A Polymer Micelle Responding to the Single Nucleotide Mutation

某些基因上的单核苷酸突变可能导致遗传性疾病和癌症。因此，建立一种简单、快速的单核苷酸差异检测方法已成为分析生物化学领域的重要课题之一。本研究采用盐析诱导浊度变化的聚合物胶束分散体系进行单核苷酸差异分析，聚合物胶束由疏水的聚（N '-异丙基丙烯酰胺）（polyNIPAAm）和亲水的寡脱氧核苷酸（ODN：9 mer）组成。ODN衍生物和NIPAAm的两亲性共聚物通过自组装形成胶束。通过甲基丙烯酰氧琥珀酰亚胺与5 '端带有氨基己基连接基的ODN偶联反应得到ODN大分子单体，与NIPAAm进行共聚，合成了两亲性共聚物。当聚合物溶液温度高于聚NIPAAm的相转变温度时，聚合物胶束自发形成并保持分散，当加入互补ODN（9 mer）时，聚合物胶束迅速聚集。相反，它们在点突变的ODN（9 mer）存在下保持分散。此外，我们还应用了一种扩展的双链体形成方法来检测较长样品ODN（39 mer）的单核苷酸突变。在该方法中，加入探针ODN（10 mer）和辅助ODN（20 mer）以补充样品ODN的剩余部分（30个碱基）。样品ODN的检测部分为10个碱基，与探针ODN互补。利用该方法，我们可以检测较长样本ODN的单核苷酸突变。这些不同的现象可以应用于基因诊断中的DNA鉴别系统。

A single nucleotide mutation on certain genes can cause heritable disorders and cancers. Consequently the development of a simple and rapid detection method for single nucleotide difference has been one of the most important subjects in analytical biochemistry. In this study, we applied a salting out-induced turbidity change of polymer micelle dispersion to single nucleotide difference assay.The polymer micelle comprises a hydrophobic core of poly(N'-isopropylacrylamide) (polyNIPAAm) and a hydrophilic shell of oligodeoxyribonucleotide (ODN : 9 mer). The micelle was constructed by self-assembly of amphiphilic copolymers of ODN derivative and NIPAAm. The amphiphilic copolymer was synthesized through copolymerization between NIPAAm and ODN macromonomer, which was obtained by coupling reaction between methacryloyloxysuccinimide and ODN having amino-hexyl linker at its 5'-end. When the copolymer solution was incubated above the phase transition temperature of polyNIPAAm, the polymer micelle was spontaneously formed and kept dispersed.The polymer micelles aggregated rapidly when the complementary ODN (9 mer) was added into the dispersion. In contrast, they kept dispersed in the presence of the point-mutated ODN (9 mer). Moreover, we have applied an extended method for the duplex formation to detect single nucleotide mutation of longer sample ODN (39 mer). In this method, the probe ODN (10 mer) and the auxiliary ODN (20 mer) were added to complement the surplus part (30 bases) of the sample ODN. The detection part of the sample ODN is 10 bases wide, which is complementary to the probe ODN. Using this method, we can detect the single nucleotide mutation of the longer sample ODN. These distinct phenomena may be applied for a DNA discrimination system in gene diagnosis.