DNA charge transport for sensing and signaling

Abstract

The DNA duplex is an exquisite macromolecular array that stores genetic information to encode proteins and regulate pathways. Its unique structure also imparts chemical function that allows it also to mediate charge transport (CT). We have utilized diverse platforms to probe DNA CT, using spectroscopic, electrochemical, and even genetic methods. These studies have established powerful features of DNA CT chemistry. DNA CT can occur over long molecular distances as long as the bases are well stacked. The perturbations in base stacking that arise with single base mismatches, DNA lesions, and the binding of some proteins that kink the DNA all inhibit DNA CT. Significantly, single molecule studies of DNA CT show that ground state CT can occur over 34 nm if the duplex is well stacked; one single base mismatch inhibits CT. The DNA duplex is an effective sensor for the integrity of the base pair stack. Moreover, the efficiency of DNA CT is what one would expect for a stack of graphite sheets: equivalent to the stack of DNA base pairs and independent of the sugar-phosphate backbone. Since DNA CT offers a means to carry out redox chemistry from a distance, we have considered how this chemistry might be used for long range biological signaling. We have taken advantage of our chemical probes and platforms to characterize DNA CT in the context of the cell. CT can occur over long distances, perhaps funneling damage to particular sites and insulating others from oxidative stress. Significantly, transcription factors that activate the genome to respond to oxidative stress can also be activated from a distance through DNA CT. Numerous proteins maintain the integrity of the genome and an increasing number of them contain [4Fe-4S] clusters that do not appear to carry out either structural or enzymatic roles. Using electrochemical methods, we find that DNA binding shifts the redox potentials of the clusters, activating them towards oxidation at physiological potentials. We have proposed a model that describes how repair proteins may utilize DNA CT to efficiently search the genome for lesions. Importantly, many of these proteins occur in low copy numbers within the cell, and thus a processive mechanism does not provide a sufficient explanation of how they find and repair lesions before the cell divides. Using atomic force microscopy and genetic assays, we show that repair proteins proficient at DNA CT can relocalize in the vicinity of DNA lesions and can cooperate in finding lesions within the cell. Conversely, proteins defective in DNA CT cannot relocalize in the vicinity of lesions and do not assist other proteins involved in repair within the cell. Moreover such genetic defects are associated with disease in human protein analogues. As we continue to unravel this chemistry and discover more proteins with redox cofactors involved in genome maintenance, we are learning more regarding opportunities for long range signaling and sensing, and more examples of DNA CT chemistry that may provide critical functions within the cell.

Figure 1

The structure of DNA facilitates charge transport. The aromatic bases (blue) of DNA stack with each other like a pile of coins and are wrapped by a sugar phosphate backbone (purple ribbon). The overlapping π orbitals of these stacked bases form a conductive core down the helical axis that mediates the flow of charge. This structure resembles that of stacked graphite sheets and, indeed, undamaged, well stacked DNA is found to have the same conductivity as that measured perpendicular to graphite. Importantly, the conformation of this structure is not static; dynamic motions within this macromolecular array contribute to the mechanistic complexity of DNA CT.

Figure 2

Platforms for the study of DNA CT. Top row, spectroscopic solution platforms: (left) photoactivated luminescence and quenching between a covalent metallointercalator and acceptor pair and (right) photoactivated fluorescence of base analog 2-aminopurine and quenching by guanine. Second row, a biochemical solution platform: photoactivated oxidation of guanine doublets by a covalently bound metallointercalator. Third row, electrochemical surface platforms: (left) DNA-modified electrodes with a covalent redox probe and (right) DNA-modified electrodes with a bound protein that contains a redox-active cofactor. Fourth row, a single molecule platform: covalent attachment of DNA across a gap in a carbon nanotube device.

Figure 3

Single molecule experiments illustrate the sensitivity of DNA CT to mismatches. Left illustration: DNA CT is measured in single molecules of DNA that covalently bridge a gap in a carbon nanotube device. One strand (blue) is covalently attached by its 3′ and 5′ ends while the other strand can be freely exchanged between well matched complements (orange) and strands that introduce a single base mismatch (green, purple). Right plot: the device was connected with a series of well matched and mismatched strands and the source-drain current (I_SD) measured at the gating voltage V_G = −3 V is shown for each. The colors and numbers of the points in the series correspond to the different strands in the left illustration. This result clearly shows that current through the device is cut off in duplexes that contain a single base mismatch and restored when the DNA in the gap is re-hybridized with its well matched complement. Adapted with permission from reference 6.

Figure 4

DNA CT triggers transcription. We have utilized a tethered metal photooxidant to oxidize bound SoxR protein (orange) from a distance.

Figure 5

Model for a DNA-mediated search by repair proteins. 1) When the cell undergoes oxidative stress, guanine radicals are formed, triggering a repair protein to bind DNA. 2) DNA-binding protein is oxidized, releasing an electron that repairs the guanine radical. 3) Another repair protein binds to a distant site. As it binds to DNA, there is a shift in the redox potential of the protein, making it more easily oxidized. 4) The protein could then send an electron through the DNA base pair stack that travels to a distally bound protein, scanning the intervening region for damage. 5) If the base pair stack is intact, charge transport occurs between proteins. The repair protein that receives the electron is reduced and dissociates. 6) If a lesion is present (red), charge transport is attenuated, and the repair proteins will remain bound in the oxidized form and slowly proceed to the site of damage.

Figure 6

Schematic illustrating the helper function assay to monitor MutY activity. When EndoIII or D138A, an EndoIII mutant that is CT proficient but glycolytically active, is available to help in the search for damage, MutY repairs lesions efficiently (green). If EndoIII is knocked out or Y82A, a mutant deficient in CT, is present, MutY efficiency decreases (yellow). When MutY is knocked out, repair is not observed (red).

Figure 7

(Top Left) Schematic displaying well matched DNA that contains a single-strand overhang, tethered to a gold electrode. The electrochemical setup is used to monitor the DNA-bound redox potential of SaXPD. (Bottom Left) Crystal structure of SaXPD with G34R mutation and the [4Fe4S] cluster shown in space filling models. (Right) ATP-dependent CT of WT (black) and G34R (red). The initial rate constant observed for G34R is lower than that for WT SaXPD. Reprinted and adapted with permission from reference 10. Copyright 2011 American Chemical Society.