to study key questions in the field of soft metaphysics, specifically, it allows to explore the behavior of semiflexible filaments and their arrangements into networks. The main advantage of this technique is that the mechanical properties of intravisive filaments are experimentally accessible and can be precisely tuned.Semiflexible polymers are defined by the special properties of the filaments which were previously only treated in the theoretical framework due to the lack of a tunable experimental systems. Though this method provides insight into the defining quantity of semiflexible polymers, it can also be applied as a new hydrogel to study cell migration in matrices of different stiffness's without changing the under laying mesh size. The smallest repetitive unit in the nanotube can be thought of as a unit ring.Several of these unit rings stack during hybridization. This leads to the formation of elongated nanotube's. To begin, re-suspend lyophilized oligonucleotides in purified water and follow the further re-suspension steps given in the corresponding manual from the company.Adjust the amount of water to obtain a final concentration of 200 micromolar. Determine the concentration's of the single DNA strands to verify the values given by the distributor since the exact stoichiometry is crucial for the assembly process. Using a micropipette, mix the DNA strands each of the same concentration in a container suitable for the thermocycler to form the desired DNA and helix tubes.Hybridize the DNA and helix tubes in a thermocycler using the parameters found in the text protocol. Store the hybridized DNA and helix tubes for up to 3 weeks at 4 degrees celsius without detectable degradation. Before performing rheology, carefully dilute the sample to the desired final concentration using the sample buffer if necessary.Choose an appropriate geometry for the dynamic shear rheometer. Then, load the sample on the rheometer. To prevent potential evaporation effects, passivate the air water interface of the sample and environment through 1 of 3 techniques.Either surround the sample with 2.5 milliliters of the sample buffer bath. Or, add a surfactant just below the micelle concentration to the sample. Alternatively, use the hamilton syringe to surround the sample with lipid to avoid direct contact of the sample with air.Seal the sample chamber using a cap equipped with wet sponges. And, if possible use an additional water bath to further suppress evaporation. Start the measurement using the rheometer specific software at room temperature allowing the sample to equilibrate for 2 hours at room temperature.The equilibration time is crucial to allow the thermoflect system to a complete isotropic randomized state since this is the only confirmation allowing comparison between the different measurements. Following measurement, process the data as described in the text protocol. Prepare 8 aliquots of 12 microliters for each sample using 1 to 4 micromolar DNA per strand, 12.5 millimolar magnesium chloride, and 1x nucleic acid gel stain.Also, make a negative control with no DNA, but include the nucleic acid gel stain. Transfer the aliquots to a real time PCR plate and seal with adhesive film. Centrifuge the plate at 100 times G for 1 minute at room temperature to remove the gas bubbles.It's critical to remove the gas bubbles because it can expand during measurements and the samples cannot be analyzed. Following centrifugation, load the sealed plate into a real time quantitative PCR system. Program an annealing program to denature the DNA at ninety degree celsius for 10 minutes and drop the temperature quickly to seventy two degrees celsius.Then the temperature should decrease slowly by 0.5 degrees celsius every thirty minutes. After the signal has decayed, accelerate the temperature ramp. Make sure that the lid of the cycler heats to at least one hundred degrees celsius to prevent the condensation of evaporated sample on the adhesive film.During the assembly, excite the samples with an argon ion laser at four hundred eighty eight nanometers and detect the fluorescence at five hundred fifty nanometers when using a CY3 dye. For other dyes, choose an appropriate excitation emission combination. Measure the fluorescence signal as often as possible to increase the overall signal quality using the built in camera.Representative raw data of a rheological measurement are shown revealing the elasticity of the network. Shown here is the time sweep during equilibration time. This plot reveals the strain dependent behavior of the network.In this plot, the frequency dependent behavior of the network is evident. At this point the values at a specific frequency are chosen. The values for the specific DNA helix tube population were plotted against the concentration.This process was repeated for all the DNA and helix tubes. The slope was then extracted in a log logged plot yielding the power law exponent. Finally, the values were replotted against the persistent length for each measured concentration.These graphs allow extraction of the scaling of the elastic plateau modulus with respect to the concentration and persistence length. After development, this technique paved the way for researchers in the field of soft matter and polymer physics to explore the effect of the filament stiffness for the class semiflexible polymers. Following this procedure, additional effects such as the recitation of the entangled networks, crossing networks in general, and structure formation process can be addressed.After watching this video you should have a good understanding on how to hybridize DNA nanotubes and how to measure the rheological properties of their networks.

Research

JoVE Journal

Bioengineering

Shape Memory Polymers for Active Cell Culture

形状记忆聚合物主动细胞培养

Shape memory polymers (SMPs) are a class of "smart" materials that have the ability to change from a fixed, temporary shape to a pre-determined permanent ...

科研

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Optical imaging offers a wide range of diagnostic modalities and has attracted a lot of interest as a tool for biomedical imaging. Despite the enormous ...

The Synthesis of RGD-functionalized Hydrogels as a Tool for Therapeutic Applications

RGD功能化水凝胶作为治疗应用的工具合成

The use of polymers as biomaterials has provided significant advantages in therapeutic applications. In particular, the possibility to modify and ...

Engineering Molecular Recognition with Bio-mimetic Polymers on Single Walled Carbon Nanotubes

工程分子识别与单壁碳纳米管仿生聚合物

Semiconducting single-wall carbon nanotubes (SWNTs) are a class of optically active nanomaterial that fluoresce in the near infrared, coinciding with the ...

A Versatile Method of Patterning Proteins and Cells

图案蛋白质和细胞的通用的方法

Substrate and cell patterning techniques are widely used in cell biology to study cell-to-cell and cell-to-substrate interactions. Conventional patterning ...