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Biochemistry

用于分析依赖帽的翻译动力学的 体外 单分子成像测定

Published: September 15, 2020
doi:
10.3791/61648
, ,
1Molecular Biology Program,Memorial Sloan Kettering Cancer Center

Summary

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。

Abstract

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。

Introduction

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。

Protocol

1. 记者 mRNA 的一代 修改DNA转录模板，通过插入N-总站表位标记编码序列来为”标记”记者mRNA生成DNA转录模板（图2A），对批量分析”未标记”的记者mRNA进行编码。注：3xFLAG/防 FLAG 交互建议用于此检测，因为它具有卓越的灵敏度和短的 3xFLAG 标记长度。但是，该检测与其他表皮/抗体对兼容。 使用线性DNA模板和 体外 转录套件合成未标记和标记的RNA（参见 <st…

Representative Results

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?…

Discussion

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）?…

Disclosures

The authors have nothing to disclose.

Acknowledgements

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。

Materials

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

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Gaba, A., Wang, H., Qu, X. An In Vitro Single-Molecule Imaging Assay for the Analysis of Cap-Dependent Translation Kinetics. J. Vis. Exp. (163), e61648, doi:10.3791/61648 (2020).
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