Theory and Applications

Theory and Applications
FRET, TR-FRET Theory and Applications

How to run on a microplate reader Advantages and limitations of FRET
FRET: contents Introduction Theory Applications How to run on a microplate reader Advantages and limitations of FRET

FRET: Introduction FRET = Fluorescence Resonance Energy Transfer
FRET is used in research laboratories typically to study molecular interactions. Like other fluorescent techniques it is used a lot in imaging applications (microscopy) Time-Resolved FRET (TR-FRET) is mostly used for screening applications in microplates (more robust than FRET, less interferences).

FRET: Theory Cy3 Cy5 Fluorescent molecules: Excitation spectrum
500 450 550 600 650 700 750 nm Fluorescent molecules: Excitation spectrum Emission spectrum Cy3: Ex max at 550 nm Em max at 565 nm Cy5: Ex max at 652 nm Em max at 667 nm Cy3 Cy5 550 nm 565 nm 652 nm 667 nm

FRET: Theory If you mix Cy3 and Cy5 in a well, they will behave as two independent dyes. Cy3 ex results in Cy3 em However, in very close proximity, FRET occurs, and Cy3 excitation results in Cy5 emission Cy3 is called the Donor Cy5 is called the Acceptor The Cy3 – Cy5 couple is called a FRET pair 550 nm 565 nm 652 nm 667 nm 550 nm 667 nm FRET

FRET: Theory FRET is the transfer of energy between a Donor molecule which is in a excited state and an Acceptor molecule This transfer happens without emission of a photon from the Donor After the transfer of energy, the excited Acceptor returns to its ground state by releasing energy as a photon 550 nm 667 nm FRET DONOR ACCEPTOR

FRET: Theory Cy3 Cy5 FRET will only occur if:
500 450 550 600 650 700 750 nm Cy3 Cy5 FRET will only occur if: The emission spectrum of the Donor (e.g. Cy3) overlaps with the excitation spectrum of the Acceptor (e.g. Cy5). Donor and Acceptor molecules are in close proximity (typically A). 550 nm 667 nm FRET 10–100 Å (1-10 nm)

FRET: Theory FRET is extremely sensitive to distance
550 nm 667 nm FRET 50 A E = 50 % FRET is extremely sensitive to distance The efficiency (E) of FRET is described by the equation: E = Ro6/(Ro6 + r6) Where Ro is the “Förster” radius, at which E = 50 % efficiency, and r is the actual distance between the Donor and the Acceptor. For the Cy3-Cy5 FRET pair, Ro is about 50 A (5 nm) 550 nm 667 nm FRET 100 A E = 1.5 % 550 nm 667 nm FRET 25 A E = 98 %

FRET: Theory The Förster radius is typically in the 1 to 10 nm range for FRET pairs 1 to 10 nm is also the typical size range for proteins. For this reason, FRET is extremely useful to study interactions between molecules, with a spatial resolution well beyond what optical microscopy can do. 5 nm 550 nm 667 nm FRET 5 nm

FRET: Theory If you label a potential ligand and a receptor, FRET allows you to detect if they bind or not. No binding would result in high Donor fluorescence and no Acceptor fluorescence. Binding would result in reduced Donor fluorescence and measurable Acceptor fluorescence. 550 nm 565 nm 667 nm

FRET: Theory FRET can be measured as a reduction of Donor emission; an increase of Acceptor emission; or a combination of both, usually expressed as a ratio. 550 nm 565 nm 667 nm

FRET: applications Example 1: Receptor – ligand binding
Example 2: Detection of nucleic acid hybridization Example 3: Real-time detection of PCR amplification Example 4: Ion channel assay Example 5: Protease assay

FRET: applications, receptor-ligand binding
The target receptor is labeled with the Donor, a positive control ligand is labeled with the Acceptor. In the absence of a competing drug candidate, FRET occurs. A potential competitor ligand is added If the competitor displaces the original ligand, FRET disappears

FRET: applications, detection of nucleic acid hybridization
In this example, the Acceptor is a quencher rather than a fluorescent molecule. It absorbs light but does not fluoresce. A FRET probe with a known sequence is mixed with the target DNA. If it hybridizes with a complementary sequence, the FRET pair is separated and the Donor fluoresces.

FRET: applications, detection of real-time PCR amplification
Primer Target DNA TaqMan In addition to the standard PCR reagents, a FRET-based TaqMan® probe is used Both the primers and the TaqMan probe hybridize with the target DNA During the Taq polymerase extension step, the probe is destroyed, FRET disappears FRET

FRET: applications, VSP ion channel assay (Invitrogen)
+ + + _ _ _ K+ Extracellular Intracellular - + + + _ _ _ FRET CC2-DMPE (Coumarin-Phospholipid) DiSBAC2(3) (hydrophobic oxonol) Copyright ©2005 Invitrogen Corporation.

FRET: applications, protease assay
A peptide that can be degraded by the HIV protease is selected. It is labeled with a FRET pair (Fluorescent Donor, Quenching Acceptor) Active HIV protease degrades the substrate and separates the FRET pair. Fluorescence is measured.

FRET: how to run on a microplate reader
The question is: is there FRET or not in a given sample. Absence of FRET means that if you excite the Donor, you should mostly see Donor emission. Presence of FRET means that if you excite the Donor, you should see a mix of Donor emission and Acceptor emission. 550 nm 565 nm 550 nm 667 nm FRET

So for a FRET measurements: Excite at the Donor’s excitation wavelength Typically measure both emission channels (Donor and Acceptor). If the Acceptor is a quencher, you just monitor the Donor’s fluorescence. 550 nm 565 nm 550 nm 667 nm FRET

Most often FRET assays are end-point assays. The signal is stable over time and the Donor and Acceptor fluorescence can be measured sequentially. In the example above, the plate is measured first with an excitation at 400/30 nm and emission at 460/40 nm (Donor channel), then measured with a 400/30 nm – 590/35 nm filter pair (Acceptor channel)

Some FRET assays have fast signal kinetics (e.g. VSP ion channel assay). In these cases, an automated reagent injector is required, and the two emission channels are measured simultaneously and kinetically for each single well.

FRET: advantages Cy3 Cy5 Cy3 Cy5
Study binding events at the molecular level Can be used for a wide variety of assays Homogeneous assays. No need to remove unbound reagents. Can be ratiometric, more robust: 500 450 550 600 650 700 750 nm Cy3 Cy5 EX EM1 EM2 750 nm 500 450 550 600 650 700 Cy3 Cy5 EX EM1 EM2 Cy5/Cy3=0.5 Cy5/Cy3=0.5

FRET: limitations Background due to non-specific excitation of Acceptor (1) and residual emission of Donor in the Acceptor’s emission range (2) (see below). This is a major limitation of FRET (limited assay window and sensitivity). Complex labeling, relatively complex assay development 500 450 550 600 650 700 750 nm Cy3 Cy5 EX EM 550 nm 565 nm Donor Acceptor 667 nm (1) 667 nm (2)

Benefits compared to FRET Limitations Applications
TR-FRET: contents Theory Benefits compared to FRET Limitations Applications How to run on a microplate reader

TR-FRET: Time Resolved Compounds
Lanthanide (e.g. Europium) chelates or cryptates are fluorescent compounds. Their lifetime after excitation is in the µs to ms range instead of ns for standard fluorescent dyes. If you excite them with a pulsed light source (e.g. Xenon Flash), wait (e.g. 20 µs) and read, all short-lived background fluorescence is gone at the time of the measurement, and the excitation light is off. H 3 C CH N Eu 3+ Time (µs) I n t e s i y 2 µs Reading

TR-FRET: Time Resolved Compounds
Time-Resolved compounds provide extremely low background compared to conventional fluorescent dyes. They can be used as Donors in FRET assays. These assays are called TR-FRET or TRET assays. HTRF® (Homogeneous Time Resolved Fluorescence) is a trademarked assay platform (Cisbio) based on TR-FRET. H 3 C CH N Eu 3+ Time (µs) I n t e s i y 2 µs Reading

TR-FRET: theory 340 nm 620 nm 633 nm 665 nm TR-compound Donor Standard dye Acceptor (ms) (ns) When excited, the TR Donor emits light over a few milliseconds. When excited directly, the Acceptor emits light over a few nanoseconds. When FRET occurs, the energy transfer occurs over a few milliseconds, thus the emission of the Acceptor occurs in the same time-frame. 340 nm 665 nm FRET TR-compound Donor Standard dye Acceptor (ms)

TR-FRET: benefits compared to FRET
Recall crosstalk problem with FRET: Some direct excitation of the Acceptor at Donor excitation wavelength Some Donor emission appears in Acceptor emission channel TR-FRET eliminates problem 1, because at the time of measurement (delayed after excitation), the unwanted emission of the free Acceptor (in the ns time scale after excitation) is gone. TR-FRET reduces assay background compared to FRET. 550 nm 565 nm 550 nm 667 nm (1) 667 nm Crosstalk issue in standard FRET (2) Donor Acceptor

In addition to non-specific Acceptor fluorescence, TR-FRET eliminates all short lived interference (fluorescence from other molecules). Time (µs) I n t e s i y 2 µs Reading Short lived fluorescence (ns time scale) Measurement window

Ro (distance between Donor and Acceptor giving a 50% FRET efficiency) can be as high as 9 or 10 nm, compared to around 5 nm for standard FRET. Allows working with large proteins Makes assays more robust (it is easier to get a signal) Makes assay development easier (label position on molecule is less critical). TR-FRET is more flexible in Acceptor concentration (because no crosstalk signal from Acceptor) TR-FRET is more forgiving for incomplete labeling In general, TR-FRET provides easier assay development and more robust assays.

TR-FRET: limitations TR fluorescent dyes are not very bright (low quantum yield). They are not efficient Donors compared to standard FRET Donors. This limits the benefit of having a reduced background level. Background due to residual emission of Donor in the Acceptor’s emission range. Complex labeling, relatively complex assay development Lanthanide labeling can be expensive.

TR-FRET: applications
Because of cost of reagents and cost of instrumentation (requires high-energy pulsed light source), TR-FRET is mostly used for drug screening assays. Reagents are sometimes available as kits (cAMP assay kits, kinase assays kits) but are mostly available as “tool boxes” for assay development laboratories. HTS preferred technologies:

TR-FRET: applications
Cisbio: HTRF® assay platform Perkin Elmer: LanceTM assay platform Invitrogen: LanthaScreenTM assay platform

TR-FRET: HTRF® Europium cryptate-based assay.
Receptor Ligand Copyright ©Cisbio International Europium cryptate-based assay. Europium cryptate is the Donor, the Acceptor is XL665 (modified allophycocyanin, standard short-lived fluorescent label).

TR-FRET: HTRF®, example of cAMP kit
620 nm TR-FRET Copyright ©Cisbio International A labeled anti-cAMP antibody and labeled cAMP are mixed. TR-FRET occurs. The TR-FRET complex is added to the sample. If cAMP is present in the sample it displaces the labeled cAMP. Presence of cAMP is detected by a decrease in the TR-FRET ratio.

TR-FRET: LanthaScreenTM, example of kinase assay
Copyright ©2005 Invitrogen Corporation Fluorescein-labeled kinase substrate peptide is incubated with kinase and ATP A Tb-labeled antibody is then added Phosphorylation (kinase activity) is detected by an increase in the TR-FRET ratio

TR-FRET: How to run on a microplate reader
TR-FRET assay are almost always end-point assays. Exact same principle as standard FRET assays. In the example above, the plate is measured first with an excitation at 340/30 nm and emission at 620/20 nm (Donor channel), then measured with a 340/30 nm – 665/20 nm filter pair (Acceptor channel). The difference is that you need a pulsed light source (Xenon Flash Lamp, Pulsed Laser…). Standard FRET can be measured with a continuous or pulsed light source.

FRET, TR-FRET: Conclusion
FRET used a lot in microscopy, In microplate format: Used in research labs on a regular basis TR-FRET very common in screening applications Like any other assay platform, has advantages that make it extremely useful for some applications, and limitations that make it less attractive in other areas.

TR-FRET: Conclusion For more details on FRET and TR-FRET, see:
(Cisbio international)

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