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Abstract

Lanthanide-based resonance energy transfer (LRET) is an established method for measuring or detecting proximity between a luminescent lanthanide (energy donor) and an organic fluorophore (energy acceptor). Because resonance energy transfer is a distance-dependent phenomenon that increases in efficiency to the 6th power of the distance between the donor and the acceptor, assay systems are often designed to minimize donor-acceptor distances. However, the authors show that because of the R⁶ relationship between transfer efficiency and sensitized emission lifetime, energy transfer can be difficult to measure in a time-gated manner when the donor-acceptor distance is small relative to the Förster radius. In such systems, the advantages inherent in time-resolved, ratiometric measurements are lost but can be regained by designing the system such that the average donor-acceptor distance is increased.

Keywords

TR-FRET lanthanides protease assay

References

Selvin PR : Principles and biophysical applications of lanthanide-based probes. Annu Rev Biophys Biomol Struct 2002;31:275-302.

Mathis G : Probing molecular interactions with homogeneous techniques based on rare earth cryptates and fluorescence energy transfer. Clin Chem 1995;41:1391-1397.

Fernandez SM , Berlin RD : Cell surface distribution of lectin receptors determined by resonance energy transfer. Nature 1976;264:411-415.

Tsien RY : Fluorescent probes of cell signaling. Annu Rev Neurosci 1989;12:227-253.

Forster T : 10th Spiers Memorial Lecture: transfer mechanisms of electronic excitation. Discuss Faraday Soc 1959;27:7-17.

Heyduk T , Heyduk E : Luminescence energy transfer with lanthanide chelates: interpretation of sensitized acceptor decay amplitudes. Anal Biochem 2001;289(1):60-67.

Li M , Selvin PR : Luminescent polyaminocarboxylate chelates of terbium and europium: the effect of chelate structure. J Amer Chem Soc 1995;117:8131-8138.

Sinha S , Anderson JP , Barbour R , Basi GS , Caccavello R , Davis D , et al : Purification and cloning of amyloid precursor protein beta-secretase from human brain. Nature 1999;402:537-540.

Citron M , Oltersdorf T , Haass C , McConlogue L , Hung AY , Seubert P , et al : Mutation of the beta-amyloid precursor protein in familial Alzheimer’s disease increases beta-protein production. Nature 1992; 360:672-674.

10.

Hendrickson WA , Pahler A , Smith JL , Satow Y , Merritt EA , Phizackerley RP : Crystal structure of core streptavidin determined from multiwave-length anomalous diffraction of synchrotron radiation. Proc Natl Acad Sci USA 1989;86:2190-2194.

11.

Harris LJ , Larson SB , Hasel KW , Day J , Greenwood A , McPherson A : The three-dimensional structure of an intact monoclonal antibody for canine lymphoma. Nature 1992;360:369-372.

12.

Thomas D , Carlsen W , Stryer L : Fluorescence energy transfer at the rapid-diffusion limit. Proc Natl Acad Sci USA 1978;75:5746-5750.

13.

Karvinen J , Elomaa A , Makinen ML , Hakala H , Mukkala VM , Peuralahti J , et al : Caspase multiplexing: simultaneous homogeneous time-resolved quenching assay (TruPoint) for caspases 1, 3, and 6. Anal Biochem 2004;325:317-325.

14.

Porcari V , et al : A continuous time-resolved fluorescence assay for identification of BACE1 inhibitors. ASSAY Drug Dev Tech 2005;3(3):287-297.

Improving Lanthanide-Based Resonance Energy Transfer Detection by Increasing Donor-Acceptor Distances

Abstract

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