Refereed journal article or data article (A1)
Distance and Temperature Dependency in Nonoverlapping and Conventional Förster Resonance Energy-Transfer
List of Authors: Johanna Vuojola, Iko Hyppänen, Marika Nummela, Jouko Kankare, Tero Soukka
Publisher: AMER CHEMICAL SOC
Publication year: 2011
Journal: Journal of Physical Chemistry B
Journal name in source: JOURNAL OF PHYSICAL CHEMISTRY B
Journal acronym: J PHYS CHEM B
Number in series: 46
Volume number: 115
Issue number: 46
Start page: 13685
End page: 13694
Number of pages: 10
ISSN: 1520-6106
DOI: http://dx.doi.org/10.1021/jp205564n
Abstract
Forster resonance energy-transfer (FRET) is a powerful and widely applied bioanalytical tool. According to the definition of FRET by Forster, for energy-transfer to take place, a substantial spectral overlap between the donor emission and acceptor excitation spectra is required. Recently also a phenomenon termed nonoverlapping FRET (nFRET) has been reported. The nFRET phenomenon is based on energy-transfer between a lanthanide chelate donor and a spectrally nonoverlapping acceptor and thus obviously differs from the conventional FRET, but the mechanism of nFRET and resulting implications to assay design have not been thoroughly examined. In this work, a homogeneous DNA-hybridization assay was constructed to study the distance and temperature dependency of both nFRET and conventional FRET. Capture oligonucleotides were labeled at the 5'-end with a Eu(III)-chelate, and these conjugates hybridized to complementary tracer oligonucleotides labeled with an organic fluorophore at various distances from the 3'-end. The distance dependency was studied with a fluorometer utilizing time-resolution, and the temperature dependency was studied using a frequency-domain (FD) luminometer. Results demonstrated a difference in both the distance and temperature dependency between conventional FRET and nFRET. On the basis of our measurements, we propose that in nFRET thermal excitation occurs from the lowest radiative state of the ion to a higher excited state that is either ionic or associated with a ligand-to-metal charge-transfer state.
Forster resonance energy-transfer (FRET) is a powerful and widely applied bioanalytical tool. According to the definition of FRET by Forster, for energy-transfer to take place, a substantial spectral overlap between the donor emission and acceptor excitation spectra is required. Recently also a phenomenon termed nonoverlapping FRET (nFRET) has been reported. The nFRET phenomenon is based on energy-transfer between a lanthanide chelate donor and a spectrally nonoverlapping acceptor and thus obviously differs from the conventional FRET, but the mechanism of nFRET and resulting implications to assay design have not been thoroughly examined. In this work, a homogeneous DNA-hybridization assay was constructed to study the distance and temperature dependency of both nFRET and conventional FRET. Capture oligonucleotides were labeled at the 5'-end with a Eu(III)-chelate, and these conjugates hybridized to complementary tracer oligonucleotides labeled with an organic fluorophore at various distances from the 3'-end. The distance dependency was studied with a fluorometer utilizing time-resolution, and the temperature dependency was studied using a frequency-domain (FD) luminometer. Results demonstrated a difference in both the distance and temperature dependency between conventional FRET and nFRET. On the basis of our measurements, we propose that in nFRET thermal excitation occurs from the lowest radiative state of the ion to a higher excited state that is either ionic or associated with a ligand-to-metal charge-transfer state.
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