Electrical Probes of DNA-Binding Proteins

Abstract

A DNA electrochemistry platform has been developed to probe proteins bound to DNA electrically. Here gold electrodes are modified with thiol-modified DNA, and DNA charge transport chemistry is used to probe DNA binding and enzymatic reaction both with redox-silent and redox-active proteins. For redox-active proteins, the electrochemistry permits the determination of redox potentials in the DNA-bound form, where comparisons to DNA-free potentials can be made using graphite electrodes without DNA modification. Importantly, electrochemistry on the DNA-modified electrodes facilitates reaction under aqueous, physiological conditions with a sensitive electrical measurement of binding and activity.

Sensing DNA through DNA Charge Transport

“…Redox molecules, either noncovalently or covalently attached to the DNA, can then be reduced or oxidized by applying a potential across the electrode surface. 3 …”

“…Even for electrochemistry experiments, probing the same DNA construct with molecules that do and do not couple to the π -stack shows a significant difference in yield. 3 It is also possible to use redox probes that selectively target mismatches or abasic sites and stack within the open site, so that a DNA-mediated signal is found only if that mismatch or abasic site is present. Critically, then, for all these experiments, DNA CT is only rapid and over long-range if the connection is truly to the base pair stack.…”

“…A wide variety of donors and acceptors were used in these experiments, ranging from transition metal complexes to purely organic moieties, base analogs, and proteins. − More recent experiments have been conducted using electrodes, typically gold or graphite, modified with a self-assembled monolayer of DNA. − Here, DNA duplexes are linked to the surface using a covalent modification on the phosphate backbone (alkane-thiols for gold or pyrene for graphite) that allows the duplexes to stand upright, facilitating interaction with DNA-binding molecules in solution. Redox molecules, either noncovalently or covalently attached to the DNA, can then be reduced or oxidized by applying a potential across the electrode surface …”

“…Here, we describe a variety of studies carried out in our laboratory probing the DNA duplex and DNA-binding partners using DNA-mediated charge transport (DNA CT). Over the past three decades, we have explored this chemistry and its application in sensing DNA. − Additionally, we have focused on how nature makes use of this chemistry for DNA sensing and for long-range signaling across the nucleus of the cell. , …”

Fe–S Clusters and MutY Base Excision Repair Glycosylases: Purification, Kinetics, and DNA Affinity Measurements

“…We also include examples of how these approaches were used in revealing important features of MutY. Of note, electrochemical approaches used to study MutY, and other Fe-S cluster containing repair and replication proteins, has recently been reported in Methods in Enzymology (Barton et al, 2017). In addition, the chapter following this one (in this volume) focuses on cellular assays used to study MutY enzymes.…”