Electrical detection of DNA binding proteins

The base pair stack of double helical DNA has proven to be an effective medium for charge transport. The pi-stacked DNA base pairs can mediate charge transport (CT) chemistry over distances as long as 20 nm, and the reaction is exquisitely sensitive to DNA sequence-dependent conformation and dynamics. This sensitivity to perturbations in DNA structure and base pair stacking makes DNA-mediated charge transport chemistry an ideal methodology for the electrical detection of base mismatches, lesions, and protein binding. Efforts toward expanding the scope of electrochemistry at DNA-modified surfaces for biosensing applications are presented here.;The first conclusive study of the single-molecule conductivity of DNA is demonstrated. A pristine single DNA duplex in its native conformation can be utilized to covalently bridge an oxidatively etched, single-walled carbon nanotube gap. The effective resistance is on the order of ∼ 1 MO, which is similar to the c-axis resistance of kish graphite. Furthermore, the inclusion of either the CA or GT mismatches within the DNA duplex is seen to increase the resistance of DNA by over two orders of magnitude. This study underscores the fact that conductivity is an intrinsic property of each individual DNA molecule and not, in fact, an ensemble property of the DNA monolayer.;The characterization of DNA monolayers at high oriented pyrolytic graphite is described. Graphite electrodes are modified with duplex DNA containing a pendant pyrene; the yield of electrochemical reduction/oxidation to DNA-bound probes on graphite provides an indicator of base stacking within the DNA. The inclusion of even a single thermodynamically stable yet poorly stacked GT mismatch dramatically attenuates CT through the monolayer. Graphite also affords a potential window which is not limited by the chemistry/stability of thiols on gold. This broad window facilitates the exploration of DNA monolayers containing either disulfides or a TEMPO moiety. In addition, graphite offers the opportunity of interrogating the electrochemistry of DNA-binding proteins containing iron sulfur clusters both in the presence and absence of DNA. Indeed, upon DNA binding, the redox potential of the Endo III, a base excision repair enzyme, shifts by -200 mV, and the redox potential of SoxR, a transcription factor, shifts by +500 mV. Such explorations underscore the importance of investigating DNA-binding enzymes in their biologically relevant conformations.;The electrochemistry of several phenoxazine- and anthraquinone-based probes of DNA structure is presented. Particular emphasis is given to a Nile Blue (NB) derivative which is connected to the base pair stack via a partially saturated linker and has a midpoint potential of ∼ 0 mV versus NHE. NB can be attached to the DNA in a simple fashion and offers the dual advantages of excellent stability and electrocatalytic activity in the presence of ferricyanide. These features facilitate the extensive exploration of DNA-mediated charge transfer to NB at DNA-modified microelectrodes, with a scanning electrochemical microscope, and in a multiplexed format. The application of NB in the detection of transcription factor binding at DNA monolayers is also presented. Nanomolar concentrations of the ubiquitous TATA binding protein transcription factor can be rapidly and sensitively detected from complicated mixtures, including whole-cell lysates.;The efficient transport of charge through self-assembled DNA monolayers on both gold and graphite therefore offers an extremely sensitive tool for investigations of DNA integrity. As such, novel methodologies for assaying protein/DNA interactions at DNA modified surfaces are now possible.