Frontier Institute for Biomolecular Engineering Research (FIBER), Konan University, 7-1-20 Minatojima-Minamimachi, Chuo-ku, Kobe, 650-0047, Japan.
Graduate School of Frontiers of Innovative Research in Science and Technology (FIRST), Konan University, 7-1-20 Minatojima-Minamimachi, Chuo-ku, Kobe, 650-0047, Japan.
Nucleic Acids Res. 2019 Apr 23;47(7):3284-3294. doi: 10.1093/nar/gkz071.
Recent advancement in nucleic acid techniques inside cells demands the knowledge of the stability of nucleic acid structures in molecular crowding. The nearest-neighbor model has been successfully used to predict thermodynamic parameters for the formation of nucleic acid duplexes, with significant accuracy in a dilute solution. However, knowledge about the applicability of the model in molecular crowding is still limited. To determine and predict the stabilities of DNA duplexes in a cell-like crowded environment, we systematically investigated the validity of the nearest-neighbor model for Watson-Crick self-complementary DNA duplexes in molecular crowding. The thermodynamic parameters for the duplex formation were measured in the presence of 40 wt% poly(ethylene glycol)200 for different self-complementary DNA oligonucleotides consisting of identical nearest-neighbors in a physiological buffer containing 0.1 M NaCl. The thermodynamic parameters as well as the melting temperatures (Tm) obtained from the UV melting studies revealed similar values for the oligonucleotides having identical nearest-neighbors, suggesting the validity of the nearest-neighbor model in the crowding condition. Linear relationships between the measured ΔG°37 and Tm in crowding condition and those predicted in dilute solutions allowed us to predict ΔG°37, Tm and nearest-neighbor parameters in molecular crowding using existing parameters in the dilute condition, which provides useful information about the thermostability of the self-complementary DNA duplexes in molecular crowding.
近年来，细胞内核酸技术的进步要求人们了解核酸结构在分子拥挤环境下的稳定性。最邻近模型已成功用于预测核酸双链体形成的热力学参数，在稀溶液中具有较高的准确性。然而，关于该模型在分子拥挤环境下的适用性的知识仍然有限。为了确定和预测类似细胞的拥挤环境中 DNA 双链体的稳定性，我们系统地研究了最邻近模型在分子拥挤环境下对沃森-克里克自我互补 DNA 双链体的适用性。在含有 0.1 M NaCl 的生理缓冲液中，用 40 wt%聚乙二醇 200 存在的情况下，测量了不同自我互补 DNA 寡核苷酸的热力学参数，这些寡核苷酸由生理缓冲液中相同的近邻组成。从紫外熔融研究中获得的热力学参数和熔解温度 (Tm) 对于具有相同近邻的寡核苷酸表现出相似的值，表明最邻近模型在拥挤条件下是有效的。在拥挤条件下测量的 ΔG°37 和 Tm 与在稀溶液中预测的值之间的线性关系允许我们使用稀溶液中现有的参数来预测分子拥挤中的 ΔG°37、Tm 和近邻参数，这为分子拥挤中自我互补 DNA 双链体的热稳定性提供了有用的信息。
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