THREE CUBE FRET METHOD (3<3>-FRET) FOR DETECTING FLUORESCENCE ENERGY TRANSFER

摘要

The invention provides a method for determining a measure of FRET comprising obtaining sequential fluorescent intensity readings from a specimen, such as a cell using three filter cubes. Simple equations manipulate readings from each of the filter sets or cubes to specify a unitless index of FRET called the FRET ratio ( FR ). FR bears a linear relation to FRET efficiency E . The method also provides for determining the fraction of acceptor-tagged molecules bound by donor-tagged molecules; the relative affinity of binding; and the strength of FRET interactions when all acceptor-tagged molecules are bound by donor. The latter determination enables estimates of the physical distance and/or orientation between interacting fluorophore molecules. The method can be used to detect analytes or inter- or intra-molecular interactions. In a preferred aspect, the method is used in an HTS assay to identify modulators of such interactions.