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An electrical probe of protein–DNA interactions on DNA-modified surfaces

Nature Biotechnology volume 20, pages 282–286 (2002)Cite this article

Abstract

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.

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Figure 1: Schematic representation of the fabrication of DNA-modified gold electrodes for electrochemical analysis of protein binding and reaction.
Figure 2: Chronocoulometry at –575 mV of DM covalently crosslinked to thiol-terminated DNA films on gold electrodes with and without bound protein (1 μM).
Figure 3: Kinetics of DNA restriction monitored electrically.

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Acknowledgements

We are grateful to the NIH for their financial support (GM61077) and for an NRSA predoctoral fellowship (E.M.B.). We thank also the Caltech SURF program for a summer undergraduate fellowship (J.E.S.) and Donato Ceres for assistance in obtaining the scanning force microscopy images.

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  1. Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, 91125, CA

    Elizabeth M. Boon, Julia E. Salas & Jacqueline K. Barton

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  1. Elizabeth M. Boon
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  3. Jacqueline K. Barton
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Correspondence to Jacqueline K. Barton.

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Jacqueline K. Barton is a Founding Director of GeneOhm Sciences Inc. in San Diego, CA. GeneOhm Sciences has exclusive rights to license Caltech patents based upon this technology.

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Boon, E., Salas, J. & Barton, J. An electrical probe of protein–DNA interactions on DNA-modified surfaces. Nat Biotechnol 20, 282–286 (2002). https://doi.org/10.1038/nbt0302-282

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