Integrated DNA Technologies - Technical Bulletins

Fluorescence Resonance Energy Transfer (FRET) is a process that shifts energy from an electronically excited molecule (the donor fluorophore) to a neighboring molecule (the acceptor or quencher), returning the donor molecule to its ground state without emission of light (i.e., fluorescence emission).

Energy transfer between different electron states of the donor and acceptor molecules is outlined in Figure 1 below:

Figure 1. Diagram of the energy levels of the donor and acceptor molecules

Horizontal lines represent discreet electron energy levels for each molecule. Energy levels are labeled as either single states (S) or triplet states (T) with subscripts numbered 0, 1, or 2, representing the ground state, first excited electronic state, or second excited electronic state. Molecules generally reside in its lowest, or ground, electronic state, S₀. A molecule may be excited to one of its higher energy levels by any of a number of processes, including light absorption and chemical reaction. Excitation of a molecule is represented in the figure by the arrows pointing upward from the ground state.

An excited donor molecule has several routes available to release its captured energy and return to a lower energy state or to the ground state. Released energy can be dissipated to the environment (as light or heat) or transferred directly to another molecular, which captures that energy and in turn itself moves to a higher energy state. These routes are marked by arrows pointing downward from the excited states; straight lines represent light emissions and wavy lines represent energy conversion without light emissions.

1) Internal energy conversions. Molecules excited into their second or higher excited states are rapidly de-excited without light to the first excited state, S₁, without interacting with other molecules. Internal conversion from S₁ to S₀ can also be rapid but in a luminescent molecule it is slow enough that de-excitation by light emission is competitive. Intersystem crossing from S₁ to the triplet state, T₁, also occurs.

2) Light emissions. Light released by the transition from S₁ to S₀ is fluorescence emission. Light released from the triplet state T₁, which generates phosphorescence, can also occur but is less common.

When a second fluorescence molecule (i.e., a quencher or FRET acceptor) is in physical proximity to an excited fluorophore, new paths for de-excitation become available.

3) Dynamic quenching. This process only occurs when the product of quencher concentration and quenching rate constant is high.

4) Static quenching. This process results from the formation of a non-emissive complex between the donor fluorescent molecule and the quencher. Static quenching reduces the donor fluorescence intensity.

5) FRET. FRET can occur when donor and acceptor molecules are in close proximity but do not require actual physical contact. In the process of FRET, de-excitation of the donor molecule is linked to excitation of the acceptor molecule. In the figure, FRET is represented by the de-excitation pathway leading from the S₁ level of the donor to the S₁ level of the acceptor. Photons of light are not involved. Once excited, the acceptor can undergo de-excitation by the same emissive and non-emissive processes described for the donor.

Primary Conditions for FRET

1) The primary requirement for FRET is that the energy lost by de-excitation of the donor molecule, S₁-S₀, be matched by the energy required for excitation of the acceptor. In other words, the absorption spectrum of the acceptor molecule must overlap the emission spectrum of the donor molecule, as shown in Figure 2.

Figure 2. Diagram of the overlapping spectrum of a pair of FRET donor and acceptor dyes.

2) Donor and acceptor molecules must be in close proximity (typically 10-100 Å). FRET is a distance-dependent energy transfer between the electronic excited states of two dye molecules. The distance at which energy transfer is 50% efficient (i.e., 50% of excited donors are deactivated by FRET) is defined by the Förster radius (Ro).

The magnitude of Ro is dependent on the spectral properties of the donor and acceptor dyes:

Table 1. Typical Values of Ro

Donor	Acceptor	Ro (Å)
Fluorescein	Tetramethylrhodamine	55
IAEDANS	Fluorescein	46
EDANS	DABCYL	33
Fluorescein	Fluorescein	44
BODIPY FL	BODIPY FL	57
Fluorescein	QSY^TM-7	61

Some typical values of Ro are listed in the table above. For good donor-acceptor pairs, Ro values of 30 to 60 Å are common.

3) Donor and acceptor transition dipole orientations must be approximately parallel.

4) Donor/Acceptor Pairs: In general, donor and acceptor are different dyes, each having unique spectral properties. Normally, a fluorophore will release light at its characteristic emission wavelength following excitation. When two suitable fluorophores are in proximity within the distance defined by the Förster radius, FRET will prevent fluorescent emission from the higher energy group. Instead, energy is transferred to the lower energy group, exciting the acceptor, and leading to fluorescence emission at a lower energy wavelength characteristic for the acceptor. If donor and acceptor are the same dye, FRET can still occur and can be detected as fluorescence depolarization. Non-fluorescent acceptors exist which will accept energy from a donor without any resulting fluorescence emission. These acceptors as a group are known as "dark quenchers", and include Dabcyl, QSY^TM-7, and BlackHole^TM dyes.

For more information on fluorescent dyes, CLICK HERE!

Applications of FRET

FRET is highly efficient within the Förster radius of the donor/acceptor pair (which is often in the 50 - 60 Å range), making it useful over distances encountered within many biological macromolecules. Further, since FRET is dependent on the inverse sixth power of the intermolecular separation, efficiency dramatically falls as donor/acceptor distance exceeds the Förster radius, making it an extremely sensitive indicator of intermolecular distance. Thus, FRET is an important technique for investigating a variety of biological phenomena where markers that track physical proximity are necessary or useful.

For example, in the TaqMan^TM 5'-nuclease assay, two fluorescent dyes (such as Fam and Tamra) are covalently linked by DNA residues in an oligonucleotide probe. When the higher-energy fluorophore (Fam) is excited at 488 nm, instead of the expected fluorescence emission at 520 nm, the captured energy is transferred to the lower energy fluorophore (Tamra) and is emitted at 580 nm (FRET has occurred); the Fam signal is quenched. Using the fluorescein/rhodamine reporter/quencher combination, FRET can occur even when the groups are separated by 25-30 bases of DNA. During the course of a TaqMan^TM assay, the two fluorophores are physically separated from each other by the 5'-exonuclease action of Taq DNA polymerase - after which 488 nm stimulation results in visible Fam emission at 520 nm (quenching is released).

Molecular Beacons are another variation on the FRET-based nucleic acid probe paradigm. In this case, a dark quencher (most commonly Dabcyl) is placed at one end of the DNA probe and a reporter dye is placed at the opposite end. The probe is designed such that a target-specific hybridization domain is positioned centrally between short sequences (unrelated to the target) that lead to hairpin formation. In the native state, the Molecular Beacon forms a hairpin structure with the reporter and quencher groups directly adjacent. When hybridized to the complementary target sequence, the hairpin structure unfolds and the reporter and quencher separate. Dabcyl has a relatively short Förster radius and will not quench the reporter dye in the open configuration. For more information about Molecular Beacons, CLICK HERE!

A new method to detect RNase activity was recently developed as a joint project between IDT and Ambion, called RNaseAlert^TM (patent pending). RNaseAlert^TM employs a fluorescence-quenching oligonucleotide probe to detect the presence of RNase activity. A fluorescein reporter group is connected to a fluorescence-quencher by several RNA residues. The precise sequence has been optimized for maximum sensitivity in detecting a wide variety of RNase activities. Upon cleavage of the RNA, the reporter and quencher separate and a fluorescent signal is revealed.

To order Dual-Labeled probes, CLICK HERE!

To order Molecular Beacons, CLICK HERE!

To order RNaseAlert^TM CLICK HERE!

Other Selected Applications of FRET

Substrates for enzyme kinetic studies¹
Structure and conformation of proteins²
Spatial distribution and assembly of protein complexes³
Receptor/ligand interactions⁴
Immunoassays⁵
Probing interactions of single molecules⁶
Structure and conformation of nucleic acids⁷
Detection of nucleic acid hybridization⁸
Primer-extension assays for detecting mutations⁹
Automated DNA sequencing¹⁰
Distribution and transport of lipids¹¹
Membrane-fusion assays¹²
Membrane-potential sensing¹³
Fluorogenic protease substrates¹⁴
Indicators for cyclic AMP¹⁵

References

1. Kelemen, B.R. Klink, T.A., Behlke, M.A., Eubanks, S.R., Leland, P.A. and Raines R.T. (1999) "Hypersensitive substrate for ribonucleases" Nucleic Acids Res., 27:3696-3701

2. Luo Y., Wu J.L., Gergely J., and Tao T. (1998) "Localization of Cys¹³³ of Rabbit Skeletal Troponin-l with Respect to Troponin-C by Resonance Energy Transfer." Biophys. J. 74:3111-3119.

3. Watson B.S., Hazlett T.L., Eccleston J.F., Davis C., Jameson D.M. and Johnson A.E.(1995) "Macromolecular Arrangement in the Aminoacyl-tRNA-Elongation Factor TuGTP Ternary Complex. A Fluorescence Energy Transfer Study." Biochemistry 34:7904-7912.

4. Berger W., Prinz H., Striessnig J., Kang H.C., Haugland R. and Glossmann H. (1994) "Complex Molecular Mechanism for Dihydropyridine Binding to L-Type Ca²⁺-Channels as Revealed by Fluorescence Resonance Energy Transfer." Biochemistry 33:11875-11883.

5. Morrison, L.E. (1988) "Time-Resolved Detection of Energy Transfer: Theory and Application to Immunoassays." Anal. Biochem. 174:101-120.

6. Ha T., Enderle T., Ogletree D.F., Chemla D.S., Selvin P.R. and Weiss S.(1996) "Probing the Interaction between Two Single Molecules: Fluorescence Resonance Energy Transfer between a Single Donor and a Singer Acceptor." Proc. Natl. Acad. Sci. USA 93:6264-6268.

7. Tòth, K., Sauermann, V. and Langowski, J. (1998) "DNA Curvature in Solution Measured by Fluorscence Resonance Energy Transfer." Biochemistry 37:8173-8179.

8. Tyagi, S., Bratu, D.P. and Kramer, F.R. (1998) "Multicolor Molecular Beacons for Allele Discrimination." Nature Biotech. 16:49-53.

9. Chen X., Zehnbauer B., Gnirke A., and Kwok P.Y.(1997) "Fluorescence Energy Transfer Detection as a Homogeneous DNA Diagnostic Method." Proc Natl Acad Sci USA 94:10756-10761.

10. Hung, S.-C., Mathies, R.A. and Glazer A.N. (1998) "Comparison of Fluorescence Energy Transfer Primers with Different Donor-Acceptor Dye Combinations." Anal. Biochem. 255:32-38.

11. Gutierrez-Merino C., Bonini de Romanelli I.C., Pietrasanta L.I. and Barrantes F.J. (1995) "Preferential Distribution of the Fluorescent Phospholipid Probes NBD-Phosphatidylcholine and Rhodamine-Phosphatidylethanolamine in the Exofacial Leaflet of Acetylcholine Receptor-Rich Membranes from Torpedo marmorata." Biochemistry 34:4846-4855.

12. Pecheur E.I., Martin I., Ruysschaert J.M., Bienvenue A. and Hoekstra D. (1998) "Membrane Fusion Induced by 11-mer Anionic and Cationic Peptides: A Structure-Function Study." Biochemistry 37:2361-2371.

13. Gonzalez J.E. and Tsien R.Y.(1995) "Voltage Sensing by Fluorescence Resonance Energy Transfer in Single Cells." Biophys. J. 69:1272-1280.

14. Kurth T., Grahn S., Thormann M., Ullmann D., Hofmann H.J., Jakubke H.D., and Hedstrom L.(1998) "Engineering the S1' Subsite of Trypsin: Design of a Protease Which Cleaves between Dibasic Residues." Biochemistry 37:11434-11440.

15. S.R. Adams, et al. (1993) "Optical Probes for Cyclic AMP." Fluorescent and Luminescent Probes for Biological Activity, W.T. Mason, Ed. , pp. 133-149.

Wei Tao, Ph.D.

Research Scientist, Molecular Genetics and Bioinformatics

Mark Behlke, M.D., Ph.D.

Vice President, Molecular Genetics and Bioinformatics

Integrated DNA Technologies

December 2000

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