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

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).

TAMRA-polypyrrole for A/T sequence visualization on DNA molecules

“…Figure 2 shows TAMRA-polypyrrole-stained λ DNA (NCBI Reference Sequence: NC_001416.1) molecules tethered to the surface of a flow cell ( 16 ). Surface-tethered DNA molecules were free-floating with a mushroom-like conformation (Figure 2A ) and were fully elongated at a flow of 100 μl/min (Figure 2B ).…”

“…A number of approaches to visualize large DNA molecules have been developed, including surface immobilization ( 15 , 36 , 37 ), DNA tethering ( 16 , 38 ), microfluidic dynamic stretching ( 14 , 39 ) and nanochannel confinement ( 17 , 40 ). Surface immobilization is practically effective because the procedure is very simple ( 37 ).…”

“…A flow chamber was prepared by placing a custom-fabricated acrylic support on an acid-cleaned cover slip with a height of 100 μm formed by double-sided tape ( 16 , 17 ). The dimensions of the flow cell were approximately 5 × 10 × 0.1 mm (L × W × H).…”

Nanochannel-Confined TAMRA-Polypyrrole Stained DNA Stretching by Varying the Ionic Strength from Micromolar to Millimolar Concentrations

“…A flow chamber was prepared as described and illustrated with a video in a previous paper [33]. Briefly, an acid-cleaned coverslip was placed on custom-made acrylic support with a height of 100 μm by double-sided tape.…”

“…4 : DNA stretch predicted from our previous calculation of F = 0.79 pN [27]. 5 : YOYO-1 stained DNA, 6~10 : FP-DBP stained DNA (6: 2(KW)2, 7: KW5, 8: K6, 9: 2HMG, 10: 2(SPRK)) [27,33]. Flow rate = 100 µL/min.…”