This work was supported by an NSF grant (MCB-1054449) and a collaborative interdisciplinary Pilot Research Program award from The RNA Institute at University at Albany to PTXL.

1，制备RNA分子的单分子如何运用眼线改变<ol><li>克隆的目的序列。克隆对应于RNA的结构到载体的DNA序列。 </li><li>合成与转录模板的净化。合成通过PCR转录模板。 [地方表1这里]接下来，净化用PCR纯化试剂盒PCR产物，集中了样品&gt; 200毫微克/微升。因为纯化的DNA用于转录，无RNA酶的水应在洗脱和浓缩步骤中使用。 </li><li> 体外转录。合成通过体外转录的RNA在37℃下过夜，以最大限度地提高RNA的产率。 [地方表2这里]转录后，加入1μl的DNase I在37℃下消化DNA模板15分钟。净化用硅胶离心柱的RNA。 </li><li>生物素化的手柄A的合成首先，通过PCR用引物Af和氩气和浓产生的未经修改的手柄上entrate扩增的DNA至&gt; 200毫微克/微升。接着，结合生物素修饰成的PCR产物使用引物延伸反应。 [地方表3这里]注:生物素化的把手A（生物素-HA），可直接使用在退火无需纯化。作为生物素-HA是用RNA进行退火，这是至关重要的，以使用无RNA酶的水在这两个步骤。 </li><li>地高辛-改性手柄B.合成的合成地高辛，通过PCR使用引物Bf与洋地黄毒苷修饰寡挖一Br（ 图3）改性手柄B（挖-11B）。接着，纯化的PCR产物和浓缩样品至&gt; 200毫微克/微升。 </li><li>退火的RNA的手柄。退火带手柄的RNA。 [地方表4和表5这里] </li><li>净化退火样品。添加3卷退火样品乙醇拌匀。在-70℃下保存该混合物至少1小时。降速在15,800 XG弃上清。干用冻干机沉淀。溶解在沉淀100μl的无RNA酶的水。注:样品准备好被使用，并且可以储存在-20℃。 </li></ol> 2，标定光镊和操作<ol><li>校准视频监控的像素大小<ol><li>使用光学陷阱陷阱与已知的直径（标准尺寸），小珠。 </li><li>使用图像处理软件捕获的珠拍摄图像（ 图4）。 </li><li>确定使用基于质心方法的图像处理软件对珠子的直径。 </li><li>重复此步骤，以确定直径大小不同的珠子。 </li><li>通过线性回归拟合珠的测定的和已知的直径，以获得像素的物理尺寸。 </li></ol></li><li>验证远程校准<ol><li>使用光学陷阱陷阱珠。 </li><li>确定其重心由图像捕获软件的像素位置，并使用Minitweezers记录其位置。 </li><li>操纵珠迪菲<html>租房位置和重复位置的确定。 </li><li>使用校准的像素尺寸转换像素的珠粒的X和Y位置，以米单位。 </li><li>比较由视频和由Minitweezers测量位置之间的X和Y。这两组数值应该同意。作为图像的分辨率是&gt; 0.1微米，将珠用1微米的测量之间，以确保准确性。如果距离校准无法媲美的一个存储在Minitweezers，重新校准仪器。 </li></ol></li><li>陷阱刚度标定<ol><li>利用光阱捕获珠，并保持不动。 </li><li>记录的陷阱使用旁路快速数据采集力的速度&gt; 5千赫，持续3秒。 </li><li>产生使用软件珠运动的记录功率谱。适合功率谱洛伦兹（ 图5）38: 542 / 51542eq1.jpg"宽度="200"/&gt;（公式1） 其中S（f）为在f的频率的电力，f c是角频率，而S 0是渐近功率。转角频率，F C，是频率在那里热运动的力量已经下降到一半的渐近值。由于F C与激光功率和珠子大小各不相同，单独校准每个被困珠。 </li><li>计算陷阱的弹簧常数，κ，从F C: <img alt="式（2）" fo:content-width="1in" src="/files/ftp_upload/51542/51542eq2.jpg" width="100" /> （式2） 其中γ是在胎圈（式3）的阻力系数。使用弹簧常数来确定力变化的RNA的延伸变化。 </li></ol></li><li>斯托克斯定律测试<ol><li>拍摄使用的光学陷阱珠。将珠子来回控制，力在不同的速度。 </li><li>记录下珠的运动，并使用Minitweezers力。 </li><li>计算胎圈的半径。 注:该摩擦力，F D，由球形粒子中的流体的运动而产生可描述由斯托克斯定律: <img alt="式（3）" fo:content-width="1.5in" src="/files/ftp_upload/51542/51542eq3.jpg" width="150" /> （式3） 其中r是该球的半径，V是速度，h是液体，它可以在参考表中找到的动态粘度。使用测得的力为F d时，胎圈的半径可以用公式进行计算。 3（ 图6）和应与该图像获取软件来确定。斯托克斯定律测试有效地测试整个校准和仪器的性能。 </li></ol></li></ol> 3，纳米操纵单个RNA分子<ol><li>使流体。 <ol><li>钻6孔（直径为2毫米的每个）在第2玻璃盖（ 图7a）和切三个狭缝（2毫米的宽度）成双面聚酰亚胺胶带（ 图7b）用激光雕刻。 </li><li>夹在2防护玻璃双面卡普顿胶带两层作流室。将胶带（ 图7c）之间的微管和两个旁路管。 </li><li>通过匹配射流孔（ 图7d）安装流动室上的金属框架。 </li><li>连接的腔室的流体通道至10ml注射器填充有通过聚乙烯管（ 图7E）的选择的缓冲液中。 </li><li>加载整个腔到两个目标之间的光学镊子。确保与流体通道的腔室的前侧朝向右侧。收回权的目标和插架（ 图7E）的铜针脚在T安装孔他镊子。拧紧螺丝固定腔的位置。 </li></ol></li><li>混合退火样品和珠子。混合用抗地高辛包被的珠子（挖珠）退火的样品，并让该混合物留在室温下搅拌5-10分钟。通常情况下，1微​​升退火的样品，用5微升挖珠在1ml缓冲液中混合。请注意:在小珠的量加载到流动室的选择应确保的珠的合理数量，由旁通管递送。退火的样品与珠子的比例不能容易地进行定量。在理想的情况下，大部分颗粒具有无RNA，使得珠子只有一小部分可以仅具有一个每珠RNA分子。作为退火的样品与珠子悬浮液难以量化，最好和唯一的方式目前是改变混合比和测试对镊子的混合物。 </li><li>交付珠的反应点流室（ 即几微米的MI的顶部cropipette尖， 图7c）。 <ol><li>负载链亲和素包被的磁珠（链球菌珠）的悬浮成1 ml注射器。 </li><li>将注射器连接到所述流体管道通向流动室（ 图7a）的底部的通道。 </li><li>推动注射器流动的链球菌珠进入腔室。 </li><li>移动用马达控制软件以放置底部旁通管（ 图7b）的开口部附近的光阱的整个腔室。然后，通过轨迹球操作光学陷阱捕捉链球菌珠。 </li><li>将珠接近使用电动机控制软件的微尖。 </li><li>拉连接到微量的珠吸入微量注射器。 </li><li>按注射器中间渠道，以冲洗掉多余的链球菌珠轻轻地涂抹流。 </li><li>类似地，提供样品和挖珠（参见步骤3.2），以在腔室的顶部通道的混合物。 </li><li>捕捉挖珠，并把它靠近使用光学陷阱链球菌珠。 </li><li>清洁中间渠道通过应用流量。注:strep-和辛珠被选择为具有不同的尺寸，以便它们能够在视觉上的微观角度下加以区分（ 图4）。 </li></ol></li><li>钓鱼"单分子对珠之间的圈养。 <ol><li>将挖珠垂直于链球菌珠（ 图4）的顶部。 </li><li>通过操纵陷阱将挖珠下来，对链球菌珠。在电脑上打开一个力 - 距离积窗口来可视化的力量变化。 </li><li>当两个珠子接触，将挖珠垂直距离链球菌珠。 </li><li>如果没有特定的接触时，该力保持几乎为零。如果两个珠粒通过分子连接时，力增大显著当两个珠分离。此过程中，通常被称为"钓鱼"，往往表现在每对珠ormed多次找到一个有效的单分子系绳。 </li></ol></li></ol>

作者什么都没有透露。

<html> 修改和故障排除中如何运用眼线改变样品的制备 选择克隆载体 。尽管此处描述的一般方案（ 图3），不需要特殊的克隆载体，该载体优选的是不具有固有的T7或T3启动子进行转录的便利性，使得启动子可通过PCR在所需的位置被引入。 序列和手柄的长度 。有没有具体的时序要求的手柄。然而，接近GC与AT比例是?...

光学镊子技术的发展早已伴随其在生物研究中的应用。当第一次被发现的光学捕获效应，亚瑟Ashkin观察到细菌污染的水可以在激光焦点1被困住。此后，细菌诱捕已经成为一个意外的，有趣的实验几代生物物理的学生，以及一个严肃的研究工具，研究微生物生理学2,3。光诱捕技术使用聚焦激光束来固定微观对象1。实际上，激光陷阱功能的光学弹簧，其中还通过从陷阱（ΔX）的中心，它的位移测量捕获对象上的力（F）。在很短的范围内，F =κ点¯xΔX，在κ是陷阱的弹簧常数。在光阱可以用来施加在picoNewton（PN）精密力为百万分之一COPIC物体并测量其与纳米精度的位置。在过去的二十年中，光学镊子已经成为生物物理学中最常用的单分子技术中的一种。该技术已用于研究的折叠和DNA 4-6，7-9的RNA和蛋白质10,11力学。光学镊子也已用于观察DNA复制12，RNA转录13，和蛋白合成14,15，以及许多其它生物分子事件16-18。 单分子的方法是采用RNA的结构研究主要探讨RNA的折叠崎岖的能源格局。的RNA序列通常可以折叠成多个稳定，互斥的结构中，由于RNA的简单的化学组合物和碱配对规则。人们早已知道，RNA结构的多态性，如发生在核糖开关19,20，起着基因调控中起重要作用。 àrecen吨全基因组的调查显示，温度变化小到几度极大地影响结构和蛋白质合成的细胞转录组21一 ​​大截。这个例子暗示的替代RNA折叠的生物学作用可能更为重要和普遍比以前承担。结构多态性，但是，带来了传统的生物化学和生物物理的方法，其中许多研究分子的平均性能是一个挑战。例如，在"开"和核开关的"关"的构象是相互排斥的。从异质结构衍生的平均结构不太可能像任生物学相关的构象异构体。此外，非编码RNA和mRNA的一般形式结构。它们与蛋白质和调控RNA的相互作用通常需要现有结构的展开作为交互的一部分。因此，研究RNA的折叠/折叠成为相关问题的RNA生物学。为了迎接这样的挑战，在单分子的方法已被用来通过研究一个分子在同一时间22-27解开RNA的结构多态性。 较流行的单分子荧光法，基于机械展开镊子提供了单个分子的构象，可以通过施加力来操纵并与纳米精度测量的优点。这种纳米操作能力可被利用来监视单个结构域，使得大量的RNA的分层折叠可解剖28的解折叠/折叠。备选地，单链，可向折叠成若干构象之一;或现有结构可以机械地诱导重新折叠成不同的构象29。根据生物的条件下，一种RNA结构可以根据温度变化或配体结合结构被改变。直接操纵分子S中的能力tructure打开一个新场地的RNA结构的研究。原则上，其它机械技术，例如原子力显微镜和磁镊子，也可以用于研究单个RNA分子的折叠。但是，这样的应用在很大程度上限于由于相对低的空间分辨率30。 光镊的就业使RNA结构受机械力来展开。 机械折叠的优点是数倍。力可以用来展现两个二级和三级结构，而金属离子和配体诱导的折叠主要限于三级结构。温度和变性剂可以显著影响水和溶质的活动。与此相反，力局部地施加于扰动的分子结构;的力对周围环境的影响是可以忽略不计。此外，热熔最好用于研究小分子RNA折叠结构热力学，而光学镊子已用于研究RNA结构，各种大小不等的7个碱基对tetraloop发卡28至400个核苷酸的核酶31。此外，RNA结构可以机械地展开在嗜温温度。与此相反，以展开的RNA中的热熔融实验中，温度通常升高远高于生理温度，这显著增加RNA的水解，特别是在Mg 2 +的离子的存在。 要注意的力的机械作用取决于它是如何施加到结构是很重要的。作用力倾斜折叠能源格局。例如，当力被施加到一个发夹，基地对被顺序地破碎，每次一个（ 图1a）。翻录叉前进沿着螺旋轴，其垂直于所施加的力。相反，当一个最小接吻络合物是在张力下，两亲碱基对，它们平行于所施加的力，共享的力负载（ 图1b）。不同几何形状的发夹，亲吻复杂相对于所施加的力导致在其不同的力学响应，这可以被用来区分二级和三级折叠28,32的。机械展开的理论方面先前已审查8,9,30。这项工作概括了基本的方法建立并执行单分子机械展开分析。 实验装置 。在我们的机械拉力实验中，对于如何运用眼线改变RNA样品由目的RNA由两个双链DNA / RNA的把手侧接（ 图2）的7。整个分子可被拴经由链霉亲和素 - 生物素和地高辛抗地高辛抗体相互作用，两个分别的表面被覆的微米尺寸的小珠（Spherotech）。一个珠子是由测力光TR举行AP，而另一种是举行微量的一角。小珠之间的相对距离可以通过操纵陷阱或移动的微量改变。使用这种方法，单个RNA分子圈养珠可以拉伸和放松。 样品的制备 。 RNA样品的合成对于如何运用眼线改变包括几个步骤（ 图3）。首先，对应于感兴趣的RNA的DNA序列，首先克隆到质粒载体中。接下来，三PCR反应进行，生成两个手柄和转录的模板。转录模板包括手柄区域和插入序列。全长RNA通过体外转录合成。最后，该RNA和化学修饰的手柄一起退火，以产生所述分子为如何运用眼线改变。 校准和镊子的操作 。在Minitweezers使用的基本设计d在此工作如下的双光束光镊33。有许多改进，Minitweezers相比第一代光学镊子显示非常稳定。一些研究小组在几个国家使用Minitweezers在他们的单分子研究14,15,34-37。建筑设备，包括教学影片，校准和操作的细节，都可以在"镊子实验室"网站（http://tweezerslab.unipr.it）。这里，需要在每日基础进行改进和校准程序进行详细说明。

<table border="0" cellpadding="0" cellspacing="0" width="745">
	<tbody>
		<tr height="20">
			<td height="20" style="height:21px;width:249px;">Minitweezers</td>
			<td style="width:211px;">Steven B. Smith Engineering</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"><a href="http://tweezerslab.unipr.it/">http://tweezerslab.unipr.it</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:249px;">Data acquisition card</td>
			<td style="width:211px;">National Instruments</td>
			<td style="width:119px;">USB-6351</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:249px;">Video card</td>
			<td style="width:211px;">National Instruments</td>
			<td style="width:119px;">PCI-1407</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:249px;">Labview programming software</td>
			<td style="width:211px;">National Instruments</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:249px;">NI Vision Builder</td>
			<td style="width:211px;">National Instruments</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:249px;">Laser engraver</td>
			<td style="width:211px;">Epilogue laser systems</td>
			<td style="width:119px;">Zing-16</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:249px;"></td>
			<td style="width:211px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:249px;">PCR purification kit</td>
			<td style="width:211px;">Qiagen</td>
			<td style="width:119px;">28104</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;width:249px;">MEGAscript T7 kit</td>
			<td style="width:211px;">Life Technologies</td>
			<td style="width:119px;">AM1334</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:249px;">MEGAclear kit</td>
			<td style="width:211px;">Life Technologies</td>
			<td style="width:119px;">AM1908</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:249px;">Biotin-11-UTP</td>
			<td style="width:211px;">Thermo Fisher</td>
			<td>FERR0081</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:249px;">T4 DNA polymerase</td>
			<td style="width:211px;">New England Labs</td>
			<td style="width:119px;">M0203</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;width:249px;">Streptavidin coated microsphere</td>
			<td style="width:211px;">Spherotech</td>
			<td style="width:119px;">SVP-20-5</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:249px;">Antidigoxigenin coated microsphere</td>
			<td style="width:211px;">Spherotech</td>
			<td style="width:119px;">DIGP-20-2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:249px;">Size standard microspheres</td>
			<td style="width:211px;">Spherotech</td>
			<td style="width:119px;">PPS-6K</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:249px;">Kapton tape</td>
			<td style="width:211px;">Kaptontape.com</td>
			<td>KPPTDE-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:249px;">No. 2 cover glass</td>
			<td style="width:211px;">Thermo Fisher</td>
			<td style="width:119px;">12-543D</td>
			<td></td>
		</tr>
	</tbody>
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nanomanipulation of single rna molecules by optical tweezers

人类基因组中有很大一部分被转录，但没有翻译。在这篇文章中基因组时代，RNA的调节功能已被证明是越来越重要。作为RNA的功能，通常取决于其采用替代的结构的能力，它是很难直接从序列预测RNA的三维结构。单分子方法展示潜能监测分子结构一个分子在同一时间，解决了RNA的结构多态性的问题。这项工作提出了一种方法，能够精​​确地操控利用光学镊子单个RNA分子的折叠和结构。首先，方法以合成适于单分子机械功分子中描述。接着，各个校准程序，以确保所述光学镊子的正确操作进行了讨论。接着，不同的实验进行说明。为了证明该技术的实用性，机械地展开发夹RNA的结果和一个RNA的亲吻克复合体作为证据使用。在这些例子中，纳米操作技术被用来研究每个结构域，其中包括二级和三级折叠，独立。最后，在限制和方法的未来的应用进行了讨论。

受空间限制，单个RNA分子的纳米操纵的证明显示的RNA发夹只是机械展开的例子，并与三级结构的RNA。 操纵单分子发夹 一旦系绳珠粒之间建立，扩展和力上的系绳可以使用用户定义的协议进行操作。四种常见的操作协议是常用的。 强制斜坡实验 。最直观和通用实验操纵的单个RNA是反复拉伸和放松在手柄的螺旋轴?...

Watch this Scientific Journal Video about Nanomanipulation of Single RNA Molecules by Optical Tweezers at JoVE.com

Nanomanipulation of Single RNA Molecules by Optical Tweezers

Optical tweezers have been used to study RNA folding by stretching individual molecules from their 5&#8217; and 3&#8217; ends. Here common procedures are described to synthesize RNA molecules for tweezing, calibration of the instrument, and methods to manipulate single molecules.

单个RNA分子的光镊纳米操作

A large portion of the human genome is transcribed but not translated. In this post genomic era, regulatory functions of RNA have been shown to be ...

nanomanipulation-of-single-rna-molecules-by-optical-tweezers

Research

JoVE Journal

Bioengineering

14.7K 次观看.  University at Albany, State University of New York. Optical tweezers have been used to study RNA folding by stretching individual molecules from their 5’ and 3’ ends. Here common procedures are described to synthesize RNA molecules for tweezing, calibration of the instrument, and methods to manipulate single molecules. 

视频: Nanomanipulation of Single RNA Molecules by Optical Tweezers

Stretching Short Sequences of DNA with Constant Force Axial Optical Tweezers

Single-molecule techniques for stretching DNA of contour lengths less than a kilobase are fraught with experimental difficulties. However, many interesting biological events such as histone binding and protein-mediated looping of DNA1,2, occur on this length scale. In recent years, the mechanical properties of DNA have been shown to play a significant role in fundamental cellular processes like the packaging of DNA into compact nucleosomes and chromatin fibers3,4. Clearly, it is then important to understand the mechanical properties of short stretches of DNA. In this paper, we provide a practical guide to a single-molecule optical tweezing technique that we have developed to study the mechanical behavior of DNA with contour lengths as short as a few hundred basepairs.
The major hurdle in stretching short segments of DNA is that conventional optical tweezers are generally designed to apply force in a direction lateral to the stage5,6, (see Fig. 1). In this geometry, the angle between the bead and the coverslip, to which the DNA is tethered, becomes very steep for submicron length DNA. The axial position must now be accounted for, which can be a challenge, and, since the extension drags the microsphere closer to the coverslip, steric effects are enhanced. Furthermore, as a result of the asymmetry of the microspheres, lateral extensions will generate varying levels of torque due to rotation of the microsphere within the optical trap since the direction of the reactive force changes during the extension.
Alternate methods for stretching submicron DNA run up against their own unique hurdles. For instance, a dual-beam optical trap is limited to stretching DNA of around a wavelength, at which point interference effects between the two traps and from light scattering between the microspheres begin to pose a significant problem. Replacing one of the traps with a micropipette would most likely suffer from similar challenges. While one could directly use the axial potential to stretch the DNA, an active feedback scheme would be needed to apply a constant force and the bandwidth of this will be quite limited, especially at low forces.
We circumvent these fundamental problems by directly pulling the DNA away from the coverslip by using a constant force axial optical tweezers7,8. This is achieved by trapping the bead in a linear region of the optical potential, where the optical force is constant-the strength of which can be tuned by adjusting the laser power. Trapping within the linear region also serves as an all optical force-clamp on the DNA that extends for nearly 350 nm in the axial direction. We simultaneously compensate for thermal and mechanical drift by finely adjusting the position of the stage so that a reference microsphere stuck to the coverslip remains at the same position and focus, allowing for a virtually limitless observation period.

We illustrate the use of a constant force axial optical tweezers to explore the mechanical properties of short DNA molecules. By stretching DNA axially, we minimize steric hindrances and artifacts arising in conventional lateral manipulation, allowing us to study DNA molecules as short as ~100 nm.

伸展的DNA短序列恒力轴向光镊

Single-molecule techniques for stretching DNA of contour lengths  less than a kilobase are fraught with experimental difficulties. However, many ...

科研

生物工程

Multiplexed Single-molecule Force Proteolysis Measurements Using Magnetic Tweezers

The generation and detection of mechanical forces is a ubiquitous aspect of cell physiology, with direct relevance to cancer metastasis1, atherogenesis2 and wound healing3. In each of these examples, cells both exert force on their surroundings and simultaneously enzymatically remodel the extracellular matrix (ECM). The effect of forces on ECM has thus become an area of considerable interest due to its likely biological and medical importance4-7.
	Single molecule techniques such as optical trapping8, atomic force microscopy9, and magnetic tweezers10,11 allow researchers to probe the function of enzymes at a molecular level by exerting forces on individual proteins. Of these techniques, magnetic tweezers (MT) are notable for their low cost and high throughput. MT exert forces in the range of ~1-100 pN and can provide millisecond temporal resolution, qualities that are well matched to the study of enzyme mechanism at the single-molecule level12. Here we report a highly parallelizable MT assay to study the effect of force on the proteolysis of single protein molecules. We present the specific example of the proteolysis of a trimeric collagen peptide by matrix metalloproteinase 1 (MMP-1); however, this assay can be easily adapted to study other substrates and proteases.

In this article we describe the use of magnetic tweezers to study the effect of force on enzymatic proteolysis at the single molecule level in a highly parallelizable manner.

警队复用单分子蛋白水解测量磁性镊子

The  generation and detection of mechanical forces is a ubiquitous aspect of cell physiology,  with direct relevance to cancer metastasis1, atherogenesis2 ...

In vitro Synthesis of Native, Fibrous Long Spacing and Segmental Long Spacing Collagen

Collagen fibrils are present in the extracellular matrix of animal tissue to provide structural scaffolding and mechanical strength. These native collagen fibrils have a characteristic banding periodicity of ~67 nm and are formed in vivo through the hierarchical assembly of Type I collagen monomers, which are 300 nm in length and 1.4 nm in diameter. In vitro, by varying the conditions to which the monomer building blocks are exposed, unique structures ranging in length scales up to 50 microns can be constructed, including not only native type fibrils, but also fibrous long spacing and segmental long spacing collagen. Herein, we present procedures for forming the three different collagen structures from a common commercially available collagen monomer. Using the protocols that we and others have published in the past to make these three types typically lead to mixtures of structures. In particular, unbanded fibrils were commonly found when making native collagen, and native fibrils were often present when making fibrous long spacing collagen. These new procedures have the advantage of producing the desired collagen fibril type almost exclusively. The formation of the desired structures is verified by imaging using an atomic force microscope.

In vitro Synthesis of Native, Fibrous Long Spacing and Segmental Long Spacing Collagen

Simple and reproducible procedures are described for making three structurally distinct collagen assemblies from a common commercially available Type I collagen monomer. Native type, fibrous long spacing or segmental long spacing collagen can be constructed by varying the conditions to which the 300 nm long and 1.4 nm diameter monomer building block is exposed.

在体外合成的原住民，纤维长间距"和"分段长间距胶原蛋白

Collagen fibrils are present in the  extracellular matrix of animal tissue to provide structural scaffolding and  mechanical strength. These native ...

Optical Detection of E. coli Bacteria by Mesoporous Silicon Biosensors

A label-free optical biosensor based on a nanostructured porous Si is designed for rapid capture and detection of Escherichia coli K12 bacteria, as a model microorganism. The biosensor relies on direct binding of the target bacteria cells onto its surface, while no pretreatment (e.g.&#160;by cell lysis) of the studied sample is required. A mesoporous Si thin film is used as the optical transducer element of the biosensor. Under white light illumination, the porous layer displays well-resolved Fabry-P&#233;rot fringe patterns in its reflectivity spectrum. Applying a fast Fourier transform (FFT) to reflectivity data results in a single peak. Changes in the intensity of the FFT peak are monitored. Thus, target bacteria capture onto the biosensor surface, through antibody-antigen interactions, induces measurable changes in the intensity of the FFT peaks, allowing for a &#39;real time&#39; observation of bacteria attachment.
The mesoporous Si film, fabricated by an electrochemical anodization process, is conjugated with monoclonal antibodies, specific to the target bacteria. The immobilization, immunoactivity and specificity of the antibodies are confirmed by fluorescent labeling experiments. Once the biosensor is exposed to the target bacteria, the cells are directly captured onto the antibody-modified porous Si surface. These specific capturing events result in intensity changes in the thin-film optical interference spectrum of the biosensor. We demonstrate that these biosensors can detect relatively low bacteria concentrations (detection limit of 104 cells/ml) in less than an hour.

Optical Detection of E. coli Bacteria by Mesoporous Silicon Biosensors

A label-free optical biosensor for rapid bacteria detection is introduced. The biosensor is based on a nanostructured porous Si, which is designed to directly capture the target bacteria cells onto its surface. We use monoclonal antibodies, immobilized onto the porous transducer, as the capture probes. Our studies demonstrate the applicability of these biosensors for the detection of low bacterial concentrations within minutes with no prior sample processing (such as cell lysis).

光学检测<em&gt; E。大肠杆菌</em&gt;由细菌介孔硅生物传感器

A label-free optical biosensor based on a nanostructured porous Si is designed for rapid capture and detection of Escherichia coli K12 bacteria, as a ...

Magnetic Tweezers for the Measurement of Twist and Torque

Single-molecule techniques make it possible to investigate the behavior of individual biological molecules in solution in real time. These techniques include so-called force spectroscopy approaches such as atomic force microscopy, optical tweezers, flow stretching, and magnetic tweezers. Amongst these approaches, magnetic tweezers have distinguished themselves by their ability to apply torque while maintaining a constant stretching force. Here, it is illustrated how such a &#8220;conventional&#8221; magnetic tweezers experimental configuration can, through a straightforward modification of its field configuration to minimize the magnitude of the transverse field, be adapted to measure the degree of twist in a biological molecule. The resulting configuration is termed the freely-orbiting magnetic tweezers. Additionally, it is shown how further modification of the field configuration can yield a transverse field with a magnitude intermediate between that of the &#8220;conventional&#8221; magnetic tweezers and the freely-orbiting magnetic tweezers, which makes it possible to directly measure the torque stored in a biological molecule. This configuration is termed the magnetic torque tweezers. The accompanying video explains in detail how the conversion of conventional magnetic tweezers into freely-orbiting magnetic tweezers and magnetic torque tweezers can be accomplished, and demonstrates the use of these techniques. These adaptations maintain all the strengths of conventional magnetic tweezers while greatly expanding the versatility of this powerful instrument.

Magnetic tweezers, a powerful single-molecule manipulation technique, can be adapted for the direct measurements of the twist (using a configuration called freely-orbiting magnetic tweezers) and torque (using a configuration termed magnetic torque tweezers) in biological macromolecules. Guidelines for performing such measurements are given, including applications to the study of DNA and associated nucleo-protein filaments.

磁性镊子的扭曲和扭矩的测量

Single-molecule techniques make it possible to investigate the behavior of individual biological molecules in solution in real time. These techniques ...

Fluorescence Biomembrane Force Probe: Concurrent Quantitation of Receptor-ligand Kinetics and Binding-induced Intracellular Signaling on a Single Cell

Membrane receptor-ligand interactions mediate many cellular functions. Binding kinetics and downstream signaling triggered by these molecular interactions are likely affected by the mechanical environment in which binding and signaling take place. A recent study demonstrated that mechanical force can regulate antigen recognition by and triggering of the T-cell receptor (TCR). This was made possible by a new technology we developed and termed fluorescence biomembrane force probe (fBFP), which combines single-molecule force spectroscopy with fluorescence microscopy. Using an ultra-soft human red blood cell as the sensitive force sensor, a high-speed camera and real-time imaging tracking techniques, the fBFP is of ~1 pN (10-12 N), ~3 nm and ~0.5 msec in force, spatial and temporal resolution. With the fBFP, one can precisely measure single receptor-ligand binding kinetics under force regulation and simultaneously image binding-triggered intracellular calcium signaling on a single live cell. This new technology can be used to study other membrane receptor-ligand interaction and signaling in other cells under mechanical regulation.

We describe a technique for concurrently measuring force-regulated single receptor-ligand binding kinetics and real-time imaging of calcium signaling in a single T lymphocyte.

生物膜荧光探针力:受体 - 配体动力学并行和定量结合诱导细胞内信号作用于单细胞

Membrane receptor-ligand interactions mediate many cellular functions. Binding kinetics and downstream signaling triggered by these molecular interactions ...

Delivery of Proteins, Peptides or Cell-impermeable Small Molecules into Live Cells by Incubation with the Endosomolytic Reagent dfTAT

Macromolecular delivery strategies typically utilize the endocytic pathway as a route of cellular entry. However, endosomal entrapment severely limits the efficiency with which macromolecules penetrate the cytosolic space of cells. Recently, we have circumvented this problem by identifying the reagent dfTAT, a disulfide bond dimer of the peptide TAT labeled with the fluorophore tetramethylrhodamine. We have generated a fluorescently labeled dimer of the prototypical cell-penetrating peptide (CPP) TAT, dfTAT, which penetrates live cells and reaches the cytosolic space of cells with a particularly high efficiency. Cytosolic delivery of dfTAT is achieved in multiple cell lines, including primary cells. Moreover, delivery does not noticeably impact cell viability, proliferation or gene expression. dfTAT can deliver small molecules, peptides, antibodies, biologically active enzymes and a transcription factor. In this report, we describe the protocols involved in dfTAT synthesis and cellular delivery. The manuscript describes how to control the amount of protein delivered to the cytosolic space of cells by varying the amount of protein administered extracellularly. Finally, the current limitations of this new technology and steps involved in validating delivery are discussed. The described protocols should be extremely useful for cell-based assays as well as for the ex vivo manipulation and reprogramming of cells.

We describe how to deliver proteins and cell-impermeable small molecules into cultured mammalian cells by a simple co-incubation protocol with a reagent that causes endocytic organelles to become leaky.

交货蛋白质，多肽或细胞不可渗透的小分子进入活细胞被孵化与核内体溶解试剂dfTAT

Macromolecular delivery strategies typically utilize the endocytic pathway as a route of cellular entry. However, endosomal entrapment severely limits the ...

Label-free Single Molecule Detection Using Microtoroid Optical Resonators

Detecting small concentrations of molecules down to the single molecule limit has impact on areas such as early detection of disease, and fundamental studies on the behavior of molecules. Single molecule detection techniques commonly utilize labels such as fluorescent tags or quantum dots, however, labels are not always available, increase cost and complexity, and can perturb the events being studied. Optical resonators have emerged as a promising means to detect single molecules without the use of labels. Currently the smallest particle detected by a non-plasmonically-enhanced bare optical resonator system in solution is a 25 nm polystyrene sphere1. We have developed a technique known as Frequency Locking Optical Whispering Evanescent Resonator (FLOWER) that can surpass this limit and achieve label-free single molecule detection in aqueous solution2. As signal strength scales with particle volume, our work represents a &#62; 100x improvement in the signal to noise ratio (SNR) over the current state of the art. Here the procedures behind FLOWER are presented in an effort to increase its usage in the field.

We have developed a label-free biosensing system based on optical resonator technology known as Frequency Locking Optical Whispering Evanescent Resonator (FLOWER) that is capable of detecting single molecules in solution. Here the procedures behind this work are described and presented.

无标记单分子检测使用微型环光学谐振器

Detecting small concentrations of molecules down to the single molecule limit has impact on areas such as early detection of disease, and fundamental ...

Optical Control of Living Cells Electrical Activity by Conjugated Polymers

Hybrid interfaces between organic semiconductors and living tissues represent a new tool for in-vitro and in-vivo applications. In particular, conjugated polymers display several optimal properties as substrates for biological systems, such as good biocompatibility, excellent mechanical properties, cheap and easy processing technology, and possibility of deposition on light, thin and flexible substrates. These materials have been employed for cellular interfaces like neural probes, transistors for excitation and recording of neural activity, biosensors and actuators for drug release. Recent experiments have also demonstrated the possibility to use conjugated polymers for all-optical modulation of the electrical activity of cells. Several in-vitro study cases have been reported, including primary neuronal networks, astrocytes and secondary line cells. Moreover, signal photo-transduction mediated by organic polymers has been shown to restore light sensitivity in degenerated retinas, suggesting that these devices may be used for artificial retinal prosthesis in the future. All in all, light sensitive conjugated polymers represent a new approach for optical modulation of cellular activity.
In this work, all the steps required to fabricate a bio-polymer interface for optical excitation of living cells are described. The function of the active interface is to transduce the light stimulus into a modulation of the cell membrane potential. As a study case, useful for in-vitro studies, a polythiophene thin film is used as the functional, light absorbing layer, and Human Embryonic Kidney (HEK-293) cells are employed as the biological component of the interface. Practical examples of successful control of the cell membrane potential upon stimulation with light pulses of different duration are provided. In particular, it is shown that both depolarizing and hyperpolarizing effects on the cell membrane can be achieved depending on the duration of the light stimulus. The reported protocol is of general validity and can be straightforwardly extended to other biological preparations.

A method for stimulation of in-vitro cell cultures electrical activity with visible light, based on the use of organic semiconducting polymers is described.

由共轭聚合物活细胞电活动的光控制

Hybrid interfaces between organic semiconductors and living tissues represent a new tool for in-vitro and in-vivo applications. In particular, conjugated ...

Development of Microfluidic Devices to Study the Elongation Capability of Tip-growing Plant Cells in Extremely Small Spaces

In vivo, tip-growing plant cells need to overcome a series of physical barriers; however, researchers lack the methodology to visualize cellular behavior in such restrictive conditions. To address this issue, we have developed growth chambers for tip-growing plant cells that contain a series of narrow, micro-fabricated gaps (~1 &#181;m) in a poly-dimethylsiloxane (PDMS) substrate. This transparent material allows the user to monitor tip elongation processes in individual cells during microgap penetration by time-lapse imaging. Using this experimental platform, we observed morphological changes in pollen tubes as they penetrated the microgap. We captured the dynamic changes in the shape of a fluorescently labeled vegetative nucleus and sperm cells in a pollen tube during this process. Furthermore, we demonstrated the capability of root hairs and moss protonemata to penetrate the 1 &#181;m gap. This in vitro platform can be used to study how individual cells respond to physically constrained spaces and may provide insights into tip-growth mechanisms.

We describe a method to investigate the capability of tip-growing plant cells, including pollen tubes, root hairs, and moss protonemata, to elongate through extremely narrow gaps (~1 &#181;m) in a microfluidic device.

微流控装置在极小空间中研究尖端生长植物细胞的伸长能力

In vivo, tip-growing plant cells need to overcome a series of physical barriers; however, researchers lack the methodology to visualize cellular behavior ...

Methods Article

单个RNA分子的光镊纳米操作