Determination of Equilibrium Constant and Relative Brightness in FRET-FCS by Including the Third-Order Correlations
Determination of Equilibrium Constant and Relative Brightness in FRET-FCS by Including the Third-Order Correlations
复制标题
通过包含三阶相关性确定 FRET-FCS 中的平衡常数和相对亮度。
DOI：
10.1021/acs.jpcb.7b09229
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发表时间：
2017-12-21
影响因子：
3.3
通讯作者：
Zhao, Xin Sheng
中科院分区：
文献类型：
--
作者：
Meng, Lingyi;He, Shanshan;Zhao, Xin Sheng
荧光相关光谱（FCS）编码关于平衡常数（K）、荧光团的相对荧光亮度（Q）以及关于物理或化学弛豫的前向和后向反应速率常数（k+和k-）的信息。然而，如何对FCS数据进行完全解析以获得热力学和动力学信息一直是一个长期存在的问题。最近，我们已经解决了荧光自相关光谱（FACS）的问题。在这里，我们将该方法扩展到荧光互相关光谱（FCCS），当FCS与荧光共振能量转移（FRET）偶联时就会出现这种情况。在12个总的二阶和三阶指前因子的弛豫过程中探测的FRET-FCS技术，3个是独立的。我们提出并讨论了3组显式解，以使用这些指前因子来计算K和Q。与弛豫时间一起，获得的K将使人们获得k+和k-，从而完全达到破译FRET-FCS数据的目的。通过大量的计算机模拟和实验测试的寡核苷酸杂交系统的理论进行了验证。
Fluorescence correlation spectroscopy (FCS) encodes the information on the equilibrium constant (K), the relative fluorescence brightness of fluorophore (Q), and the forward and backward reaction rate constants (k+ and k-) on a physical or chemical relaxation. However, it has been a long-standing problem to completely resolve the FCS data to get the thermodynamic and kinetic information. Recently, we have solved the problem for fluorescence autocorrelation spectroscopy (FACS). Here, we extend the method to fluorescence cross-correlation spectroscopy (FCCS), which appears when FCS is coupled with fluorescence resonance energy transfer (FRET). Among 12 total second-order and third-order pre-exponential factors in a relaxation process probed by the FRET-FCS technique, 3 are independent. We presented and discussed 3 sets of explicit solutions to use these pre-exponential factors to calculate K and Q. Together with the relaxation time, the acquired K will allow people to obtain k+ and k-, so that the goal of deciphering the FRET-FCS data will be fully reached. The theory is verified by extensive computer simulations and tested experimentally on a system of oligonucleotide hybridization.