Methylene blue (MB′), covalently attached to DNA through a flexible C12 alkyl linker, provides a sensitive redox reporter in DNA electrochemistry measurements. Tethered, intercalated MB′ is reduced through DNA-mediated charge transport; the incorporation of a single base mismatch at position 3, 10, or 14 of a 17-mer causes an attenuation of the signal to 62 ± 3% of the well-matched DNA, irrespective of position in the duplex. The redox signal intensity for MB′–DNA is found to be least 3-fold larger than that of Nile blue (NB)–DNA, indicating that MB′ is even more strongly coupled to the π-stack. The signal attenuation due to an intervening mismatch does, however, depend on DNA film density and the backfilling agent used to passivate the surface. These results highlight two mechanisms for reduction of MB′ on the DNA-modified electrode: reduction mediated by the DNA base pair stack and direct surface reduction of MB′ at the electrode. These two mechanisms are distinguished by their rates of electron transfer that differ by 20-fold. The extent of direct reduction at the surface can be controlled by assembly and buffer conditions.

Here we describe a multiplexed electrochemical characterization of DNA-bound proteins containing [4Fe-4S] clusters. DNA-modified electrodes have become an essential tool for the characterization of the redox chemistry of DNA repair proteins containing redox cofactors, and multiplexing offers a means to probe different complex samples and substrates in parallel to elucidate this chemistry. Multiplexed analysis of EndonucleaseIII (EndoIII), a DNA repair protein containing a [4Fe-4S] cluster known to be accessible via DNA-mediated charge transport, shows subtle differences in the electrochemical behavior as a function of DNA morphology. The peak splitting, signal broadness, sensitivity to π-stack perturbations, and kinetics were all characterized for the DNA-bound reduction of EndoIII on both closely and loosely packed DNA films. DNA-bound EndoIII is seen to have two different electron transfer pathways for reduction, either through the DNA base stack or through direct surface reduction; closely packed DNA films, where the protein has limited surface accessibility, produce electrochemical signals reflecting electron transfer that is DNA-mediated. Multiplexing furthermore permits the comparison of the electrochemistry of EndoIII mutants, including a new family of mutations altering the electrostatics surrounding the [4Fe-4S] cluster. While little change in the midpoint potential was found for this family of mutants, significant variations in the efficiency of DNA-mediated electron transfer were apparent. Based on the stability of these proteins, examined by circular dichroism, we propose that the electron transfer pathway can be perturbed not only by the removal of aromatic residues but also through changes in solvation near the cluster.

Intraduplex DNA-mediated reduction is established as a general mechanism for the reduction of distally bound stacked redox-active species covalently tethered to DNA through flexible alkane linkages. Methylene Blue (MB), Nile Blue (NB), and Anthraquinone (AQ) were covalently tethered to DNA with three different covalent linkages. Using these reporters DNA electrochemistry was shown to be both DNA-mediated and intra-, rather than inter-, duplex. Significantly, the charge transport pathway occurring through the DNA π-stack is established by using an intervening AC mismatch to break this path. The fact that the DNA-mediated reduction of MB occurs primarily via intraduplex intercalation is established through varying the proximity and integrity of the neighboring duplex DNA.DNA-modified electrodes are extensively used in both the development of next generation diagnostic sensors (1-6) and the characterization of ground-state DNA-mediated electrochemistry (7-15). Many different electrochemical reporters have been developed for DNA-modified electrodes, including: DNA-binding compounds (1, 5), quantum dots (4), and metallization (15,16). Two classes of DNA-based devices have since emerged using the same covalently tethered intercalative compounds but different mechanisms: DNA conformation (3,(17)(18)(19)(20)(21)(22) and DNA-mediated charge transfer (DNA CT) (6-14).The DNA-mediated reduction of both freely diffusing and covalently tethered redox-active reporters has long been established on these DNA-modified electrodes (23)(24)(25). The strategy of covalently tethering redox-active reporters to the DNA, as opposed to the use of freely diffusing reporters, has been adopted to significantly diminish non-specific signals (11,17). Redox-active reporters covalently tethered to DNA have been shown to electronically couple to the π-stack by a variety of mechanisms: end capping (13), intercalation (11,12), and direct conjugation (14). However, the recent characterization of covalently tethered Methylene Blue (MB), an extensively used reporter for DNA-conformation based assays, has spurred a new debate with regards to the mechanism of its reduction (12,(19)(20)(21).The capacity of these redox reporters to be reduced via an intraduplex DNA-mediated pathway has been brought into question with alternative mechanisms such as the duplex tilting to the surface, the charge traveling along the counter ions associated with the sugar phosphate backbone, or the reporter intercalating in a neighboring duplex (19)(20)(21) demonstrate the generality of DNA-mediated reduction of covalently tethered reporters by varying both the redox-active species and the covalent linkage. Critical to these assays is the use of an intervening AC mismatch to establish that the charge transport pathway is through the duplex base pair stack. Beyond demonstrating that the reduction is DNA-mediated, we also provide evidence supporting an intraduplex process by varying the identity and proximity of neighboring duplex DNA.The electronic coupling of redox-act...

Electrocatalysis offers a means of electrochemical signal amplification, yet in DNA-based sensors, electrocatalysis has required highdensity DNA films and strict assembly and passivation conditions. Here, we describe the use of hemoglobin as a robust and effective electron sink for electrocatalysis in DNA sensing on low-density DNA films. Protein shielding of the heme redox center minimizes direct reduction at the electrode surface and permits assays on low-density DNA films. Electrocatalysis with methylene blue that is covalently tethered to the DNA by a flexible alkyl chain linkage allows for efficient interactions with both the base stack and hemoglobin. Consistent suppression of the redox signal upon incorporation of a single cytosine-adenine (CA) mismatch in the DNA oligomer demonstrates that both the unamplified and the electrocatalytically amplified redox signals are generated through DNAmediated charge transport. Electrocatalysis with hemoglobin is robust: It is stable to pH and temperature variations. The utility and applicability of electrocatalysis with hemoglobin is demonstrated through restriction enzyme detection, and an enhancement in sensitivity permits femtomole DNA sampling.DNA charge transport | DNA sensors | mismatch detection