Förster resonance energy transfer (FRET) technology and TR-FRET assays are used in laboratories globally for a variety of applications ranging from disease detection, to drug discovery, to monitoring genetic differences between individuals and populations. The brilliance of this technology is due to the sensitivity provided by the spectral overlap and the proximity of the acceptor and donor fluorophores involved in the reaction.
A targeted biomolecule is labeled with a donor fluorophore that excites at a specific wavelength and, when near its acceptor (or sometimes called a quencher) fluorophore, emits a measurable signal. 1,2 Time-resolved FRET (TR-FRET) uses long-lived fluorophores that cause a delay between excitation and emission detection compared to traditional FRET assays. Since artificial (false-positive) fluorescence is short-lived, one of the benefits of TR-FRET is the decreased background noise.2
Often used in HTS assays are fluorophores consisting of lanthanides. Europium and Terbium are the most common donors. A variety of acceptor fluors are used. In one example, Tb is excited by an energy source, typically a laser or flash lamp with a wavelength of 320 nm. The Tb will then emit at a variety of wavelengths including 620 nm. This will, in turn, excite the HiLyte 647 fluor which can be read at 665 nm.
Due to its homogenous, one-step reaction capabilities, TR-FRET is ideal for high throughput screening (HTS), bringing efficiency to drug discovery. Currently, there are a plethora of multimode microplate readers on the market to select from in which the fluorescent signal obtained by the proximity of donor and acceptor fluorophore can be detected.
Competitive vs. Sandwich TR-FRET Assays
Depending on what you are attempting to achieve, various TR-FRET assay technology exists. The sandwich immunoassay approach detects a target of interest via energy transfer from two labeled antibodies, a labeled donor and labeled acceptor that recognize the target at different regions. Opposed to a traditional sandwich ELISA where multiple steps are needed including wash steps, the TR-FRET assay includes all reagents necessary per well, providing a simple mix-and-read solution.1
Sandwich TR-FRET assay. Two antibodies specific to different epitopes of the same antigen are used. One antibody is labeled with a Tb donor while the other antibody is labeled with an acceptor fluorophore. In the presence of analyte the donor and acceptor or in close enough proximity to produce a signal when excited.
Competitive TR-FRET assays acceptor probe is also labeled with the target so that it competes with the labeled donor, which ultimately decreases the signal.1
Competitive assays like Transcreener, use a donor labeled antibody and a fluorescently labeled tracer. When no analyte is present FRET signal is produced. In the presence of analyte, the tracer is competed off and no FRET occurs.
The Transcreener® ADP2 TR-FRET Assay is one example of using competitive TR-FRET technology for drug discovery. Since the assay is selective for ADP rather than a specific substrate, it can be used with any enzyme that converts ATP to ADP. Therefore, researchers studying an assortment of ADP producing targets, including kinases, ATPases, and DNA helicases, can all use the same assay technology. Furthermore, it’s perfect for drug discovery as each well in an HTS well plate provides a meaningful data point in a matter of hours.
TR-FRET technology continues to show improvement throughout the years. New assay methods are produced and new acceptor-doner pairs are developed, making drug discovery faster and more efficient than ever before.
- Degorce, F., Card, A., Soh, S., Trinquet, E., Knapik, G. P., & Xie, B. (2009). HTRF: A Technology Tailored for Drug Discovery – A Review of Theoretical Aspects and Recent Applications HTRF: A Technology Tailored for Drug Discovery – A Review of Theoretical Aspects and Recent Applications, 2(October 2015). https://doi.org/10.2174/1875397300903010022
- Ergin, E., Dogan, A., Parmaksiz, M., E Elcin, A., & M Elcin, Y. (2016). Time-resolved fluorescence resonance energy transfer [TR-FRET] assays for biochemical processes. Current pharmaceutical biotechnology, 17(14), 1222-1230. https://www.ncbi.nlm.nih.gov/pubmed/27604358
- Transcreener ADP² TR-FRET Technical Manual (2020), 1–13. https://www.bellbrooklabs.com/wp-content/uploads/2018/04/Tech-Manual-ADP2-TRFRET-RED-v12717.pdf