Quantitative fluorescence resonance energy transfer measurements using microarray technology

Abstract

Fluorescence resonance energy transfer (FRET) has found wide use in structural biology, biochemistry, and cell biology for measuring intra- and inter-molecular distances in the 1-10 nm range and for obtaining quantitative spatial and temporal information about the interaction of proteins, lipids, and DNA. The measurements of distances and interactions are based on the calculation of the fluorescence transfer efficiency using some algorithms to process the acquired images from several different filter sets. However, FRET measurements can suffer from several sources of distortion because of cross talk between donor and acceptor fluorophores. In this paper, we measured the FRET efficiency on glass coverslips using microarray technology and described an algorithm to analyze the FRET data obtained, which is corrected for the cross talk due to spectral overlap of donor and acceptor molecules. Measurement of the interaction of the donor and acceptor, which are mixed together or coupled to the respective 3'-end and 5'-end of a single-strand DNA are shown to document the accuracy of the approach, and allow one to estimate cross talk between the different filter units and to reveal the relationship of the FRET efficiencies of these two samples relative to the donor and acceptor concentrations.