这项工作得到了国家卫生研究院[R01GM121847]的支持;纪念斯隆·凯特林癌症中心（MSKCC）支持赠款/核心赠款（P30 CA008748）;和 MSKCC 功能基因组学倡议。

作者没有什么可透露的。

与典型的体外TIRF 单分子实验相比，此处描述的检测的单分子成像由于使用细胞提取物和高浓度荧光标记抗体而异常复杂。与一轮表面PEGylation的更常见做法相比，第二轮PEGylation（第2步）大大减少了非特异性抗体对检测表面15的结合。扩散荧光抗体的高浓度会导致极高的荧光背景，掩盖单分子检测抗体结合。为了减少这种荧光背景，激光事件角度远高于临界角（第 3.4 步）。抗体检测也因氟磷不稳定性过大而减弱，这会限制量化居住时间的能力。遇到此问题时，用更可拍照的荧光剂替换氟磷，例如与三重状态淬火47结合的氟磷，或降低激光照明强度。虽然氧气清理系统常用于体外单分子实验，以抑制氟磷光吸收48，49，这里描述的检测可以提供一个非常慷慨的检测窗口，而无需执行任何氧气清理系统15。此外，氧气清理系统可以大大降低真核细胞无翻译效率。因此，氧气清理系统在使用此检测之前应仔细评估。
需要指出的是，由于试剂的单分子分辨率，检测检测到试剂制剂的微小变异。例如，复制的翻译提取物制剂可以显示可能通过批量方法检测到或无法检测的不同活动。此外，翻译提取物和其他试剂的冻结-解冻周期会迅速损害翻译活动。我们建议在计划进行系统研究之前，先用易于获得的材料进行小规模的试验，以测试条件。为了进行系统研究，建议大规模制备翻译试剂、冷冻/解冻敏感试剂的一次性别名，以及执行协议步骤的一致性。在液氮中闪烁并储存在 -80 °C 的转化提取物显示至少两年内活动损失最小。这些措施的应用可以有效抑制实验变异，提高单分子测定的稳健性。
由于该方法结合了现有的无体积细胞翻译系统和 体外 单分子成像技术，因此此处没有讨论这两个领域的通用问题的故障排除。此类通用问题的例子包括记者 mRNA 或 PEGylated 封面的低质量制备和细胞提取物的低翻译活性。在这里，我们将重点讨论此方法的具体问题。除了协议中讨论的几个技术问题外，另一个潜在的问题可能是流道中的抗体结合率低或没有抗体结合，因为翻译条件预计具有良好的活动性。有几个可能性可以排除故障。生物基化mRNA固定到检测表面的效率可以通过将补充和氟化DNA寡聚物与固定的mRNA和成像荧光DNA:RNA复式成像来评估。翻译组合和生物素化记者 mRNA 的质量可以通过运行批量翻译检测来检验。此外，在 mRNA 固定流动通道中执行翻译反应后，翻译反应解决方案可从通道中传出，并用于记者活动分析，以确认通道内翻译的发生。如有必要，可使用过高的 mRNA 表面密度进行通道内转换反应，以便记者活动检测更容易检测。
此测定的主要限制是其启动动力学的分辨率。在此测定中，直接实验输出（第一次到达时间）包含第一轮启动时间和肽拉长时间，用于翻译表皮和 35 个额外的 codon。虽然肽拉长时间的贡献可以纠正（步骤6.9.），因此，这个检测是最敏感的测量启动动力学发生在分钟数的顺序，这似乎是一个通用属性的上限依赖启动15。在实验中，很容易适应这种分析来研究其他启动机制。但是，如果启动机制在秒数顺序上发生得非常快，则此检测不太可能解决启动动力学问题。
此处描述的单分子方法结合了单分子成像和基于提取物的翻译，可实时和在无活性细胞转换期间测量单个依赖于上限的翻译事件。该检测允许从批量翻译分析轻松过渡到单分子检测，同时保留批量翻译动力学。因此，单分子动力学观测可以直接参考相应的批量结果进行解释。先前的方法，包括最近开发的体内方法11、12、13、14，缺乏对单个翻译启动事件的动能测量的分辨率，需要对翻译机制的假设从实验观测中提取启动时间平均值。此处介绍的单分子方法为量化主动依赖上限翻译的平均启动时间提供了第一个无假设方法。此外，虽然利用发光检测的散装动能测量方法，为46、50年代迄今依赖上限的转化动力学提供最定量和最系统的批量特征，但此处描述的单分子测定测量平均启动动能变化，其灵敏度高于散装测定（图7）。此外，单分子数据还保留了单 mRNA 转换随机性和异质性，这些属性在批量测量中平均值。翻译动力学的单分子分辨率可用于系统动能分析，揭示翻译机制与其他方法所无法企及的见解。
此检测与用于翻译研究的现有生化和遗传方法兼容。例如，可以通过生成因子耗尽提取物并补充野生型或突变因子来研究特定因子的转化功能。此外，通过补充荧光标记因子，研究可以潜在地研究因子结合和翻译进展之间的动能关系。使用两种不同的表位/抗体对，此协议可能会从单色检测扩展到双色检测，从而能够同时检测两个不同的编码序列。这种适应将允许跟踪不同阅读帧的翻译，并可用于帧转换和开始选址研究。或者，双色系统可用于检测由上行和下游开放读取帧编码的标记多肽的抗体相互作用，以监控单个 mRNA 上的上下游翻译事件。这些扩展可以支持广泛的机械研究，研究依赖于上限的翻译及其法规。

在真核系统的翻译主要通过7甲基瓜诺辛（m7G）帽依赖通路1发生。研究表明，真核翻译的启动步骤是限速的，也是第2、3、4条规则的共同目标。利用遗传5、生化6、7、8、结构9和基因组10大块方法，对上限依赖转化机制进行了广泛的研究。虽然这些方法确定了调节依赖上限启动的多种机制，但其分辨率仅限于异质和异步启动事件信号的合奏平均值。最近，在体内翻译事件中，通过测量荧光抗体与新生多肽11、12、13、14上的表皮结合的方法，对个体进行了可视化。然而，这些新方法在解决单个启动事件的能力方面也受到限制，因为多个荧光抗体必须结合一种新生肽，以便从高细胞内荧光背景中解决单个转化事件。在许多生物相互作用中，解决的个体动能事件为理解在分子水平上无法同步的复杂多步骤和重复的生物过程提供了重要的见解。需要采用新的方法来跟踪各个翻译事件的动态，以便更好地了解依赖上限的启动和监管。
我们最近开发了一种体外测定，用单分子分辨率15测量依赖上限的启动动力学。考虑到这个启动途径3、16中涉及大量已知和未知的蛋白质因子，单分子测定被开发为与现有的体外无细胞翻译系统兼容，从而受益于细胞因子的保存和强大的翻译活性17、18、19、20、21、22、23、24、25。此外，使用无细胞翻译系统可以更兼容地比较单分子观测结果和以前的批量结果。这种方法将新的单分子动力学见解直接整合到现有的依赖上限启动的机械框架中。为了建立单分子测定，传统的无细胞翻译系统被修改为三种方式:在记者mRNA的开放读取帧（ORF）的开头插入表位编码序列:记者mRNA的3+末端是生物素化，以促进mRNA末端系绳到单分子检测表面:和荧光标签抗体补充翻译提取物。这些修改只需要基本的分子生物学技术和常用的试剂。此外，这些修饰和单分子成像条件保留了无体细胞翻译反应的翻译动力学15。
在此测定（图1），5+端封顶和3+端生物素化记者mRNA被固定在流室的链球菌涂层检测表面。然后，流室中将填充一种无细胞的转化混合物，并辅以荧光标记的抗体。在mRNA翻译发生在表位序列26、27下游的大约30-40个子元之后，表位从核糖体出口隧道中浮现出来，并变得能够与荧光标记的抗体相互作用。这种相互作用是快速的，它通过单分子荧光成像技术进行检测，能够在无活性细胞转化过程中跟踪具有单分子分辨率的翻译动力学。这种检测在体外研究依赖上限的翻译动力学及其调节方面应大有裨益，特别是对于体外分析中具有工作量的系统。
建立这种单分子检测的先决条件是工作散装无细胞翻译分析，这可以通过翻译提取物实现，无论是商业上可用的或按照先前描述的方法28准备。真核转化提取物可以从不同的细胞中获取，包括真菌、哺乳动物和植物28。对于成像，此测定需要配备可调谐激光强度和事件角度的 TIRF 显微镜、电动样品阶段、机动流体系统和样品温度控制装置。这种要求对于现代 体外 单分子TIRF实验来说是通用的，而且可能以不同的方式实现。这里介绍的实验使用了一个客观类型的TIRF系统，该系统由商用显微镜、软件和配件组成，全部列在 材料表中。

<table>
	<tbody>
		<tr>
			<td>100X oil objective, N.A. 1.49</td>
			<td>Olympus</td>
			<td>UAPON 100XOTIRF</td>
			<td></td>
		</tr>
		<tr>
			<td>Acryamide/bis (40%, 19:1)</td>
			<td>Bio-Rad</td>
			<td>161-0144</td>
			<td></td>
		</tr>
		<tr>
			<td>Alkaline liquid detergent</td>
			<td>Decon</td>
			<td>5332</td>
			<td></td>
		</tr>
		<tr>
			<td>Aminosilane (N-(2-Aminoethyl)-3-Aminopropyltrimethoxysilane)</td>
			<td>UCT Specialties, LLC</td>
			<td>A0700</td>
			<td></td>
		</tr>
		<tr>
			<td>Andor ixon Ultra DU 897V EMCCD</td>
			<td>Andor</td>
			<td>DU-897U-CSO-#BV</td>
			<td></td>
		</tr>
		<tr>
			<td>Andor Solis software</td>
			<td>Andor</td>
			<td></td>
			<td>For controlling the Andor EMCCD</td>
		</tr>
		<tr>
			<td>Band-pass filter</td>
			<td>Chroma</td>
			<td>532/640/25</td>
			<td></td>
		</tr>
		<tr>
			<td>Band-pass filter</td>
			<td>Chroma</td>
			<td>NF03-405/488/532/635E-25</td>
			<td></td>
		</tr>
		<tr>
			<td>Biotin-PEG-SVA</td>
			<td>Laysan Bio Inc</td>
			<td>Biotin-PEG-SVA</td>
			<td></td>
		</tr>
		<tr>
			<td>Coenzyme A free acid</td>
			<td>Prolume</td>
			<td>309-250</td>
			<td></td>
		</tr>
		<tr>
			<td>Coolterm software</td>
			<td></td>
			<td></td>
			<td>For controlling the syringe pump</td>
		</tr>
		<tr>
			<td>Desktop computer</td>
			<td>Dell</td>
			<td></td>
			<td>For controlling the microscope, camera, stage, and pump.</td>
		</tr>
		<tr>
			<td>Dichroic mirror</td>
			<td>Semrock</td>
			<td>R405/488/532/635</td>
			<td></td>
		</tr>
		<tr>
			<td>Direct-zol RNA microprep 50RNX</td>
			<td>Fisher Scientific</td>
			<td>NC1139450</td>
			<td></td>
		</tr>
		<tr>
			<td>Dual-Luciferase Reporter Assay System</td>
			<td>Promega</td>
			<td>E1910</td>
			<td></td>
		</tr>
		<tr>
			<td>Epoxy</td>
			<td>Devcon</td>
			<td>14250</td>
			<td></td>
		</tr>
		<tr>
			<td>Firefly luciferin D-Luciferin free acid</td>
			<td>Prolume</td>
			<td>306-250</td>
			<td></td>
		</tr>
		<tr>
			<td>Glacial acetic acid</td>
			<td>Fisher Scientific</td>
			<td>BP1185500</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrogen perioxide</td>
			<td>Sigma-Aldrich</td>
			<td>216763-500ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Immersion oil</td>
			<td>Olympus</td>
			<td>Z-81226A</td>
			<td>Low auto-fluorescence</td>
		</tr>
		<tr>
			<td>Luciferase Assay System</td>
			<td>Promega</td>
			<td>E1500</td>
			<td></td>
		</tr>
		<tr>
			<td>MEGASCRIPT T7 Transcription Kit</td>
			<td>Thermo fisher</td>
			<td>AM1334</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Fisher Scientific</td>
			<td>MMX04751</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>Olympus</td>
			<td>IX83</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope slide</td>
			<td>Thermo Scientific</td>
			<td>3048</td>
			<td></td>
		</tr>
		<tr>
			<td>Monoclonal anti-FLAG M2-Cy3</td>
			<td>Sigma-Aldrich</td>
			<td>A9594</td>
			<td></td>
		</tr>
		<tr>
			<td>mPEG-SVA</td>
			<td>Laysan Bio Inc</td>
			<td>mPEG-SVA-5000</td>
			<td></td>
		</tr>
		<tr>
			<td>MS(PEG)4</td>
			<td>Thermo Scientific</td>
			<td>22341</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl (5M)</td>
			<td>Thermo Scientific</td>
			<td>AM9760G</td>
			<td></td>
		</tr>
		<tr>
			<td>No 1.5 microscope Cover glass</td>
			<td>Fisherband</td>
			<td>12-544-C</td>
			<td></td>
		</tr>
		<tr>
			<td>Olympus Laser, 532nm 100mM</td>
			<td>Olympus digital Laser system</td>
			<td>CMR-LAS 532nm 100mW</td>
			<td></td>
		</tr>
		<tr>
			<td>Olympus TirfCtrl software</td>
			<td>Olympus</td>
			<td></td>
			<td>For controlling the laser intensity and incident angle</td>
		</tr>
		<tr>
			<td>Optical table</td>
			<td>TMC vibration control</td>
			<td>63-563</td>
			<td>With vibration isolation</td>
		</tr>
		<tr>
			<td>Phenol chloroform isoamyl alcohol mix</td>
			<td>Sigma-Aldrich</td>
			<td>77617-100ml</td>
			<td></td>
		</tr>
		<tr>
			<td>Pierce RNA 3&#39; End Biotinylation Kit</td>
			<td>Thermo Scientific</td>
			<td>20160</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium hydroxide pellets</td>
			<td>Sigma-Aldrich</td>
			<td>P1767-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Prior motorized XY translation stage</td>
			<td>Prior</td>
			<td>PS3J100</td>
			<td></td>
		</tr>
		<tr>
			<td>Prior PriorTest software</td>
			<td>Prior</td>
			<td></td>
			<td>For controlling the Prior motorized stage</td>
		</tr>
		<tr>
			<td>Recombinant RNasin RNase Inhibitor</td>
			<td>Promega</td>
			<td>N2515</td>
			<td></td>
		</tr>
		<tr>
			<td>Stage top Incubator</td>
			<td>In vivo scientific (world precision Instruments)</td>
			<td>98710-1</td>
			<td>With a custom built acrylic cage</td>
		</tr>
		<tr>
			<td>Staining jar</td>
			<td>Fisher Scientific</td>
			<td>08-817</td>
			<td></td>
		</tr>
		<tr>
			<td>Streptavidin</td>
			<td>Thermo Scientific</td>
			<td>43-4301</td>
			<td></td>
		</tr>
		<tr>
			<td>Sulfuric acid</td>
			<td>Fisher Scientific</td>
			<td>A300212</td>
			<td></td>
		</tr>
		<tr>
			<td>SYBR green II</td>
			<td>Fisher Scientific</td>
			<td>S7564</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe</td>
			<td>Hamilton</td>
			<td>1725RN</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe pump</td>
			<td>Harvard apparatus</td>
			<td>55-3333</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris (1M), pH = 7.0</td>
			<td>Thermo Scientific</td>
			<td>AM9850G</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultrasonic Bath</td>
			<td>Branson</td>
			<td>CPX1800H</td>
			<td></td>
		</tr>
		<tr>
			<td>Urea</td>
			<td>Sigma-Aldrich</td>
			<td>U5378-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Vaccinia Capping system</td>
			<td>New England Biolabs</td>
			<td>M2080S</td>
			<td></td>
		</tr>
		<tr>
			<td>Zymo-Spin IC Columns</td>
			<td>Zymo Research</td>
			<td>C1004</td>
			<td></td>
		</tr>
	</tbody>
</table>

an in vitro single-molecule imaging assay for the analysis of cap-dependent translation kinetics

依赖上限的蛋白质合成是真核细胞的主要转化途径。虽然各种生化和遗传方法允许对依赖上限的翻译及其调节进行广泛的研究，但这种翻译途径的高分辨率动能特征仍然缺乏。最近，我们开发了一种 体外 测定法，以单分子分辨率测量依赖上限的转化动力学。该检测基于荧光标记的抗体结合到新生的表皮标记的多肽。通过成像抗体与新生肽（核糖体）mRNA复合物的结合和分离，可以跟踪单个 mRNA 的翻译进展。在这里，我们提出了建立此测定的协议，包括 mRNA 和 PEGylated 幻灯片制备、翻译的实时成像以及单分子轨迹分析。此分析能够跟踪单个依赖上限的翻译事件，并解决关键的翻译动力学，如启动和拉长率。该测定可广泛应用于不同的翻译系统，在依赖上限的翻译动力学和转化控制机制的 体外研究中 应大有裨益。

按照所描述的协议，在3'末端系记者mRNA（图1）的活性无细胞翻译过程中，能够与新生的N终端标记多肽进行单个抗体相互作用的成像，具有单分子分辨率。报告使用三种合成 mRNA 进行最小演示实验:LUC（编码未标记的路西法酶）、LUCFLAG（编码 3xFLAG 标记的路西法酶）和hp-LUCFLAG（LUC FLAG，在 5' 领先区域具有稳定的茎环）（图2C）。使用荧光酶编码 mRNA 可比较单分子观测和散装发光测量。3'生物基化mRNA的完整性通过去硝化聚丙烯酰胺凝胶电泳（PAGE）和RNA染色来评估。高质量的 mRNA 显示单个干净的波段，不应显示额外的快速迁移信号 （图 3A）。糖精塞雷维西亚翻译提取物在这里使用，并准备遵循协议，以前描述30和修改15。无细胞转化反应中路西法酶活性与酵母提取物和生物基化LUCFLAG mRNA 的批量测量，这些元素缺少或包含 m7G 上限，显示了LUCFLAG mRNA 翻译的上限刺激（图3B）。
对于单分子成像，翻译提取物辅以 67 nM 的 Cy3 标记抗 FLAG 抗体 （Cy3-αFLAG）。这种抗体具有高标签比2-7染料每个抗体。532 nm 激光器在目标时被设置为 10 μW。激光事件角度设置为 71.5°，高于我们设置的 63.8° 临界角。低水平的荧光是从含有mRNA但缺乏翻译组合的检测表面（图4A）中成像的。对于大约 2 毫米（宽）x 50 μm（深度）x 24 mm（长度）的通道尺寸，150 μl/min 的流量输送速率为 3 - 4 秒的试剂交换时间。在5s的翻译混合交付，背景荧光水平增加，由于扩散荧光抗体。大约2分钟后，翻译混合交付到 LUCFLAG mRNA包含通道，Cy3点开始出现在视野中，并继续积累约30分钟（图4B）。非特异性抗体结合到表面，使用3xFLAG缺乏 LUC mRNA（图2C）测量，保持在非常低的水平15。mRNA/核糖体/肽绑定Cy3-βFLAG的信号在优化的激光事件角度下清晰可见，但可以通过低激光事件角度的高荧光背景（图4C）遮盖。
为了分析 Cy3-αFLAG 绑定事件，选定的 Cy3-αFLAG 点（图 5A）的荧光强度按帧计算整个电影，然后连接形成强度轨迹 （图 5B）。原始轨迹可能显得嘈杂，可能需要使用非线性前向后过滤器进行改进，以减少背景噪音41。使用步进检测算法对轨迹42进行数字化，可以进一步改进。对于所有轨迹，由于翻译组合中的荧光抗体扩散（图 5B），试剂交换期间会出现普遍背景荧光水平增加。抗体与新生的聚肽结合导致荧光瞬间增加，而抗体/聚肽与mRNA分离会导致瞬时荧光减少（图5B）。
抗体结合的停留时间可用于测量打开读取帧的总解码时间。LUCFLAG mRNA 的常住时间直方图适合日志正常分布（图 6A），并产生肽合成率 2.5 ± 0.1 氨基酸每秒15。第一次到达时间直方图适合移动 （3 参数） 日志正常函数 （图 6B）。第一次到达直方图的广泛传播显示了单mRNA翻译活动中高度的异质性。直方图表明，单LUCFLAG mRNA 分子上的第一个肽合成事件最早可在翻译混合交付后 2 分钟和最晚 20 分钟开始（图 6B）。与LUCFLAG mRNA 的翻译相比，使用hp-LUCFLAG mRNA 翻译的第一次到达时间直方图显示抗体结合速度较慢（图7A）。然而，使用与这些相同的mRNA无细胞翻译反应的散装发光测量并不能解决翻译动力学（图7B，C）的差异。这些结果表明，与用于检测启动动力学中微小变化的散装动能路西法酶活动测量相比，我们方法的分辨率更高。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61648/61648fig01.jpg"> 图1:协议流程图。显示了盖片和幻灯片制备、单分子室组装、TIRF 成像和数据采集以及数据分析步骤的示意图表示。TIRF 成像步骤包括 mRNA 固定化的示意图描述和流通道中的翻译。指示检测表面组件、荧光标记抗体和细胞提取物组件。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61648/61648fig01large.jpg" target="_blank">请单击此处查看此图的更大版本。</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61648/61648fig2v2.jpg"> 图2:单分子测定mRNA设计。（A） 为了使批量翻译分析适应单分子测定，散装记者ORF的DNA模板通过N端表位标记序列插入进行修改，以生成标记记者编码ORF。指示ORF和N端标记编码区域。（B） mRNA 为 5+m7G 上限，允许依赖上限的翻译和 3+生物素化，使其固定到单分子检测表面。5+m7G 上限和 3+生物素在未标记和标记的 ORF RNA 上注明。（C） 路西法酶编码 mRNA 用于演示单分子测定。 LUC 和 LUC标志 mRNA 分别编码缺乏并包含 N 端 3xFLAG 标签的路西法酶。 hp-LUC标志 mRNA 包含额外的插入，在 LUC标志 mRNA 5+-领导者中引入发夹二级结构。表示发夹恒温性、3+-多（A）尾和3+生物素。所有三个 mRNA 都包括一个 30 nt 3+-poly（A） 尾部，用于更高效的依赖上限的翻译。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61648/61648fig02large.jpg" target="_blank">请单击此处查看此图的更大版本。</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61648/61648fig3v2.jpg"> 图3:单分子记者mRNA完整性和批量翻译。（A） 单分子记者 mRNA 的代表否认 PAGE 成像。5+m7G 封顶和 3+生物素化 LUCFLAG mRNA 被加载到 RNA 梯子旁边的 10% 聚丙烯酰胺凝胶上。（B） 单分子记者 mRNA 在无散装细胞的翻译反应中保留了 5' 的上限依赖性。3~生物素化 LUCFLAG mRNA，要么缺少 （+帽），要么包含 （+ 帽） 5+m7G 帽，在 25 °C 的 S. cerevisiae 翻译提取物中翻译了 30 分钟。路西法酶活动从两个独立反应中测量，并与 -cap mRNA 活动正常化，该活动被任意设置为 1.0。标准偏差与平均值的偏差由错误条表示。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61648/61648fig03large.jpg" target="_blank">请单击此处查看此图的更大版本。</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/61648/61648fig04.jpg"> 图4:对含有固定mRNA的检测表面进行成像。（A） 在没有翻译组合的情况下背景荧光。（B） Cy3 荧光点在翻译混合交付后 30 分钟在视场中出现，并具有优化的激光事件角度。（C） 翻译混合交付后 30 分钟的图像视场，激光事件角度较低。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61648/61648fig04large.jpg" target="_blank">请单击此处查看此图的更大版本。</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/61648/61648fig05.jpg"> 图5:图像分析。（A） Cy3 点在没有（左面板）或（右面板）选址的视野中。（B） 选定 Cy3 点的单分子轨迹示例。黑色箭头表示由于在翻译组合交付过程中扩散抗体，背景荧光升高。绿色箭头表示由于 Cy3-αFLAG 绑定导致荧光强度突然增加。红色箭头表示由于 Cy3-αFLAG/鼻肽与 mRNA 分离而突然降低荧光强度。Cy3-αFLAG 绑定事件的停留时间用双头箭头表示。指示原始、过滤和数字化数据。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61648/61648fig05large.jpg" target="_blank">请单击此处查看此图的更大版本。</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/61648/61648fig06.jpg"> 图6:Cy3-αFLAG与 LUC标志 mRNA翻译结合的动能分析。（A） Cy3-αFLAG 绑定居住时间直方图适合日志正常分布。（B） Cy3-αFLAG 绑定第一次到达时间直方图适合移动 （3 参数） 日志正常函数。改编自王等人15 与许可。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61648/61648fig06large.jpg" target="_blank">请单击此处查看此图的更大版本。</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/61648/61648fig7v3.jpg"> 图7:单分子测定比散装发光测定具有更高的启动动力学分辨率。（A） Cy3-αFLAG 的首次到达时间直方图与LUCFLAG和hp-LUCFLAG mRNA 的翻译结合显示，mRNA 5+领导中的一个小发夹结构导致启动速度较慢。N = 10650 轨迹（LUCFLAG），由 3 个数据集和 N = 4079 轨迹（hp-LUCFLAG）组合而为 2 个数据集。（B ， C）散装路西法酶活性检测动能测量没有解决LUCFLAG （N = 9 数据集） 和hp-LUCFLAG （N = 6 数据集） mRNA 翻译的启动动力学差异。（B） 散装发光动力学。（C） 瓦西连科等人描述的（B）中发光动力学曲线第二衍生物高斯安装确定的第一轮翻译时间的散射图。46.（C）中的红圆和条形分别表示计算的第一轮翻译时间的平均值和标准偏差。改编自王等人15与许可。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61648/61648fig07large.jpg" target="_blank">请单击此处查看此图的更大版本。</a>

Watch this Scientific Journal Video about An In Vitro Single-Molecule Imaging Assay for the Analysis of Cap-Dependent Translation Kinetics at JoVE.com

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

跟踪单个翻译事件允许对依赖上限的翻译机制进行高分辨率动能研究。在这里，我们演示了基于荧光标记抗体和表皮标记的新生肽之间的成像相互作用的 体外 单分子检测。该方法使启动和肽伸长动力学的单分子特征 在主动体外 帽依赖翻译。

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 ...

an-vitro-single-molecule-imaging-assay-for-analysis-cap-dependent

Nanyang Technological University

Research

JoVE Journal

Biochemistry

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

3.0K 次观看.  Memorial Sloan Kettering Cancer Center. 跟踪单个翻译事件允许对依赖上限的翻译机制进行高分辨率动能研究。在这里，我们演示了基于荧光标记抗体和表皮标记的新生肽之间的成像相互作用的 体外 单分子检测。该方法使启动和肽伸长动力学的单分子特征 在主动体外 帽依赖翻译。

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

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

Phosphoinositides in the cell membrane are signaling lipids with multiple cellular functions. Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) is a determinant phosphoinositide of the plasma membrane (PM), and it is required to modulate ion channels, actin dynamics, exocytosis, endocytosis, intracellular signaling, and many other cellular processes. However, the spatial organization of PI(4,5)P2 in the PM is controversial, and its nanoscale distribution is poorly understood due to the technical limitations of research approaches. Here by utilizing single molecule localization microscopy and the Pleckstrin Homology (PH) domain based dual color fluorescent probes, we describe a novel method to visualize the nanoscale distribution of PI(4,5)P2 in the PM in fixed membrane sheets as well as live cells.

PI(4,5)P2 regulates various cellular functions, but its nanoscale organization in the cell plasma membrane is poorly understood. By labeling PI(4,5)P2 with a dual-color fluorescent probe fused to the Pleckstrin Homology domain, we describe a novel approach to study the PI(4,5)P2 spatial distribution in the plasma membrane at the nanometer scale.

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

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 ...

Toeprinting Analysis of Translation Initiation Complex Formation on Mammalian mRNAs

Translation initiation is the rate-limiting step of protein synthesis and represents a key point at which cells regulate their protein output. Regulation of protein synthesis is the key to cellular stress-response, and dysregulation is central to many disease states, such as cancer. For instance, although cellular stress leads to the inhibition of global translation by attenuating cap-dependent initiation, certain stress-response proteins are selectively translated in a cap-independent manner. Discreet RNA regulatory elements, such as cellular internal ribosome entry sites (IRESes), allow for the translation of these specific mRNAs. Identification of such mRNAs, and the characterization of their regulatory mechanisms, have been a key area in molecular biology. Toeprinting is a method for the study of RNA structure and function as it pertains to translation initiation. The goal of toeprinting is to assess the ability of in vitro transcribed RNA to form stable complexes with ribosomes under a variety of conditions, in order to determine which sequences, structural elements, or accessory factors are involved in ribosome binding&#8212;a pre-cursor for efficient translation initiation. Alongside other techniques, such as western analysis and polysome profiling, toeprinting allows for a robust characterization of mechanisms for the regulation of translation initiation.

Toeprinting aims to measure the ability of in vitro transcribed RNA to form translation initiation complexes with ribosomes under a variety of conditions. This protocol describes a method for toeprinting mammalian RNA and can be used to study both cap-dependent and IRES-driven translation.

哺乳动物基因翻译起始复合体形成的 Toeprinting 分析

Translation initiation is the rate-limiting step of protein synthesis and represents a key point at which cells regulate their protein output. Regulation ...

Covalent Immobilization of Proteins for the Single Molecule Force Spectroscopy

In recent years, atomic force microscopy (AFM) based single molecule force spectroscopy (SMFS) extended our understanding of molecular properties and functions. It gave us the opportunity to explore a multiplicity of biophysical mechanisms, e.g., how bacterial adhesins bind to host surface receptors in more detail. Among other factors, the success of SMFS experiments depends on the functional and native immobilization of the biomolecules of interest on solid surfaces and AFM tips. Here, we describe a straightforward protocol for the covalent coupling of proteins to silicon surfaces using silane-PEG-carboxyls and the well-established N-hydroxysuccinimid/1-ethyl-3-(3-dimethyl-aminopropyl)carbodiimid (EDC/NHS) chemistry in order to explore the interaction of pilus-1 adhesin RrgA from the Gram-positive bacterium Streptococcus pneumoniae (S. pneumoniae) with the extracellular matrix protein fibronectin (Fn). Our results show that the surface functionalization leads to a homogenous distribution of Fn on the glass surface and to an appropriate concentration of RrgA on the AFM cantilever tip, apparent by the target value of up to 20% of interaction events during SMFS measurements and revealed that RrgA binds to Fn with a mean force of 52 pN. The protocol can be adjusted to couple via site specific free thiol groups. This results in a predefined protein or molecule orientation and is suitable for other biophysical applications besides the SMFS.

This protocol describes the covalent immobilization of proteins with a heterobifunctional silane coupling agent to silicon-oxide surfaces designed for the atomic force microscopy based single molecule force spectroscopy which is exemplified by the interaction of RrgA (pilus-1 tip adhesin of S. pneumoniae) with fibronectin.

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

Cell proteomes are often characterized using electrophoresis assays, where all species of proteins in the cells are non-specifically labeled with a fluorescent dye and are spotted by a photodetector following their separation. Single molecule fluorescence imaging can provide ultrasensitive protein detection with its ability for visualizing individual fluorescent molecules. However, the application of this powerful imaging method to electrophoresis assays is hampered by the lack of ways to characterize the homogeneity of fluorescent labeling of each protein species across the proteome. Here, we developed a method to evaluate the labeling homogeneity across the proteome based on a single molecule fluorescence imaging assay. In our measurement using a HeLa cell sample, the proportion of proteins labeled with at least one dye, which we termed &#8216;labeling occupancy&#8217; (LO), was determined to range from 50% to 90%, supporting the high potential of the application of single molecule imaging to sensitive and precise proteome analysis.

Here, we present a protocol to assess the labeling homogeneity for each protein species in a complex protein sample 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

Cytoskeletal motors are responsible for a wide variety of functions in eukaryotic cells, including mitosis, cargo transport, cellular motility, and others. Many of these functions require motors to operate in ensembles. Despite a wealth of knowledge about the mechanisms of individual cytoskeletal motors, comparatively less is known about the mechanisms and emergent behaviors of motor ensembles, examples of which include changes to ensemble processivity and velocity with changing motor number, location, and configuration. Structural DNA nanotechnology, and the specific technique of DNA origami, enables the molecular construction of well-defined architectures of motor ensembles. The shape of cargo structures as well as the type, number and placement of motors on the structure can all be controlled. Here, we provide detailed protocols for producing these ensembles and observing them using total internal reflection fluorescence microscopy. Although these techniques have been specifically applied for cytoskeletal motors, the methods are generalizable to other proteins that assemble in complexes to accomplish their tasks. Overall, the DNA origami method for creating well-defined ensembles of motor proteins provides a powerful tool for dissecting the mechanisms that lead to emergent motile behavior.

The goal of this protocol is to form ensembles of molecular motors on DNA origami nanostructures and observe the ensemble motility using total internal reflection fluorescence microscopy.

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 ...

Single Cell Analysis Of Transcriptionally Active Alleles By Single Molecule FISH

Gene transcription is an essential process in cell biology, and allows cells to interpret and respond to internal and external cues. Traditional bulk population methods (Northern blot, PCR, and RNAseq) that measure mRNA levels lack the ability to provide information on cell-to-cell variation in responses. Precise single cell and allelic visualization and quantification is possible via single molecule RNA fluorescence in situ hybridization (smFISH). RNA-FISH is performed by hybridizing target RNAs with labeled oligonucleotide probes. These can be imaged in medium/high throughput modalities, and, through image analysis pipelines, provide quantitative data on both mature and nascent RNAs, all at the single cell level. The fixation, permeabilization, hybridization and imaging steps have been optimized in the lab over many years using the model system described herein, which results in successful and robust single cell analysis of smFISH labeling. The main goal with sample preparation and processing is to produce high quality images characterized by a high signal-to-noise ratio to reduce false positives and provide data that are more accurate. Here, we present a protocol describing the pipeline from sample preparation to data analysis in conjunction with suggestions and optimization steps to tailor to specific samples.&#160;

Single molecule RNA fluorescence in situ hybridization (smFISH) is a method to accurately quantify levels and localization of specific RNAs at the single cell level. Here, we report our validated lab protocols for wet-bench processing, imaging and image analysis for single cell quantification of specific RNAs.

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

This novel total internal reflection fluorescence microscopy-based assay facilitates the simultaneous measurement of the length of the catalytic cycle for hundreds of individual restriction endonuclease (REase) molecules in one experiment. This assay does not require protein labeling and can be carried out with a single imaging channel. In addition, the results of multiple individual experiments can be pooled to generate well-populated dwell-time distributions. Analysis of the resulting dwell-time distributions can help elucidate the DNA cleavage mechanism by revealing the presence of kinetic steps that cannot be directly observed. Example data collected using this assay with the well-studied REase, EcoRV - a dimeric Type IIP restriction endonuclease that cleaves the palindromic sequence GAT&#8595;ATC (where &#8595; is the cut site) - are in agreement with prior studies. These results suggest that there are at least three steps in the pathway to DNA cleavage that is initiated by introducing magnesium after EcoRV binds DNA in its absence, with an average rate of 0.17 s-1 for each step.

Using quantum-dot-labeled DNA and total internal reflection fluorescence microscopy, we can investigate the reaction mechanism of restriction endonucleases while using unlabeled protein. This single-molecule technique allows for massively multiplexed observation of individual protein-DNA interactions, and data can be pooled to generate well-populated dwell-time distributions.

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 Imaging of EWS-FLI1 Condensates Assembling on DNA

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 (FET) family of fusion oncoproteins, is one of the most frequently involved fusion genes in Ewing sarcoma. These FET family fusion proteins typically harbor a low-complexity domain (LCD) of FET protein at their N-terminus and a DNA-binding domain (DBD) at their C-terminus. EWS-FLI1 has been confirmed to form biomolecular condensates at its target binding loci due to LCD-LCD and LCD-DBD interactions, and these condensates can recruit RNA polymerase II to enhance gene transcription. However, how these condensates are assembled at their binding sites remains unclear. Recently, a single-molecule biophysics method-DNA Curtains-was applied to visualize these assembling processes of EWS-FLI1 condensates. Here, the detailed experimental protocol and data analysis approaches are discussed for the application of DNA Curtains in studying the biomolecular condensates assembling on target DNA.

Here, we describe the use of the single-molecule imaging method, DNA Curtains, to study the biophysical mechanism of EWS-FLI1 condensates assembling on DNA.

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 ...

An Optimized Single-Molecule Pull-Down Assay for Quantification of Protein Phosphorylation

Phosphorylation is a necessary posttranslational modification that regulates protein function and directs cell signaling outcomes. Current methods to measure protein phosphorylation cannot preserve the heterogeneity in phosphorylation across individual proteins. The single-molecule pull-down (SiMPull) assay was developed to investigate the composition of macromolecular complexes via immunoprecipitation of proteins on a glass coverslip followed by single-molecule imaging. The current technique is an adaptation of SiMPull that provides robust quantification of the phosphorylation state of full-length membrane receptors at the single-molecule level. Imaging thousands of individual receptors in this way allows for quantifying protein phosphorylation patterns. The present protocol details the optimized SiMPull procedure, from sample preparation to imaging. Optimization of glass preparation and antibody fixation protocols further enhances data quality. The current protocol provides code for the single-molecule data analysis that calculates the fraction of receptors phosphorylated within a sample. While this work focuses on phosphorylation of the epidermal growth factor receptor (EGFR), the protocol can be generalized to other membrane receptors and cytosolic signaling molecules.

The present protocol describes sample preparation and data analysis to quantify protein phosphorylation using an improved single-molecule pull-down (SiMPull) assay.

用于蛋白质磷酸化定量的优化单分子下拉测定

Phosphorylation is a necessary posttranslational modification that regulates protein function and directs cell signaling outcomes. Current methods to ...

用于分析依赖帽的翻译动力学的 体外 单分子成像测定

用于分析依赖帽的翻译动力学的体外单分子成像测定