作者要感谢S.道格拉斯极有价值的讨论和建议，以及巴切莱特实验室的所有成员有益的讨论和工作。这项工作是由生命科学学院和纳米技术及先进材料研究所在巴伊兰大学的资助。

1.准备的斯台普斯池混合<ol><li>如表 1中所列的96孔板顺序冻干的DNA纳米机器人订书钉（ 见材料），并归一至10纳摩尔。将DNA纳米机器人的设计和结构的详细描述，请参见本- Ishay 等人 14和Douglas 等人15）。 </li><li>重构各订书钉以及用DNA酶/无RNase超纯至100μM的浓度。主食归到10纳摩尔，重组与100微升超纯水。 </li><li>池一起20微升各订书钉的使用多通道移液器和无菌55毫升解决方案盆地。 注:由于纳米机器人形状是由254短 ​​链（包括核心，边缘，手柄和指南序列， 表1），每个U形钉池中的订书钉的浓度为394纳米。从订书钉池中取出钉，或添加一些不同的卷超微电极，可降低产率相当，或者以其他方式导致展开聚集体。 </li></ol> 2.制备制作反应液<ol><li>对于一个标准的反应混合物用40微升的M13mp18病毒基因组的环状单链DNA中，对应于4皮摩尔脚手架的DNA（100nM的原液， 见材料）。支架在制造混合物的最终浓度为20nM的，最终体积200微升。 <ol><li>调整根据具体需要脚手架DNA的量;然而，保持在反应混合物中的DNA支架的最终浓度在恒定的20纳米。 </li></ol></li><li>添加订书钉达到支架为1订书钉比率至10。对于20纳米支架DNA浓度，每254订书钉&#39;最终浓度是200nM的。对于200微升最终反应体积加上102微升斯台普斯游泳池混合物（第1.2节）的。 <ol><li>添加特定的门序列，分别为thË折叠反应混合物在这个时候。这些寡单独订购，并要求HPLC纯化。确保门寡核苷酸存在于1:10支架到门序列比， 即 200nM的每种寡核苷酸的20nM的脚手架浓度。对于200微升折叠反应体积，在100微米的股票浓度增加0.4微升每个四门寡。 </li></ol></li><li>添加10倍TAE储备缓冲到达1×TAE（40mM的Tris-醋酸，1mM EDTA）中的最终浓度。对于200微升折叠反应体积，加入20微升的10X TAE。 </li><li>添加1M的MgCl 2的，以10mM的终浓度。对于200微升折叠反应体积，加入2微升的1M 氯化镁 。 </li><li>添加36微升DNA酶/ RNA酶游离超纯水，使达到200微升的最终体积。 </li><li>涡和分装100微升样品到PCR小瓶。 注:请有关最大反应体积中使用热循环仪规范。减少体积，以满足这些限制将不降低获得的收益率。反应体积上述规定将危及收益率的最大值。 </li></ol>制作反应3.温度退火斜坡<ol><li>程序热循环仪如下: <ol><li>坡道85℃至60℃，在5分钟/℃的速率。 </li><li>斜坡60℃至4℃下以75分钟/℃的速率。 </li><li>保持在4℃下去。 </li></ol></li><li>经过加工已经结束商店样品在-20℃。 </li></ol> 4.去除多余史泰博<ol><li>加入100微升的折叠反应混合物与0.5 ml的离心过滤器100 kDa的的MWCO。保存纳米机器人预纯化供以后分析的10微升样品。 </li><li>离心机在9,600×g的10分钟。 </li><li>加入400微升的折叠缓冲液（1×TAE，10毫米氯化镁2）。 </li><li>重复步骤4.2和4.3的两倍多。 </li><li>离心5分钟，在9,600×g的。 </li><li>通过将过滤器倒置在一个干净的离心管中，自旋为1分钟，在9600×g下恢复该浓缩物。最终体积可以根据制造反应混合物的初始体积被放入过滤器的不同而不同。通常浓缩纳米机器人样品的25-60微升最终体积被获得。 </li><li>测量通过分光光度计在样品中的DNA的浓度在260纳米。计算摩尔浓度的纳米机器人的样品时，应采用5.3微克/皮摩尔分子量。 注:摩尔消光系数为双链DNA，50微克/ OD 260，足以满足大多数应用。 48微克/ OD 260考虑到聚胸腺嘧啶的ssDNA延伸在边缘处缝合钉。 </li></ol>折叠纳米机器人的5琼脂糖凝胶电泳分析<ol><li>补充了10mM的MgCl 2 0.5×TBE（45毫摩尔Tris-硼酸盐，1mM EDTA中），2％琼脂糖凝胶的制备</suB&gt;（改编自埃内斯托·卡斯特罗等 [21]。）: <ol><li>在118.75双蒸2 O.毫升准备0.5X TBE缓冲液稀释6.25毫升10倍TBE储备缓冲</li><li>溶解2.5克琼脂糖在125毫升的0.5×TBE缓冲液。 </li><li>沸腾在微波直到琼脂糖完全溶解。 </li><li>加入1.25毫升的1M的MgCl 2的 10mM的最终浓度。 </li><li>加7微升10毫克/毫升的溴化乙锭。 </li><li>等待解决方案，以偏凉和琼脂糖凝胶固化前，填补了凝胶托盘。立即安装所需的梳子。 </li><li>制备1升0.5×TBE缓冲液加入50毫升10×TBE储备缓冲和10ml的1M MgCl 2的940至双蒸2 O中的溶液</li><li>一旦凝胶是固体加载运行缓冲液的电泳设备，并把设备在冰浴中。 </li></ol></li><li>在纳米机器人预净化负载1微克总DNA（步骤3.2），和纯化后（步骤4.7），旁边一个支架DNA样品和1 kb的DNA标记，凝胶上。给出一个例子在图2中。样品的实际量取决于纯化后得到的浓度。每个样品装入在1装载缓冲液:6终体积比。 </li><li>设置电源到80伏，并运行凝胶3小时。运行在装满冰和水的浴中的凝胶。纳米机器人将展开，如果琼脂糖凝胶电泳过程中加热出现一抹黑。电泳过程中添加额外的冰块，以保持设备的加热。 </li><li> （ 图2）紫外线表视图凝胶。 </li></ol>纳米机器人6.用铀甲酸盐负染色<ol><li> 2％的铀甲酸盐原液制备（改编自卡斯特罗等23）: <ol><li>权衡的双氧铀酸盐粉末100毫克到15毫升管。 </li><li>煮沸DNA酶/ RNA酶的超纯水进行3分钟脱含氧化合物。 </li><li>添加5毫升脱氧热水（〜60℃）入15ml试管中含有双氧铀酸盐粉末（来自之前的步骤）。紧紧合上机盖，包装在铝箔和涡严格的10分钟（系管涡流）。溶液应出现浑浊带有黄色。 </li><li>通过0.2微米注射器滤波器过滤解决方案。解决方法应该很清楚。 </li><li>分装200微升的溶液倒入离心管中。 </li><li>离心机中以最大速度为5分钟在台式离心机以池的样品在管的底部。 </li></ol></li><li>样品电网和消极染色加载: <ol><li>除霜2％铀甲酸盐溶液（6.1节准备）200微升等分。加入10微升0.5M的NaOH溶液和涡旋严格3分钟。离心机中以最大速度用台式离心机5分钟。 </li><li>同时，辉光放电网格使用室内空气在0.2毫巴和25 mA的30秒。 </li><li>添加15微升为2nM纳米机器人样品的纯化后（secti上4.7）到电网的顶侧（用镊子保持），并让吸收1分钟。使用滤波器纸张放置在网格上的滤纸的顶部以排出多余的液体。请勿触摸网格表面。 </li><li>加入10微升2％的铀甲酸盐（从步骤6.2.1）的网格上的顶面（用钳子举行）。 </li><li>快速使用滤波器纸张放置在网格上的滤纸的顶部以排出多余的液体。请勿触摸网格表面。 </li><li>重复步骤6.2.4和6.2.5。 </li><li>加入10微升2％的铀甲酸盐（从步骤6.2.1）的网格上的顶面（用钳子举行）。让染色溶液吸收30秒。 </li><li>使用滤波器纸张放置在网格上的滤纸的顶部以排出多余的液体。请勿触摸网格表面。 </li><li>让格完全干燥至少30分钟，面朝上的滤纸，注射入的TEM之前。 </li></ol></li></ol>

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作者什么都没有透露。

我们介绍的加工，净化，和可视化的DNA纳米机器人的。继装置的六边形底盘的制造中，所述纳米机器人的功能进行编程时将简单介绍的特定货物和传感链到机器人容易地找到它们的指定的位置，由于氢键互补可用单链对接位点14 ，15,22。 描述的制造协议使用缓慢退火坡道，其通常使用在我们的实验室折叠广泛折纸形状。如果生产时间成为关键因素的其它协议，如由Sobczak 等描述的快速折叠协议24可以使用。该协议被报告以实现高产率折纸折叠，然而它需要校准每个折纸形状。 自旋过滤被用来净化从过量订书钉的机器人。当装载在S与样品或缓冲销柱，应注意不要损伤膜枪头。该膜可以潜在破裂从而显着降低了产量。明智的做法是不丢弃流过，直到纳米机器人是由AGE可视化。 对于某些应用程序更高的净化率需要;这可以通过使用原始旋转柱重复过滤来实现。对于甚至更高的纯度，一个新的旋转柱可以使用，然而，这将对收率率产生重大的负面影响。其它方法用于纯化的DNA折纸结构进行了测试，如从电泳和过量订书钉透析随后琼脂糖凝胶切除。这些方法导致了无论是产量很低或与描述的协议相比，差的净化率。其他方法，如基于PEG的纯化25和速率纬向超离心26未测试。这些方法被报告给ACHI前夜高净化率，但他们要么导致不良的收益率（利率纬向超速离心），或需要沉淀（PEG为基础的），这可能会损害中空的纳米机器人的形状。 纳米机器人被制造与指南订书钉其锁定在关闭位置的形状，以增加制造yeilds 15。它通过添加指南去除订书钉为纳米机器人响应于所设计的刺激有效开放（PDGF在所描述的协议），以除去这些订书钉是重要的。导向钉都设计有一个立足点单链区域的其中通过链置换的18。这些订书钉应在10被加入一个过程释放出引导订书钉导向去除订书钉对接:1的摩尔比在过量的纯化的端订书钉"阶段（步骤4.7），并温育2小时，在室温下在端超过端振荡器。 可视化用TEM进行了说明，其中包吴铀甲酸盐2％的负染色。用醋酸双氧铀相比，铀，甲酸产生更细的颗粒结构，该结构能更好地解决了DNA折纸设计。应注意作为双氧铀酸盐将凝固如果等分试样冷冻长时间。最好是使用染色新鲜制备的2％的双氧铀酸盐溶液。更好的分辨率是可以实现与使用低温TEM 27。 在DNA纳米机器人的基本体系结构设计和制造协议的最终统一和简单。然而，大范围的灵活性的特定货物的混合物和感测链的形式提供。此外，在一个单一的人口许多亚型可以引入和它们的相对化学计量编程和优化的，以适应特定的需要。在每个限速步骤的具体动力学的更大的微调是可以实现的增加/减少的双链杂交或长度引进的不匹配，在具体的关键岗位。这种高度的轻松构建灵活度使得能够在一个生物相关环境进行多种多样复杂的任务的纳米器件的工程。

的用途核酸纳米技术是惊人。在Watson-Crick碱基配对的易处理性以及大规模合成的定制寡核苷酸2的易用性和相对低的成本已生成的应用3中的DNA纳米技术领域的爆炸和研究。结构DNA纳米，基于所述不动的塞曼结4,5-作为基本构建块利用的DNA，作为任意形状6-8的结构的自组装基本单位。 脚手架DNA折纸9技术的最新发展使得复杂的2D / 3D纳米结构10-12亚纳米级精度的建设，是建设新的功能对象日益增长的复杂性和惊人的多样性的有效途径。施工过程是基于一个长脚手架单链DNA，通常是从病毒染色体组衍生即，它可以通过几百短单链DNA寡核苷酸的杂交被折叠称为订书钉。通过这种技术获得的高结构分辨率是DNA双螺旋的自然尺寸的直接结果，而制造的再现性剪裁短单链订书钉序列以促进最大的氢键互补达到的结果。与使用慢温度退火的斜坡设计最低能量，热力学优选的纳米结构中达到高的产率和保真度。在计算机代码轻松实现连接处的设计规则允许的CAD工具，如caDNAno 13的发展，也极其简化设计包含数百个路口相连的大，结构复杂的任务。 先前我们描述的DNA纳米机器人的设计与caDNAno工具14,15的助剂。在这里，我们描述的制造和可视化，通过透射电子显微镜（TEM），所述纳米机器人，一个三维空心六角纳米器件，具有尺寸为35×35×50纳米的三件设计成经受到预定刺激和本具体货物中，响应一个主要的构象变化，例如蛋白质或核酸的寡核苷酸，隔离内部。虽然12装载站可在空心机箱内部，束缚货物的实际数量与不同尺寸的货物。货物分子范围从小型DNA分子的酶，抗体和5-10纳米金纳米颗粒。 Cargocan要么是均匀的或异质的，使得每个纳米机器人包含不同的分子的混合物。感测通过两个双螺旋锁定门设计实现成感测蛋白质，核酸或其它化学品，或者基于适体传感器16,17或DNA链置换18的技术。在适体选择的协议19-21最近的事态发展使纳米机器人的设计响应到分子和细胞类型的不断增加的范围。 早期的工作表明一个纳米机器人携带的特异性抗体，其在结合其抗原可以中继既抑制性或多产的信号，以特定的细胞类型的内部以混合细胞群15。这些纳米器件的一个令人兴奋的特性是他们与一个单一的群体推出不同的纳米机器人亚型的执行更为复杂的任务和逻辑控制能力。最近，我们证明了在执行作为正或负调节纳米机器人的特定亚型，控制含活性货物分子22的效应器群体。 这里介绍的协议描述门控与选择性结合的PDGF以促进纳米机器人15,22的开口适体传感器序列的纳米机器人的制造中，纯化和成像。中描述的制造过程类似于n个anorobot制造工艺最初由Douglas 等人 15的变化旨在减少整个过程持续时间所描绘的，同时增加了产率和纯化率。

<table>
	<tbody>
		<tr>
			<td>DNase/RNase free distilled water</td>
			<td>Gibco</td>
			<td>10977</td>
		</tr>
		<tr>
			<td>M13mp18 ssDNA scaffold</td>
			<td>NEB</td>
			<td>N4040S</td>
		</tr>
		<tr>
			<td>10x TAE</td>
			<td>Gibco</td>
			<td>15558-042</td>
		</tr>
		<tr>
			<td>1 M MgCl2</td>
			<td>Ambion</td>
			<td>AM9530G</td>
		</tr>
		<tr>
			<td>Amicon Ultra 0.5 mL centrifugal filter 100K MWCO</td>
			<td>Amicon</td>
			<td>UFC510024</td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>Promega</td>
			<td>V3125</td>
		</tr>
		<tr>
			<td>TBE buffer</td>
			<td>Promega</td>
			<td>V4251</td>
		</tr>
		<tr>
			<td>Ethidium bromide 10mg/ml solution&#160;</td>
			<td>Sigma Aldrich</td>
			<td>E1510</td>
		</tr>
		<tr>
			<td>1 kb DNA marker</td>
			<td>NEB</td>
			<td>N3232S</td>
		</tr>
		<tr>
			<td>Loading Dye</td>
			<td>NEB</td>
			<td>B7021S</td>
		</tr>
		<tr>
			<td>uranyl formate</td>
			<td>polysciences</td>
			<td>24762</td>
		</tr>
		<tr>
			<td>carbon-coated TEM grids&#160;</td>
			<td>Science services</td>
			<td>EFCF400-Cu-50</td>
		</tr>
		<tr>
			<td>Thermal Cycler c1000 Touch</td>
			<td>Bio-Rad</td>
			<td></td>
		</tr>
		<tr>
			<td>Glow Discharge K100X</td>
			<td>Emitech</td>
			<td></td>
		</tr>
		<tr>
			<td>UV table Gel Doc EZ Imager</td>
			<td>Bio-Rad</td>
			<td></td>
		</tr>
		<tr>
			<td>NanoDrop 2000c</td>
			<td>Thermo Scientific</td>
			<td></td>
		</tr>
		<tr>
			<td>TEM FEI-G12</td>
			<td>Tecnai</td>
			<td></td>
		</tr>
	</tbody>
</table>

folding and characterization of a bio-responsive robot from dna origami

该DNA纳米机器人是一个中空的六角纳米器件，设计成响应于特定的刺激和本货物内部隔离开。既刺激和货物可以根据具体需要量身定做。在这里，我们描述了DNA纳米机器人制造协议，与使用DNA折纸技术。该步骤首先短单链DNA订书钉混合到原料混合物中，然后加入到长圆形，单链DNA的支架中的折叠缓冲液中的存在。一个标准的热循环仪被编程，以逐渐降低所述混合反应温度，方便钉到脚手架退火，这是纳米机器人的折叠背后的导向力。一旦60小时折叠反应完成后，过剩的钉被使用离心过滤器，接着经由可视琼脂糖凝胶电泳（AGE）丢弃。最后，将纳米机器人的制造成功是由透射电子显微镜检验（TEM）与使用双氧铀酸盐作为负染色。

Watch this Scientific Journal Video about Folding and Characterization of a Bio-responsive Robot from DNA Origami at JoVE.com

DNA 折纸生物响应机器人的折叠和表征 |化学|视频

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

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 ...

folding-characterization-bio-responsive-robot-from-dna

Nanyang Technological University

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DNA Structure and Function

SCIENTISTS IN ACTION

14.4K 次观看.  Bar-Ilan University. 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). 

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

Origami Inspired Self-assembly of Patterned and Reconfigurable Particles

There are numerous techniques such as photolithography, electron-beam lithography and soft-lithography that can be used to precisely pattern two dimensional (2D) structures. These technologies are mature, offer high precision and many of them can be implemented in a high-throughput manner. We leverage the advantages of planar lithography and combine them with self-folding methods1-20 wherein physical forces derived from surface tension or residual stress, are used to curve or fold planar structures into three dimensional (3D) structures. In doing so, we make it possible to mass produce precisely patterned static and reconfigurable particles that are challenging to synthesize.
In this paper, we detail visualized experimental protocols to create patterned particles, notably, (a) permanently bonded, hollow, polyhedra that self-assemble and self-seal due to the minimization of surface energy of liquefied hinges21-23 and (b) grippers that self-fold due to residual stress powered hinges24,25. The specific protocol described can be used to create particles with overall sizes ranging from the micrometer to the centimeter length scales. Further, arbitrary patterns can be defined on the surfaces of the particles of importance in colloidal science, electronics, optics and medicine. More generally, the concept of self-assembling mechanically rigid particles with self-sealing hinges is applicable, with some process modifications, to the creation of particles at even smaller, 100 nm length scales22, 26 and with a range of materials including metals21, semiconductors9 and polymers27. With respect to residual stress powered actuation of reconfigurable grasping devices, our specific protocol utilizes chromium hinges of relevance to devices with sizes ranging from 100 &mu;m to 2.5 mm. However, more generally, the concept of such tether-free residual stress powered actuation can be used with alternate high-stress materials such as heteroepitaxially deposited semiconductor films5,7 to possibly create even smaller nanoscale grasping devices.

We describe experimental details of the synthesis of patterned and reconfigurable particles from two dimensional (2D) precursors. This methodology can be used to create particles in a variety of shapes including polyhedra and grasping devices at length scales ranging from the micro to centimeter scale.

折纸灵感的图案和可重构颗粒自组装

There are numerous techniques such as photolithography, electron-beam lithography and soft-lithography that can be used to precisely pattern two ...

科研

化学

Isolation and Chemical Characterization of Lipid A from Gram-negative Bacteria

Lipopolysaccharide (LPS) is the major cell surface molecule of gram-negative bacteria, deposited on the outer leaflet of the outer membrane bilayer. LPS can be subdivided into three domains: the distal O-polysaccharide, a core oligosaccharide, and the lipid A domain consisting of a lipid A molecular species and 3-deoxy-D-manno-oct-2-ulosonic acid residues (Kdo). The lipid A domain is the only component essential for bacterial cell survival. Following its synthesis, lipid A is chemically modified in response to environmental stresses such as pH or temperature, to promote resistance to antibiotic compounds, and to evade recognition by mediators of the host innate immune response. The following protocol details the small- and large-scale isolation of lipid A from gram-negative bacteria. Isolated material is then chemically characterized by thin layer chromatography (TLC) or mass-spectrometry (MS). In addition to matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) MS, we also describe tandem MS protocols for analyzing lipid A molecular species using electrospray ionization (ESI) coupled to collision induced dissociation (CID) and newly employed ultraviolet photodissociation (UVPD) methods. Our MS protocols allow for unequivocal determination of chemical structure, paramount to characterization of lipid A molecules that contain unique or novel chemical modifications. We also describe the radioisotopic labeling, and subsequent isolation, of lipid A from bacterial cells for analysis by TLC. Relative to MS-based protocols, TLC provides a more economical and rapid characterization method, but cannot be used to unambiguously assign lipid A chemical structures without the use of standards of known chemical structure. Over the last two decades isolation and characterization of lipid A has led to numerous exciting discoveries that have improved our understanding of the physiology of gram-negative bacteria, mechanisms of antibiotic resistance, the human innate immune response, and have provided many new targets in the development of antibacterial compounds.

Isolation and characterization of the lipid A domain of lipopolysaccharide (LPS) from gram-negative bacteria provides insight into cell surface based mechanisms of antibiotic resistance, bacterial survival and fitness, and how chemically diverse lipid A molecular species differentially modulate host innate immune responses.

隔离和脂质A的化学表征革兰氏阴性菌

Lipopolysaccharide (LPS) is the major cell surface molecule of gram-negative bacteria, deposited on the outer leaflet of the outer membrane bilayer. LPS ...

Nucleoside Triphosphates - From Synthesis to Biochemical Characterization

The traditional strategy for the introduction of chemical functionalities is the use of solid-phase synthesis by appending suitably modified phosphoramidite precursors to the nascent chain. However, the conditions used during the synthesis and the restriction to rather short sequences hamper the applicability of this methodology. On the other hand, modified nucleoside triphosphates are activated building blocks that have been employed for the mild introduction of numerous functional groups into nucleic acids, a strategy that paves the way for the use of modified nucleic acids in a wide-ranging palette of practical applications such as functional tagging and generation of ribozymes and DNAzymes. One of the major challenges resides in the intricacy of the methodology leading to the isolation and characterization of these nucleoside analogues.
	
	In this video article, we present a detailed protocol for the synthesis of these modified analogues using phosphorous(III)-based reagents. In addition, the procedure for their biochemical characterization is divulged, with a special emphasis on primer extension reactions and TdT tailing polymerization. This detailed protocol will be of use for the crafting of modified dNTPs and their further use in chemical biology.

The protocol described herein aims to explain and abridge the numerous obstacles in the way of the intricate route leading to modified nucleoside triphosphates. Consequently, this protocol facilitates both the synthesis of these activated building-blocks and their availability for practical applications.

核苷三磷酸 - 从合成到生化特征

The  traditional strategy for the introduction of chemical functionalities is the  use of solid-phase synthesis by appending suitably modified ...

Synthesis and Characterization of Functionalized Metal-organic Frameworks

Metal-organic frameworks have attracted extraordinary amounts of research attention, as they are attractive candidates for numerous industrial and technological applications. Their signature property is their ultrahigh porosity, which however imparts a series of challenges when it comes to both constructing them and working with them. Securing desired MOF chemical and physical functionality by linker/node assembly into a highly porous framework of choice can pose difficulties, as less porous and more thermodynamically stable congeners (e.g., other crystalline polymorphs, catenated analogues) are often preferentially obtained by conventional synthesis methods. Once the desired product is obtained, its characterization often requires specialized techniques that address complications potentially arising from, for example, guest-molecule loss or preferential orientation of microcrystallites. Finally, accessing the large voids inside the MOFs for use in applications that involve gases can be problematic, as frameworks may be subject to collapse during removal of solvent molecules (remnants of solvothermal synthesis). In this paper, we describe synthesis and characterization methods routinely utilized in our lab either to solve or circumvent these issues. The methods include solvent-assisted linker exchange, powder X-ray diffraction in capillaries, and materials activation (cavity evacuation) by supercritical CO2 drying. Finally, we provide a protocol for determining a suitable pressure region for applying the Brunauer-Emmett-Teller analysis to nitrogen isotherms, so as to estimate surface area of MOFs with good accuracy.

Synthesis, activation, and characterization of intentionally designed metal-organic framework materials is challenging, especially when building blocks are incompatible or unwanted polymorphs are thermodynamically favored over desired forms. We describe how applications of solvent-assisted linker exchange, powder X-ray diffraction in capillaries and activation via supercritical CO2 drying, can address some of these challenges.

功能化的合成与表征金属 - 有机骨架

Metal-organic frameworks have attracted extraordinary amounts of research attention, as they are attractive candidates for numerous industrial and ...

Preparation of Mica and Silicon Substrates for DNA Origami Analysis and Experimentation

The designed nature and controlled, one-pot synthesis of DNA origami provides exciting opportunities in many fields, particularly nanoelectronics. Many of these applications require interaction with and adhesion of DNA nanostructures to a substrate. Due to its atomically flat and easily cleaned nature, mica has been the substrate of choice for DNA origami experiments. However, the practical applications of mica are relatively limited compared to those of semiconductor substrates. For this reason, a straightforward, stable, and repeatable process for DNA origami adhesion on derivatized silicon oxide is presented here. To promote the adhesion of DNA nanostructures to silicon oxide surface, a self-assembled monolayer of 3-aminopropyltriethoxysilane (APTES) is deposited from an aqueous solution that is compatible with many photoresists. The substrate must be cleaned of all organic and metal contaminants using Radio Corporation of America (RCA) cleaning processes and the native oxide layer must be etched to ensure a flat, functionalizable surface. Cleanrooms are equipped with facilities for silicon cleaning, however many components of DNA origami buffers and solutions are often not allowed in them due to contamination concerns. This manuscript describes the set-up and protocol for in-lab, small-scale silicon cleaning for researchers who do not have access to a cleanroom or would like to incorporate processes that could cause contamination of a cleanroom CMOS clean bench. Additionally, variables for regulating coverage are discussed and how to recognize and avoid common sample preparation problems is described.

Reproducible cleaning processes for substrates used in DNA origami research are described, including bench-top RCA cleaning and derivatization of silicon oxide. Protocols for surface preparation, DNA origami deposition, drying parameters, and simple experimental set-ups are illustrated.

对于DNA折纸分析和实验准备云母和硅衬底

The designed nature and controlled, one-pot synthesis of DNA origami provides exciting opportunities in many fields, particularly nanoelectronics. Many of ...

Preparation and Characterization of Individual and Multi-drug Loaded Physically Entrapped Polymeric Micelles

Amphiphilic block copolymers like polyethyleneglycol-block-polylactic acid (PEG-b-PLA) can self-assemble into micelles above their critical micellar concentration forming hydrophobic cores surrounded by hydrophilic shells in aqueous environments. The core of these micelles can be utilized to load hydrophobic, poorly water soluble drugs like docetaxel (DTX) and everolimus (EVR). Systematic characterization of the micelle structure and drug loading capabilities are important before in vitro and in vivo studies can be conducted. The goal of the protocol described herein is to provide the necessary characterization steps to achieve standardized micellar products. DTX and EVR have intrinsic solubilities of 1.9 and 9.6 &#181;g/ml respectively Preparation of these micelles can be achieved through solvent casting which increases the aqueous solubility of DTX and EVR to 1.86 and 1.85 mg/ml, respectively. Drug stability in micelles evaluated at room temperature over 48 hr indicates that 97% or more of the drugs are retained in solution. Micelle size was assessed using dynamic light scattering and indicated that the size of these micelles was below 50 nm and depended on the molecular weight of the polymer. Drug release from the micelles was assessed using dialysis under sink conditions at pH 7.4 at 37 oC over 48 hr. Curve fitting results indicate that drug release is driven by a first order process indicating that it is diffusion driven.

The goal of this protocol is to describe the preparation and characterization of physically entrapped, poorly water soluble drugs in micellar drug delivery systems composed of amphiphilic block copolymers.

制备及个体表征和多载药物理截留聚合物胶束

Amphiphilic block copolymers like polyethyleneglycol-block-polylactic acid (PEG-b-PLA) can self-assemble into micelles above their critical micellar ...

Synthesis and Characterization of Supramolecular Colloids

Control over colloidal assembly is of utmost importance for the development of functional colloidal materials with tailored structural and mechanical properties for applications in photonics, drug delivery and coating technology. Here we present a new family of colloidal building blocks, coined supramolecular colloids, whose self-assembly is controlled through surface-functionalization with a benzene-1,3,5-tricarboxamide (BTA) derived supramolecular moiety. Such BTAs interact via directional, strong, yet reversible hydrogen-bonds with other identical BTAs. Herein, a protocol is presented that describes how to couple these BTAs to colloids and how to quantify the number of coupling sites, which determines the multivalency of the supramolecular colloids. Light scattering measurements show that the refractive index of the colloids is almost matched with that of the solvent, which strongly reduces the van der Waals forces between the colloids. Before photo-activation, the colloids remain well dispersed, as the BTAs are equipped with a photo-labile group that blocks the formation of hydrogen-bonds. Controlled deprotection with UV-light activates the short-range hydrogen-bonds between the BTAs, which triggers the colloidal self-assembly. The evolution from the dispersed state to the clustered state is monitored by confocal microscopy. These results are further quantified by image analysis with simple routines using ImageJ and Matlab. This merger of supramolecular chemistry and colloidal science offers a direct route towards light- and thermo-responsive colloidal assembly encoded in the surface-grafted monolayer.

A protocol for the synthesis and characterization of colloids coated with supramolecular moieties is described. These supramolecular colloids undergo self-assembly upon the activation of the hydrogen-bonds between the surface-anchored molecules by UV-light.

超分子胶体的合成与表征

Control over colloidal assembly is of utmost importance for the development of functional colloidal materials with tailored structural and mechanical ...

Phthalic Acid Ester-Binding DNA Aptamer Selection, Characterization, and Application to an Electrochemical Aptasensor

Phthalic acid esters (PAEs) areone of the major groups of persistent organic pollutants. The group-specific detection of PAEs is highly desired due to the rapid growing of congeners. DNA aptamers have been increasingly applied as recognition elements on biosensor platforms, but selecting aptamers toward highly hydrophobic small molecule targets, such as PAEs, is rarely reported. This work describes a bead-based method designed to select group-specific DNA aptamers to PAEs. The amino group functionalized dibutyl phthalate (DBP-NH2) as the anchor target was synthesized and immobilized on the epoxy-activated agarose beads, allowing the display of the phthalic ester group at the surface of the immobilization matrix, and therefore the selection of the group-specific binders. We determined the dissociation constants of the aptamer candidates by quantitative polymerization chain reaction coupled with magnetic separation. The relative affinities and selectivity of the aptamers to other PAEs were determined by the competitive assays, where the aptamer candidates were pre-bounded to the DBP-NH2 attached magnetic beads and released to the supernatant upon incubation with the tested PAEs or other potential interfering substances. The competitive assay was applied because it provided a facile affinity comparison among PAEs that had no functional groups for surface immobilization. Finally, we demonstrated the fabrication of an electrochemical aptasensor and used it for ultrasensitive and selective detection of bis(2-ethylhexyl) phthalate. This protocol provides insights for the aptamer discovery of other hydrophobic small molecules.

A protocol for the in vitro selection and characterization of group-specific phthalic acid ester- binding DNA aptamers is presented. The application of the selected aptamer in an electrochemical aptasensor is also included.

邻苯二甲酸酯结合 DNA 核酸适体的选择、表征及其在电化学 Aptasensor 中的应用

Phthalic acid esters (PAEs) areone of the major groups of persistent organic pollutants. The group-specific detection of PAEs is highly desired due to the ...

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 ...

Gene-therapy Inspired Polycation Coating for Protection of DNA Origami Nanostructures

DNA origami nanostructures hold an immense potential to be used for biological and medical applications. However, low-salt conditions and nucleases in physiological fluids induce denaturation and degradation of self-assembled DNA nanostructures. In non-viral gene delivery, enzymatic degradation of DNA is overcome by the encapsulation of the negatively charged DNA in a cationic shell. Herein, inspired by gene delivery advancements, a simple, one-step and robust methodology is presented for the stabilization of DNA origami nanostructures by coating them with chitosan and linear polyethyleneimine. The polycation coating efficiently protects DNA origami nanostructures in Mg-depleted and nuclease-rich media. This method also preserves the full addressability of enzyme- and aptamer-based functionalization of DNA nanostructures.

Here, a protocol for the protection of DNA origami nanostructures in Mg-depleted and nuclease-rich media using natural cationic polysaccharide chitosan and synthetic linear polyethyleneimine (LPEI) coatings is presented.

基因治疗诱导聚化涂层保护 dna 折纸纳米结构

DNA origami nanostructures hold an immense potential to be used for biological and medical applications. However, low-salt conditions and nucleases in ...