Pulsed IR Heating Studies of Single-Molecule DNA Duplex Dissociation Kinetics and Thermodynamics
Pulsed IR Heating Studies of Single-Molecule DNA Duplex Dissociation Kinetics and Thermodynamics
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DOI：
10.1016/j.bpj.2013.11.008
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发表时间：
2014-01-07
影响因子：
3.4
通讯作者：
Nesbitt, David J.
中科院分区：
文献类型：
--
作者：
Holmstrom, Erik D.;Dupuis, Nicholas F.;Nesbitt, David J.
单分子荧光光谱是一种强大的技术，它使人们有可能观察与生物分子过程相关的构象动力学。在这些实验中加入精确的温度控制可以得到有关平衡和动力学速率常数的有价值的热力学信息。为了实现这一点，我们开发了一种基于红外激光泛音/组合带吸收的显微镜技术，以加热小体积（约10（-11）升）的水。该技术的详细实验表征揭示了与常规阶段加热方法相比的三个主要优点：1）较大的稳态温度范围（20-100摄氏度）; 2）基本上上级空间（10 bp）DNA双链体。这些测量已被扩展到探索温度依赖性动力学的21 bp的构建体，这允许确定单分子的激活的DNA双链体解离的熵和熵。
Single-molecule fluorescence spectroscopy is a powerful technique that makes it possible to observe the conformational dynamics associated with biomolecular processes. The addition of precise temperature control to these experiments can yield valuable thermodynamic information about equilibrium and kinetic rate constants. To accomplish this, we have developed a microscopy technique based on infrared laser overtone/combination band absorption to heat small (approximate to 10(-11) liter) volumes of water. Detailed experimental characterization of this technique reveals three major advantages over conventional stage heating methods: 1), a larger range of steady-state temperatures (20-100 degrees C); 2), substantially superior spatial ( 10 bp) DNA duplexes. These measurements have been extended to explore temperature-dependent kinetics for the 21 bp construct, which permit determination of single-molecule activation enthalpies and entropies for DNA duplex dissociation.