title: Extending Förster resonance energy transfer measurements beyond 100 Å using common organic fluorophores: enhanced transfer in the presence of multiple acceptors.
abstract: Using commercially available organic fluorophores, the current applications of Förster (fluorescence) resonance energy transfer (FRET) are limited to about 80 Å. However, many essential activities in cells are spatially and/or temporally dependent on the assembly/disassembly of transient complexes consisting of large-size macromolecules that are frequently separated by distances greater than 100 Å. Expanding the accessible range for FRET to 150 Å would open up many cellular interactions to fluorescence and fluorescence-lifetime imaging. Here, we demonstrate that the use of multiple randomly distributed acceptors on proteins/antibodies, rather than the use of a single localized acceptor, makes it possible to significantly enhance FRET and detect interactions between the donor fluorophore and the acceptor-labeled protein at distances greater than 100 Å. A simple theoretical model for spherical bodies that have been randomly labeled with acceptors has been developed. To test the theoretical predictions of this system, we carried out FRET studies using a 30-mer oligonucleotide-avidin system that was labeled with the acceptors DyLight649 or Dylight750. The opposite 5'-end of the oligonucleotide was labeled with the Alexa568 donor. We observed significantly enhanced energy transfer due to presence of multiple acceptors on the avidin protein. The results and simulation indicate that use of a nanosized body that has been randomly labeled with multiple acceptors allows FRET measurements to be extended to over 150 Å when using commercially available probes and established protein-labeling protocols.
authors.affiliation: University of North Texas Health Science Center, Department of Molecular Biology and Immunology, Center for Commercialization of Fluorescence Technologies, 3500 Camp Bowie Boulevard, Fort Worth, Texas 76106, USA.
authors.forename: Badri P
authors.initials: BP
authors.lastname: Maliwal
authors.forename: Sangram
authors.initials: S
authors.lastname: Raut
authors.forename: Rafal
authors.initials: R
authors.lastname: Fudala
authors.forename: Sabato
authors.initials: S
authors.lastname: D'Auria
authors.forename: Vincenzo M
authors.initials: VM
authors.lastname: Marzullo
authors.forename: Alberto
authors.initials: A
authors.lastname: Luini
authors.forename: Ignacy
authors.initials: I
authors.lastname: Gryczynski
authors.forename: Zygmunt
authors.initials: Z
authors.lastname: Gryczynski
mesh_terms: D001360:Avidin / Q000737:chemistry; D001710:Biotin / Q000737:chemistry; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry*; D008956:Models, Chemical; D009841:Oligonucleotides / Q000737:chemistry
chemical_list: D005456:Fluorescent Dyes; D009841:Oligonucleotides; D001360:Avidin; D001710:Biotin