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Study of DNA-Ferrocene Conjugate for Application to Gene Targeting

DNA-二茂铁缀合物在基因打靶中的应用研究

基本信息

批准号：
11650831
负责人：
NAKANO Koji
金额：
$ 2.3万
依托单位：
KYUSHU UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1999
资助国家：
日本
起止时间：
1999 至 2000
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-11650831/
关键词：
gene sensor biosensor chemically-modified electrode labeling photo cross-linking psoralen ferrocene DNA DNAセンサ酸化還元

项目摘要

Two examples of electrochemical gene sensor are described. We first introduced a ferrocene derivative with psoralen moiety. A gel-shift assay using a plasmid DNA (pBR322) showed that the compound binds irreversibly to DNA double strands upon UV radiation. This is due to photoadduct formation between the psoralen moiety and nucleases in the DNA duplex. In cyclic voltammetric measurements, the compound gave reversible redox waves, which were ascribed to the electrode reaction of the ferrocene moiety. Thus, the compound was expected to be useful for redox labeling of double-helical DNA ; the reagent which is bound covalently to DNA gives the double-strand a redox-active function. Based on these results, a possible application to electrochemical gene targeting was demonstrated in a combined use in a DNA-modified electrode system ; a gold electrode modified with a DNA probe was first treated with the target DNA to form the DNA double-strand gave redox waves upon a subsequent treatment with … More the labeling reagent while the redox activity for the DNA-modified electrode without the hybridization procedure (single-stranded DNA) was about one-half. Next, a DNA modified electrode, which can report DNA hybridization, was introduced. We have been investigating various DNA-modified electrode systems. By taking advantage of the principle of an "ion-channel sensor", some of those have been applying to electrochemical sensing of DNA-binding molecules and ions as well as DN-binding proteins. Here, the chemoreceptive surface adlayer was improved to a DNA/ferrocene bilayer format to give an analyte-dependent electrochemical signal. Electrode preparation was made as follows ; a gold electrode was first modified with 11-aminoalkanethiol and was subsequently reacted with 1, 1-ferrocendicarboxylic acid. Reaction with an oligodeoxyribonucleic acid with an amino function gave a modified electrode with a DNA/ferrocene interfacial structure. Cyclic voltammetric measurements revealed those reversible redox waves due to the electrode reaction of ferrocene suppressed to one-half upon formation of the DNA layer. If the electrode was treated with an aqueous solution of DNA having specific base sequence to the immobilized DNA, the redox waves were further suppressed to one-tenth. These results suggest that the electrode system can be applicable to the electrochemical detection of DNA hybridization without any kind of reporter molecules. Less

介绍了电化学基因传感器的两个实例。我们首先引入了一种具有补骨脂素片段的二茂铁衍生物。使用质粒DNA （pBR322）的凝胶转移试验表明，该化合物在紫外线辐射下与DNA双链不可逆地结合。这是由于在DNA双链中补骨脂素部分和核酸酶之间形成的光加合物。在循环伏安测量中，化合物给出可逆的氧化还原波，这归因于二茂铁部分的电极反应。因此，该化合物有望用于双螺旋DNA的氧化还原标记；与DNA共价结合的试剂使双链具有氧化还原活性功能。基于这些结果，展示了在dna修饰电极系统中联合使用电化学基因靶向的可能应用；用DNA探针修饰的金电极首先用目标DNA处理，形成DNA双链，随后用标记试剂处理，产生氧化还原波，而未经杂交处理的DNA修饰电极（单链DNA）的氧化还原活性约为一半。其次，介绍了一种DNA修饰电极，它可以报道DNA杂交。我们一直在研究各种dna修饰电极系统。利用“离子通道传感器”的原理，其中一些已经应用于dna结合分子和离子以及dn结合蛋白的电化学传感。在这里，化学感受表面层被改进为DNA/二茂铁双层格式，以提供依赖于分析物的电化学信号。电极制备步骤如下；首先用11-氨基烷硫醇修饰金电极，然后与1,1 -二茂铁二羧酸反应。与具有氨基功能的低聚脱氧核糖核酸反应得到具有DNA/二茂铁界面结构的修饰电极。循环伏安测量揭示了由于二茂铁电极反应在DNA层形成时抑制到一半而产生的可逆氧化还原波。如果电极与固定DNA具有特定碱基序列的DNA水溶液处理，则氧化还原波进一步被抑制到十分之一。这些结果表明，该电极系统可适用于不含任何报告分子的DNA杂交电化学检测。少