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Bioquímica

光学镊子研究翻译调控中的RNA-蛋白质相互作用

Published: February 12, 2022
doi:
10.3791/62589
, ,
1Helmholtz Institute for RNA-based Infection Research (HIRI),Helmholtz Zentrum für Infektionsforschung (Helmholtz Centre for Infection Research), 2Medical Faculty,Julius-Maximilians University Würzburg

Summary

该协议为使用光学镊子研究RNA-蛋白质相互作用提供了完整的实验工作流程。概述了几种可能的实验设置，包括光学镊子与共聚焦显微镜的组合。

Abstract

RNA采用不同的结构褶皱，这对其功能至关重要，因此可以影响细胞中的不同过程。此外，RNA的结构和功能可以通过各种 反式作用因子（例如蛋白质，代谢物或其他RNA）来调节。例如，移码RNA分子是位于编码区域的调节RNA，它们直接将核糖体翻译成替代的开放阅读框，从而充当基因开关。在与蛋白质或其他 反式因子结合后，它们也可能采用不同的褶皱。为了剖析RNA结合蛋白在翻译中的作用以及它们如何调节RNA结构和稳定性，同时研究这些RNA-蛋白复合物的相互作用和力学特征至关重要。这项工作说明了如何利用单分子荧光耦合光学镊子以高分辨率探索RNA-蛋白质复合物的构象和热力学景观。例如，详细阐述了SARS-CoV-2程序性核糖体移码元件与锌指抗病毒蛋白 的反式作用因子短同种型的相互作用。此外，使用共聚焦单元监测荧光标记的核糖体，这最终将实现翻译伸长率的研究。荧光偶联OT测定可广泛用于探索各种RNA-蛋白复合物或调节翻译的 跨作用因子，并有助于研究基于RNA的基因调控。

Introduction

通过mRNA将遗传信息从DNA传递到蛋白质是一个复杂的生化过程，通过细胞内的大分子相互作用在各个层面上进行精确调节。对于翻译调控，RNA-蛋白相互作用赋予了对各种刺激和信号快速反应的关键作用1，2。一些RNA-蛋白质相互作用会影响mRNA的稳定性，从而改变RNA翻译活性的时间。其他RNA-蛋白质相互作用与重新编码机制有关，例如停止密码子读通，旁路或程序化核糖体移码（PRF）3，4，5，6，7。最近，许多RNA结合蛋白（RMP）已被证明与刺激性mRNA元件和翻译机制相互作用，以决定何时以及在细胞中发生多少重新编码7，8，9，10，11。因此，为了剖析RNA结合蛋白在翻译中的作用以及它们如何调节RNA结构和稳定性，详细研究这些RNA-蛋白复合物的相互作用原理和力学性质至关重要。

数十年的工作为研究翻译的多步骤和多组分过程奠定了基础，该过程依赖于翻译机器的RNA和蛋白质组分之间的复杂通信来实现速度和准确性12，13，14。理解复杂调节事件的关键下一步是在高精度平移过程中确定力、时间尺度和结构决定因素12，15，16，17。单分子工具的出现进一步阐明了RNA构象动力学的研究，特别是反式辅助因子在翻译过程中如何作用于RNA结构，这些工具的出现进一步阐明了16，17，18，19，20，21，22，23，24，25，26.

光学镊子（OT）代表了一种高度精确的单分子技术，已被应用于研究多种RNA依赖性动态过程，包括转录和翻译26，27，28，29，30，31，32。光学镊子的使用允许详细探测这些过程的分子相互作用，核酸结构以及热力学性质，动力学和能量学16，17，22，33，34，35，36，37，38，39.光学镊子测定基于用聚焦激光束捕获微观物体。在典型的OT实验中，感兴趣的分子被拴在两个透明（通常是聚苯乙烯）珠子之间（图1A）27。然后，这些珠子被光学陷阱捕获，其行为类似于弹簧。因此，施加在分子上的力可以基于磁珠从聚焦激光束中心（陷阱中心）的位移来计算。最近，光学镊子与共聚焦显微镜相结合（图1B），可实现荧光或Förster共振能量转移（FRET）测量40，41，42。这开辟了一个全新的可能实验领域，允许同时测量，从而精确地将力光谱和荧光数据联系起来。

在这里，我们展示了使用光学镊子结合共聚焦显微镜的实验，以研究调节翻译移帧的蛋白质 – RNA相互作用。在物镜和冷凝器之间，具有五个通道的流通池可实现层流连续的样品应用。通过微流体通道，可以直接注射各种组分，这减少了动手时间，并且在整个实验过程中允许非常少的样品消耗。

首先，提出了辅助OT实验设计的基本指南，并讨论了各种设置的优点和缺点。接下来，描述了样品的制备和实验工作流程，并提供了数据分析方案。为了代表一个例子，我们概述了从RNA拉伸实验中获得的结果，以研究SARS-CoV-2移码RNA元件（图2A）， 其反式因子锌指抗病毒蛋白（ZAP）的短同种型改变了病毒RNA从替代阅读框架的翻译43。此外，还证明荧光标记的核糖体可用于这种OT共聚焦测定，这对于监测翻译机制的处理能力和速度是有用的。这里介绍的方法可用于快速测试不同缓冲液，配体或其他细胞组分的作用，以研究翻译的各个方面。最后，讨论了常见的实验陷阱以及如何排除故障。下面，概述了实验设计中的一些关键点。

施工设计
原则上，有两种常见的方法来创建OT兼容的RNA构建体。第一种方法采用长RNA分子，该分子与互补的DNA手柄杂交，从而产生由两个RNA / DNA杂交区域组成的构建体，中间是单链RNA序列的两侧（图2B）。大多数OT RNA实验都采用了这种方法33，44，45。

第二种方法利用具有短悬伸（约20 nt）的dsDNA句柄15，17。然后将这些悬垂与RNA分子杂交。虽然设计上更复杂，但dsDNA手柄的使用克服了DNA/RNA杂交系统的一些局限性。原则上，即使是非常长的手柄（>10kb）也可以实现，这对于共聚焦测量更方便。此外，RNA分子可以连接到DNA手柄上，以提高系留稳定性。

最终标签策略
该结构必须通过强分子相互作用拴附到珠子上。虽然有一些方法可用于手柄与珠子的共价键合46，但强但非共价的相互作用，如链霉亲和素 – 生物素和地高辛 – 抗体通常用于OT实验15，33，35，45。在所描述的方案中，构建体用生物素或地高丝宁标记，并且珠子分别涂有链霉亲和素或针对地高辛的抗体（图1A）。这种方法适用于施加高达约60 pN（每系绳）的力47。此外，使用不同的5’和3’标记策略可以确定珠子之间形成的系绳的方向17。

用于荧光测量的蛋白质标记
对于共聚焦成像，有几种常用的荧光标记方法。例如，荧光团可以共价附着在蛋白质中的氨基酸残基上，或者通过反应性有机基团通过位点导向诱变引入。硫醇或胺反应性染料可分别用于半胱氨酸和赖氨酸残基的标记。有几种可逆的保护方法可以增加标记的特异性48，49，但是天然蛋白质通常会在多个残基处标记。虽然荧光团的小尺寸可能带来优势，但非特异性标记可能会干扰蛋白质活性，因此信号强度可能会变化49。此外，根据标记效率的不同，不同实验之间的信号强度可能会有所不同。因此，应在实验之前执行活动检查。

如果感兴趣的蛋白质含有N端或C端标签，例如His标签或链球菌标签，则这些标签的特定标记代表了另一种流行的方法。此外，标签靶向标记可降低荧光团干扰蛋白质活性的机会，并可提高溶解度49。然而，标签特异性标记通常产生单荧光团标记的蛋白质，这可能很难检测。另一种特异性标记方法可以通过使用抗体来完成。

微流体设置
OT与微流体系统的结合允许在不同的实验条件之间快速过渡。此外，电流系统利用了维持流通池内部的层流，这排除了相对于流动方向的垂直方向上来自其他通道的液体的混合。因此，层流对于实验设计特别有利。目前，通常使用多达5个通道的流通池（图3）。

Protocol

1. 样品制备 将感兴趣的序列克隆到含有Lambda DNA片段的载体中，这些载体用作手柄序列（图2）43，50。 首先生成DNA模板，用于随后通过PCR 进行体外 转录（图2B;反应1）。在此PCR步骤中，T7启动子被添加到感测DNA分子的5’末端32，33<sup…

Representative Results

在本节中，重点主要放在荧光光学镊子对RNA-蛋白质/配体相互作用的测量上。有关一般RNA光学镊子实验的描述和相应的代表性结果，请参见32。有关RNA / DNA – 蛋白质相互作用的更详细讨论，另见1，2，26，59，60。 原则上，RBP或任何其他 <e…

Discussion

在这里，我们展示了使用荧光耦合光学镊子来研究RNA分子与各种配体的相互作用和动态行为。下面，将讨论当前技术的关键步骤和局限性。

协议中的关键步骤
与许多其他方法一样，样品的质量对于获得可靠数据至关重要。因此，为了获得尽可能高质量的样品，值得花时间优化样品制备的程序。优化步骤包括正确的引物设计、退火温度、RNA 和蛋白质纯化步骤。<…

Declarações

The authors have nothing to disclose.

Acknowledgements

我们感谢Anuja Kibe和Jun. Prof. Redmond Smyth对手稿的批判性审查。我们感谢塔季扬娜·科赫的专家技术援助。我们感谢Kristyna Pekarkova帮助录制实验视频。我们实验室的工作得到了亥姆霍兹协会的支持，并得到了欧洲研究理事会（ERC）Grant Nr. 948636（到NC）的资助。

Materials

Bacterial Strains
E. coli HB101 lab collection N/A cloning of the vectors
Chemicals and enzymes
Sodium chloride Sigma-Aldrich 31424 Buffers
Biotin-16-dUTP Roche 11093070910 Biotinylation
BSA Sigma-Aldrich A4737 Buffers
Catalase Lumicks N/A Oxygen scavanger system
Dithiothreitol (DTT) Melford Labs D11000 Buffers
DNAse I from bovine pancreas Sigma-Aldrich D4527 in vitro transcription
dNTPs Th.Geyer 11786181 PCR
EDTA Sigma-Aldrich E9884 Buffers
Formamide Sigma-Aldrich 11814320001 Buffers
Glucose Sigma-Aldrich G8270-1KG Oxygen scavanger system
Glucose-oxidase Lumicks N/A Oxygen scavanger system
HEPES Carl Roth HN78.3 Buffers
Magnesium chloride Carl Roth 2189.1 Buffers
Phusion DNA polymerase NEB M0530L Gibson assembly, cloning
Potassium chloride Merck 529552-1KG Buffers
PrimeSTAR GXL DNA Polymerase Takara Bio Clontech R050A PCR
Pyrophosphotase, thermostabile, inorganic NEB M0296L in vitro transcription
RNase Inhibitor Molox 1000379515 Buffers
rNTPS life technologies R0481 in vitro transcription
Sodium thiosulophate Sigma-Aldrich S6672-500G Bleach deactivation
Sytox Green Lumicks N/A confocal measurements
T4 DNA Polymerase NEB M0203S Biotinylation
T5 exonuclease NEB M0363S Gibson assembly, cloning
T7 RNA polymerase Produced in-house N/A in vitro transcription
Taq DNA polymerase NEB M0267S PCR
Taq ligase Biozym L6060L Gibson assembly, cloning
TWEEN 20 BioXtra Sigma-Aldrich P7949 Buffers
Kits
Monolith Protein Labeling Kit RED-NHS 2nd Generation (Amine Reactive) Nanotemper MO-L011 Used for ribosome labeling
Purefrex 2.0 GeneFrontier PF201-0.25-EX Ribosomes used for the labeling
Oligonucleotides
5' handle T7 forward Microsynth custom order 5’ – CTTAATACGACTCACTATAGGTC
CTTTCTGTGGACGCC – 3’, used to generate OT in vitro transcription template in PCR 1
3’ handle reverse Microsynth custom order 5' -  GTCAAAGTGCGCCCCGTTATCC – 3', used to generate OT in vitro transcription template in PCR 1
5' handle forward Microsynth custom order 5' – TCCTTTCTGTGGACGCCGC – 3' , used to generate 5' handle in PCR 2
5’ handle reverse Microsynth custom order 5’ – CATAAATACCTCTTTACTAATATA
TATACCTTCGTAAGCTAGCGT – 3’, used to generate 5' handle in PCR 2
3’ handle forward Microsynth custom order 5' – ATCCTGCAACCTGCTCTTCGCC
AG – 3', used to generate 3' handle in PCR 3
3’ handle reverse 5’labeled with digoxigenin Microsynth custom order 5' -[Dig]-GTCAAAGTGCGCCCCGTTATCC – 3', used to generate 3' handle in PCR 3
DNA vectors
pMZ_OT produced in-house N/A further description in "Structural studies of Cardiovirus 2A protein reveal the molecular basis for RNA recognition and translational control"
Chris H. Hill, Sawsan Napthine, Lukas Pekarek, Anuja Kibe, Andrew E. Firth, Stephen C. Graham, Neva Caliskan, Ian Brierley
bioRxiv 2020.08.11.245035; doi: https://doi.org/10.1101/2020.08.11.245035
Software and Algorithms
Atom https://atom.io/packages/ide-python N/A
Bluelake Lumicks N/A
Graphpad https://www.graphpad.com/ N/A
InkScape 0.92.3 https://inkscape.org/ N/A
Matlab https://www.mathworks.com/products/matlab.html N/A
POTATO https://github.com/lpekarek/POTATO.git N/A
RNAstructure https://rna.urmc.rochester.edu/RNAstructure.html N/A
Spyder https://www.spyder-ide.org/ N/A
Outro
Streptavidin Coated Polystyrene Particles, 1.5-1.9 µm, 5 ml, 1.0% w/v Spherotech SVP-15-5
Anti-digoxigenin Coated Polystyrene Particles, 2.0-2.4 µm, 2 ml, 0.1% w/v Spherotech DIGP-20-2
Syringes VWR TERUMO SS+03L1
Devices
C-trap Lumicks N/A  optical tweezers coupled with confocal microscopy
DOWNLOAD MATERIALS LIST

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Optical TweezersRNA-protein InteractionsTranslation RegulationSingle Molecule TechniquesFluorescence Assisted Optical TweezersForce-ramp ExperimentsConstant Force MeasurementsReal-time VisualizationDNA-RNA ConstructIn-vitro TranscriptionPCR Reactions
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Pekarek, L., Buck, S., Caliskan, N. Optical Tweezers to Study RNA-Protein Interactions in Translation Regulation. J. Vis. Exp. (180), e62589, doi:10.3791/62589 (2022).
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