FRET Analysis

In fact, FRET (Forster resonant energy transfer) analysis has the same basis as the energy transfer described in the above section. It is used either in simple bioanalyses or to detect protein interactions and DNA hybridization. Its principle is shown on Fig. 14 in the case of a homogeneous immunoassay [43]. 

In homogeneous immunoassays, the analyte is biochemically coupled to two specific antibodies labeled one with a LLB and the other by an organic acceptor. Emission from the organic acceptor is detected in time-resolved mode because the population of its excited state by intramolecular transfer from the LLB shifts its lifetime in the millisecond range. In this way, it is easy to discriminate between the luminescence emitted by uncoupled and coupled antibody molecules labeled with A similarly, since the luminescence of A is spectrally different from that of the LLB, interference from Ln luminescence emitted by the uncoupled antibody labeled with the Ln chelate is also discriminated. There is, therefore, no need to wash out unused reactants. A method using FRET for the 

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Takanishi CL, Bykova EA, Cheng W, Zheng J (2006) GFP-based FRET analysis in live cells. Brain Res 1091 132-139... 

Ruiz-Velasco, V. and Ikeda, S. R. (2001). Functional expression and FRET analysis of green fluorescent proteins fused to G-protein subunits in rat sympathetic neurons. J. Physiol. 537, 679-92. 

Ecker, R. C., de Martin, R., Steiner, G. E. and Schmid, J. A. (2004). Application of spectral imaging microscopy in cytomics and fluorescence resonance energy transfer (FRET) analysis. Cytometry A 59, 172-81. 

Kohl, T., Heinze, K. G., Kuhlemann, R., Koltermann, A. and Schwille, P. (2002). A protease assay for two-photon crosscorrelation and FRET analysis based solely on fluorescent proteins. Proc. Natl Acad. Sci. USA 99, 12161-6. 

Padilla-Parra, S, Auduge, N, Coppey-Moisan, M and Tramier, M. (2008). Quantitative FRET analysis by fast acquisition time domain FLIM at high spatial resolution in living cells. Biophys. J DOI 10.1529/bio-... 

Wallrabe, H., Chen, Y., Periasamy, A. and Barroso, M. (2006). Issues in confocal microscopy for quantitative FRET analysis. Microsc. Res. Tech. 69, 196-206. 

Vereb G, Nagy P, SzoUosi J (2011) Flow cytometric FRET analysis of protein interaction. Methods Mol Biol 699 371-392... 

Figure 8.7 Steady-state FRET analysis of a 4H four-way junction derived from the 2HSj2HS2 junction of the HCV IRES (Melcher et al., 2003). The central sequence of the junction is shown. The four arms are sequentially named A, B, C and S. Donor-acceptor-labeled vectors for FRET analysis are constructed with. -terminally attached fluorescein (donor) and Cy3 (acceptor) on selected helical arms, named according to those arms in that order. Thus, BA is labeled with donor on the end of arm B, and acceptor on A. (A) Fiistogram of the FRET efficiencies of the six end-to-end vectors.

Nienhaus GU. Exploring protein structure and dynamics under denaturing conditions by single-molecule FRET analysis. Macro-mol. Biosci. 2006 6 907-922. 

There are also a number of new dye-modified analogues for use in FRET analysis. A new four colour set of FRET dideoxy 5 -triphosphate terminators has been developed involving a rigid, linear ET cassette linker chemistry, and formulated with Thermo Sequenase II DNA polymerase. An alternative approach for a four colour set of FRET dyes has been reported where the dyes are incorporated into ODNs as phosphoramidite derivatives. ... 

Dimethylaminonaphthamide and fluorescein have each been attached to the C position of 2 -amino-2 -deoxyuridine as fluorescence energy donor and acceptor for FRET analysis. The analogues were incorporated at the termini of DNA, and it was shown that the presence of the bulky groups at the 2 -position did not affect duplex stability or conformation of DNA duplexes, and they were effective in FRET analysis. 

Nienhaus, G. U., Exploring Protein Structure and Dynamics under Denaturing Conditions by Single molecule FRET Analysis, Macromol. Biosci. 2006, 6, 907 922. 

A. Y. Kobitski, A. Nierth, M. Helm, A. Jaschke, G. U. Nienhaus, Mg -dependent folding of a Diels-Alderase ribozyme probed by single-molecule FRET analysis. Nucleic Acids Res., 2007, 35, 2047-2059. 

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