Chen Chunlai, Wang Wenjuan, Wang Zhang, Wei Fang, Zhao Xin Sheng
Beijing National Laboratory for Molecular Sciences, and Department of Chemical Biology, College of Chemistry and Molecular Engineering, Peking University, Beijing 100871, China.
Nucleic Acids Res. 2007;35(9):2875-84. doi: 10.1093/nar/gkm177. Epub 2007 Apr 11.
Hybridization of nucleic acids with secondary structure is involved in many biological processes and technological applications. To gain more insight into its mechanism, we have investigated the kinetics of DNA hybridization/denaturation via fluorescence resonance energy transfer (FRET) on perfectly matched and single-base-mismatched DNA strands. DNA hybridization shows non-Arrhenius behavior. At high temperature, the apparent activation energies of DNA hybridization are negative and independent of secondary structure. In contrast, when temperature decreases, the apparent activation energies of DNA hybridization change to positive and become structure dependent. The large unfavorable enthalpy of secondary structure melting is compensated for by concomitant duplex formation. Based on our results, we propose a reaction mechanism about how the melting of secondary structure influences the hybridization process. A significant point in the mechanism is that the rate-limiting step switches along with temperature variation in the hybridization process of structured DNA, because the free energy profile of hybridization in structured DNA varies with the variation in temperature.
具有二级结构的核酸杂交参与了许多生物过程和技术应用。为了更深入地了解其机制，我们通过荧光共振能量转移（FRET）研究了完全匹配和单碱基错配DNA链上DNA杂交/变性的动力学。DNA杂交表现出非阿累尼乌斯行为。在高温下，DNA杂交的表观活化能为负且与二级结构无关。相反，当温度降低时，DNA杂交的表观活化能变为正且与结构有关。二级结构解链的大量不利焓变被伴随的双链形成所补偿。基于我们的结果，我们提出了一个关于二级结构解链如何影响杂交过程的反应机制。该机制中的一个重要点是，在结构化DNA的杂交过程中，限速步骤会随着温度变化而切换，因为结构化DNA中杂交的自由能分布会随温度变化而变化。
