结合单分子操纵和成像蛋白-DNA相互作用的研究
Summary
在这里，我们描述了仪器和方法，用于检测单荧光标记的蛋白质分子与悬浮两层光学俘获微球之间的单个DNA分子的相互作用。
Abstract
The paper describes the combination of optical tweezers and single molecule fluorescence detection for the study of protein-DNA interaction. The method offers the opportunity of investigating interactions occurring in solution (thus avoiding problems due to closeby surfaces as in other single molecule methods), controlling the DNA extension and tracking interaction dynamics as a function of both mechanical parameters and DNA sequence. The methods for establishing successful optical trapping and nanometer localization of single molecules are illustrated. We illustrate the experimental conditions allowing the study of interaction of lactose repressor (lacI), labeled with Atto532, with a DNA molecule containing specific target sequences (operators) for LacI binding. The method allows the observation of specific interactions at the operators, as well as one-dimensional diffusion of the protein during the process of target search. The method is broadly applicable to the study of protein-DNA interactions but also to molecular motors, where control of the tension applied to the partner track polymer (for example actin or microtubules) is desirable.
Introduction
单分子（SM）的技术有了很大的发展，在过去三十年来克服一些传统的，本体溶液测量1-3的限制，需要作出回应。单一生物分子的操纵创造了机会，以测量生物聚合物4的机械性能和控制的蛋白-蛋白5和蛋白质-DNA相互作用6,7的机械参数。 SM荧光检测，另一方面，代表一个非常灵活的工具用于研究蛋白质的活性在体外和体内 ，导致定位和跟踪单分子与纳米精度的可能性。通过仪器的点扩散函数与SM图像的拟合，实际上，可以实现定位与精度主要取决于信号噪声比（SNR）和达到约一纳米8,9的限制。这些方法找到强大应用在马达蛋白的动力学的研究，以及对底层中的DNA结合蛋白的目标搜索的扩散过程。确定扩散常数为DNA序列上的目标函数，停留时间和精确地测量在一维扩散事件探索了DNA的长度的能力，代表一个功能强大的工具，蛋白质-DNA相互作用动力学的研究和调查的特定目标的搜索机制。
最近，这两种技术的结合，产生了新一代实验装置10-14实现同时操控的生物基质（如肌动蛋白丝或DNA分子）的相互作用伙伴酶检测/定位（例如肌凝蛋白或DNA结合蛋白）。这些技术的优点主要停留在施加机械控制被困聚合物的可能性，从而ENA金光闪闪的相互作用动力学与力或力矩的研究。此外，该方法允许从表面为止测量的生化反应，避免了经典的SM的方法， 即，需要对所研究的分子固定在表面上（玻璃载玻片或微球体）的主要限制之一。
两个单分子技术的结合需要克服几个技术难题，所产生的主要是从机械稳定性和足够的信噪比（SNR）（特别是当需要定位以纳米精度）15的要求。特别地，耦合SM荧光检测用光学镊子，噪声的降低时，并从捕获红外激光器16和生化缓冲液的生物复合物的装配和实验测量11的性能的控制漂白是最重要的。在这里，我们描述了方法，用于执行成功测量双套色/ SM荧光定位设置。该方法被示出与含有特定了LacI结合序列（ 例如，运营商）乳糖阻遏蛋白（了LacI）的荧光标记（用Atto532）和检测到的，因为它结合到DNA分子（两个光学镊子之间的残留）的例子。我们证明在检测了LacI的DNA和扩散约束力沿其在目标搜索过程中轮廓的方法的有效性。该方法适用于DNA序列和DNA结合蛋白中的任意组合，以及与其他系统（微管或肌动蛋白丝和马达蛋白与它们进行交互）。
Protocol
Representative Results
Discussion
在过去的十年中，单分子操纵和成像技术已经看到在空间和时间分辨率方面很大的进步。的操纵和成像技术的组合是在的强大工具的基础，现在允许一个单一的生物聚合物的机械条件，如DNA，RNA或细胞骨架细丝的控制，和单一的蛋白质同时定位与同一聚合物相互作用。控制困聚合物的机械条件是特别感兴趣的。事实上，在活细胞中，核酸被连续经历引起相互作用的酶和蛋白质的机械应力;同样的，…
Disclosures
The authors have nothing to disclose.
Acknowledgements
我们感谢Gijs Wuite，欧文JG彼得曼，彼得总的帮助，微流体和阿莱西娅Tempestini的帮助，样品制备。这项研究是根据资助协议资金由欧盟第七框架计划（FP7 / 2007-2013）N°284464和意大利外交部，教育，大学和研究外国投资审查委员会2011 RBAP11X42L006，清新汽油在Ricerca 2013 RBFR13V4M2，并在框架该旗舰项目NANOMAX。
Materials
| Name of Material/ Equipment | Company | Catalog Number | Comments/Description | Web Address |
| elastomeric isolators | Newport | Newdamp | Choose the appropriate Newdamp elastomer depending on the microscope weight and resonance frequencies | http://www.newport.com |
| optical isolator | Optics for Research | IO-3-YAG-VHP | http://www.ofr.com | |
| Nd:YAG laser, 1064 nm wavelength | Spectra-Physics | Millennia IR | http://www.newport.com/ | |
| acousto-optic deflectors (AODs) | A&A optoelectronic | DTS-XY 250 | http://www.aaoptoelectronic.com/ | |
| Direct Digital Synthesizers | Analog Devices | http://www.analog.com/ | ||
| quadrant detector photodiodes | OSI optoelectronics | SPOT-15-YAG | http://www.osioptoelectronics.com | |
| DIO and FPGA board | National Instruments | NI-PCI-7830R | http://www.ni.com | |
| Halogen lamp | Schott | KL 1500 LCD | http://www.schott.com | |
| Condenser | Olympus | U-AAC 1.4NA Aplanat Apchromat | http://www.olympus-global.com/en/ | |
| Objective | Nikon | CFI Plan Apochromat 60x 1.2NA water immersion | http://www.nikoninstruments.com | |
| 532 nm laser | Coherent | Sapphire | http://www.coherent.com | |
| CCD 200X and 2000X | Hamamatsu | XC-ST70 CE | http://www.hamamatsu.com | |
| electron-multiplied CCD | Hamamatsu | C9100-13 | http://www.hamamatsu.com/ | |
| piezo stage with nm-accuracy | Physik Instrumente | P-527.2CL | http://www.physikinstrumente.com/ | |
| Emission Filter | Chroma Technologies | 600/100m | http://www.chroma.com | |
| silica beads (1.54 mm) | Bangs Laboratories | SS04N/5303 | http://www.bangslabs.com/ | |
| Albumin from bovine serum (BSA) | Sigma Aldrich | B4287 | http://www.sigmaaldrich.com/ | |
| pentyl acetate | Sigma Aldrich | 46022 | Flammable liquid and vapour (No 1272/2008) | http://www.sigmaaldrich.com/ |
| nitrocellulose | Sigma Aldrich | N8267-5EA | Flammable solid (No 1272/2008) | http://www.sigmaaldrich.com/ |
| heat block | MPM Instruments Srl | M502-HBD | with 2 removable blocks; preheated at 120° C | http://www.mpminstruments.com |
| NanoPort assemblies | Upchurch Scientific Inc. | N-333 | http://www.upchurch.com/ | |
| polyetheretherketone tubing | Upchurch Scientific Inc. | 1535 | http://www.upchurch.com/ | |
| home-made metallic holder for the assembly of the flow-chamber pressure reservoir made of Plexiglass | ||||
| luer lock-tip syringes 2.5 mL | Terumo | SS 02LZ1 | http://www.terumomedical.com | |
| shut-off valves | Upchurch Scientific, Inc. | P-732 | http://www.upchurch.com/ | |
| flangeless fittings | Upchurch Scientific, Inc. | LT-111 | http://www.upchurch.com/ | |
| fluorinated ethylene propylene tubing | Upchurch Scientific, Inc. | 1549 | http://www.upchurch.com/ | |
| two computer-controlled solenoid valves | Clippard, Cincinnati, USA | ET-2-H-M5 | http://www.clippard.com | |
| pressure transducer | Druck LTD | PTX 1400 | ||
| biotin-14-dCTP | Life Technologies | 19518-018 | http://www.lifetechnologies.com/ | |
| Terminal deoxynucleotidyl Transferase (TdT) | Thermoscientific | EP0161 | http://www.thermoscientificbio.com/ | |
| ATTO532 maleimide | Sigma Aldrich | 68499 | http://www.sigmaaldrich.com/ | |
| N,N-dimethylformamide (DMF) | Sigma Aldrich | 227056 | Combustible Liquid, Harmful by skin absorption., Irritant, Teratogen. H226; H303; H312; H316; H319; H331; H360; P201; P261; P280;P305; P351; P338; P311 | http://www.sigmaaldrich.com/ |
| Tris-(2-carboxyethyl)phosphine hydrochloride (TCEP) | Sigma Aldrich | C4706 | http://www.sigmaaldrich.com/ | |
| L-Glutathione reduced (GSH) | Sigma Aldrich | G4251 | Acute toxicity, Oral (Category 5), H303 | http://www.sigmaaldrich.com/ |
| Amicon Ultra-15, PLQK Ultracel-PL Membrane, 10 kDa cutoff spin concentrators | Merck Millipore | UFC901024 | http://www.merckmillipore.it/ | |
| streptavidin-coated polystyrene beads 1,87 µm | Spherotech, Inc. | SVP-15-5 | http://www.spherotech.com/ |
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