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所引起的热力学稳定性差异太小，难以区分同源目标链和其他同源目标链，因此用DNA芯片分析很难确定最佳条件并获得定量信息。本研究的目的是开发“超功能”DNA芯片，在其表面附着新的功能分子，作为普通分子的替代装置。首先，定量分析了三链、四链和双链的热力学稳定性，为双链或三链微阵列系统的分析提供了信息。此外，还量化了pH、金属离子和其他共溶质对稳定性的依赖关系。例如，d（G_4T_4G_4）的构象发生了剧烈的变化，即反平行的g -四重结构在“细胞模拟”溶液中变为平行取向。这一现象表明，稀释溶液中的发现可能不适用于细胞提取物或其他浓缩溶液中分析物的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。第二，将我实验室开发的脱氧核酶应用于DNA芯片策略。将该脱氧核酶芯片作为表面等离子体共振（SPR）分析的传感芯片，可以在传感图中监测底物的识别和裂解过程。这种人工芯片绝对是一种超功能的DNA芯片，除了具有普通DNA芯片的正常功能外，还同时具有多种特殊功能，严格识别底物的一级和二级结构，酶切底物。

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.