An electrical probe of protein-DNA interactions on DNA-modified surfaces.
DNA charge transport chemistry is found to provide a sensitive method for probing protein-dependent changes in DNA structure and enzymatic reactions. Here we describe the development of an electrochemical assay of protein binding to DNA-modified electrodes based upon the detection of associated perturbations in DNA base stacking. Gold electrode surfaces that were modified with loosely packed DNA duplexes, covalently crosslinked to a redox-active intercalator and containing the binding site of the test protein, were constructed. Charge transport through DNA as a function of protein binding was then assayed. Substantial attenuation in current is seen in the presence of the base-flipping enzymes HhaI methylase and uracil DNA glycosylase, as well as with TATA-binding protein. When restriction endonuclease PvuII (R.PvuII) binds to its methylated target, little base-stacking perturbation occurs and little diminution in current flow is observed. Importantly, the kinetics of restriction by R.PvuII of its nonmethylated target is also easily monitored electrochemically. This approach should be generally applicable to assaying protein--DNA interactions and reactions on surfaces.
Boon
Elizabeth M
EM
Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, CA 91125, USA.
Salas
Julia E
JE
Barton
Jacqueline K
JK
D001483:Base Sequence; D001665:Binding Sites; D001709:Biotechnology / Q000379:methods; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D019175:DNA Methylation; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004563:Electrochemistry; D004566:Electrodes; D006046:Gold / Q000737:chemistry; D007700:Kinetics; D008956:Models, Chemical; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D011485:Protein Binding; D013997:Time Factors
D004268:DNA-Binding Proteins; D006046:Gold; D004247:DNA