表面拴DNA大分子的可视化与荧光蛋白的DNA结合肽
Summary
We present an approach for visualizing fluorescent protein DNA binding peptide (FP-DBP)-stained large DNA molecules tethered on the polyethylene glycol (PEG) and avidin-coated glass surface and stretched with microfluidic shear flows.
Abstract
Large DNA molecules tethered on the functionalized glass surface have been utilized in polymer physics and biochemistry particularly for investigating interactions between DNA and its binding proteins. Here, we report a method that uses fluorescent microscopy for visualizing large DNA molecules tethered on the surface. First, glass coverslips are biotinylated and passivated by coating with biotinylated polyethylene glycol, which specifically binds biotinylated DNA via avidin protein linkers and significantly reduces undesirable binding from non-specific interactions of proteins or DNA molecules on the surface. Second, the DNA molecules are biotinylated by two different methods depending on their terminals. The blunt ended DNA is tagged with biotinylated dUTP at its 3′ hydroxyl terminus, by terminal transferase, while the sticky ended DNA is hybridized with biotinylated complimentary oligonucleotides by DNA ligase. Finally, a microfluidic shear flow makes single DNA molecules stretch to their full contour lengths after being stained with fluorescent protein-DNA binding peptide (FP-DBP).
Introduction
拴在玻璃或珠表面大DNA分子可视化的已被用于研究DNA底物，1,2-和高分子物理上DNA-蛋白质相互作用，蛋白质的动态。3,4-一种单-拴系的大的DNA分子平台有几个不同的相对于其他的DNA固定化方法的优点。5首先，拴表面上的大的DNA分子具有无剪切流，这是一种DNA结合蛋白识别其结合位点至关重要的自然随机卷曲构象。第二，这是很容易改变的周围的DNA分子的化学环境中的一系列流室酶反应。第三，微流体剪切流动诱导DNA分子伸展到全轮廓长度，这是非常困难的使用替代的DNA伸长达到100％的方法，如表面固定6和纳通道禁闭。7的充分stretcheD核酸分子也可提供一种可用于基因组图谱上监测酶促运动有用的位置信息。
然而，该DNA束缚方法在该嵌入染料的一个关键缺点如YOYO-1通常会导致拴系的DNA分子，以通过荧光激励光可容易地断裂。一般地，大DNA分子必须与用于可视化在荧光显微镜下的荧光染料染色。为了这个目的，YOYO-1或其它TOTO系染料主要使用，因为当它们相互插入双链DNA这些染料仅发出荧光。8然而，这是众所周知的，双-嵌入染导致光诱导的DNA光裂解因为荧光的嵌入。9。此外，拴在表面上的荧光染色的DNA分子更脆弱，因为剪切流动能发挥上自由移动的DNA分子破坏的力量。因此，我们开发的FP-DBP作为一种新颖的ðNA-染色染料蛋白成像拴在表面上大DNA分子。使用FP-DBP的优点是，它不会导致其所结合的DNA分子的光裂解。10此外，FP-DBP不增加DNA的轮廓长度，而双-嵌入染料增加轮廓长度由约33％。
此视频方法引入了对拴系大DNA分子的PEG生物素表面的实验方法。 图1示出了具有平端和粘性末端圈养的DNA的不同方法。因此，这种染色方法可以适用于任何类型的DNA分子： 图2示出了流室组件，其可通过注射泵来控制，以产生剪切流伸展的DNA分子以及加载的化学和酶的示意图的解决方案。 图3显示拴在PEGylat完全拉伸的DNA分子的显微照片ED表面11和FP-DBP染色。
Protocol
Representative Results
Discussion
在这里，我们提出了一个可视化的生物素锚定在表面上长的DNA分子的平台。我们已经报道了拴亲和素蛋白涂层表面与生物素牛血清白蛋白对DNA分子的方法。6在前面的方法，我们发现拴在造成染污DNA分子双插染料DNA光裂解的关键问题表面。由于这些不断激发荧光团有很高的概率攻击DNA磷酸骨架，9激发光功率必须最小化，以避免光裂解。
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Divulgazioni
The authors have nothing to disclose.
Acknowledgements
This work was supported by the Sogang University Research Grant of 201410036.
Materials
| 1. DNA Biotinylation | |||
| 1.1) Biotinylation of blunt-end DNA using Terminal Transferase (TdT) | |||
| Terminal Transferase | New England Biolabs | M0315S | Provided with 10x reaction buffer, 2.5 mM Cobalt chloride |
| Biotin-11-dUTP | Invitrogen | R0081 | Biotin-ddNTP is also available |
| T4GT7 Phage DNA | Nippon Gene | 318-03971 | |
| Ethylenediaminetetraacetic acid | Sigma-Aldrich | E6758 | EDTA |
| 1.2) Biotinylation of sticky end DNA Using DNA Ligase | |||
| T4 DNA Ligase | New England Biolabs | M0202S | Provided with 10x reaction buffer |
| Lambda Phage DNA | Bioneer | D-2510 | Also available at New England Biolabs |
| 2. Functionalized Surface Derivatization | |||
| 2.1) Piranha Cleaning | |||
| Coverslip | Marienfeld-Superior | 0101050 | 22×22 mm, No. 1 Thickness |
| Teflon rack | Custom Fabrication | ||
| PTFE Thread Seal Tape | Han Yang Chemical Co. Ltd. | 3032292 | Teflon™ tape |
| Sulferic acid | Jin Chemical Co. Ltd. | S280823 | H2SO4, 95 % Purity |
| Hydrogen peroxide | Jin Chemical Co. Ltd. | H290423 | H2O2, 35 % in water |
| Sonicator | Daihan Scientific Co. Ltd. | WUC-A02H | Table-top Ultrasonic Cleaner |
| 2.2) Aminosilanization on Glass Surface | |||
| N-[3-(Trimethoxysilyl)propyl] ethylenediamine |
Sigma-Aldrich | 104884 | |
| Glacial Acetic Acid | Duksan Chemicals | 414 | 99 % Purity |
| Methyl Alcohol | Jin Chemical Co. Ltd. | M300318 | 99.9 % Purity |
| Polypropylene Container | Qorpak | PLC-04907 | |
| Ethyl Alcohol | Jin Chemical Co. Ltd. | A300202 | 99.9 % Purity |
| 2.3) PEGylation of the coverslip | |||
| Sodium Bicarbonate | Sigma-Aldrich | S5761 | |
| Syringe Filter | Sartorius | 16534———-K | |
| Biotin-PEG-SC | Laysan Bio | Biotin-PEG-SC-5000 | |
| mPEG-SVG | Laysan Bio | MPEG-SVA-5000 | |
| Acetone | Jin Chemical Co. Ltd. | A300129 | 99 % Purity |
| Microscope Slides | Marienfeld-Superior | 1000612 | ~76x26x1 mm |
| 3. Assembling a Flow Chamber | |||
| Acrylic Support | Custom Fabrication | ||
| Double-sided Tape | 3M | Transparent type | |
| Quick-dry Epoxy | 3M | ||
| Polyethylene Tubing | Cole-Parmer | 06417-11, 06417-21 | |
| Gas Tight 250 µl Syringe | Hamilton | 81165 | |
| Syringe Pump | New Era Pump Systems Inc. | NE-1000 | |
| 4. Sample Loading into Flow Chamber | |||
| Neutravidin | Thermo Scientific | 31000 | |
| Tris base | Sigma-Aldrich | T1503-5KG | Trizma base |
| Microscope | Olympus | IX70 | |
| EMCCD Camera | Q Imaging | Rolera EM-C2 | |
| Solid-state Laser (488 nm) | Oxxius | LBX488 | |
| Alconox | Alconox Inc. | ||
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