Analysis of DNA Binding and Nucleotide Flipping Kinetics Using Two-Color Two-Photon Fluorescence Lifetime Imaging Microscopy

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• 作者：Tom Robinson ; Prashant Valluri ; Gordon Kennedy ; Alessandro Sardini ; Christopher Dunsby ; Mark A. A. Neil ; Geoff S. Baldwin ; Paul M. W. French ; Andrew J. de Mello
• 刊名：Analytical Chemistry
• 出版年：2014
• 出版时间：November 4, 2014
• 年：2014
• 卷：86
• 期：21
• 页码：10732-10740
• 全文大小：531K
• ISSN：1520-6882

文摘

Uracil DNA glycosylase plays a key role in DNA maintenance via base excision repair. Its role is to bind to DNA, locate unwanted uracil, and remove it using a base flipping mechanism. To date, kinetic analysis of this complex process has been achieved using stopped-flow analysis but, due to limitations in instrumental dead-times, discrimination of the 鈥渂inding鈥?and 鈥渂ase flipping鈥?steps is compromised. Herein we present a novel approach for analyzing base flipping using a microfluidic mixer and two-color two-photon (2c2p) fluorescence lifetime imaging microscopy (FLIM). We demonstrate that 2c2p FLIM can simultaneously monitor binding and base flipping kinetics within the continuous flow microfluidic mixer, with results showing good agreement with computational fluid dynamics simulations.