Photocurrent response after enzymatic treatment of DNA duplexes immobilized on gold electrodes: electrochemical discrimination of 5-methylcytosine modification in DNA

Abstract

We demonstrate a photoelectrochemical approach to the detection of the methylation status of cytosine bases in DNA. We prepared anthraquinone (AQ) photosensitizer-tethered oligodeoxynucleotide (ODN) duplexes bearing 5-methylcytosine (mC) or the corresponding cytosine (C) at a restriction site of the ODN strand immobilized on gold electrodes, and measured their photocurrent responses arising from hole transport after enzymatic digestion. Treatment with HapII or HhaI of the duplexes bearing normal C led to strand cleavage, and the photosensitizer unit was eliminated from the ODN strand immobilized on the gold electrode, exclusively reducing the photocurrent density. With a similar treatment, the duplexes bearing mC showed higher photocurrent responses arising from hole transport through the duplex. This significant difference in the photocurrent response between mC and normal C residues in DNA on the gold electrodes is potentially applicable to the detection of mC modification in DNA.

Electrochemical Assay for the Signal-On Detection of Human DNA Methyltransferase Activity

“…Reported electrochemical strategies include the direct oxidation of individual DNA bases to detect 5-methylcytosine 27 and several methods that use methylation-sensitive restriction enzymes. These include restriction-based signal modulation with DNA-functionalized gold nanoparticles, 28 restriction-facilitated binding of redox-active moieties such as carbon nantubes, 29 probe-modified DNA, 30 and redox-active enzymes, 31 and DNA monolayers with methylation-sensitive restriction sites that bear either electrochemical 32–35 or photoelectrochemical 36 reporters. Though diverse, these strategies are limited in that they are either demonstrated with synthetic 5-methylcytosine alone and not enzymatic methylation or they are only applicable to the detection of bacterial methyltransferase activity.…”

Methods for detection of cytosine and thymine modifications in DNA

“…90 Direct electrochemical oxidation of mC is also a useful detection method. 91,92 A number of other methods based on electrochemical detection, [93][94][95][96] and FRET based methods for the detection of mC have also been reported. [97][98][99] Derivatisation of DNA with O-allylhydroxylamine has been used by Carell and co-workers to detect mC.…”

Nucleotides and Nucleic Acids; Oligo- and Polynucleotides

“…519 Charge transport on gold electrodes using anthraquinone as the photosensitiser when combined with enzymatic digestion is able to differentiate cytosine from 5-methylcytosine. 475 Detection of a duplex mismatch by charge transfer experiments is efficient, as a mismatch disrupts charge transport leading to considerably slower charge transport rates as measured on electrodes. 93,520 The fluorophore Redmond Red (66) has been used as a probe for an abasic site by charge transfer as an efficient electrochemical response is only achieved when (66) is properly stacked within the duplex occupying the space of a normal DNA base pair.…”

“…Gold is also widely used as an electrode, and there are many applications for the detection of changes to oligonucleotides attached to them. Oligonucleotides attached to gold electrodes have been used to detect duplex melting, 471 hybridisation, 472,473 as an electrical relay in conjunction with an oligonucleotide-conjugated quantum dot, 474 for the detection of 5-methylcytosine following oxidation by anthraquinone, 475 as a method for the detection and amplification of telomerase 476 and, in conjunction with an enzyme assay, a sensitive (femtomolar) method for detecting target DNA. 477 Gold-functionalised nanoparticles are more widely used, and are useful as they undergo a colour change from red to purple on aggregation, as well as possessing surface plasmon properties.…”