DNA-Mediated Electrochemistry

The base pair stack of DNA has been demonstrated as a medium for long-range charge transport chemistry both in solution and at DNA-modified surfaces. This chemistry is exquisitely sensitive to structural perturbations in the base pair stack as occur with lesions, single base mismatches, and protein binding. We have exploited this sensitivity for the development of reliable electrochemical assays based on DNA charge transport at self-assembled DNA monolayers. Here, we discuss the characteristic features, applications, and advantages of DNA-mediated electrochemistry

DNA-Mediated Electrochemistry of Disulfides on Graphite

The electrochemistry of disulfides incorporated into the sugar−phosphate backbone of pyrene-modified DNA has been examined on highly oriented pyrolytic graphite (HOPG). Two signals, one irreversible, one reversible, are observed at −160 and −290 mV versus normal hydrogen electrode, respectively. The inclusion of a single base mismatch in the DNA duplex below the location of the disulfide leads to a significant attenuation in the electrochemical signal, while the inclusion of a mismatch above the disulfide has little effect on the electrochemistry observed. Thus disulfide reduction in the DNA backbone appears to be DNA-mediated. The redox couples found show a strong pH dependence consistent with formation of a disulfide radical anion or 2e- reduction of the disulfide to the two thiols. These data demonstrate that DNA electrochemistry can be utilized to promote disulfide reduction at a distance mediated by the DNA duplex

Functionalized hyperbranched polymers via olefin metathesis

Hyperbranched polymers are highly branched, three-dimensional macromolecules which are closely related to dendrimers and are typically prepared via a one-pot polycondensation of AB_(n≥2) monomers.^1 Although hyperbranched macromolecules lack the uniformity of monodisperse dendrimers, they still possess many attractive dendritic features such as good solubility, low solution viscosity, globular structure, and multiple end groups.^1-3 Furthermore, the usually inexpensive, one-pot synthesis of these polymers makes them particularly desirable candidates for bulk-material and specialty applications. Toward this end, hyperbranched polymers have been investigated as both rheology-modifying additives to conventional polymers and as substrate-carrying supports or multifunctional macroinitiators, where a large number of functional sites within a compact space becomes beneficial

Multiplexed DNA-Modified Electrodes

We report the use of silicon chips with 16 DNA-modified electrodes (DME chips) utilizing DNA-mediated charge transport for multiplexed detection of DNA and DNA-binding protein targets. Four DNA sequences were simultaneously distinguished on a single DME chip with 4-fold redundancy, including one incorporating a single base mismatch. These chips also enabled investigation of the sequence-specific activity of the restriction enzyme Alu1. DME chips supported dense DNA monolayer formation with high reproducibility, as confirmed by statistical comparison to commercially available rod electrodes. The working electrode areas on the chips were reduced to 10 μm in diameter, revealing microelectrode behavior that is beneficial for high sensitivity and rapid kinetic analysis. These results illustrate how DME chips facilitate sensitive and selective detection of DNA and DNA-binding protein targets in a robust and internally standardized multiplexed format

Scanning Electrochemical Microscopy of DNA Monolayers Modified with Nile Blue

Scanning electrochemical microscopy (SECM) is used to probe long-range charge transport (CT) through DNA monolayers containing the redox-active Nile Blue (NB) intercalator covalently affixed at a specific location in the DNA film. At substrate potentials negative of the formal potential of covalently attached NB, the electrocatalytic reduction of Fe(CN)63− generated at the SECM tip is observed only when NB is located at the DNA/solution interface; for DNA films containing NB in close proximity to the DNA/electrode interface, the electrocatalytic effect is absent. This behavior is consistent with both rapid DNA-mediated CT between the NB intercalator and the gold electrode as well as a rate-limiting electron transfer between NB and the solution phase Fe(CN)63−. The DNA-mediated nature of the catalytic cycle is confirmed through sequence-specific and localized detection of attomoles of TATA-binding protein, a transcription factor that severely distorts DNA upon binding. Importantly, the strategy outlined here is general and allows for the local investigation of the surface characteristics of DNA monolayers both in the absence and in the presence of DNA binding proteins. These experiments highlight the utility of DNA-modified electrodes as versatile platforms for SECM detection schemes that take advantage of CT mediated by the DNA base pair stack

Direct Electrochemistry of Endonuclease III in the Presence and Absence of DNA

The electrochemistry of the base excision repair enzyme Endonuclease III (Endo III) in the presence and absence of DNA has been examined on highly oriented pyrolytic graphite (HOPG). At the surface modified with pyrenated DNA, a reversible signal is observed at 20 mV versus NHE for the [4Fe−4S]^(3+/2+) couple of Endo III, similar to Au. Without DNA modification, oxidative and reductive signals for the [4Fe−4S] cluster of Endo III are found on bare HOPG, allowing a direct comparison between DNA-bound and free redox potentials. These data indicate a shift of approximately −200 mV in the 3+/2+ couple upon binding of Endo III to DNA. This potential shift reflects a difference in affinity for DNA of more than 3 orders of magnitude between the oxidized 3+ and reduced 2+ protein and provides quantitative support for our model utilizing DNA-mediated charge transport to redistribute base excision repair enzymes in the vicinity of damaged DNA

Transducing methyltransferase activity into electrical signals in a carbon nanotube–DNA device

This study creates a device where the DNA is electronically integrated to serve as both the biological target and electrical transducer in a CNT–DNA–CNT device. We detect DNA binding and methylation by the methyltransferase M.SssI at the single molecule level. We demonstrate sequence-specific, reversible binding of M.SssI and protein-catalyzed methylation that alters the protein-binding affinity of the device. This device, which relies on the exquisite electrical sensitivity of DNA, represents a unique route for the specific, single molecule detection of enzymatic activity

Coupling into the Base Pair Stack Is Necessary for DNA-Mediated Electrochemistry

The electrochemistry of DNA films modified with different redox probes linked to DNA through saturated and conjugated tethers was investigated. Experiments feature two redox probes bound to DNA on two surfaces:  anthraquinone (AQ)-modified uridines incorporated into thiolated DNA on gold (Au) and 2,2,6,6-tetramethylpiperidine 1-oxyl (TEMPO)-modified uridines in pyrene-labeled DNA on highly oriented pyrolytic graphite (HOPG). The electrochemistry of these labels when incorporated into DNA has been examined in DNA films containing both well matched and mismatched DNA. DNA-mediated electrochemistry is found to be effective for the TEMPO probe linked with an acetylene linker but not for a saturated TEMPO connected through an ethylenediamine linker. For the AQ probe, DNA-mediated electrochemistry is found with an acetylene linker to uridine but not with an alkyl chain to the 5‘ terminus of the oligonucleotide. Large electrochemical signals and effective discrimination of intervening base mismatches are achieved for the probes connected through the acetylene linkages, while probes connected through saturated linkages exhibit small electrochemical signals associated only with direct surface to probe charge transfer and poor mismatch discrimination. Thus DNA electrochemistry with these probes is dramatically influenced by the chemical nature of their linkage to DNA. These results highlight the importance of effective coupling into the π-stack for long-range DNA-mediated electrochemistry