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Development of novel gene-chips with unnatural nucleic acids

使用非天然核酸开发新型基因芯片

基本信息

批准号：
12555231
负责人：
SUGIMOTO Naoki
金额：
$ 8.06万
依托单位：
Konan University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2000
资助国家：
日本
起止时间：
2000 至 2003
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-12555231/
关键词：
unnatural nucleic acid deoxyribozyme DNA chip ribozyme higher order structure gene chip peptide nucleic acid RNA cleavage SNPs解析情報変換デオキシリボサイムリボサイム RNA切断活性アビジン-ビオチン相互作用カルシウムイオン金属イオン

项目摘要

Microarray systems with gene chips(DNA chips) are powerful tools for many researches in post-genome era. However, it is difficult to determine the optimum condition and to get quantitative information by the analysis with a DNA chip because the difference of the thermodynamic stability arisen from one nucleotide difference in a nucleic acid duplex is too small to distinguish the cognate target strand from the others.The aim of this study is development of "supra-functional" DNA chips on which surface novel functional molecules are attached as an alternative device of the common ones. First of all, thermodynamic stability of triplexes and quadruplexes as well as duplexes was analyzed quantitatively, providing the information for the microarray system that deals with duplex or triplex strands as the analyte. Furthermore, the dependence of the stability was quantified by pH, metal ion, and other co-solutes. For example, a drastic conformational change was found, i.e. antiparallel G-quadruplex structure of d(G_4T_4G_4) changed to parallel orientation in the "cell-mimetic" solution. This phenomenon indicates that the findings in the diluted solution may not be applicable to the DNA chip analysis with the analytes in cell extracts or other concentrated solutions, and therefore, our energy parameters obtained in this study would be the indispensable reference for such a DNA chip system.Second, the deoxyribozyme developed in my lab was applied to the DNA chip strategy. When the deoxyribozyme chip was used as the sensorchip of the surface plasmon resonance(SPR) analysis, the recognition and the cleavage process of the substrate could be monitored in the sensorgram as expected. This artificial chip is definitely a supra-functional DNA chip exhibits multiple special functions at a time, strict recognition of primary and secondary structure of the substrate, enzymatic cleavage of the substrate, in addition to the normal function of ordinary DNA chips.

基因芯片（DNA芯片）是后基因组时代许多研究的有力工具。然而，在这方面，由于核酸双链体中一个核苷酸的差异所引起的热力学稳定性的差异太小，无法区分同源的靶链和其他链，因此很难确定最佳条件，也很难通过DNA芯片分析得到定量信息。DNA芯片，其表面附着有新型功能分子，作为普通装置的替代装置。首先，对三链体和四链体以及双链体的热力学稳定性进行了定量分析，为以双链体或三链体作为分析物的微阵列系统提供了信息。此外，通过pH、金属离子和其他共溶质定量了稳定性的依赖性。例如，在“细胞模拟物”溶液中，d（G_4T_4G_4）的反平行G-四链体结构变为平行取向。这一现象表明，在稀释溶液中的研究结果可能不适用于在细胞提取物或其他浓缩溶液中分析的DNA芯片分析，因此，本研究所获得的能量参数将是这种DNA芯片系统不可或缺的参考。第二，将本实验室开发的脱氧核酶应用于DNA芯片策略。将脱氧核酶芯片用作表面等离子体共振（SPR）分析的传感芯片，可以在传感图中监测底物的识别和切割过程。这种人工芯片绝对是一种超功能DNA芯片，它除了具有普通DNA芯片的正常功能外，还同时表现出多种特殊功能，严格识别底物的一级和二级结构，酶促切割底物。