Visualisering af Surface-tøjret store DNA-molekyler med et fluorescerende protein DNA-bindende peptid
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
Visualisering af store DNA-molekyler forankret på glas eller perle overflader er blevet udnyttet til undersøgelse DNA-protein interaktioner, protein dynamik på DNA substrat, 1,2 og polymer fysik. 3,4 En platform for enkelt-tøjrede store DNA-molekyler har et par særskilt fordele i forhold til andre DNA immobiliseringsmetoder. 5 Første, et stort DNA-molekyle bindes på overfladen har en naturlig random-coil konformation uden en forskydningshastighed flow, hvilket er kritisk vigtigt for et DNA-bindende protein til at genkende dets bindingssted. For det andet er det meget nemt at ændre det kemiske miljø omkring DNA-molekyler for en serie af enzymatiske reaktioner i et flow kammer. Tredje, en mikrofluid forskydningsstrømning inducerer DNA molekylær strækker sig op til 100% af fuld kontur længde, som er meget vanskelig at opnå ved anvendelse af alternative DNA-forlængelse tilgange såsom overfladen immobilisering 6 og nanochannel indespærring. 7 En fuldt stretched DNA-molekyle indeholder også positionsinformation, som kan være nyttig til overvågning enzymatiske bevægelser på det genomiske kort.
Alligevel DNA tethering tilgang har en kritisk mangel i at interkalerende farvestof, såsom YOYO-1 generelt forårsager tøjrede DNA-molekyler let kan brydes ved hjælp af fluorescens excitationslys. Generelt store DNA molekyler skal farves med et fluorescerende farvestof til visualisering under et fluorescensmikroskop. Til dette formål YOYO-1 eller andre TOTO serie farvestoffer anvendes primært fordi disse farvestoffer fluorescerer, når de interkalerer dobbeltstrenget DNA. 8. Det er imidlertid velkendt, at bis-interkalerende farvestof forårsager lysinduceret DNA foto-spaltning, fordi af interkalationselektroden af fluoroforer. 9 yderligere, fluorescerende farvede DNA-molekyler forankret på overfladen er mere skrøbelige, idet shear strømme kan udøve rev kræfter på frit bevægelige DNA-molekyler. Derfor har vi udviklet FP-DBP som en ny DNA-farvning protein farvestof til afbildning store DNA-molekyler forankret på overfladen. Fordelen ved at anvende FP-DBP er, at det ikke forårsager foto-spaltning af DNA-molekylerne, som det binder. 10. Hertil kommer, at FP-DBP ikke øge kontur længde af DNA, mens bis-interkalerende farvestoffer forøge konturlængde med omkring 33%.
Denne video metode introducerer den eksperimentelle tilgang til tethering store DNA-molekyler til en PEG-biotin overflade. Figur 1 illustrerer forskellige tilgange af tethering DNA med stumpe ender og klæbrige ender. Således kan denne farvningsmetode anvendes på enhver type af DNA-molekylet. Figur 2 afbilder en skematisk repræsentation af strømningskammeret samling, der kan styres af en sprøjtepumpe at generere forskydningskræfter strømme til at strække DNA-molekyler såvel som at indlæse kemiske og enzym løsninger. Figur 3 viser mikrografier af fuldt strakte DNA-molekyler forankret på PEGylated overflade 11 og farvet med FP-DBP.
Protocol
Representative Results
Discussion
Her præsenterer vi en platform til at visualisere lange DNA-molekyler biotinyleret til forankring på overflader. Vi har rapporteret en fremgangsmåde til DNA-molekyler forankret på et avidin protein overflade med biotinyleret bovint serumalbumin. 6 I den tidligere fremgangsmåde, fandt vi et afgørende spørgsmål om DNA foto-spaltning forårsaget af bis-interkalationstoppe farvestoffer pletten DNA-molekyler bindes på overflade. Da disse løbende exciterede fluoroforer har en høj sandsynlighed for at ang…
Divulgations
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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