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Biology

Visualization of Surface-tethered Large DNA Molecules with a Fluorescent Protein DNA Binding Peptide

Published: June 23rd, 2016

DOI:

10.3791/54141

1Department of Chemistry and Interdisciplinary Program of Integrated Biotechnology, Sogang University

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.

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).

Visualization of large DNA molecules tethered on glass or bead surfaces has been utilized for investigating DNA-protein interactions, protein dynamics on DNA substrate,1,2 and polymer physics.3,4 A platform for single-tethered large DNA molecules has a few distinct advantages compared to other DNA immobilization methods.5 First, a large DNA molecule tethered on the surface has a natural random-coil conformation without a shear flow, which is critically important for a DNA-binding protein to recognize its binding site. Second, it is very easy to change the chemical environment around DNA molecules for a series of enzymatic reactions in ....

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1. DNA Biotinylation

  1. Biotinylation of blunt ended DNA using Terminal Transferase (TdT)
    Note: Use T4 DNA (166 kbp), which is a blunt ended DNA.
    1. Add 5 µl of 2.5 mM CoCl2, 5 µl of 10× reaction buffer, 0.5 µl of 10 mM biotin-11-dUTP, 0.5 µl of terminal transferase (10 units), and 0.5 µl of T4 DNA (0.5 µg/µl) to the reaction mixture. Make to a final volume of 50 µl by adding 38.5 µl of water.
    2. Incubate the re.......

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Figure 1 shows two different DNA tethering methods depending on terminal structures of the DNA molecule. Figure 1a illustrates how the sticky ended DNA molecules are hybridized with complementary biotinylated oligonucleotides, which are immobilized on the avidin-coated PEG surface. Figure 1b shows the addition of biotinylated ddNTP or dNTP to the 3' hydroxyl group of a blunt ended DNA by terminal transferase. We added flexible linkers.......

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Here we present a platform for visualizing long DNA molecules biotinylated for anchoring on surfaces. We have reported an approach for DNA molecules tethered on an avidin protein coated surface with biotinylated bovine serum albumin.6 In the earlier approach, we found a crucial issue of DNA photo-cleavage caused by bis-intercalation dyes that stain DNA molecules tethered on the surface. As these continually excited fluorophores have a high probability to attack a DNA phosphate backbone,9 the excitat.......

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This work was supported by the Sogang University Research Grant of 201410036.

....

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Name Company Catalog Number Comments
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 22x22 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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