Marc McWilliams

DNA as a Molecular Wire: Distance and Sequence Dependence

Functional nanowires and nanoelectronics are sought for their use in next generation integrated circuits, but several challenges limit the use of most nanoscale devices on large scales. DNA has great potential for use as a molecular wire due to high yield synthesis, near-unity purification, and nanoscale self-organization. Nonetheless, a thorough understanding of ground state DNA charge transport (CT) in electronic configurations under biologically relevant conditions, where the fully base-paired, double-helical structure is preserved, is lacking. Here, we explore the fundamentals of CT through double-stranded DNA monolayers on gold by assessing 17 base pair bridges at discrete points with a redox active probe conjugated to a modified thymine. This assessment is performed under temperature-controlled and biologically relevant conditions with cyclic and square wave voltammetry, and redox peaks are analyzed to assess transfer rate and yield. We demonstrate that the yield of transport is strongly tied to the stability of the duplex, linearly correlating with the melting temperature. Transfer rate is found to be temperature-activated and to follow an inverse distance dependence, consistent with a hopping mechanism of transport. These results establish the governing factors of charge transfer speed and throughput in DNA molecular wires for device configurations, guiding subsequent application for nanoscale electronics.

Sensitive and selective real-time electrochemical monitoring of DNA repair

Unrepaired DNA damage can lead to mutation, cancer, and death of cells or organisms. However, due to the subtlety of DNA damage, it is difficult to sense the presence of damage repair with high selectivity and sensitivity. We have shown sensitive and selective electrochemical sensing of 8-oxoguanine and uracil repair glycosylase activity within DNA monolayers on gold by multiplexed analysis with silicon chips and low-cost electrospun nanofibers. Our approach compared the electrochemical signal of electroactive, probe-modified DNA monolayers containing a base defect versus the rational control of defect-free monolayers. We found damage-specific sensitivity thresholds on the order of femtomoles of proteins and dynamic ranges of over two orders of magnitude for each target. Temperature-dependent kinetics were extracted, showing exponential signal loss with time constants of seconds. Damage specific detection in a mixture of enzymes and in response to environmental oxidative damage was also demonstrated. Nanofibers were shown to behave similarly to conventional gold-on-silicon devices, showing the potential of these low-cost devices for sensing applications. This device approach achieves a sensitive, selective, and rapid assay of repair protein activity, enabling a biological interrogation of DNA damage repair.

Temperature Dependence of Electrochemical DNA Charge Transport: Influence of a Mismatch

Charge transfer through DNA is of interest as DNA is both the quintessential biomolecule of all living organisms and a self-organizing element in bioelectronic circuits and sensing applications. Here, we report the temperature-dependent properties of DNA charge transport in an electronically relevant arrangement of DNA monolayers on gold under biologically relevant conditions, and we track the effects of incorporating a CA single base pair mismatch. Charge transfer (CT) through double stranded, 17mer monolayers was monitored by following the yield of electrochemical reduction of a Nile blue redox probe conjugated to a modified thymine. Analysis with cyclic voltammetry and square wave voltammetry shows that DNA CT increases significantly with temperature, indicative of more DNA bridges becoming active for transport. The mismatch was found to attenuate DNA CT at lower temperatures, but the effect of the mismatch diminished as temperature was increased. Voltammograms were analyzed to extract the electron transfer rate k(0), the electron transfer coefficient α, and the redox-active surface coverage Γ*. Arrhenius behavior was observed, with activation energies of 100 meV for electron transfer through well-matched DNA. Single CA mismatches increased the activation energy by 60 meV. These results have clear implications for sensing applications and are evaluated with respect to the prominent models of DNA CT.

The Electronic Influence of Abasic Sites in DNA.

Abasic sites in DNA are prevalent as both naturally forming defects and as synthetic inclusions for biosensing applications. The electronic impact of these defects in DNA sensor and device configurations has yet to be clarified. Here we report the effect of an abasic site on the rate and yield of charge transport through temperature-controlled analysis of DNA duplex monolayers on multiplexed devices. Transport yield through the abasic site monolayer strongly increases with temperature, but the yield relative to an undamaged monolayer decreases with temperature. This is opposite to the increasing relative yield with temperature from a mismatched base pair, and these effects are accounted for by the unique structural impact of each defect. Notably, the effect of the abasic site is nearly doubled when heated from room temperature to 37 °C. The rate of transport is largely unaffected by the abasic site, showing Arrhenius-type behavior with an activation energy of ∼300 meV. Detailed abasic site investigation elucidates the electrical impact of these biologically spontaneous defects and aids development of biological sensors.

Using DNA devices to track anticancer drug activity

It is beneficial to develop systems that reproduce complex reactions of biological systems while maintaining control over specific factors involved in such processes. We demonstrated a DNA device for following the repair of DNA damage produced by a redox-cycling anticancer drug, beta-lapachone (β-lap). These chips supported ß-lap-induced biological redox cycle and tracked subsequent DNA damage repair activity with redox-modified DNA monolayers on gold. We observed drug-specific changes in square wave voltammetry from these chips at therapeutic ß-lap concentrations of high statistical significance over drug-free control. We also demonstrated a high correlation of this change with the specific ß-lap-induced redox cycle using rational controls. The concentration dependence of ß-lap revealed significant signal changes at levels of high clinical significance as well as sensitivity to sub-lethal levels of ß-lap. Catalase, an enzyme decomposing peroxide, was found to suppress DNA damage at a NQO1/catalase ratio found in healthy cells, but was clearly overcome at a higher NQO1/catalase ratio consistent with cancer cells. We found that it was necessary to reproduce key features of the cellular environment to observe this activity. Thus, this chip-based platform enabled tracking of ß-lap-induced DNA damage repair when biological criteria were met, providing a unique synthetic platform for uncovering activity normally confined to inside cells.

Application of Electrochemical Devices to Characterize the Dynamic Actions of Helicases on DNA

Much remains to be understood about the kinetics and thermodynamics of DNA helicase binding and activity. Here, we utilize probe-modified DNA monolayers on multiplexed gold electrodes as a sensitive recognition element and morphologically responsive transducer of helicase-DNA interactions. The electrochemical signals from these devices are highly sensitive to structural distortion of the DNA produced by the helicases. We used this DNA electrochemistry to distinguish the details of the DNA interactions of three distinct XPB helicases, which belong to the superfamily-2 of helicases. Clear changes in DNA melting temperature and duplex stability were observed upon helicase binding, shifts that could not be observed with conventional UV-visible absorption measurements. Binding dissociation constants were estimated in the range from 10 to 50 nM and correlated with observations of activity. ATP-stimulated DNA unwinding activity was also followed, revealing exponential time scales and distinct time constants associated with conventional and molecular wrench modes of operation further confirmed by crystal structures. These devices thus provide a sensitive measure of the structural thermodynamics and kinetics of helicase-DNA interactions.

Sensitive and selective real-time electrochemical monitoring of DNA repair (Presentation Recording)

Unrepaired DNA damage can lead to mutation, cancer, and death of cells or organisms. However, due to the subtlety of DNA damage, it is difficult to sense the repair of damage products with high selectivity and sensitivity. Here, we show sensitive and selective electrochemical sensing of the repair activity of 8-oxoguanine and uracil glycosylases within DNA monolayers on gold by multiplexed analysis with silicon chips and low-cost electrospun nanofibers. Our approach involves comparing the electrochemical signal of redox probe modified monolayers containing the defect versus the rational control of defect-free monolayers. We find sequence-specific sensitivity thresholds on the order of femtomoles of proteins and dynamic ranges of over two orders of magnitude for each target. For 8-oxoguanine repair, temperature-dependent kinetics are extracted, showing exponential signal loss with time constants of seconds. Electrospun fibers are shown to behave similarly to conventional gold-on-silicon devices, showing the potential of these low-cost devices for sensing applications.