DNA Electrochemistry Research Articles

Perspective – Leveraging the Versatility of DNA Origami and Electrochemistry for New Sensing Modalities

Abstract Electrochemical biosensors are uniquely positioned to offer real-time in vivo molecular sensing due to their robustness to both biofluids and contaminants found in biofluids, and their adaptability for the detection of different analytes by their use of oligonucleotides or proteins as binding moiety. DNA Origami, the folding of a long DNA scaffold by hundreds of shorter oligonucleotide “staple” strands, allows the construction of nanoscale molecular objects of essentially arbitrary form, flexibility and functionality. We describe work at the intersection of these two fields and their – hopefully – bright future together.

Measuring DNA Charge Transport on a Surface Using a Redox Modulated Fluorescence Intensity Strategy with DNA SAMs

The double stranded DNA helix has drawn great attention across different research areas. Various types of platforms have been used to study DNA charge transfer (CT) system. Two types of DNA CT platforms are considered as the most effective: the photoinduced CT in solution with assembled donor and acceptor and electrochemistry on ground state CT in a DNA self-assembled monolayer (SAM)1. We will show a new approach for the study of DNA CT on gold surface by studying the redox modulated fluorescence signal of a co-deposited double-stranded DNA assemblies labeled with Methylene Blue (MB) or AlexaFluoro488 (AF488). We demonstrate a method that can optically distinguish between the two mechanisms of the MB redox on DNA-modified surface: namely either DNA CT mediated reduction or the direct reduction of MB by the gold surface2. Given the fact that the studies on DNA-mediated electrochemistry on surface has been mainly using electrochemical measurements and we still do not fully understand the full picture of the DNA CT, we believe that this study will provide a fresh opinion and facilitate understanding on the DNA CT mechanism on electrode surface.Reference: Arnold, A. R.; Grodick, M. A.; Barton, J. K. DNA Charge Transport: From Chemical Principles to the Cell. Cell Chem. Biol. 2016, 23 (1), 183–197.Pheeney, C. G.; Barton, J. K. DNA Electrochemistry with Tethered Methylene Blue. Langmuir 2012, 28 (17), 7063–7070.Muren, N. B.; Olmon, E. D.; Barton, J. K. Solution, Surface, and Single Molecule Platforms for the Study of DNA-Mediated Charge Transport. Chem. Chem. Phys. 2012, 14 (40), 13754–13771.

The [4Fe4S] Cluster of Yeast DNA Polymerase ε Is Redox Active and Can Undergo DNA-Mediated Signaling.

Many DNA replication and DNA repair enzymes have been found to carry [4Fe4S] clusters. The major leading strand polymerase, DNA polymerase ε (Pol ε) from Saccharomyces cerevisiae, was recently reported to have a [4Fe4S] cluster located within the catalytic domain of the largest subunit, Pol2. Here the redox characteristics of the [4Fe4S] cluster in the context of that domain, Pol2CORE, are explored using DNA electrochemistry, and the effects of oxidation and rereduction on polymerase activity are examined. The exonuclease deficient variant D290A/E292A, Pol2COREexo–, was used to limit DNA degradation. While no redox signal is apparent for Pol2COREexo– on DNA-modified electrodes, a large cathodic signal centered at −140 mV vs NHE is observed after bulk oxidation. A double cysteine to serine mutant (C665S/C668S) of Pol2COREexo–, which lacks the [4Fe4S] cluster, shows no similar redox signal upon oxidation. Significantly, protein oxidation yields a sharp decrease in polymerization, while rereduction restores activity almost to the level of untreated enzyme. Moreover, the addition of reduced EndoIII, a bacterial DNA repair enzyme containing [4Fe4S]2+, to oxidized Pol2COREexo– bound to its DNA substrate also significantly restores polymerase activity. In contrast, parallel experiments with EndoIIIY82A, a variant of EndoIII, defective in DNA charge transport (CT), does not show restoration of activity of Pol2COREexo–. We propose a model in which EndoIII bound to the DNA duplex may shuttle electrons through DNA to the DNA-bound oxidized Pol2COREexo– via DNA CT and that this DNA CT signaling offers a means to modulate the redox state and replication by Pol ε.

DNA Electrochemistry: Charge-Transport Pathways through DNA Films on Gold.

Over the past 25 years, collective evidence has demonstrated that the DNA base-pair stack serves as a medium for charge transport chemistry in solution and on DNA-modified gold surfaces. Since this charge transport depends sensitively upon the integrity of the DNA base pair stack, perturbations in base stacking, as may occur with DNA base mismatches, lesions, and protein binding, interrupt DNA charge transport (DNA CT). This sensitivity has led to the development of powerful DNA electrochemical sensors. Given the utility of DNA electrochemistry for sensing and in response to recent literature, we describe critical protocols and characterizations necessary for performing DNA-mediated electrochemistry. We demonstrate DNA electrochemistry with a fully AT DNA sequence using a thiolated preformed DNA duplex and distinguish this DNA-mediated chemistry from that of electrochemistry of largely single-stranded DNA adsorbed to the surface. We also demonstrate the dependence of DNA CT on a fully stacked duplex. An increase in the percentage of mismatches within the DNA monolayer leads to a linear decrease in current flow for a DNA-bound intercalator, where the reaction is DNA-mediated; in contrast, for ruthenium hexammine, which binds electrostatically to DNA and the redox chemistry is not DNA-mediated, there is no effect on current flow with mismatches. We find that, with DNA as a well hybridized duplex, upon assembly, a DNA-mediated pathway facilitates the electron transfer between a well coupled redox probe and the gold surface. Overall, this report highlights critical points to be emphasized when utilizing DNA electrochemistry and offers explanations and controls for analyzing confounding results.

Advanced electrochemical detection of nitrogenous bases, synthetic oligonucleotides, and single-stranded DNA through flow injection analysis and catalytic oxidation on Prussian Blue

The effect of Prussian Blue (PB, ferric hexacyanoferrate) on oxidation of free nucleobases, synthetic oligonucleotides, single- and double-stranded DNA (ssDNA and dsDNA) was evaluated by cyclic voltammetry (CV) and flow injection analysis (FIA) on carbon screen printed electrodes, both bare (SPE) and PB modified (SPE/PB). It has been found that electrocatalytic oxidation of nucleobases, namely guanine, adenine, thymine, and cytosine, or nucleobase residues takes place via electrochemically generated Berlin Green (BG), fully oxidized form of PB. The constant potential FIA allowed one to register electrooxidation of all DNA nitrogenous bases at 0.95 V and three nitrogenous bases (except for cytosine) at 0.70 V on SPE and SPE/PB. The modification of electrode surface with PB resulted in a shift of oxidation potential to less positive values and enhanced (up to 8 times) detection sensitivity for DNA nucleobases. The pronounced catalytic effect of electrogenerated BG on their oxidation has also been observed for synthetic oligonucleotides and degraded ssDNA of natural origin. By fractionating ssDNA, it has been found that the main contributor to the total oxidation signal is the fraction of lowest molecular weight (< 3 kDa). At the same time, no oxidation signal was observed for large molecular weight native dsDNA in the potential range from 0 to 1 V on bare SPE and SPE/PB in both CV and FIA measurement modes. Therefore, the advanced electrochemical detection by FIA with SPE/PB was achieved for all nucleobases, synthetic oligonucleotides, and ssDNA of natural origin. Moreover, the obtained results expand the contemporary understanding of DNA electrochemistry.

Rational Design of Amphiphilic Diblock Copolymer/MWCNT Surface Modifiers and Their Application for Direct Electrochemical Sensing of DNA.

We demonstrate the application of amphiphilic ionic poly(n-butylmethacrylate)-block- poly(2-(dimethylamino)ethyl methacrylate) diblock copolymers (PnBMA40-b-PDMAEMA40, PnBMA40-b-PDMAEMA120, PnBMA70-b-PDMAEMA120) for dispersing multiwalled carbon nanotubes (MWCNTs) in aqueous media, a subsequent efficient surface modification of screen-printed electrodes (SPEs), and the application of the modified SPEs for DNA electrochemistry. Stable and fine aqueous dispersions of MWCNTs were obtained with PnBMAx-b-PDMAEMAy diblock copolymers, regardless of the structure of the copolymer and the amount of MWCNTs in the dispersions. The effect of the diblock copolymer structure was important when the dispersions of MWCNTs were deposited as modifying layers on surfaces of SPEs, resulting in considerable increases of the electroactive surface areas and great acceleration of the electron transfer rate. The SPE/(PnBMAx-b-PDMAEMAy + MWCNT) constructs were further exploited for direct electrochemical oxidation of the guanine (G) and adenine (A) residues in a model salmon sperm double-stranded DNA (dsDNA). Two well-defined irreversible oxidation peaks were observed at about +600 and +900 mV, corresponding to the electrochemical oxidation of G and A residues, respectively. A multi-parametric optimization of dsDNA electrochemistry enables one to get the limits of detection (LOD) as low as 5 μg/mL (0.25 μM) and 1 μg/mL (0.05 μM) for G and A residues, respectively. The achieved sensitivity of DNA assay enables quantification of the A and G residues of dsDNA in the presence of human serum and DNA in isolated human leukocytes.

UvrC Coordinates an O2-Sensitive [4Fe4S] Cofactor.

Recent advances have led to numerous landmark discoveries of [4Fe4S] clusters coordinated by essential enzymes in repair, replication, and transcription across all domains of life. The cofactor has notably been challenging to observe for many nucleic acid processing enzymes due to several factors, including a weak bioinformatic signature of the coordinating cysteines and lability of the metal cofactor. To overcome these challenges, we have used sequence alignments, an anaerobic purification method, iron quantification, and UV-visible and electron paramagnetic resonance spectroscopies to investigate UvrC, the dual-incision endonuclease in the bacterial nucleotide excision repair (NER) pathway. The characteristics of UvrC are consistent with [4Fe4S] coordination with 60-70% cofactor incorporation, and additionally, we show that, bound to UvrC, the [4Fe4S] cofactor is susceptible to oxidative degradation with aggregation of apo species. Importantly, in its holo form with the cofactor bound, UvrC forms high affinity complexes with duplexed DNA substrates; the apparent dissociation constants to well-matched and damaged duplex substrates are 100 ± 20 nM and 80 ± 30 nM, respectively. This high affinity DNA binding contrasts reports made for isolated protein lacking the cofactor. Moreover, using DNA electrochemistry, we find that the cluster coordinated by UvrC is redox-active and participates in DNA-mediated charge transport chemistry with a DNA-bound midpoint potential of 90 mV vs NHE. This work highlights that the [4Fe4S] center is critical to UvrC.

Surpassing the detection limit and accuracy of the electrochemical DNA sensor through the application of CRISPR Cas systems

Robust developments of personalized medicine for next-generation healthcare highlight the need for sensitive and accurate point-of-care platforms for quantification of disease biomarkers. Broad presentations of clustered regularly interspaced short palindromic repeats (CRISPR) as an accurate gene editing tool also indicate that the high-specificity and programmability of CRISPR system can be utilized for the development of biosensing systems. Herein, we present a CRISPR Cas system enhanced electrochemical DNA (E-DNA) sensor with unprecedented sensitivity and accuracy. The principle of the E-DNA sensor is the target induced conformational change of the surface signaling probe (containing an electrochemical tag), leading to the variation of the electron transfer rate of the electrochemical tag. With the introduction of CRISPR cleavage activity into the E-DNA sensor, a more apparent signal change between with and without the presence of the target can be achieved. We compared the performance of Cas9 and Cas12a enhanced E-DNA sensor and optimized the chemical environment of CRISPR, achieving a femto-molar detection limit without enzymatic amplification. Moreover, we correlated the CRISPR cleavage signal with the original E-DNA signal as a strategy to indicate potential mismatches in the target sequence. Comparing with classic DNA electrochemistry based mutation detection strategy, CRISPR enhanced E-DNA sensor can determine the presence of a single mutation at an unknown concentration condition. Overall, we believe that the CRISPR enhanced E-DNA sensing strategy will be of especially high utility for point-of-care systems owing to the programmability, modularity, high-sensitivity and high-accuracy.

Redox Chemistry in the Genome: Emergence of the [4Fe4S] Cofactor in Repair and Replication.

Many DNA-processing enzymes have been shown to contain a [4Fe4S] cluster, a common redox cofactor in biology. Using DNA electrochemistry, we find that binding of the DNA polyanion promotes a negative shift in [4Fe4S] cluster potential, which corresponds thermodynamically to a ∼500-fold increase in DNA-binding affinity for the oxidized [4Fe4S]3+ cluster versus the reduced [4Fe4S]2+ cluster. This redox switch can be activated from a distance using DNA charge transport (DNA CT) chemistry. DNA-processing proteins containing the [4Fe4S] cluster are enumerated, with possible roles for the redox switch highlighted. A model is described where repair proteins may signal one another using DNA-mediated charge transport as a first step in their search for lesions. The redox switch in eukaryotic DNA primases appears to regulate polymerase handoff, and in DNA polymerase δ, the redox switch provides a means to modulate replication in response to oxidative stress. We thus describe redox signaling interactions of DNA-processing [4Fe4S] enzymes, as well as the most interesting potential players to consider in delineating new DNA-mediated redox signaling networks.

Substrate Binding Regulates Redox Signaling in Human DNA Primase.

Generation of daughter strands during DNA replication requires the action of DNA primase to synthesize an initial short RNA primer on the single-stranded DNA template. Primase is a heterodimeric enzyme containing two domains whose activity must be coordinated during primer synthesis: an RNA polymerase domain in the small subunit (p48) and a [4Fe4S] cluster-containing C-terminal domain of the large subunit (p58C). Here we examine the redox switching properties of the [4Fe4S] cluster in the full p48/p58 heterodimer using DNA electrochemistry. Unlike with isolated p58C, robust redox signaling in the primase heterodimer requires binding of both DNA and NTPs; NTP binding shifts the p48/p58 cluster redox potential into the physiological range, generating a signal near 160 mV vs NHE. Preloading of primase with NTPs enhances catalytic activity on primed DNA, suggesting that primase configurations promoting activity are more highly populated in the NTP-bound protein. We propose that p48/p58 binding of anionic DNA and NTPs affects the redox properties of the [4Fe4S] cluster; this electrostatic change is likely influenced by the alignment of primase subunits during activity because the configuration affects the [4Fe4S] cluster environment and coupling to DNA bases for redox signaling. Thus, both binding of polyanionic substrates and configurational dynamics appear to influence [4Fe4S] redox signaling properties. These results suggest that these factors should be considered generally in characterizing signaling networks of large, multisubunit DNA-processing [4Fe4S] enzymes.

Nitric Oxide Modulates Endonuclease III Redox Activity by a 800 mV Negative Shift upon [Fe4S4] Cluster Nitrosylation.

Here we characterize the [Fe4S4] cluster nitrosylation of a DNA repair enzyme, endonuclease III (EndoIII), using DNA-modified gold electrochemistry and protein film voltammetry, electrophoretic mobility shift assays, mass spectrometry of whole and trypsin-digested protein, and a variety of spectroscopies. Exposure of EndoIII to nitric oxide under anaerobic conditions transforms the [Fe4S4] cluster into a dinitrosyl iron complex, [(Cys)2Fe(NO)2]-, and Roussin's red ester, [(μ-Cys)2Fe2(NO)4], in a 1:1 ratio with an average retention of 3.05 ± 0.01 Fe per nitrosylated cluster. The formation of the dinitrosyl iron complex is consistent with previous reports, but the Roussin's red ester is an unreported product of EndoIII nitrosylation. Hyperfine sublevel correlation (HYSCORE) pulse EPR spectroscopy detects two distinct classes of NO with 14N hyperfine couplings consistent with the dinitrosyl iron complex and reduced Roussin's red ester. Whole-protein mass spectrometry of EndoIII nitrosylated with 14NO and 15NO support the assignment of a protein-bound [(μ-Cys)2Fe2(NO)4] Roussin's red ester. The [Fe4S4]2+/3+ redox couple of DNA-bound EndoIII is observable using DNA-modified gold electrochemistry, but nitrosylated EndoIII does not display observable redox activity using DNA electrochemistry on gold despite having a similar DNA-binding affinity as the native protein. However, direct electrochemistry of protein films on graphite reveals the reduction potential of native and nitrosylated EndoIII to be 127 ± 6 and -674 ± 8 mV vs NHE, respectively, corresponding to a shift of approximately -800 mV with cluster nitrosylation. Collectively, these data demonstrate that DNA-bound redox activity, and by extension DNA-mediated charge transport, is modulated by [Fe4S4] cluster nitrosylation.

A human MUTYH variant linking colonic polyposis to redox degradation of the [4Fe4S]2+ cluster.

The human DNA repair enzyme MUTYH excises mispaired adenine residues in oxidized DNA. Homozygous MUTYH mutations underlie the autosomal, recessive cancer syndrome MUTYH-associated polyposis. We report a MUTYH variant, p.C306W (c.918C>G), with a tryptophan residue in place of native cysteine, that ligates the [4Fe4S] cluster in a patient with colonic polyposis and family history of early age colon cancer. In bacterial MutY, the [4Fe4S] cluster is redox active, allowing rapid localization to target lesions by long-range, DNA-mediated signalling. In the current study, using DNA electrochemistry, we determine that wild-type MUTYH is similarly redox-active, but MUTYH C306W undergoes rapid oxidative degradation of its cluster to [3Fe4S]+, with loss of redox signalling. In MUTYH C306W, oxidative cluster degradation leads to decreased DNA binding and enzyme function. This study confirms redox activity in eukaryotic DNA repair proteins and establishes MUTYH C306W as a pathogenic variant, highlighting the essential role of redox signalling by the [4Fe4S] cluster.

DNA Electrochemistry and Electrochemical Sensors for Nucleic Acids.

Sensitive, specific, and fast analysis of nucleic acids (NAs) is strongly needed in medicine, environmental science, biodefence, and agriculture for the study of bacterial contamination of food and beverages and genetically modified organisms. Electrochemistry offers accurate, simple, inexpensive, and robust tools for the development of such analytical platforms that can successfully compete with other approaches for NA detection. Here, electrode reactions of DNA, basic principles of electrochemical NA analysis, and their relevance for practical applications are reviewed and critically discussed.

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.

The [4Fe4S] cluster of human DNA primase functions as a redox switch using DNA charge transport.

DNA charge transport chemistry offers a means of long-range, rapid redox signaling. We demonstrate that the [4Fe4S] cluster in human DNA primase can make use of this chemistry to coordinate the first steps of DNA synthesis. Using DNA electrochemistry, we found that a change in oxidation state of the [4Fe4S] cluster acts as a switch for DNA binding. Single-atom mutations that inhibit this charge transfer hinder primase initiation without affecting primase structure or polymerization. Generating a single base mismatch in the growing primer duplex, which attenuates DNA charge transport, inhibits primer truncation. Thus, redox signaling by [4Fe4S] clusters using DNA charge transport regulates primase binding to DNA and illustrates chemistry that may efficiently drive substrate handoff between polymerases during DNA replication.

Electrical Probes of DNA-Binding Proteins.

A DNA electrochemistry platform has been developed to probe proteins bound to DNA electrically. Here gold electrodes are modified with thiol-modified DNA, and DNA charge transport chemistry is used to probe DNA binding and enzymatic reaction both with redox-silent and redox-active proteins. For redox-active proteins, the electrochemistry permits the determination of redox potentials in the DNA-bound form, where comparisons to DNA-free potentials can be made using graphite electrodes without DNA modification. Importantly, electrochemistry on the DNA-modified electrodes facilitates reaction under aqueous, physiological conditions with a sensitive electrical measurement of binding and activity.

The Osmium Tetroxide Bipyridine‐labeled DNA Probe: Hairpin Conformations and Characterization of Redox‐label Behavior

AbstractOsmium tetroxide bipyridine ([OsO4(bpy)]) is a versatile label for DNA electrochemistry. Here we report our efforts to create an osmium tetroxide‐labeled immobilized DNA probe for use in biosensing experiments. Our label is applied in‐house, as opposed to many other covalent redox labels used with DNA. We developed an on‐electrode labeling method that was able to avoid attack of our disulfide‐based linker by the [OsO4(bpy)]. Our results include two different hairpin‐based signal‐off hybridization detection assays, each with robust, reproducible signal decrease on binding to complementary target. We also found that 6‐mercapto‐1‐hexanol was able to interact with [OsO4(bpy)] in a stable manner when assembled at the surface, but was able to remove excess [OsO4(bpy)], which was found to adsorb strongly to the gold surface, and avoid MCH transformation, when applied after the labeling process. Furthermore, we found an increased adsorption affinity of [OsO4(bpy)]‐labeled DNA onto gold, characterized the redox behavior of adsorbed [OsO4(bpy)], and determined voltammetric signals on the HMDE of our intact disulfide linker while verifying that this linker is attacked by [OsO4(bpy)] when in solution.

DNA Electrochemistry Shows DNMT1 Methyltransferase Hyperactivity in Colorectal Tumors

DNMT1, the most abundant human methyltransferase, is responsible for translating the correct methylation pattern during DNA replication, and aberrant methylation by DNMT1 has been linked to tumorigenesis. We have developed a sensitive signal-on electrochemical assay for the measurement of DNMT1 activity in crude tissue lysates. We have further analyzed ten tumor sets and have found a direct correlation between DNMT1 hyperactivity and tumorous tissue. In the majority of samples analyzed, the tumorous tissue has significantly higher DNMT1 activity than the healthy adjacent tissue. No such correlation is observed in measurements of DNMT1 expression by qPCR, DNMT1 protein abundance by western blotting, or DNMT1 activity using a radiometric DNA labeling assay. DNMT1 hyperactivity can result from both protein overexpression and enzyme hyperactivity. DNMT1 activity measured electrochemically provides a direct measure of activity in cell lysates and, as a result, provides a sensitive and early indication of cancerous transformation.

Synthesis, DNA binding properties and electrochemistry towards an electrode-bound DNA of a novel anthracene–viologen conjugate

We have synthesized herein a novel molecular conjugate integrating an anthracene chromophore and a viologen moiety in the same molecular skeleton, joined together by a p-xylene spacer. This conjugate preserves both the DNA intercalative properties of the anthracene polycycle and the reversible electrochemical behavior of the viologen unit. The simultaneous presence of these components in one single molecular structure increases the affinity towards DNA in solution with respect to that observed for the isolated functional units, probably due to a co-operation of intercalative and electrostatic binding modes. The molecular conjugate interacts with both single and double-stranded DNA absorbed on ITO surfaces. It is shown that the presence of the anthracene unit plays a key role in increasing the affinity for the electrode-bound DNA also for the reduced, monocationic form of the conjugate. The application of the synthesized compound as an electroactive indicator able to discriminate between non-hybridized and hybridized surface-immobilized DNA has been investigated.

Intraduplex DNA-mediated electrochemistry of covalently tethered redox-active reporters.

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.