Kumamoto Satoshi, Watanabe Mariko, Kawakami Naoko, Nakamura Mitsunobu, Yamana Kazushige
Department of Materials Science and Chemistry, Graduate School of Engineering, University of Hyogo, 2167 Shosha, Himeji, Hyogo 671-2201, Japan.
Bioconjug Chem. 2008 Jan;19(1):65-9. doi: 10.1021/bc070097f. Epub 2007 Nov 8.
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.
我们之前制备了通过2'-糖位上的一个碳连接子与蒽醌（AQ）基团共轭的寡核苷酸（ODN）。当这些修饰的ODN与cDNA序列结合时，AQ部分可插入双链DNA的预定碱基对口袋中。在本文中，2'-AQ修饰的ODN被证明是一种出色的电化学探针，可用于阐明错配碱基对通过DNA的电荷转移（CT）的影响。通过将二硫键末端的2'-AQ-ODN双链体组装到金电极上构建了两种类型的DNA修饰电极。一种类型的电极（系统I）在氧化还原中心与电极之间的双链DNA中包含完全匹配的碱基对或单碱基错配。另一种（系统II）由错配组成，但在氧化还原中心之外。通过循环伏安法分析修饰电极，以估计通过双链DNA的CT速率。在系统I中，发现完全匹配的AQ-ODN双链体的CT速率约为每秒50次（-1），而错配DNA的CT速率比完全匹配的DNA慢得多。在系统II中，AQ-ODN双链体对于完全匹配的DNA和错配的DNA显示出几乎相似的CT速率（约每秒50次（-1））。然后通过计时库仑法（CC）进行单碱基错配的检测。与完全匹配的DNA相比，系统I中所有包含错配碱基的DNA双链体的电化学响应均降低。特别是，包括G--A错配的错配DNA无需使用任何电化学催化剂即可与完全匹配的DNA区分开来。我们进一步测试了固定在金电极上的单链（ss）AQ-ODN在通过杂交测定法电化学检测单碱基错配中的实用性。将ss-AQ-ODN电极在室温下浸入含靶标的缓冲液中，并进行CC测量以观察积分电荷的变化。在60分钟内，通过CC差异可以清楚地区分错配DNA与完全匹配的靶标。因此，电化学杂交测定法为DNA突变提供了一种简便的检测方法，无需任何额外的试剂、催化剂、靶标标记和洗涤步骤。
