Investigations of the molecular crowding effect on nucleic acid structures and development of nucleotide switches

这项研究的目的是构建一个核苷酸相互作用的数据库，并开发可应用于非水介质(如细胞和传感器表面)中的分子传感的功能核苷酸。我们发现金纳米颗粒上的DNA杂交能不同于在均相水溶液中获得的DNA杂交能。用聚乙二醇和其他共溶质构建了一个模拟细胞和金纳米颗粒上分子拥挤环境的模型体系。在水溶液中加入大量的共溶体会大大降低水的活度，而大尺寸的共溶体会产生很大的排阻体积效应。对共溶体溶液中寡核苷酸结构及其热力学参数的研究表明，聚乙二醇或其他共溶质的分子拥挤降低了沃森-克里克碱基对的稳定性，但稳定了三链、G-四链和LEF…更多的T形双面(Z形)结构。为了了解分子拥挤效应，我们对热力学和动力学参数进行了分析，得出在分子拥挤条件下，参与核苷酸结构形成的水分子对热力学稳定性和动力学有显著影响。根据核苷酸相互作用的数据，我们开发了当与多胺或碱性多肽结合时改变构象的DNA分子，这些分子通过转移到分子拥挤介质中形成DNA导线。此外，我们还构建了具有响应分子环境的逻辑门功能的DNA分子。我们还证明了分子拥挤效应被用于酶活性的上调或下调以及富含GC的DNA序列的高效PCR扩增。我们还成功地合成了几种类型的功能核苷酸。从医学和工程的角度出发，对分子拥挤的研究具有重要的意义。较少

The purposes of this study are construction of a database for nucleotide interactions and development of functional nucleotides that can be applied for a molecular sensing in the non-aqueous media such as in a cell and on a sensor surface. We found the DNA hybridization energy on a gold nanoparticle differed from that obtained in a homogeneous aqueous solution. A model system that mimics the molecular crowding environment supposedly occurring in a cell and on a gold nanoparticle was constructed by using PEG (polyethylene glycol) and other cosolutes. A large amount of cosolutes added in an aqueous solution substantially decreases the water activity and large-sized cosolutes generate large excluded volume effect. Investigations of oligonucleotide structures and their thermodynamic parameters in the cosolute-containing solutions revealed that the molecular crowding by PEG or other cosolute decreased the stability of Watson-Crick base pairs but stabilized the triplex, G-quadruplex, and lef … More t-handed duplex (Z-form) structures. To understand the molecular crowding effect, we analyzed the thermodynamic and kinetic parameters and obtained the notations that water molecules participating in a nucleotide structure formation significantly influence the thermodynamic stability and the kinetics under the molecular crowding conditions. According to the nucleotide interaction data, we developed DNA molecules that changed the conformation when binding to polyamines or basic peptides, and those formed a DNA-wire by transferring into the molecular crowding media. Moreover, we constructed the DNA molecules with a logic gate function that responded to the molecular environment. We also demonstrated that the molecular crowding effect was used for up-or down-regulation of an enzyme activity and an efficient PCR amplification of a GC-rich DNA sequence. We also successfully synthesized several types of functional nucleotides. The researches that focus on the molecular crowding are very important from the standpoint of medical and engineering aspects. Less