This work was supported by the Sogang University Research Grant of 201410036.

1. DNA生物素<ol><li> 使用终端转移钝端DNA的生物素化（TDT） 注意:使用T 4 DNA（166千碱基），它是一个平端的DNA。 <ol><li>添加2.5mM的5微升氯化钴，5微升10×反应缓冲液，0.5微升10毫摩尔生物素11-dUTP的的，末端转移0.5微升（10单位）和0.5微升的T4 DNA（0.5微克/微升）到反应混合物中。通过加入38.5微升的水使至50微升的最终体积。 </li><li>孵育在37℃下将反应混合物1小时。 注:扩展反应时间可以得到两端-拴系的DNA， 即 ，它是可能的生物素的标签的两端。 </li><li>停止加入5微升的0.5M EDTA，pH为8的反应。 </li><li>保持管在4℃下。 </li></ol></li><li> 粘端的DNA的生物素化利用DNA连接酶 注意:使用λ的DNA（48.5 kbp的），这是一种粘性末端的DNA。 &lt;OL&gt; <li>加入1μlT4 DNA连接酶，5微升10×连接缓冲液，1μl的25纳克/微升λ噬菌体DNA，并加入43微升的水，使50微升的最终体积。 </li><li>保持管在4℃下。 </li></ol></li></ol> 2.功能化表面衍生注意:为了系绳在玻璃表面的DNA分子的末端，伯胺基团上的盖玻片随后生物素PEG涂层硅烷化， 如图1该PEG化过程是对单分子的DNA成像重要的，因为它可以显著减少通过在表面上不希望的分子的附着产生的随机噪声。 <ol><li> 食人鱼清洗 注:食人鱼解决方案有机材料剧烈反应，因此应小心处理，遵循正确的安全准则。 <ol><li>地方盖玻片上的聚四氟乙烯（PTFE）架，并把它们býPTFE螺纹密封带纵向，一半的表面的边缘和半机架上。包装后，留下带长片（约5厘米）的清洁过程中搬运机架。 </li><li>填充1升的玻璃烧杯用350ml的H 2 SO 4和150ml 的 H 2 O 2，使在通风橱食人鱼溶液。放置在食人鱼溶液机架2小时。 </li><li>从烧杯中空食人鱼溶液，并冲洗盖玻片用去离子水彻底直至烧杯中的pH值的水达到中性。使用pH试纸条。 </li><li>超声处理盖玻片的齿条中的含烧杯水，30分钟。空水从烧杯中刚氨基硅烷化之前。使用75瓦超声功率的清洁和衍生的玻璃基板。 </li></ol></li><li> Aminosilanization在玻璃表面 <ol><li>加的N 2毫升- [3-（三甲氧基甲硅烷基）丙基]乙二胺和10毫升冰醋酸的200毫升在制备aminosilanization溶液清洁聚丙烯容器甲醇。 </li><li>放置清洗盖玻片在聚丙烯容器中。在100rpm和室温下搅拌30分钟，摇动它们。 </li><li>超声处理以衍生盖玻片烧杯15分钟，在75 W·摇动它们在100rpm和室温下至少30分钟。 </li><li>用乙醇清空从烧杯中的溶液，并用甲醇小心冲洗盖玻片，一次，两次。在乙醇商店盖玻片并用它们在两周内。 </li></ol></li><li> 盖玻片的PEG化 <ol><li>打10毫升0.1N 1M碳酸氢钠的（ 碳酸氢钠 ）。筛选具有0.22μm的注射器过滤该溶液。 </li><li>溶解2mg的生物素的PEG琥珀酰亚胺基碳酸酯（生物素PEG-SC）和在350微升的NaHCO 3的80毫克聚乙二醇琥珀酰亚胺基戊酸（MPEG-SVG）的。使用光保护管。 </li><li>涡管大力为10秒，和离心机它以10,000×g的1分钟以除去气泡。 </li><li>冲洗用丙酮载玻片，随后用乙醇漂洗。允许幻灯片空气完全干燥。 </li><li>将PEG溶液对干净的载玻片上滴（50微升）。与氨基硅烷化盖玻片轻轻盖上液滴，而不会产生任何气泡。 </li><li> 3小时到位幻灯片一夜之间在一个黑暗的，在室温下以及层次湿热试验箱。在底部用空枪头保持具腔室，用水。密封在管的残余PEG溶液，并保持它在4℃下。 </li><li>用去离子水彻底冲洗PEG化盖玻片。在阴暗干燥处，直至使用存储。 </li></ol></li></ol> 3.组装流室<ol><li>制造冲孔丙烯酸支架，如在图2中 。确保油管刀片的直径是0.762毫米，并指定一个孔是入口，而另一个是的出口（ 图2）。 </li><li>放置双面胶带条上的丙烯酸类保持器（宽5-6毫米）（ 图2），垂直对准的入口和出口孔，不扰乱任何通道的孔。使用这些条带多通道的墙壁作室。磨砂使用吸管尖磁带。 </li><li>放置在顶部的PEG化的盖玻片，使流动室，用PEG化面朝下放置。 </li><li>为了防止任何溶液泄漏，按一个盖玻片在何处双面胶带被放置的区域的顶部。添加快干环氧树脂胶的顶部和底部（黄色图2），以关闭该室的边缘。 </li><li>管（外径，OD，0.042"）的一段短（2.5厘米）连接到气密注射器和密封用环氧树脂胶关节。 </li><li>长柔性管（外径:0.03"）连接到连接于注射器管和密封用环氧树脂胶关节。 </li><li>填写TUBI纳克挂用DI水注射器。确保有没有气泡。 </li><li>插入管子与环氧胶密封流动室的孔中。 </li><li>放置一个黄色尖端（200微升尖端）上的另一孔作为贮存器。 </li></ol> 4.样品装载到流动腔注意:中性亲可以与其它抗生物素蛋白的蛋白质，如链霉抗代替。所有的反应可在室温下进行，除非它被提及。取在一黄色尖端样品溶液，并安装与溶液的前端到丙烯酸类保持器（ 图2b）的孔中。 <ol><li>以50μl/分钟设定的注射​​泵的流速。负载20微升抗生物素蛋白（在T50溶液25微克/毫升，三的10mM，氯化钠的50mM，pH8）中，并保持10分钟。 </li><li>负载20微升生物素化寡核苷酸（100微米1×TE），并保持10分钟。如果使用终端转移，跳过加载寡核苷酸的。 </li><li>负载20微升以10微升/分钟的流速从第1步到流动室DNA溶液，并保持30分钟。 </li><li>用1×TE洗净流室，然后装入40微升FP-10 DBP（〜80纳米）。 </li><li>在1×TE染色分子的连续流上的60X物镜的荧光显微镜下观察DNA。使用488纳米固态激光器FP（EGFP）-DBP的激励。对DNA分子的充分伸展，根据使用的DNA长度应用不同的流速。例如，应用50微升/分钟为48.5千碱基λ的DNA，和100微升/分钟的T4 DNA的166千碱基。 <ol><li>对于丙烯酸架 ​​回收，浸泡组装流室在家用洗涤剂溶液12。取出胶带和环氧用刀片擦用手完全把他们赶走。疏通与注射器针头孔。保持在去离子水中保持器，直到进一步使用。 </li></ol></li></ol>

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The authors have nothing to disclose.

在这里，我们提出了一个可视化的生物素锚定在表面上长的DNA分子的平台。我们已经报道了拴亲和素蛋白涂层表面与生物素牛血清白蛋白对DNA分子的方法。6在前面的方法，我们发现拴在造成染污DNA分子双插染料DNA光裂解的关键问题表面。由于这些不断激发荧光团有很高的概率攻击DNA磷酸骨架，9激发光功率必须最小化，以避免光裂解。 为了克服这种不便，我?...

拴在玻璃或珠表面大DNA分子可视化的已被用于研究DNA底物，1,2-和高分子物理上DNA-蛋白质相互作用，蛋白质的动态。3,4-一种单-拴系的大的DNA分子平台有几个不同的相对于其他的DNA固定化方法的优点。5首先，拴表面上的大的DNA分子具有无剪切流，这是一种DNA结合蛋白识别其结合位点至关重要的自然随机卷曲构象。第二，这是很容易改变的周围的DNA分子的化学环境中的一系列流室酶反应。第三，微流体剪切流动诱导DNA分子伸展到全轮廓长度，这是非常困难的使用替代的DNA伸长达到100％的方法，如表面固定6和纳通道禁闭。7的充分stretcheD核酸分子也可提供一种可用于基因组图谱上监测酶促运动有用的位置信息。 然而，该DNA束缚方法在该嵌入染料的一个关键缺点如YOYO-1通常会导致拴系的DNA分子，以通过荧光激励光可容易地断裂。一般地，大DNA分子必须与用于可视化在荧光显微镜下的荧光染料染色。为了这个目的，YOYO-1或其它TOTO系染料主要使用，因为当它们相互插入双链DNA这些染料仅发出荧光。8然而，这是众所周知的，双-嵌入染导致光诱导的DNA光裂解因为荧光的嵌入。9。此外，拴在表面上的荧光染色的DNA分子更脆弱，因为剪切流动能发挥上自由移动的DNA分子破坏的力量。因此，我们开发的FP-DBP作为一种新颖的ðNA-染色染料蛋白成像拴在表面上大DNA分子。使用FP-DBP的优点是，它不会导致其所结合的DNA分子的光裂解。10此外，FP-DBP不增加DNA的轮廓长度，而双-嵌入染料增加轮廓长度由约33％。 此视频方法引入了对拴系大DNA分子的PEG生物素表面的实验方法。 图1示出了具有平端和粘性末端圈养的DNA的不同方法。因此，这种染色方法可以适用于任何类型的DNA分子: 图2示出了流室组件，其可通过注射泵来控制，以产生剪切流伸展的DNA分子以及加载的化学和酶的示意图的解决方案。 图3显示拴在PEGylat完全拉伸的DNA分子的显微照片ED表面11和FP-DBP染色。

<table><tbody><tr><td colspan="4">&lt;strong&gt;1. DNA Biotinylation&lt;/strong&gt;</td></tr><tr><td colspan="4">&lt;strong&gt;1.1) Biotinylation of blunt-end DNA using Terminal Transferase (TdT)&lt;/strong&gt;</td></tr><tr><td>Terminal Transferase</td><td>New England Biolabs</td><td>M0315S</td><td>Provided with 10x reaction buffer, 2.5 mM cobalt chloride</td></tr><tr><td>Biotin-11-dUTP</td><td>Invitrogen</td><td>R0081</td><td>Biotin-ddNTP is also available</td></tr><tr><td>T4GT7 Phage DNA</td><td>Nippon Gene</td><td>318-03971</td><td></td></tr><tr><td>Ethylenediaminetetraacetic acid</td><td>Sigma-Aldrich</td><td>E6758</td><td>EDTA</td></tr><tr><td colspan="4">&lt;strong&gt;1.2) Biotinylation of sticky end DNA Using DNA Ligase&lt;/strong&gt;</td></tr><tr><td>T4 DNA Ligase</td><td>New England Biolabs</td><td>M0202S</td><td>Provided with 10x reaction buffer</td></tr><tr><td>Lambda Phage DNA</td><td>Bioneer</td><td>D-2510</td><td>Also available at New England Biolabs</td></tr><tr><td colspan="4">&lt;strong&gt;2. Functionalized Surface Derivatization&lt;/strong&gt;</td></tr><tr><td colspan="4">&lt;strong&gt;2.1) Piranha Cleaning&lt;/strong&gt;</td></tr><tr><td>Coverslip</td><td>Marienfeld-Superior</td><td>0101050</td><td>22 mm x 22 mm, No. 1 Thickness</td></tr><tr><td>Teflon rack</td><td></td><td></td><td>Custom Fabrication</td></tr><tr><td>PTFE Thread Seal Tape</td><td>Han Yang Chemical Co. Ltd.</td><td>3032292</td><td>Teflon&amp;trade; tape</td></tr><tr><td>Sulfuric acid</td><td>Jin Chemical Co. Ltd.</td><td>S280823</td><td>H&lt;sub&gt;2&lt;/sub&gt;SO&lt;sub&gt;4&lt;/sub&gt;, 95% Purity</td></tr><tr><td>Hydrogen peroxide</td><td>Jin Chemical Co. Ltd.</td><td>H290423</td><td>H&lt;sub&gt;2&lt;/sub&gt;O&lt;sub&gt;2&lt;/sub&gt;, 35% in water</td></tr><tr><td>Sonicator</td><td>Daihan Scientific Co. Ltd.</td><td>WUC-A02H</td><td>Table-top Ultrasonic Cleaner</td></tr><tr><td colspan="4">&lt;strong&gt;2.2) Aminosilanization on Glass Surface&lt;/strong&gt;</td></tr><tr><td>&lt;em&gt;N&lt;/em&gt;-[3-(Trimethoxysilyl)propyl]&lt;br/&gt; ethylenediamine</td><td>Sigma-Aldrich</td><td>104884</td><td></td></tr><tr><td>Glacial Acetic Acid</td><td>Duksan Chemicals</td><td>414</td><td>99% Purity</td></tr><tr><td>Methyl Alcohol</td><td>Jin Chemical Co. Ltd.</td><td>M300318</td><td>99.9% Purity</td></tr><tr><td>Polypropylene Container</td><td>Qorpak</td><td>PLC-04907</td><td></td></tr><tr><td>Ethyl Alcohol</td><td>Jin Chemical Co. Ltd.</td><td>A300202</td><td>99.9% Purity</td></tr><tr><td colspan="4">&lt;strong&gt;2.3) PEGylation of the coverslip&lt;/strong&gt;</td></tr><tr><td>Sodium Bicarbonate</td><td>Sigma-Aldrich</td><td>S5761</td><td></td></tr><tr><td>Syringe Filter</td><td>Sartorius</td><td>16534----------K</td><td></td></tr><tr><td>Biotin-PEG-SC</td><td>Laysan Bio</td><td>Biotin-PEG-SC-5000</td><td></td></tr><tr><td>mPEG-SVG</td><td>Laysan Bio</td><td>MPEG-SVA-5000</td><td></td></tr><tr><td>Acetone</td><td>Jin Chemical Co. Ltd.</td><td>A300129</td><td>99% Purity</td></tr><tr><td>Microscope Slides</td><td>Marienfeld-Superior</td><td>1000612</td><td>~76 mm x 26 mm x 1&amp;nbsp;mm</td></tr><tr><td colspan="4">&lt;strong&gt;3. Assembling a Flow Chamber&lt;/strong&gt;</td></tr><tr><td>Acrylic Support</td><td></td><td></td><td>Custom Fabrication</td></tr><tr><td>Double-sided Tape</td><td>3M</td><td></td><td>Transparent type</td></tr><tr><td>Quick-dry Epoxy</td><td>3M</td><td></td><td></td></tr><tr><td>Polyethylene Tubing</td><td>Cole-Parmer</td><td>06417-11, 06417-21</td><td></td></tr><tr><td>Gas Tight 250 &amp;micro;l Syringe</td><td>Hamilton</td><td>81165</td><td></td></tr><tr><td>Syringe Pump</td><td>New Era Pump Systems Inc.</td><td>NE-1000</td><td></td></tr><tr><td colspan="4">&lt;strong&gt;4. Sample Loading into Flow Chamber&lt;/strong&gt;</td></tr><tr><td>Neutravidin</td><td>Thermo Scientific</td><td>31000</td><td></td></tr><tr><td>Tris base</td><td>Sigma-Aldrich</td><td>T1503-5KG</td><td>Trizma base</td></tr><tr><td>Microscope</td><td>Olympus</td><td>IX70</td><td></td></tr><tr><td>EMCCD Camera</td><td>Q Imaging</td><td>Rolera EM-C2</td><td></td></tr><tr><td>Solid-state Laser (488&amp;nbsp;nm)</td><td>Oxxius</td><td>LBX488</td><td></td></tr><tr><td>Alconox</td><td>Alconox Inc.</td><td></td><td></td></tr></tbody></table>

visualization of surface-tethered large dna molecules with a fluorescent protein dna binding peptide

Large DNA molecules tethered on the functionalized glass surface have been utilized in polymer physics and biochemistry particularly for investigating interactions between DNA and its binding proteins. Here, we report a method that uses fluorescent microscopy for visualizing large DNA molecules tethered on the surface. First, glass coverslips are biotinylated and passivated by coating with biotinylated polyethylene glycol, which specifically binds biotinylated DNA via avidin protein linkers and significantly reduces undesirable binding from non-specific interactions of proteins or DNA molecules on the surface. Second, the DNA molecules are biotinylated by two different methods depending on their terminals. The blunt ended DNA is tagged with biotinylated dUTP at its 3' hydroxyl terminus, by terminal transferase, while the sticky ended DNA is hybridized with biotinylated complimentary oligonucleotides by DNA ligase. Finally, a microfluidic shear flow makes single DNA molecules stretch to their full contour lengths after being stained with fluorescent protein-DNA binding peptide (FP-DBP).

图1示出了两个不同的DNA束缚方法依赖于DNA分子的末端结构。 图1a示出了粘端的DNA分子是如何与互补的生物素化的寡核苷酸，其被固定在亲和素包被的PEG表面上进行杂交。 图1b，示出了另外的生物素化的ddNTP或的dNTP的3&#39;羟基通过末端转移酶钝端DNA的基团。我们增加了DNA分子和生物素之间的柔性连接体。这增加了结扎和抗生物素蛋白-?...

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Visualization of Surface-tethered Large DNA Molecules with a Fluorescent Protein DNA Binding Peptide

We present an approach for visualizing fluorescent protein DNA binding peptide (FP-DBP)-stained large DNA molecules tethered on the polyethylene glycol (PEG) and avidin-coated glass surface and stretched with microfluidic shear flows.

表面拴DNA大分子的可视化与荧光蛋白的DNA结合肽

Large DNA molecules tethered on the functionalized glass surface have been utilized in polymer physics and biochemistry particularly for investigating ...

visualization-surface-tethered-large-dna-molecules-with-fluorescent

Research

JoVE Journal

Biology

10.7K 次观看.  Sogang University. We present an approach for visualizing fluorescent protein DNA binding peptide (FP-DBP)-stained large DNA molecules tethered on the polyethylene glycol (PEG) and avidin-coated glass surface and stretched with microfluidic shear flows. 

视频: Visualization of Surface-tethered Large DNA Molecules with a Fluorescent Protein DNA Binding Peptide

Single-molecule Manipulation of G-quadruplexes by Magnetic Tweezers

Non-canonical nucleic acid secondary structure G-quadruplexes (G4) are involved in diverse cellular processes, such as DNA replication, transcription, RNA processing, and telomere elongation. During these processes, various proteins bind and resolve G4 structures to perform their function. As the function of G4 often depends on the stability of its folded structure, it is important to investigate how G4 binding proteins regulate the stability of G4. This work presents a method to manipulate single G4 molecules using magnetic tweezers, which enables studies of the regulation of G4 binding proteins on a single G4 molecule in real time. In general, this method is suitable for a wide scope of applications in studies for proteins/ligands interactions and regulations on various DNA or RNA secondary structures.

A single-molecule magnetic tweezers platform to manipulate G-quadruplexes is reported, which allows for the study of G4 stability and regulation by various proteins.

磁性镊子 quadruplexes 单分子操作

Non-canonical nucleic acid secondary structure G-quadruplexes (G4) are involved in diverse cellular processes, such as DNA replication, transcription, RNA ...

科研

生物化学

DNA Curtains Shed Light on Complex Molecular Systems During Homologous Recombination

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

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

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

遗传学

Visualizing Protein-DNA Interactions in Live Bacterial Cells Using Photoactivated Single-molecule Tracking

Protein-DNA interactions are at the heart of many fundamental cellular processes. For example, DNA replication, transcription, repair, and chromosome organization are governed by DNA-binding proteins that recognize specific DNA structures or sequences. In vitro experiments have helped to generate detailed models for the function of many types of DNA-binding proteins, yet, the exact mechanisms of these processes and their organization in the complex environment of the living cell remain far less understood. We recently introduced a method for quantifying DNA-repair activities in live Escherichia coli cells using Photoactivated Localization Microscopy (PALM) combined with single-molecule tracking. Our general approach identifies individual DNA-binding events by the change in the mobility of a single protein upon association with the chromosome. The fraction of bound molecules provides a direct quantitative measure for the protein activity and abundance of substrates or binding sites at the single-cell level. Here, we describe the concept of the method and demonstrate sample preparation, data acquisition, and data analysis procedures.

Photoactivated localization microscopy (PALM) combined with single-molecule tracking allows direct observation and quantification of protein-DNA interactions in live Escherichia coli cells.

可视化的活细菌细胞蛋白-DNA相互作用使用光活化的单分子跟踪

Protein-DNA interactions are at the heart of many fundamental cellular processes. For example, DNA replication, transcription, repair, and chromosome ...

免疫与感染

Combining QD-FRET and Microfluidics to Monitor DNA Nanocomplex Self-Assembly in Real-Time

Advances in genomics continue to fuel the development of therapeutics that can target pathogenesis at the cellular and molecular level. Typically functional inside the cell, nucleic acid-based therapeutics require an efficient intracellular delivery system. One widely adopted approach is to complex DNA with a gene carrier to form nanocomplexes via electrostatic self-assembly, facilitating cellular uptake of DNA while protecting it against degradation. The challenge lies in the rational design of efficient gene carriers, since premature dissociation or overly stable binding would be detrimental to the cellular uptake and therapeutic efficacy. Nanocomplexes synthesized by bulk mixing showed a diverse range of intracellular unpacking and trafficking behavior, which was attributed to the heterogeneity in size and stability of nanocomplexes. Such heterogeneity hinders the accurate assessment of the self-assembly kinetics and adds to the difficulty in correlating their physical properties to transfection efficiencies or bioactivities. We present a novel convergence of nanophotonics (i.e. QD-FRET) and microfluidics to characterize the real-time kinetics of the nanocomplex self-assembly under laminar flow. QD-FRET provides a highly sensitive indication of the onset of molecular interactions and quantitative measure throughout the synthesis process, whereas microfluidics offers a well-controlled microenvironment to spatially analyze the process with high temporal resolution (~milliseconds). For the model system of polymeric nanocomplexes, two distinct stages in the self-assembly process were captured by this analytic platform. The kinetic aspect of the self-assembly process obtained at the microscale would be particularly valuable for microreactor-based reactions which are relevant to many micro- and nano-scale applications. Further, nanocomplexes may be customized through proper design of microfludic devices, and the resulting QD-FRET polymeric DNA nanocomplexes could be readily applied for establishing structure-function relationships. 

We present a novel and powerful integration of nanophotonics (QD-FRET) and microfluidics to investigate the formation of polyelectrolyte polyplexes, which is expected to provide better control and synthesis of uniform and customizable polyplexes for future nucleic acid-based therapeutics.

结合QD - FRET技术和微流体实时监测DN​​A纳米复合自组装

Advances in genomics continue to fuel the development of therapeutics that can target pathogenesis at the cellular and molecular level. Typically ...

生物学

Visualizing Single Molecular Complexes In Vivo Using Advanced Fluorescence Microscopy

Full insight into the mechanisms of living cells can be achieved only by investigating the key processes that elicit and direct events at a cellular level. To date the shear complexity of biological systems has caused precise single-molecule experimentation to be far too demanding, instead focusing on studies of single systems using relatively crude bulk ensemble-average measurements. However, many important processes occur in the living cell at the level of just one or a few molecules; ensemble measurements generally mask the stochastic and heterogeneous nature of these events. Here, using advanced optical microscopy and analytical image analysis tools we demonstrate how to monitor proteins within a single living bacterial cell to a precision of single molecules and how we can observe dynamics within molecular complexes in functioning biological machines. The techniques are directly relevant physiologically. They are minimally-perturbative and non-invasive to the biological sample under study and are fully attuned for investigations in living material, features not readily available to other single-molecule approaches of biophysics. In addition, the biological specimens studied all produce fluorescently-tagged protein at levels which are almost identical to the unmodified cell strains ("genomic encoding"), as opposed to the more common but less ideal approach for generating significantly more protein than would occur naturally ('plasmid expression'). Thus, the actual biological samples which will be investigated are significantly closer to the natural organisms, and therefore the observations more relevant to real physiological processes.

Visualizing Single Molecular Complexes In Vivo Using Advanced Fluorescence Microscopy

Here we demonstrate the protocols for performing single-molecule fluorescence microscopy on living bacterial cells to enable functional molecular complexes to be detected, tracked and quantified.

可视化单分子络合物在体内使用高级荧光显微

Full insight into the mechanisms of living cells can be achieved only by investigating the key processes that elicit and direct events at a cellular ...

Functional Surface-immobilization of Genes Using Multistep Strand Displacement Lithography

Immobilization of genes on lithographically structured surfaces allows the study of compartmentalized gene expression processes in an open microfluidic bioreactor system. In contrast to other approaches towards artificial cellular systems, such a setup allows for a continuous supply with gene expression reagents and simultaneous draining of waste products. This facilitates the implementation of cell-free gene expression processes over extended periods of time, which is important for the realization of dynamic gene regulatory feedback systems. Here we provide a detailed protocol for the fabrication of genetic biochips using a simple-to-use lithographic technique based on DNA strand displacement reactions, which exclusively uses commercially available components. We also provide a protocol on the integration of compartmentalized genes with a polydimethylsiloxane (PDMS)-based microfluidic system. Furthermore, we show that the system is compatible with total internal reflection fluorescence (TIRF) microscopy, which can be used for the direct observation of molecular interactions between DNA and molecules contained in the expression mix.

We describe a simple lithographic procedure for the immobilization of gene-length DNA molecules on a surface, which can be used to perform cell-free gene expression experiments on biochips.

基于多步法链位移光刻的基因功能表面固定化研究

Immobilization of genes on lithographically structured surfaces allows the study of compartmentalized gene expression processes in an open microfluidic ...

生物工程

<meta charset="utf8"></head><body>用于单分子研究的原位核小体重构

In Situ Nucleosome Assembly for Single-Molecule Correlative Force and Fluorescence Microscopy

Nucleosomes constitute the primary unit of eukaryotic chromatin and have been the focus of numerous informative single-molecule investigations regarding their biophysical properties and interactions with chromatin-binding proteins. Nucleosome reconstitution on DNA for these studies typically involves a salt dialysis procedure that provides precise control over the placement and number of nucleosomes formed along a DNA tether. However, this protocol is time-consuming and requires a substantial amount of DNA and histone octamers as inputs. To offer an alternative strategy, an in situ nucleosome reconstitution method for single-molecule force and fluorescence microscopy that utilizes the histone chaperone Nap1 is described. This method enables users to assemble nucleosomes on any DNA template without the need for strong nucleosome positioning sequences, adjust nucleosome density on demand, and use fewer reagents. In situ nucleosome formation occurs within seconds, offering a simpler experimental workflow and a convenient transition into single-molecule measurements. Examples of two downstream assays for probing nucleosome mechanics and visualizing the behavior of individual proteins on chromatin are further described.

<meta charset="utf8"></head><body>作者聚焦：用于单分子技术的高效核小体重构

This article presents a detailed experimental procedure for reconstituting nucleosome-containing DNA tethers for single-molecule correlative force and fluorescence microscopy. It further describes several downstream experiments that can be conducted to visualize the binding behavior of chromatin-interacting proteins and analyze changes in the physical properties of nucleosomes.

用于单分子相关力和荧光显微镜的原位核小体组装

Nucleosomes constitute the primary unit of eukaryotic chromatin and have been the focus of numerous informative single-molecule investigations regarding ...

<meta charset="utf8"></head><body>通过 ssDNA-RPA 束生长观察到的 CMG 介导的 DNA 解旋

Single-Molecule Real-Time Visualization of DNA Unwinding by CMG Helicase

Faithful genome duplication is essential for preserving the genetic stability of dividing cells. DNA replication is carried out during the S phase by a dynamic complex of proteins termed the replisome. At the heart of the replisome is the&#160;CDC45-MCM2-7-GINS (CMG) helicase, which separates the two strands of the DNA double helix such that DNA polymerases can copy each strand. During genome duplication, replisomes must overcome a plethora of obstacles and challenges. Each of these threatens genome stability, as failure to replicate DNA completely and accurately can lead to mutations, diseases, or cell death. Therefore, it is of great interest to understand how CMG functions in the replisome during both normal replication and replication stress. Here, we describe a total internal reflection fluorescence (TIRF) microscopy assay using recombinant purified proteins, which allows for real-time visualization of surface-tethered stretched DNA molecules by individual CMG complexes. This assay provides a powerful platform to investigate CMG behavior at the single-molecule level, allowing helicase dynamics to be directly observed with real-time control over reaction conditions.

<meta charset="utf8"></head><body>作者聚焦:通过单分子可视化揭示真核 DNA 复制的动力学

This protocol demonstrates performing a single-molecule assay for live visualization of DNA unwinding by CMG helicase. It describes (1) preparing a DNA substrate, (2) purifying fluorescently labeled Drosophila melanogaster CMG helicase, (3) preparing a microfluidic flow cell for total internal reflection fluorescence (TIRF) microscopy, and (4) the single-molecule DNA unwinding assay.

通过 CMG 解旋酶对 DNA 解旋进行单分子实时可视化

Faithful genome duplication is essential for preserving the genetic stability of dividing cells. DNA replication is carried out during the S phase by a ...

表面拴DNA大分子的可视化与荧光蛋白的DNA结合肽

摘要

探索更多视频

此视频中的章节

磁性镊子 quadruplexes 单分子操作

DNA Curtains Shed Light on Complex Molecular Systems During Homologous Recombination

可视化的活细菌细胞蛋白-DNA相互作用使用光活化的单分子跟踪

结合QD - FRET技术和微流体实时监测DNA纳米复合自组装

可视化单分子络合物在体内使用高级荧光显微

基于多步法链位移光刻的基因功能表面固定化研究

用于单分子相关力和荧光显微镜的原位核小体组装

用于单分子相关力和荧光显微镜的原位核小体组装

用于单分子相关力和荧光显微镜的原位核小体组装

通过 CMG 解旋酶对 DNA 解旋进行单分子实时可视化

表面拴DNA大分子的可视化与荧光蛋白的DNA结合肽

摘要

探索更多视频

此视频中的章节

磁性镊子 quadruplexes 单分子操作

DNA Curtains Shed Light on Complex Molecular Systems During Homologous Recombination

可视化的活细菌细胞蛋白-DNA相互作用使用光活化的单分子跟踪

结合QD - FRET技术和微流体实时监测DN​​A纳米复合自组装

可视化单分子络合物在体内使用高级荧光显微

基于多步法链位移光刻的基因功能表面固定化研究

用于单分子相关力和荧光显微镜的原位核小体组装

用于单分子相关力和荧光显微镜的原位核小体组装

用于单分子相关力和荧光显微镜的原位核小体组装

通过 CMG 解旋酶对 DNA 解旋进行单分子实时可视化

结合QD - FRET技术和微流体实时监测DNA纳米复合自组装