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New techniques revive Fluorescence Resonance Energy Transfer (FRET)

1st April 2013


Fluorescence energy resonance transfer (FRET) is widely usedin the study of molecular interactions and reaction pathways.As Peter Roberts shows, the introduction of new probes and labelling techniques will make the technique and even more powerful laboratory tool.

Fluorescence resonance energy transfer (FRET) is a method applicable to the investigation of biological phenomena that produce changes in molecular proximity.

The recent introduction of new probes and labelling techniques has revived interest in FRET as the most powerful fluorescence tool for this area of research.

This article provides an introduction to FRET, describing the mechanism by which energy transfer between compatible fluorophores (fluorescent dyes) takes place, and highlighting two of FRET's many applications: real-time polymerase chain reaction ­ one of its major uses ­ and the fluorometric measurement of HIV protease activity, an important factor in the maturation of infectious virus particles.

How it works

Fluorescence is a powerful tool used extensively in the study of molecular interactions and reaction pathways. Recent advances in FRET technologies make this the most reliable technique for clearly determining changes in molecular proximity.

When FRET is used as a contrast medium, co-localisation of proteins and other molecules can be imaged with spatial resolution far beyond the limits of conventional optical microscopy. When fluorophores are less than 200 nm apart, for instance, the space between them cannot be resolved using a light microscope.

FRET uses the transfer of energy between molecules to analyse intermolecular distances. It is a distance-dependent interaction between the electronic excited states of two dye molecules, in which excitation is transferred from a donor to an acceptor molecule without emission of a photon. When the quantum is transferred, the electron acceptor is raised to a higher state and the photo-excited electron in the donor returns to the ground state.

For the process to succeed, the two molecules must meet certain criteria, relating to distance, spectral overlap, and their orientation to each other, ie they must be in close proximity; the absorption spectrum of the acceptor must overlap with the emission spectrum of the donor (Fig. 1); and the donor and acceptor transition dipole orientations must be approximately parallel.

Where the acceptor and donor molecules are different, FRET can be detected by the fluorescence of the acceptor molecule, or the quenching of donor fluorescence. When donor and acceptor are identical, FRET can be detected by the resulting fluorescence depolarisation.

A variety of new probes is available for FRET applications courtesy of Molecular Probes, a company based in Leiden, The Netherlands. These have been produced by covalently linking fluorescent phycobiliproteins to selected Alexa Fluor dyes, creating tandem fluorescent constructs that act as donor-acceptor pairs for FRET. These tandem fluorescent dyes are optimised for energy transfer efficiency to compensate for fluorescence emission from the donor dye ­ a common problem with conventional tandem dyes.

In the Alexa Fluor 647-R-PE construct, for instance, the energy of a photon is absorbed by the R-phycoerythrin (R-PE) member of the pair, putting the R-PE into an electronic excited state. The excitation is transferred without photon emission to the paired dye, which then emits a photon at about 688 nm. The same wavelength of excitation light creates fluorescence colours in response to the particular acceptor dye of the tandem construct (Fig. 2).

All the R-PE tandem fluorescent dyes permit simultaneous multi-colour labelling and detection using a single excitation source: the 488 nm spectral line of the argon-ion laser. This is particularly important in flow cytometry applications, where it enables multiple detection capabilities within the same experimental samples.

FRET offers considerable advantages over traditional end-point methods when performingreal-time PCR analyses.

Real-time PCR measures the kinetics of the reaction in the early phase of PCR, providing several distinct improvements over traditional PCR, which relies on the use of gels to detect PCR amplification at the end-point of the reaction.

End-point PCR is more time-consuming thanreal-time PCR; its results are based on size discrimination, and may therefore be less precise; and resolution on agarose gels is poor. In contrast, real-time PCR can detect as little as a two-fold change; it can quantitate reaction products for every sample in the cycle without intervention or the need for replicates; and is suitable for high throughput analysis.

Real-time PCR requires the use of a fluorescent reporter to identify and quantitate signals produced by a reaction in the PCR product. SYBR Green I dye (Fig. 3) was one of the first fluorescent dyes to significantly improve the capabilities of real-time PCR by performing well in both these respects.

It binds to dsDNA and emits light upon excitation, its fluorescence increasing as the PCR product accumulates. Sensitive, easy to use and inexpensive, real-time PCR using SYBR Green dye has been used in the development of reliable and simple diagnostic assays for detecting genetic mutations.

These include duplications and deletions in mosquito drug resistance genes, chromosomal translocations in human disease genes, and base substitutions. It has also been used for the unequivocal identification of viral, bacterial and fungalpathogens and for quantitative reverse transcription PCR.

HIV-protease activity

Another valuable use of FRET is in the fluorometric analysis of HIV-protease (HIV-PR) activity.

Mammalian retroviruses encode a 10-12kD aspartic protease (PR) that is expressed as part of the Prgag-pol precursor. Retroviral PR is required for the processing of both Prgag and Prgag-pol precursor polyproteins at specific cleavage sites. These modifications are necessary for maturation of infectious particles of human immunodeficiency virus 1 (HIV-1).Measuring HIV-PR activity by FRET requires a synthetic peptide substrate for the enzyme. The sequence of this substrate includes the HIV-PR cleavage site, together with two covalently modified amino acid residues: one that has been linked to a fluorophore (5-(aminoethyl) aminonaphthalene sulfonate, EDANS); and theother to an acceptor chromophore(4'-dimethylaminobenzene-4-carboxylate, dabcyl).

The acceptor fluorophore is selected for maximal overlap of its absorbance with the emission spectrum of the fluorophore. This allows quenching of the nearby fluorophore through resonance energy transfer.

For the assay, synthetic HIV-PR substrate is dispensed into UV-pass fluorescence cuvettes. Excitation and emission monochromators of a spectrofluorometer are set to 340 nm and 490 nm respectively, enabling detection of FRET between the fluorophore and the chromophore. The reaction is started by the addition of HIV-PR, and the initial rate of cleavage of fluorogenic substrate is measured by monitoring the increase in fluorescence signal at 490 nm.

Once the substrate is cleaved, EDANS fluorescence ceases to be quenched by dabcyl, since the molecules are no longer in close proximity.

Without FRET technology, retroviral PR activity must be measured by immunoblot analysis of the gag protein and its cleavage products, combinedwith HPLC or TLC analysis of synthetic peptide cleavage products. This method is labour-intensive and does not allow kinetic measurement of enzyme activity ­ two disadvantages overcome through the use of FRET.

Conclusions

FRET offers considerable advances compared with traditional end-point methods in performingreal-time PCR analyses, notably superior sensitivity and accuracy, suitability for high throughput screening, affordability and ease of use.

It also delivers high quality results. With recent advances in FRET technology, it is now regarded as the most powerful fluorescence tool for investigating changes in molecular proximity and is expected to become the technique of choice across a wide range of research areas. The use of FRET in measuring HIV-PR activity provides a good example of the mechanics of the technique and highlights its technical advantages.

Dr Peter Roberts is Marketing Manager with Molecular Probes Europe BV, Leiden, The Netherlands.Tel:+31 71 524 1894, Fax: +31 71 524 1883.email peter.roberts@probes.nl





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