Development of novel gene-chips with unnatural nucleic acids

基因芯片(DNA芯片)微阵列系统是后基因组时代许多研究的有力工具。然而，由于核酸双链中一个核苷酸差异引起的热力学稳定性差异太小，不能区分同源靶链和其他同源靶链，用DNA芯片分析很难确定最佳条件和获得定量信息。本研究的目的是开发表面新型功能分子连接在其上的超功能DNA芯片，作为普通DNA芯片的替代装置。首先，定量分析了三链、四链和双链的热力学稳定性，为以双链或三链为分析物的微阵列系统提供了信息。此外，还用pH、金属离子和其他共溶质对稳定性的依赖性进行了量化。例如，d(G_4T_4G_4)的反平行G-四链结构在“拟细胞”溶液中发生了剧烈的构象变化，变为平行取向。这种现象表明，稀释液中的结果可能不适用于细胞提取液或其他浓缩液中的分析物进行DNA芯片分析，因此，我们在本研究中获得的能量参数将是此类DNA芯片系统不可或缺的参考。当脱氧核酶芯片作为表面等离子体共振(SPR)分析的传感芯片时，可以在传感图中如期监测底物的识别和切割过程。这种人造芯片绝对是超功能的DNA芯片，除了普通DNA芯片的正常功能外，还同时展示了多种特殊功能，对底物的一级和二级结构的严格识别，底物的酶促切割。

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.