Parallel, quantitative measurement of protein binding to a 120-element double-stranded DNA array in real time using surface plasmon resonance microscopy.
Quantitative, real-time measurement of kinetics of sequence-specific binding of DNA-binding proteins to double-stranded DNA (dsDNA) immobilized in a 10 x 12 array on a planar gold surface is demonstrated using surface plasmon resonance (SPR) microscopy. This binding of the yeast transcription factor Gal4 to a 120-spot dsDNA array made with alternating 200-microm spots of its dsDNA operator sequence and an unrelated DNA sequence proves that this method could be used to simultaneously monitor the kinetics of binding of proteins to 120 different dsDNA sequences with a sensitivity to approximately 0.5 pg (<2 x 10(7) molecules) of bound protein in each array spot at a time resolution of 1 s. The method is label free and also allows absolute quantitative determination of the binding stoichiometry (i.e., the number of proteins bound per dsDNA) at each time. The dsDNA array was fabricated using a robotic microspotting system to deliver nanoliter droplets of biotinylated dsDNA solutions onto a streptavidin linker layer immobilized with biotinylated alkylthiols on a thin gold film. Simultaneous monitoring of binding to the many array elements allows the use of reference spots (i.e., array elements with unrelated dsDNA sequences) to correct the signal for nonspecific protein-DNA binding and changes in bulk refractive index of the solutions in the SPR microscope's flow cell. This allows high-throughput analyses of the kinetics and equilibrium of protein-dsDNA binding.
Shumaker-Parry
Jennifer S
JS
Department of Chemistry, University of Washington, Seattle, WA 98195-1700, USA.
Aebersold
Ruedi
R
Campbell
Charles T
CT
D000818:Animals; D001483:Base Sequence; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000032:analysis*; D006046:Gold / Q000737:chemistry / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D020349:Surface Plasmon Resonance / Q000379:methods*; D013499:Surface Properties; D013997:Time Factors
D004268:DNA-Binding Proteins; D006046:Gold; D004247:DNA