Name: single-molecule imaging of ews-fli1 condensates assembling on dna
Uploaded: 2021-09-08T15:00:00
Description: Watch this Scientific Journal Video about Single-Molecule Imaging of EWS-FLI1 Condensates Assembling on DNA at JoVE.com

这项工作得到了国家自然科学基金第31670762号资助（Z.Q.）的支持。

1. 脂质双层预混液的制备
<ol><li>用双蒸水（ddH2O）和99%乙醇冲洗玻璃小瓶，并在60°C干燥炉中干燥。</li>
	<li>通过将 1 g 1，2-二油酰基-sn-甘油-3-磷酸胆碱 （DOPC）、100 mg 聚乙二醇反应（聚乙二醇化）脂质（1，2-二油酰基-sn-甘油-3-磷酸乙醇胺-N-[甲氧基（聚乙二醇）-2000]（铵盐）（PEG2000 原液）和 25 mg 生物素化脂质（1，2-二油酰基-sn-甘油-3-磷酸乙醇胺-N-（帽生物素基）（钠盐）（生物素帽原...

<ol>
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由于单分子方法对反应系统的内容物极其敏感，因此在DNA幕实验期间必须投入额外的精力来确保所有材料和溶液的良好质量，特别是在第1和第2节中制备的脂质以及第5节中使用的缓冲液。必须使用更高纯度的试剂来制备缓冲液，并且必须为单分子测定新鲜制备缓冲液
当500 nM mCherry标记的EWS-FLI1被冲洗到腔室中时，含有25×个GGAA序列的Lambda DNA上出现了几个洋红色点。请注意，即?...

转录调控是活细胞中精确表达基因的关键步骤。许多因素，如染色体修饰、转录因子（TF）和非编码RNA，都参与了这一复杂的过程1，2，3。在这些因素中，TF通过识别和结合称为启动子或增强子的特定DNA序列并随后招募其他功能蛋白来激活或抑制转录4，5，6，7，从而有助于转录调控的特异性。几十年来，这些TF如何设法在人类基因组中搜索其靶位点并与涂有组蛋白和非组蛋白DNA结合蛋白的DNA相互作用，这一直困扰着科学家们。在过去的几年中，已经建立了几个用于TF目标搜索机制的经典模型来描述它们如何沿着DNA链8，9，10，11“滑动”，“跳跃”，“跳跃”或“片段间转移”。这些模型侧重于单个TF分子DNA上的搜索行为。然而，最近的研究表明，一些TF在细胞核中单独或与介质复合物一起进行液-液相分离（LLPS）12。观察到的TFs液滴与启动子或增强子区域相关，突出了生物分子冷凝物形成在转录和三维基因组中的作用13，14，15。这些生物分子缩合物与体内和体外缺乏膜的隔室相连。它们通过LLPS形成，其中模块化生物大分子和蛋白质的固有无序区域（IDR）是多价相互作用的两个主要驱动力16。因此，TF不仅可以搜索DNA，还可以在这些凝聚物4，17，18中协同发挥作用。迄今为止，这些转录凝聚物在DNA上的生物物理特性尚不清楚。
因此，本研究旨在应用单分子方法——DNA幕，在体外直接成像TFs在DNA上形成的转录凝聚物的形成和动力学。DNA窗帘是一种用于研究蛋白质和DNA之间相互作用的高通量体外成像平台，已应用于DNA修复19，20，21，靶标搜索22和LLPS17，23，24。DNA幕的流通池涂有生物素化的脂质双层，以钝化表面并允许生物分子在表面上扩散。纳米制造的锯齿形图案限制了DNA的运动。生物素化的Lambda DNA底物可以沿着屏障边缘排列，并被定向缓冲流拉伸。所有分子的相同起始序列和结束序列允许跟踪DNA上的蛋白质并描述结合事件的位置分布25，26。此外，DNA幕与全内反射荧光显微镜（TIRFM）的组合有助于最大限度地减少背景噪声并在单分子水平上检测信号。因此，DNA幕布可能是研究DNA基序上转录凝聚物形成的动力学的一种有前途的方法。本文描述了由染色体易位产生的FUS/EWS/TAF15（FET）家族融合癌蛋白EWS-FLI1的例子。含有25×GGAA（EWS-FLI127的结合序列）的Lambda DNA被用作DNA幕实验中的DNA底物，以观察EWS-FLI1分子如何在DNA上经历LLPS。本文详细讨论了实验方案和数据分析方法。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>488 nm diodepumped solid-state laser</td>
			<td>Coherent</td>
			<td>OBIS488LS</td>
			<td></td>
		</tr>
		<tr>
			<td>561 nm diodepumped solid-state laser</td>
			<td>Coherent</td>
			<td>OBIS561LS</td>
			<td></td>
		</tr>
		<tr>
			<td>Agar</td>
			<td>Rhawn</td>
			<td>R003215-50g</td>
			<td></td>
		</tr>
		<tr>
			<td>biotinylated DOPE</td>
			<td>Avanti</td>
			<td>870273P</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>Sigma</td>
			<td>A7030</td>
			<td></td>
		</tr>
		<tr>
			<td>Chloroform</td>
			<td>Amresco</td>
			<td>1595C027</td>
			<td></td>
		</tr>
		<tr>
			<td>Coating Electra 92</td>
			<td>Allresist GmbH</td>
			<td>AR-PC 5090.02</td>
			<td>The conductive protective coating</td>
		</tr>
		<tr>
			<td>Deoxyribonuclease I bovine</td>
			<td>Sigma</td>
			<td>D5139-2MG</td>
			<td></td>
		</tr>
		<tr>
			<td>DOPC</td>
			<td>Avanti</td>
			<td>850375P</td>
			<td></td>
		</tr>
		<tr>
			<td>DTT</td>
			<td>Sigma</td>
			<td>D9779</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass coverslip</td>
			<td>Fisher Scientific</td>
			<td>12-544-7</td>
			<td></td>
		</tr>
		<tr>
			<td>Hellmanex III</td>
			<td>Sigma</td>
			<td>Z805939-1EA</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Sigma</td>
			<td>60130</td>
			<td></td>
		</tr>
		<tr>
			<td>Lambda DNA</td>
			<td>NEB</td>
			<td>N3013S</td>
			<td></td>
		</tr>
		<tr>
			<td>Lambda Packing Extracts</td>
			<td>Epicentre</td>
			<td>MP5120</td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Sigma</td>
			<td>M2670</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma</td>
			<td>s3014</td>
			<td></td>
		</tr>
		<tr>
			<td>Nanoport</td>
			<td>Idex</td>
			<td>N-333-01</td>
			<td></td>
		</tr>
		<tr>
			<td>NheI-HF</td>
			<td>NEB</td>
			<td>R3131S</td>
			<td></td>
		</tr>
		<tr>
			<td>Nikon Inverted Microscope</td>
			<td>Nikon</td>
			<td>Eclipse Ti</td>
			<td></td>
		</tr>
		<tr>
			<td>NZCYM Broth</td>
			<td>Sigma</td>
			<td>N3643-250G</td>
			<td></td>
		</tr>
		<tr>
			<td>PEG-2000 DOPE</td>
			<td>Avanti</td>
			<td>880130P-1G</td>
			<td></td>
		</tr>
		<tr>
			<td>PEG-8000</td>
			<td>Amresco</td>
			<td>25322-68-3</td>
			<td></td>
		</tr>
		<tr>
			<td>PMMA 200K, ETHYL LACTATE 4%</td>
			<td>Allresist GmbH</td>
			<td>AR-P 649.04</td>
			<td></td>
		</tr>
		<tr>
			<td>PMMA 950K, ANISOLE 2%</td>
			<td>Allresist GmbH</td>
			<td>AR-P 672.02</td>
			<td></td>
		</tr>
		<tr>
			<td>Prime 95B Scientific CMOS camera</td>
			<td>PHOTOMETRICS</td>
			<td>Prime95B</td>
			<td></td>
		</tr>
		<tr>
			<td>proteinase K</td>
			<td>NEB</td>
			<td>P8107S</td>
			<td></td>
		</tr>
		<tr>
			<td>Silica glass slide</td>
			<td>G.Finkenbeiner</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Six-way injection valve</td>
			<td>Idex</td>
			<td>MXP9900-000</td>
			<td></td>
		</tr>
		<tr>
			<td>Streptavidin</td>
			<td>Thermo</td>
			<td>S888</td>
			<td>Diluted with ddH2O</td>
		</tr>
		<tr>
			<td>Syringe pump</td>
			<td>Harvard Apparatus</td>
			<td>Pump11 Elite</td>
			<td></td>
		</tr>
		<tr>
			<td>T4 DNA Ligase</td>
			<td>NEB</td>
			<td>M0202S</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris base</td>
			<td>Sigma</td>
			<td>T6066</td>
			<td></td>
		</tr>
		<tr>
			<td>XhoI</td>
			<td>NEB</td>
			<td>R0146V</td>
			<td></td>
		</tr>
		<tr>
			<td>YOYO-1 Iodide (491/509)</td>
			<td>Invitrogen</td>
			<td>Y3601</td>
			<td>Diluted with DMSO</td>
		</tr>
	</tbody>
</table>

single-molecule imaging of ews-fli1 condensates assembling on dna

由染色体易位产生的融合基因已在许多实体瘤或白血病中发现。EWS-FLI1属于融合癌蛋白的FUS/EWS/TAF15（FET）家族，是尤文肉瘤中最常涉及的融合基因之一。这些FET家族融合蛋白通常在其N末端具有FET蛋白的低复杂性结构域（LCD），在其C末端具有DNA结合结构域（DBD）。由于LCD-LCD和LCD-DBD的相互作用，EWS-FLI1已被证实在其靶结合位点形成生物分子缩合物，这些缩合物可以募集RNA聚合酶II以增强基因转录。然而，这些冷凝物如何在其结合位点组装仍不清楚。最近，一种单分子生物物理学方法——DNA窗帘——被应用于可视化EWS-FLI1凝聚物的这些组装过程。本文讨论了DNA幕在靶DNA上生物分子凝聚物组装中应用的详细实验方案和数据分析方法。

DNA幕的示意图如图1A、图1B和图1D所示。在尤因肉瘤的 NORB1 启动子中发现了包含 25 个不间断重复的 GGAA 的克隆靶序列。该目标序列对于EWS-FLI1招募至关重要28。通过检测用561nm激光获得的mCherry标记的EWS-FLI1信号来可视化EWS-FLI1分子（图1C和图1E）。在建立DNA幕实验?...

Watch this Scientific Journal Video about Single-Molecule Imaging of EWS-FLI1 Condensates Assembling on DNA at JoVE.com

Single-Molecule Imaging of EWS-FLI1 Condensates Assembling on DNA

在这里，我们描述了使用单分子成像方法DNA窗帘来研究EWS-FLI1凝聚物在DNA上组装的生物物理机制。

EWS-FLI1凝聚物在DNA上组装的单分子成像

The fusion genes resulting from chromosomal translocation have been found in many solid tumors or leukemia. EWS-FLI1, which belongs to the FUS/EWS/TAF15 ...

我们提供单分子测定来观察DNA上转录因子的相分离该技术允许实时实现转录因子组装为DNA上的液滴的动态过程。EWS-FL1是尤因肉瘤肿瘤发生中最常参与的融合基因之一。我们的研究可以确定DNA基序数与EWS-FL1凝聚物形成之间的关系，这与肿瘤细胞中贫瘠的基因转录有关，可能有助于预后。该方法可用于体外研究任何转录缩合物或复合物，如P53和MED1。要制备具有锯齿形纳米制造屏障的流通池用于DNA幕实验，请以正确的方向连接输入管和输出管。然后使用两个三毫升注射器用2.5毫升脂质缓冲液清洗流通池，确保流通池中没有气泡。从输出中取出注射器，注射一毫升脂质体溶液三次，每次注射之间孵育五分钟。为了愈合，用2.5毫升脂质缓冲液缓慢重新洗涤流通池，并在室温下孵育30分钟。在孵育过程中，按照文本手稿中所述制备BSA缓冲液。然后在愈合30分钟后，从输出侧用2.5毫升BSA缓冲液洗涤流通池。接下来，分两步从输入侧将 800 μL 链霉亲和素缓冲液注入流通池中，每步后孵育 10 分钟。然后用2.5毫升BSA缓冲液洗涤流通池以去除所有游离链霉亲和素分子。接下来，使用BSA缓冲液用克隆基序稀释生物素Lambda DNA，并以五分钟的间隔缓慢注射四次。在注射过程中，打开科学互补金属氧化物半导体系统中的显微镜。然后用10毫升双蒸水清洗管道，并用水，2%液体比色皿清洁剂和99%乙醇冲洗棱镜和管道连接器。接下来，将至少20毫升的成像缓冲液吸入30毫升的注射器中。在显微镜载物台上设置流通池并将其连接到微流体系统。使用每分钟0.03毫升的流速，将DNA分子冲洗到屏障上10分钟。然后关闭微流体系统并孵育30分钟。随着流动停止，允许DNA横向扩散。要对DNA幕上的EWS-FLI1凝聚形成进行成像，请打开成像软件，在明场下找到并标记九种锯齿形图案的位置。然后以每分钟0.2毫升的速度打开流量，用双链DNA染料染色DNA10分钟。接下来，用成像缓冲液在 100 微升中以 100 纳摩尔的浓度稀释 mCherry EWS-FLI1 蛋白。然后用100微升玻璃注射器通过阀门加载蛋白质样品，并将流速更改为每分钟0.4毫升。打开488纳米激光后，预扫描每个区域以检查DNA分布状态并选择DNA分子均匀分布的区域。然后将488纳米激光器的激光功率设置为10%，将561纳米激光器的激光功率设置为20%。并使用功率计，测量棱镜附近的实际激光功率。开始以两秒的间隔同时使用488和561纳米激光器采集图像。然后将阀门从手动模式更改为进样模式，让成像缓冲液在60秒后将蛋白质样品冲洗到流通池中。要取出游离的 EWS-FLI1，请在仅打开 561 纳米激光器的情况下，继续用成像缓冲液清洗流通池五分钟。然后停止流动并在 37 摄氏度下孵育 10 分钟。10分钟后，以每分钟0.4毫升的速度开启流动，让DNA在不同帧之间以两秒的间隔延伸和获取图像，以获得EWS-FLI1凝合物形成的高通量数据。通过检测用561纳米激光获得的mCherry标记的EWS-FLI1信号来可视化EWS-FLI1分子。可以直接看到DNA底物中25个GGAA重复位点处EWS-FLI1缩合物的体外形成。DNA幕中使用的mCherry EWS-FLI1的特异性通过电泳迁移率位移测定得到证实，该测定使用分别具有和不具有25个GGAA重复序列的DNA模板。当EWS-FLI1浓度从20纳摩尔滴定到500纳摩尔时，EWS-FLI1强度急剧增加。然而，当蛋白质饱和以覆盖GGAA重复序列时，FLI一DBD强度的变化可以忽略不计，这表明EWS-FLI1在DNA上形成缩合物。注射应该非常缓慢和柔软，以便脂质体和DNA可以均匀地分布在流通池上。重要的是进行MSA以确认纯化的转录因子在体外与靶序列特异性结合。这种技术使人们能够探索凝聚物是否会促进转录因子的靶标搜索，以及它对转录的影响。

Research

JoVE Journal

Biochemistry

Single-molecule Super-resolution Imaging of Phosphatidylinositol 4,5-bisphosphate in the Plasma Membrane with Novel Fluorescent Probes

在质膜用新型荧光探针磷脂酰-4,5-二磷酸的单分子超分辨率成像

Phosphoinositides in the cell membrane are signaling lipids with multiple cellular functions. Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) is a ...

科研

生物化学

A Simple, Robust, and High Throughput Single Molecule Flow Stretching Assay Implementation for Studying Transport of Molecules Along DNA

一种简单、健壮且高通量的单分子流动拉伸试验研究分子沿 DNA 运移的方法

We describe a simple, robust and high throughput single molecule flow-stretching assay for studying 1D diffusion of molecules along DNA. In this assay, ...

Single-molecule Manipulation of G-quadruplexes by Magnetic Tweezers

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 ...

Covalent Immobilization of Proteins for the Single Molecule Force Spectroscopy

单分子力谱中蛋白质共价固定化的研究

In recent years, atomic force microscopy (AFM) based single molecule force spectroscopy (SMFS) extended our understanding of molecular properties and ...

Proteome-wide Quantification of Labeling Homogeneity at the Single Molecule Level

单分子水平标记均匀性的全蛋白质定量

Cell proteomes are often characterized using electrophoresis assays, where all species of proteins in the cells are non-specifically labeled with a ...

Production of Dynein and Kinesin Motor Ensembles on DNA Origami Nanostructures for Single Molecule Observation

生产用于单分子观察的DNA折纸纳米结构的Dynein和Kinesin电机组合

Cytoskeletal motors are responsible for a wide variety of functions in eukaryotic cells, including mitosis, cargo transport, cellular motility, and ...

An In Vitro Single-Molecule Imaging Assay for the Analysis of Cap-Dependent Translation Kinetics

An In Vitro Single-Molecule Imaging Assay for the Analysis of Cap-Dependent Translation Kinetics

用于分析依赖帽的翻译动力学的 体外 单分子成像测定

Cap-dependent protein synthesis is the predominant translation pathway in eukaryotic cells. While various biochemical and genetic approaches have allowed ...

Single Cell Analysis Of Transcriptionally Active Alleles By Single Molecule FISH

单分子鱼的转录活性等位基因单细胞分析

Gene transcription is an essential process in cell biology, and allows cells to interpret and respond to internal and external cues. Traditional bulk ...

Single-Molecule Dwell-Time Analysis of Restriction Endonuclease-Mediated DNA Cleavage

限制性核酸内切酶介导的 DNA 切割的单分子停留时间分析

This novel total internal reflection fluorescence microscopy-based assay facilitates the simultaneous measurement of the length of the catalytic cycle for ...

Single Molecule Fluorescence Energy Transfer Study of Ribosome Protein Synthesis

单分子荧光能量转移研究核糖体蛋白质合成

The ribosome is a large ribonucleoprotein complex that assembles proteins processively along mRNA templates. The diameter of the ribosome is approximately ...