研究DNA环接了单分子荧光共振能量转移
Summary
这项研究提出了一个详细的实验步骤，使用单分子荧光共振能量转移（FRET）来衡量的双链DNA循环动力学。该协议还描述了如何提取称为歼因子循环概率密度。
Abstract
弯曲的双链DNA（dsDNA）的是与许多重要的生物学过程，如DNA-蛋白质识别和DNA包装成核小体相关联。双链DNA弯曲的热力学研究了它依赖于DNA连接酶共价连接一个双链DNA的短粘性末端的方法称为环化。然而，连接效率可受许多不相关的双链DNA循环如围绕接合的粘性末端的DNA结构，和连接酶的因素也可以通过诸如非特异性结合的影响明显的循环速率。在这里，我们将展示如何通过检测瞬态DNA环的形成由FRET（荧光共振能量转移）来衡量双链DNA循环动力学无连接酶。双链DNA分子使用的是简单的基于PCR的协议与FRET对和生物素连接器构成。称为歼因子的循环概率密度是从两个断路之间的循环速率和退火速率提取特德粘性末端。通过测试2 dsDNAs具有不同本征曲率，我们表明，歼因子是对双链DNA的固有形状敏感。
Introduction
了解双链DNA的力学性能是极为重要的基础科学和工程应用。双链DNA的结构比直螺旋阶梯更加复杂，因为连续的碱基对之间滚动，倾斜和扭转角可以与序列有所不同。热波动可能会导致双链DNA，以进行构象波动的不同模式，如弯曲，扭转和伸展。过渡，例如熔化和扭结，也可能发生在极端条件下。
在这些议案，双链DNA弯曲有最明显的生物学影响1。双链DNA弯曲与基因抑制或激活通过将两个遥远接近彼此关联。它也起着在细胞核内的DNA包装或病毒衣壳中起重要作用。双链DNA的弯曲变形可以通过实验由高分辨率显微镜（AFM 2和TEM 3），以及thermodyn进行可视化AMICS和动力学可以通过循环检测，其化学连接的双链DNA并列的网站进行研究。
一个这样的实验是连接酶依赖的环化4。在该试验中，双链DNA分子的“粘性”（聚）端部发送或通过DNA连接酶二聚化。通过比较圆和二聚物的形成速率，可以得到在其另一端，这是已知的歼因子附近的DNA的一端的有效摩尔浓度。这j数目是二维等价于求该DNA的一端从另一端，在短距离的概率密度，从而反映了DNA的灵活性。测量歼因子的DNA长度的函数揭示了DNA的机制包括持续长度4,5诸多特点。
该蠕虫状链（WLC）模型已被广泛认为是基于其在expla成功双链DNA力学的规范聚合物模型伊宁DNA的拉动实验6，正确预测dsDNAs的歼因素大于200 bp的7所得到的力-延伸曲线。但是，使用双链DNA上的分子越短100 bp的环化法，克卢捷和Widom测量歼因素是几个数量级比WLC模型预测高8。一年后，杜等人 。生产Ĵ因素与使用环化检测与低连接酶的浓度在WLC模型协议，并归因于异常结果从Widom组用9高连接酶的浓度。使用传统的分析9时，这场争论体现了DNA连接酶环化上动力学不可避免的影响。此外，DNA连接酶还可以通过非特异性结合10,11影响DNA结构和刚度。
为了消除蛋白依赖的循环试验的技术问题，我们最近展示了普罗特基于荧光共振能量转移（FRET）12 EIN自由循环试验。在该方法中，环的构象是通过FRET附近的DNA分子的粘性末端的供体和受体之间进行检测。客观型全内反射荧光显微镜（TIRFM）是用来记录可逆循环，并从表面固定的单个DNA分子为长时间unlooping事件的轨迹。此方法具有的DNA分子的PCR为基础的组件以产生错配的游离DNA分子，这是通过由Vafabakhsh和哈13类似的方法的一个关键的改进。此协议的单分子方面允许除了分布测量到总体均值而FRET方面允许一个反复从同一分子测定DNA循环动力学，甚至在可能损害连接酶活性的条件。
该TIRFM设置如图1。自定义设计的样品载物台放置在奥林巴斯IX61显微镜体。 532纳米和640纳米激光器从侧面引入并通过微小的椭圆镜14进入高NA物镜被反射来实现入射临界角在盖玻片-水界面。我们注意到，更广泛地通过目标使用二向色反射镜或棱镜型TIR TIR设置也可用于本申请FRET。由显微镜所形成的荧光图像通过分色镜分成供体和受体的图像。然后将它们重新成像到一个EMCCD的两半。附加的长通发射滤波器被用于减少背景信号。
温度控制是获得可重复的动力学数据是必不可少的。对于温度控制，目标是从镜身，以减少热传递，并从温度控制冷却器/加热器水的分离物镜转换器通过一个黄铜项圈紧密配合循环围绕目标外套下的内部金属。这种设置能够实现强大的温度控制在（ 图2），15至50℃的盖玻片表面。在这项工作中，将样品的温度保持在24℃。
以下协议给出了一步一步的过程进行DNA施工，估计DNA的形状，单分子实验，和J的因素决定。
Protocol
Representative Results
Discussion
基于FRET一个简单的单分子实验来研究不同的内在形状DNA的循环动力学。弯曲的DNA可以通过重复一个10 – 聚体序列中的相位与10.5沸点螺旋周期来制备，并且其曲率可以用PAGE进行估计。这些dsDNAs都设计有粘性末端，让短暂的环路稳定性。我们从指数上升的环的分子数随时间提取的循环速率。断开连接的粘性末端段之间的退火速率常数是用来确定该浓度相当于该环形概率密度，这是被称为歼因子。 </p…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
我们感谢詹姆斯·沃特斯，山墙沃兹沃斯和博Broadwater的紧要阅读手稿。我们也感谢4匿名审稿人提供有用的意见。我们承认从乔治亚理工学院，宝来惠康基金事业奖在科学界面，并从生命系统的NSF物理学学生研究网络赠款资金支持。
Materials
| Small DNA FRAG Extract Kit-100PR | VWR | 97060-558 | |
| Acrylamide 40% solution 500 mL | VWR | 97064-522 | |
| Bis-acrylamide 2% (w/v) solution 500 mL | VWR | 97063-948 | |
| GeneRuler 100 bp DNA Ladder, 100-1000 bp | Fermentas | SM0241 | |
| Mini Vertical PAGE System | VWR | 89032-300 | |
| Syringe filter 0.2um CS50 | VWR | A2666 | |
| Trolox | Sigma-Aldrich | 238813-1G | triplet state quencher |
| Protocatechuic acid (PCA) | Sigma-Aldrich | 08992-50MG | oxygen scavenging system |
| Protocatechuate 3,4-Dioxygenase (PCD) | Sigma-Aldrich | P8279-25UN | oxygen scavenging system |
| mPEG-silane, MW 2000 1g | Laysan Bio | MPEG-SIL-2000-1g | |
| Biotin-PEG-Silane, MW 3400 | Laysan Bio | Biotin-PEG-SIL-3400-1g | |
| Avidin, NeutrAvidin Biotin-binding Protein | Invitrogen | A2666 | |
| Phusion Hot Start High-Fidelity DNA Polymerase | New England Biolabs | F-540L | |
| Gel/PCR DNA Fragments Extraction Kit | IBI Scientific | IB47020 | |
| Premium plain glass microscope slides | Fisher Scientific | 12-544-1 | |
| VWR micro cover glass, rectangular, no. 1 | VWR | 48404-456 | |
| Fisher Scientific Isotemp 1006s Recirculating Chiller/Heater | Fisher Scientific | temperature control | |
| Objective Cooling Collar | Bioptechs | 150303 | temperature control |
| KMI53 Biological Micrometer Measuring Stage | Semprex | KMI53 | |
| High Performance DPSS Laser 532nm 50mW | Edmund optics | NT66-968 | Cy3 excitation |
| CUBE Fiber Pigtailed 640 nm, 30mW, Fiber, FC/APC Connector | Coherent | 1139604 | Cy5 excitation |
| 650 nm BrightLine Dichroic Beamsplitter | Semrock | FF650-Di01-25×36 | splitting dichroic |
| LaserMUX Beam Combiner, reflects 514.5, 532, & 543.5 nm lasers, 25 mm | Semrock | LM01-552-25 | combining dichroic |
| Brightline Fluorescence Filter 593/40 | Semrock | FF01-593/40-25 | Cy3 emission filter |
| 635 nm EdgeBasic LWP longpass Filter, 25 mm | Semrock | BLP01-635R-25 | Cy5 emission filter |
| EMCCD iXon+ | Andor Technology | DU-897E-CS0-#BV | |
| IX51 inverted microscope frame | Olympus | ||
| Objective UApo N 100x/1.49 Oil TIRF | Olympus | ||
| Immersion oil type-F for fluorescence microscopy | Olympus | IMMOIL-F30CC | |
| 2mm Diameter 45° Rod Lens Aluminum Coated | Edmund optics | 54-092 | miniature mirror |
| 1/4" Travel Single-Axis Translation Stage | Thorlabs | MS-1 | translation of miniature mirror |
| Ø1" Achromatic Doublet, ARC: 400-700 nm, f=200 mm | Thorlabs | AC254-200-A | focusing lens |
| Adjustable Mechanical Slit | Thorlabs | VA100 | |
| Dielectric Mirror | Thorlabs | BB1-E02 | |
| Ø1" Achromatic Doublet, f = 100 mm | Thorlabs | AC254-100-A | relay lens |
| Lens Mount for Ø1" Optics | Thorlabs | LMR1 | |
| Dichroic Filter Mount | Thorlabs | FFM1 | |
| Fixed Cage Cube Platform | Thorlabs | B3C | |
| Kinematic Mount for Ø1" Optics | Thorlabs | KM100 | |
| N-BK7 Plano-Convex Lens, Ø1", f = 40 mm | Thorlabs | LA1422-A | collimating lens |
| N-BK7 Plano-Convex Lense, Ø6.0 mm, f = 15 mm | Thorlabs | LA1222-A | telescope lens |
| N-BK7 Plano-Convex Lense, Ø6.0 mm, f = 150 mm | Thorlabs | LA1433-A | telescope lens |
Riferimenti
- Garcia, H. G., et al. Biological consequences of tightly bent DNA: The other life of a macromolecular celebrity. Biopolymers. 85, 115-130 (2007).
- Wiggins, P. A., et al. High flexibility of DNA on short length scales probed by atomic force microscopy. Nature Nanotechnology. 1, (2006).
- Lionberger, T. A., et al. Cooperative kinking at distant sites in mechanically stressed DNA. Nucleic Acids Research. 41, 6785-6792 (2011).
- Shore, D., et al. DNA flexibility studied by covalent closure of short fragments into circles. Proc Natl Acad Sci U S A. 78, 4833-4837 (1981).
- Geggier, S., Vologodskii, A. Sequence dependence of DNA bending rigidity. Proc Nat Acad Sci U S A. 107, 15421-15426 (1992).
- Smith, S. B., et al. Direct mechanical measurements of the elasticity of single DNA molecules by using magnetic beads. Science. 258, 1122-1126 (1992).
- Peters, J. P., Maher, L. J. DNA curvature and flexibility in vitro and in vivo. Quarterly Reviews of Biophysics. 43, 23-63 (2010).
- Cloutier, T. E., Widom, J. Spontaneous sharp bending of double-stranded DNA. Molecular Cell. 14, 355-362 (2004).
- Du, Q., et al. Cyclization of short DNA fragments and bending fluctuations of the double helix. Proc Natl Acad Sci U S A. 102, 5397-5402 (2005).
- Yuan, C., et al. T4 DNA ligase is more than an effective trap of cyclized dsDNA. Nucl. Acids Res. 35, 5294-5302 (2007).
- Manzo, C., et al. The effect of nonspecific binding of lambda repressor on DNA looping dynamics. Biophysical Journal. 103, 1753-1761 (2012).
- Le, T. T., Kim, H. D. Measuring shape-dependent looping probability of DNA. Biophys. J. 104, 2068-2076 (2013).
- Vafabakhsh, R., Ha, T. Extreme bendability of DNA less than 100 base pairs long revealed by single-molecule cyclization. Science. 337, 1097-1101 (2012).
- Friedman, L., et al. Viewing dynamic assembly of molecular complexes by multi-wavelength single-molecule fluorescence. Biophysical Journal. 91, 1023-1031 (2006).
- Kaplan, N., et al. The DNA-encoded nucleosome organization of a eukaryotic genome. Nature. 458, 362-366 (2009).
- Koo, H. S., Crothers, D. M. Calibration of DNA curvature and a unified description of sequence-directed bending. Proc Nat Acad Sci U S A. 85, 1763-1767 (1988).
- Prosseda, G., et al. A temperature-induced narrow DNA curvature range sustains the maximum activity of a bacterial promoter in vitro. Biochimica. 49, 2778-2785 (2010).
- Rasnik, I., et al. Nonblinking and long-lasting single-molecule fluorescence imaging. Nature Methods. 3, 891-893 (2006).
- Cordes, T., et al. On the mechanism of Trolox as antiblinking and antibleaching reagent. J. Am. Chem. Soc. 131, 5018-5019 (2009).
- Aitken, C. E., et al. An oxygen scavenging system for improvement of dye stability in single-molecule fluorescence experiments. Biophys J. 94, 1826-1835 (2008).
- Taylor, W. H., Hagerman, P. J. Application of the method of phage T4 DNA ligase-catalyzed ring-closure to the study of DNA structure: II. NaCl-dependence of DNA flexibility and helical repeat. Journal of Molecular Biology. 212, 363-376 (1990).
- Bolshoy, A., et al. Curved DNA without A-A: experimental estimation of all 16 DNA wedge angles. Proc Natl Acad Sci U S A. 88, 2312-2316 (1991).
- Gibson, D. G. Synthesis of DNA fragments in yeast by one-step assembly of overlapping oligonucleotides. Nucl. Acids Res. 37, 6984-6990 (2009).
- Vologodskii, A., et al. Bending of short DNA helices. Artif DNA PNA XNA. 4, (2013).
- Hoover, D. M., Lubkowski, J. DNAWorks: an automated method for designing oligonucleotides for PCR-based gene synthesis. Nucl. Acids Res. 30, (2002).
- Waters, J. T., Kim, H. D. Equilibrium Statistics of a Surface-Pinned Semiflexible Polymer. Macromolecules. 46, 6659-6666 (2013).
- Mills, J. B., et al. Electrophoretic evidence that single-stranded regions of 1 or more nucleotides dramatically increase the flexibility of DNA. Biochimica. 33, 1797-1803 (1994).
