2'-anthraquinone-conjugated oligonucleotide as an electrochemical probe for DNA mismatch.
We previously prepared the oligonucleotides (ODNs) conjugated to an anthraquinone (AQ) group via one carbon linker at the 2'-sugar position. When these modified ODNs bind to cDNA sequences, the AQ moiety can be intercalated into the predetermined base-pair pocket of a duplex DNA. In this paper, 2'-AQ-modified ODNs are shown to be an excellent electrochemical probe to clarify the effect of a mismatch base on the charge transfer (CT) though DNA. Two types of DNA-modified electrodes were constructed by assembly of disulfide-terminated 2'-AQ-ODN duplexes onto gold electrodes. One type of electrodes (system I) contains fully matched base pairs or a single-base mismatch in duplex DNA between the redox center and the electrode. The other (system II) consists of the mismatch but at the outside of the redox center. The modified electrodes were analyzed by cyclic voltammetry to estimate the CT rate through duplex DNA. In system I, the CT rate was found to be approximately 50 s (-1) for the fully matched AQ-ODN duplexes, while the CT rates of the mismatched DNA were considerably slower than that of the fully matched DNA. In system II, the AQ-ODN duplexes showed almost similar CT rates ( approximately 50 s (-1)) for the fully matched DNA and for the mismatched DNAs. The detection of a single-base mismatch was then performed by chronocoulometry (CC). All the DNA duplexes containing a mismatch base in system I gave the reduced electrochemical responses when compared to the fully matched DNA. In particular, the mismatched DNAs including G--A mismatch can be differentiated from fully matched DNA without using any electrochemical catalyst. We further tested the usefulness of single-stranded (ss) AQ-ODN immobilized on a gold electrode in the electrochemical detection of a single-base mismatch through hybridization assay. The ss-AQ-ODN electrodes were immersed in target-containing buffer at room temperature, and the CC measurements were carried out to see the changes in the integrated charge. Within 60 min, the mismatched DNA was clearly distinguishable by the CC differences from the fully matched target. Thus, the electrochemical hybridization assay provides an easy and convenient detection for DNA mutation that does not require any extra reagents, catalyst, target labeling, and washing steps.
Kumamoto
Satoshi
S
Department of Materials Science and Chemistry, Graduate School of Engineering, University of Hyogo, 2167 Shosha, Himeji, Hyogo 671-2201, Japan.
Watanabe
Mariko
M
Kawakami
Naoko
N
Nakamura
Mitsunobu
M
Yamana
Kazushige
K
D000880:Anthraquinones / Q000737:chemistry*; D020137:Base Pair Mismatch; D001483:Base Sequence; D004247:DNA / Q000235:genetics*; D004252:DNA Mutational Analysis; D004563:Electrochemistry; D004566:Electrodes; D015335:Molecular Probes / Q000032:analysis* / Q000737:chemistry; D009841:Oligonucleotides / Q000032:analysis* / Q000737:chemistry* / Q000235:genetics; D020641:Polymorphism, Single Nucleotide
D000880:Anthraquinones; D015335:Molecular Probes; D009841:Oligonucleotides; D004247:DNA