荧光RNA适配体 外和细胞 开启动力学的测定
Summary
该协议提供了两种确定荧光RNA适配体菠菜2和西兰花动力学的方法。第一种方法描述了如何使用酶标仪 在体外 测量荧光适配体动力学，而第二种方法详细介绍了通过流式细胞术测量细胞中荧光适配体动力学。
Abstract
荧光RNA适配体已被应用于活细胞中，以标记和可视化RNA，报告基因表达，并激活荧光生物传感器，以检测代谢物和信号分子的水平。为了研究每个系统中的动态变化，需要获得实时测量，但测量的准确性取决于荧光反应的动力学比采样频率快。在这里，我们描述了分别使用配备样品进样器和流式细胞仪的酶标仪来确定荧光RNA适配体的体外和细胞开启动力学的方法。我们表明，Spinach2和西兰花适配体荧光活化的体外动力学可以建模为两相反合反应，并且具有不同的快速相速率常数0.56 s-1和0.35 s-1。此外，我们表明，大肠杆菌中Spinach2荧光激活的细胞动力学仍然足够快，可以在分钟时间尺度上实现准确的采样频率。这些分析荧光活化动力学的方法适用于已开发的其他荧光RNA适配体。
Introduction
荧光反应是产生荧光信号的化学反应。荧光RNA适配体通常通过结合小分子染料来提高其荧光量子产率来执行此功能（图1A）1。已经开发了不同的荧光RNA适配体系统，由特定的RNA适配体序列和相应的染料配体1组成。荧光RNA适配体已作为荧光标签附加到RNA转录本中，可实现mRNA和非编码RNA2，3，4的活细胞成像。它们也被放置在启动子序列之后作为基因表达的荧光报告基因，类似于使用绿色荧光蛋白（GFP）作为报告基因，只是报告功能在RNA水平5，6。最后，荧光RNA适配体已被掺入基于RNA的荧光生物传感器中，其设计用于响应特定的小分子触发荧光反应。基于RNA的荧光生物传感器已被开发用于各种非荧光代谢物和信号分子的活细胞成像7，8，9，10，11。
人们越来越关注荧光RNA适配体的开发，以可视化RNA定位，基因表达和小分子信号的动态变化。对于这些应用中的每一个，都需要获得实时测量，但测量的准确性取决于荧光反应的动力学是否快于采样频率。在这里，我们描述了使用配备样品进样器的酶标仪确定荧光RNA适配体Spinach2 12和Broccoli13的体外动力学的方法，并使用流式细胞仪确定在大肠杆菌中表达的Spinach2的细胞开启动力学的方法。之所以选择这两种RNA适配体，是因为它们已被应用于研究RNA定位2，3，4，它们已被用于报告5，6和生物传感器7，8，9，10，11，并且相应的染料配体（DFHBI或DFHBI-1T）已上市。表14，13，14给出了文献中确定的它们的体外特性的摘要，这为方案开发提供了信息（例如，使用的波长和染料浓度）。这些结果表明，受RNA适配体影响的荧光反应是快速的，不应妨碍所需细胞生物学应用的准确测量。
Protocol
1. 体外 动力学实验 通过PCR制备DNA模板设置PCR反应：要制备PCR反应，请将以下试剂合并到薄壁PCR管中：33 μL 双蒸水 （ddH2O）10 μL 5x 缓冲液，用于高保真 DNA 聚合酶5 μL 脱氧核糖核苷三磷酸 （dNTP） 各 2 mM0.5 μL 40 μM 正向引物0.5 μL 40 μM 反向引物0.5 μL （10-100 ng） DNA 模板（仅适用于菠菜2 PCR;西兰花底漆重叠）0.5 μL 高保真 DNA 聚合酶（最?…
Representative Results
体外 动力学作为合成寡核苷酸购买的DNA模板和引物的序列如 表2所示， 试剂配方如 补充文件1所示。PCR扩增用于扩大带有T7启动子的DNA模板的量，这是随后的 体外 转录（IVT）反应所必需的。此外，PCR扩增可用于同一反应中的两个目的：通过引物延伸生成全长西兰花DNA模板，以及放大DNA模板。 在IVT反应合成RNA?…
Discussion
对于体外动力学实验，可以修改相同的通用方案以测量含有配体结合域和荧光团结合结构域的基于RNA的荧光生物传感器的体外动力学8。在这种情况下，RNA应在注射配体后进行测量之前与荧光团一起孵育，以获得配体反应动力学。如果在重复之间观察到高变异性，则可以通过在测量前检查每个样品是否允许在96孔板中平衡相同的时间来排除故障。每个样品或重复样品?…
Declarações
The authors have nothing to disclose.
Acknowledgements
这项工作得到了MCH的以下资助：NSF-BSF 1815508和NIH R01 GM124589。MRM得到了NIH T32 GM122740培训资助的部分支持。
Materials
| Agarose | Thermo Fischer Scientific | BP160500 | |
| Agarose gel electrophoresis equipment | Thermo Fischer Scientific | B1A-BP | |
| Alpha D-(+)-lactose monohydrate | Thermo Fischer Scientific | 18-600-440 | |
| Amber 1.5 mL microcentrifuge tubes | Thermo Fischer Scientific | 22431021 | |
| Ammonium persulfate (APS) | Sigma-Aldrich | A3678 | |
| Ammonium sulfate ((NH4)2SO4) | Sigma-Aldrich | A4418 | |
| Attune NxT Flow cytometer | Thermo Fischer Scientific | A24861 | |
| Attune 1x Focusing Fluid | Thermo Fischer Scientific | A24904 | |
| Attune Shutdown Solution | Thermo Fischer Scientific | A24975 | |
| Attune Performance Tracking Beads | Thermo Fischer Scientific | 4449754 | |
| Attune Wash Solution | Thermo Fischer Scientific | J24974 | |
| Boric acid | Sigma-Aldrich | B6768 | |
| Bromophenol blue | Sigma-Aldrich | B0126 | |
| Carbenicillin disodium salt | Sigma-Aldrich | C3416 | |
| Chlorine Bleach | Amazon | B07J6FJR8D | |
| Corning Costar 96-well plate | Daigger Scientific | EF86610A | |
| Culture Tubes, 12 mm x 75 mm, 5 mL with attached dual position cap | Globe Scientific | 05-402-31 | |
| DFHBI | Sigma-Aldrich | SML1627 | |
| DFHBI-1T | Sigma-Aldrich | SML2697 | |
| D-Glucose (anhydrous) | Acros Organics | AC410955000 | |
| Dimethyl sulfoxide (DMSO) | Sigma-Aldrich | D8418 | |
| Dithiothreitol (DTT) | Sigma-Aldrich | DTT-RO | |
| DNA loading dye | New England Biolabs | B7025S | |
| DNA LoBind Tubes (2.0 mL) | Eppendorf | 22431048 | |
| dNTPs: dATP, dCTP, dGTP, dTTP | New England Biolabs | N0446S | |
| EDTA, pH 8.0 | Gibco, Life Technologies | AM9260G | |
| Ethanol (EtOH) | Sigma-Aldrich | E7023 | |
| Filter-tip micropipettor tips | Thermo Fischer Scientific | AM12635, AM12648, AM12655, AM12665 | |
| FlowJo Software | BD Biosciences | N/A | FlowJo v10 Software |
| Fluorescent plate reader with heating control | VWR | 10014-924 | |
| Gel electrophoresis power supply | Thermo Fischer Scientific | EC3000XL2 | |
| Glycerol | Sigma-Aldrich | G5516 | |
| Glycogen AM95010 | Thermo Fischer Scientific | AM95010 | |
| GraphPad Prism | Dotmatics | N/A | Analysis software from Academic Group License |
| Heat block | Thomas Scientific | 1159Z11 | |
| HEPES | Sigma-Aldrich | H-4034 | |
| Inorganic pyrophosphatase | Sigma-Aldrich | I1643-500UN | |
| Low Molecular Weight DNA Ladder | New England Biolabs | N3233L | Supplied with free vial of Gel Loading Dye, Purple (6x), no SDS (NEB #B7025). |
| Magnesium chloride hexahydrate (MgCl2) | Sigma-Aldrich | M2670 | |
| Magnesium sulfate (MgSO4) | Fisher Scientific | MFCD00011110 | |
| Microcentrifuge tubes (1.5 mL) | Eppendorf | 22363204 | |
| Microcentrifuge with temperature control | Marshall Scientific | EP-5415R | |
| Micropipettors | Gilson | FA10001M, FA10003M, FA10005M, FA10006M | |
| Micropipettor tips | Sigma-Aldrich | Z369004, AXYT200CR, AXYT1000CR | |
| Millipore water filter with BioPak unit | Sigma-Aldrich | CDUFBI001, ZRQSVR3WW | |
| Narrow micropipettor pipette tips | DOT Scientific | RN005R-LRS | |
| PBS, 10x | Thermo Fischer Scientific | BP39920 | |
| PCR clean-up kit | Qiagen | 28181 | |
| PCR primers and templates | Integrated DNA technologies | ||
| PCR thermocycler for thin-walled PCR tubes | Bio-Rad | 1851148 | |
| PCR thermocycler for 0.5 mL tubes | Techne | 5PRIME/C | |
| pET31b-T7-Spinach2 Plasmid | Addgene | Plasmid #79783 | |
| Phusion High-Fidelity DNA polymerase | New England Biolabs | M0530L | Purchase of Phusion High-Fideldity Enzyme is supplied with 5x Phusion HF Buffer, 5x Phusion GC Buffer, and MgCl2 and DMSO solutions. |
| Polyacrylamide gel electrophoresis gel comb, C.B.S. Scientific | C.B.S. Scientific | VGC-1508 | |
| Polyacrylamide gel electrophoresis equipment | C.B.S. Scientific | ASG-250 | |
| Potassium chloride (KCl) | Sigma-Aldrich | P9333 | |
| Potassium phosphate monobasic | Sigma-Aldrich | P5655 | |
| Razor blades | Genesee Scientific | 38-101 | |
| rNTPs: ATP, CTP, GTP, UTP | New England Biolabs | N0450L | |
| SDS | Sigma-Aldrich | L3771 | |
| Short wave UV light source | Thermo Fischer Scientific | 11758221 | |
| Sodium carbonate (Na2CO3) | Sigma-Aldrich | S7795 | |
| Sodium chloride (NaCl) | Sigma-Aldrich | S7653 | |
| Sodium hydroxide (NaOH) | Sigma-Aldrich | S8045 | |
| Sodium phosphate dibasic, anhydrous | Thermo Fischer Scientific | S375-500 | |
| SoftMax Pro | Molecular Devices | N/A | SoftMax Pro 6.5.1 (platereader software) obtained through Academic Group License |
| Sterile filter units | Thermo Fischer Scientific | 09-741-88 | |
| Sucrose | Sigma-Aldrich | S0389 | |
| SYBR Safe DNA gel stain | Thermo Fischer Scientific | S33102 | |
| TAE buffer for agarose gel electrophoresis | Thermo Fischer Scientific | AM9869 | |
| Tetramethylethylenediamine (TEMED) | Sigma-Aldrich | T9281 | |
| Tris base | Sigma-Aldrich | TRIS-RO | |
| Tryptone (granulated) | Thermo Fischer Scientific | M0251S | |
| T7 RNA polymerase | New England Biolabs | M0251S | |
| Urea-PAGE Gel system | National Diagnostics | EC-833 | |
| UV fluorescent TLC plate | Sigma-Aldrich | 1.05789.0001 | |
| UV/Vis spectrophotometer | Thermo Fischer Scientific | ND-8000-GL | |
| Vortex mixer | Thermo Fischer Scientific | 2215415 | |
| Xylene cyanol | Sigma-Aldrich | X4126 | |
| Yeast Extract (Granulated) | Thermo Fischer Scientific | BP9727-2 |
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Citar este artigo
Mumbleau, M. M., Meyer, M. R., Hammond, M. C. Determination of In Vitro and Cellular Turn-on Kinetics for Fluorogenic RNA Aptamers. J. Vis. Exp. (186), e64367, doi:10.3791/64367 (2022).