Abstract
Förster resonant energy transfer (FRET) is extensively used to probe macromolecular interactions and conformation changes. The established FRET lifetime analysis method measures the FRET process through its effect on the donor lifetime. In this paper we present a method that directly probes the time-resolved FRET signal with frequency domain Fourier lifetime excitation-emission matrix (FLEEM) measurements. FLEEM separates fluorescent signals by their different phonon energy pathways from excitation to emission. The FRET process generates a unique signal channel that is initiated by donor excitation but ends with acceptor emission. Time-resolved analysis of the FRET EEM channel allows direct measurements on the FRET process, unaffected by free fluorophores that might be present in the sample. Together with time-resolved analysis on non-FRET channels, i.e. donor and acceptor EEM channels, time resolved EEM analysis allows precise quantification of FRET in the presence of free fluorophores. The method is extended to three-color FRET processes, where quantification with traditional methods remains challenging because of the significantly increased complexity in the three-way FRET interactions. We demonstrate the time-resolved EEM analysis method with quantification of three-color FRET in incompletely hybridized triple-labeled DNA oligonucleotides. Quantitative measurements of the three-color FRET process in triple-labeled dsDNA are obtained in the presence of free single-labeled ssDNA and double-labeled dsDNA. The results establish a quantification method for studying multi-color FRET between multiple macromolecules in biochemical equilibrium.
Highlights
First established by Theodor Förster in the 1940s [1], Förster resonant energy transfer (FRET) is widely used as a fluorescence spectroscopy method to measure distances between fluorophores on the nanometer scale
The EEM measurements are based on Fourier lifetime excitation-emission matrix spectroscopy (FLEEM), a frequency domain lifetime technique we previously developed, which performs fluorescence intensity and lifetime measurements in all EEM channels simultaneously [19]
We first tested the time-resolved EEM analysis with double-labeled double-strand DNA (dsDNA) after complete hybridization. 20 base-pair long complimentary sequences of oligonucleotides were labeled on each 5′-end with Alexa 488 and Alexa 546
Summary
First established by Theodor Förster in the 1940s [1], Förster resonant energy transfer (FRET) is widely used as a fluorescence spectroscopy method to measure distances between fluorophores on the nanometer scale. FRET occurs when an excited donor fluorophore transfers its energy to an adjacent ground-state acceptor fluorophore through dipole coupling. This process depends strongly on the distance between molecules in the 1-10 nm range, and can be exploited as a “spectroscopic ruler” [2]. With recent advances in fluorescence proteins, organic dyes and instrumentation, FRET has found an ever increasing range of applications in biological studies, ranging from tracking protein-protein interactions in cellular processes [3], probing DNA/RNA regulations and dynamics [4], to highthroughput drug screening [5]
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