Michael G. Hill

Electrochemical DNA sensors

Electrochemistry-based sensors offer sensitivity, selectivity and low cost for the detection of selected DNA sequences or mutated genes associated with human disease. DNA-based electrochemical sensors exploit a range of different chemistries, but all take advantage of nanoscale interactions between the target in solution, the recognition layer and a solid electrode surface. Numerous approaches to electrochemical detection have been developed, including direct electrochemistry of DNA, electrochemistry at polymer-modified electrodes, electrochemistry of DNA-specific redox reporters, electrochemical amplifications with nanoparticles, and electrochemical devices based on DNA-mediated charge transport chemistry.

Mutation detection by electrocatalysis at DNA-modified electrodes

Detection of mutations and damaged DNA bases is important for the early diagnosis of genetic disease. Here we describe an electrocatalytic method for the detection of single-base mismatches as well as DNA base lesions in fully hybridized duplexes, based on charge transport through DNA films. Gold electrodes modified with preassembled DNA duplexes are used to monitor the electrocatalytic signal of methylene blue, a redox-active DNA intercalator, coupled to [Fe(CN)6]3−. The presence of mismatched or damaged DNA bases substantially diminishes the electrocatalytic signal. Because this assay is not a measure of differential hybridization, all single-base mismatches, including thermodynamically stable GT and GA mismatches, can be detected without stringent hybridization conditions. Furthermore, many common DNA lesions and “hot spot” mutations in the human p53 genome can be distinguished from perfect duplexes. Finally, we have demonstrated the application of this technology in a chip-based format. This system provides a sensitive method for probing the integrity of DNA sequences and a completely new approach to single-base mismatch detection.

Long‐Range Electron Transfer through DNA Films

Regardless of its position within the DNA film, cross-linked daunomycin (DM) is efficiently reduced electrochemically, indicating that the electron transfer exhibits a shallow distance dependence. Upon the introduction of an intervening cytosine-adenine (CA) mismatch, the electrochemical response is dramatically attenuated (shown schematically). Therefore, the DNA double helix can facilitate long-range electron transfer, but only in the presence of a well-stacked pathway.

Total synthesis of cytochrome b562 by native chemical ligation using a removable auxiliary

We have completed the total chemical synthesis of cytochrome b562 and an axial ligand analogue, [SeMet7]cyt b562, by thioester-mediated chemical ligation of unprotected peptide segments. A novel auxiliary-mediated native chemical ligation that enables peptide ligation to be applied to protein sequences lacking cysteine was used. A cleavable thiol-containing auxiliary group, 1-phenyl-2-mercaptoethyl, was added to the α-amino group of one peptide segment to facilitate amide bond-forming ligation. The amine-linked 1-phenyl-2-mercaptoethyl auxiliary was stable to anhydrous hydrogen fluoride used to cleave and deprotect peptides after solid-phase peptide synthesis. Following native chemical ligation with a thioester-containing segment, the auxiliary group was cleanly removed from the newly formed amide bond by treatment with anhydrous hydrogen fluoride, yielding a full-length unmodified polypeptide product. The resulting polypeptide was reconstituted with heme and folded to form the functional protein molecule. Synthetic wild-type cyt b562 exhibited spectroscopic and electrochemical properties identical to the recombinant protein, whereas the engineered [SeMet7]cyt b562 analogue protein was spectroscopically and functionally distinct, with a reduction potential shifted by ≈45 mV. The use of the 1-phenyl-2-mercaptoethyl removable auxiliary reported here will greatly expand the applicability of total protein synthesis by native chemical ligation of unprotected peptide segments.

Charge transport through a molecular π-stack: double helical DNA

Abstract Double helical DNA, containing a π-stacked array of base pairs within its interior, can be considered as a molecular analogue of solid state π-stacked arrays. Like the solid state materials, the DNA base pair stack provides a medium to facilitate charge transport. However, owing to the dynamical motions of the base pairs within the molecular stack, as well as sequence-dependent inhomogeneities in energetics and base–base couplings, DNA charge transport differs considerably from that in solid state π-stacked materials. Here we review some of the experimental techniques and chemical assemblies used to probe charge transport in DNA. We focus on those parameters that distinguish charge transport within the molecular base pair stack and highlight the sensitivity of DNA charge transport to dynamical variations in base stacking and couplings. Exploiting the sensitivity of DNA charge transport to these sequence- and structure-dependent variations in stacking provides a route to the design of DNA-based nanoscale sensors. Certainly the application of DNA in molecular electronic devices must take into consideration those factors that promote and inhibit DNA-mediated charge transport.

Tricarbonyl(1,10-phenanthroline) (imidazole) rhenium(I): a powerful photooxidant for investigations of electron tunneling in proteins☆

Abstract The structure of [Re(CO)3(phen)(im)]2SO4·4H2O has been determined by X-ray crystallography. The yellow crystals are orthorhombic, space group Pccn (No. 56), with a=17.456(6), b=18.194(5), c=12.646(4) A , R=0.063 for F o 2 >0, R=0.032 for F o 2 >3σ . The compound, which also has been characterized by IR, 1H NMR, and UVVis spectroscopies, exhibits room temperature luminescence in aqueous solution (τ=120 ns) as well as reversible oxidation and reduction in acetonitrile solution (1.85 and −1.30 V versus SCE). The redox properties of the excited state of the complex (E 0 ( Re +∗/0 = 1.2; E 0 ( Re 2+/+∗ ) = −0.7 V ) are being exploited in studies of laser-induced electron tunneling in Re(CO)3(phen)(histidine)-modified proteins.

Electron transfer in ruthenium-modified proteins

Photochemical techniques have been used to measure the kinetics of intramolecular electron transfer in Ru(bpy)2(im)(His)2+-modified (bpy = 2,2′-bipyridine; im = imidazole) cytochromec and azurin. A driving-force study with the His33 derivatives of cytochromec indicates that the reorganization energy (γ) for Fe2+→Ru3+ ET reactions is 0.8 eV. Reductions of the ferriheme by either an excited complex,*Ru2+, or a reduced complex, Ru+, are anomalously fast and may involve formation of an electronically excited ferroheme. The distance dependence of Fe2+→Ru3+ and Cu+→Ru3+ electron transfer in 12 different Ru-modified cytochromes and azurins has been analyzed using a tunneling-pathway model. The ET rates in 10 of the 12 systems exhibit an exponential dependence on metal-metal separation (decay constant of 1.06 å−1) that is consistent with predictions of the pathway model.

Reduction potentials of blue and purple copper proteins in their unfolded states: a closer look at rack-induced coordination

Abstract Cyclic voltammetry has been used to determine the reduction potentials of blue (Pseudomonas aeruginosa azurin) and purple (Thermus thermophilus CuA domain) copper proteins unfolded by guanidine hydrochloride. These Cu(II/I) potentials [456 (azurin); 453 (CuA) mV vs., NHE] are higher than those of the folded proteins. The downshift of the potential in the folded state can be accounted for by assuming that rack-induced axial coordination stabilizes Cu(II) relative to Cu(I) in a protein-encapsulated active site.

Electrochemical Patterning and Detection of DNA Arrays on a Two-Electrode Platform

We report a novel method of DNA array formation that is electrochemically formed and addressed with a two-electrode platform. Electrochemical activation of a copper catalyst, patterned with one electrode, enables precise placement of multiple sequences of DNA onto a second electrode surface. The two-electrode patterning and detection platform allows for both spatial resolution of the patterned DNA array and optimization of detection through DNA-mediated charge transport with electrocatalysis. This two-electrode platform has been used to form arrays that enable differentiation between well-matched and mismatched sequences, the detection of TATA-binding protein, and sequence-selective DNA hybridization.

Label-free electrochemical detection of human methyltransferase from tumors

Significance Epigenetic modifications, including DNA methylation, govern gene expression. Aberrant methylation by DNA methyltransferases can lead to tumorigenesis, so that efficient detection of methyltransferase activity provides an early cancer diagnostic. Current methods, requiring fluorescence or radioactivity, are cumbersome; electrochemical platforms, in contrast, offer high portability, sensitivity, and ease of use. We have developed a label-free electrochemical platform to detect the activity of the most abundant human methyltransferase, DNA(cytosine-5)-methyltransferase1 (DNMT1), and have applied this method in detecting DNMT1 in crude lysates from both cultured human colorectal cancer cells (HCT116) and colorectal tissue samples. The role of abnormal DNA methyltransferase activity in the development and progression of cancer is an essential and rapidly growing area of research, both for improved diagnosis and treatment. However, current technologies for the assessment of methyltransferase activity, particularly from crude tumor samples, limit this work because they rely on radioactivity or fluorescence and require bulky instrumentation. Here, we report an electrochemical platform that overcomes these limitations for the label-free detection of human DNA(cytosine-5)-methyltransferase1 (DNMT1) methyltransferase activity, enabling measurements from crude cultured colorectal cancer cell lysates (HCT116) and biopsied tumor tissues. Our multiplexed detection system involving patterning and detection from a secondary electrode array combines low-density DNA monolayer patterning and electrocatalytically amplified DNA charge transport chemistry to measure selectively and sensitively DNMT1 activity within these complex and congested cellular samples. Based on differences in DNMT1 activity measured with this assay, we distinguish colorectal tumor tissue from healthy adjacent tissue, illustrating the effectiveness of this two-electrode platform for clinical applications.