What are HTRF/FRET screens and what are its applications?

Author: Daiza Norman

 

Learning Outcomes:

  1. Describe how HTRF/FRET screens work.
  2. Identify the applications of HTRF/FRET screens in research.
  3. Evaluate the Pros and Cons of doing an HTRF/FRET Screen.
  4. Identify the components that are required to perform an HTRF/FRET Screen.

 

Graphical Abstract 

Figure 1: The purpose of this article is to give an overview of what HTRF/FRET screens are, how these screens work, and what its applications are. HTRF/FRET screens are a target-based screen that are used by the pharmaceutical industry and the biotechnology industry. This technology utilizes energy transfer to identify target proteins and protein interactions in a sample (1,5). HTRF/FRET screens have many applications in science today and are a very useful tool in research. Image created with Canva Pro.

 

What are HTRF/FRET screens?

Homogeneous time-resolved Förster Resonance Energy Transfer (HTRF) technology relies on the energy transfer from a donor fluorophore to an acceptor fluorophore when the two fluorophores are in close proximity of each other (1,5). This technology is commonly used in screens to identify how much of a protein of interest is in a sample (1,5). Two different antibodies are added to a sample: one antibody is coupled to a donor fluorophore, such as Europium or Terbium Citrate, and the other is coupled to an acceptor fluorophore, such as fluorophore d2 or XL665 (1,5). Once the antibodies bind to the protein of interest, the sample is exposed to a light that can activate fluorescence in the donor fluorophore. The donor fluorophore then transfers its energy to the acceptor fluorophore, and this causes the acceptor fluorophore to fluoresce. The donor and the acceptor fluorophore must be between 20 to 90 Å apart because this allows for weak electronic coupling to occur (6). The fluorescence of the acceptor fluorophore is then measured using a fluorescence spectrophotometer, and the fluorescence of the sample is directly proportional to the amount of the target protein in the sample (6).

Advantages

HTRF screens are fast compared to many other screens because they do not require a lot of processing (1). They are also cost-effective because they do not require wash buffers (1). They are reproducible and they are a robust assay (1). In addition, there is a time delay after the donor fluorophore is activated which makes it easier to measure the fluorescence of the acceptor fluorophore (1). The time delay also allows there to be enough time for the background fluorescence to dissipate before the fluorescence of the acceptor fluorophore is measured (1). This makes the readout highly specific when the fluorescence of the samples is measured using a fluorescence spectrophotometer (1).

Disadvantages

This screen requires two monoclonal antibodies to work (2). Monoclonal antibodies are antibodies that only bind to one site on the target molecule (7). Monoclonal antibodies are expensive compared to polyclonal antibodies (7). In addition, researchers must be knowledgeable about the target molecules, so they can choose the right monoclonal antibodies for the specific assay being done (3). Monoclonal antibodies can also be unstable, which means that the assay needs to be validated before performing an experiment because this can affect the results (7).

What are the applications of HTRF/FRET screens?

This assay has been heavily used to identify potential drugs. Specifically, this assay can be used to identify whether a drug has bound to a particular receptor (6). Since the acceptor fluorophore only fluoresces when it is between 20 to 90 Å from the donor fluorophore, this technique can be used to identify whether a drug binds to a particular receptor and this is very useful in pharmaceutical research (6). Using this same concept, this assay can also be used to help identify protein-protein interaction, protein-DNA/RNA interactions, protein peptide interactions, protein kinase activity, biomarker detection, viral protein interactions with human proteins, and cell surface protein interactions (6). By doing this, elements in a biological pathway can be identified which can lead to new scientific discoveries (6).

 

Audio Recordings

Part 1 ( 2:00 min)

Part 2 (1:41 min)

Part 3 (1:37 min)

Part 4 (1:15 min)

Part 5 (0:45 min)

 

References

  1. Aslanoglou, D., George, E. W., Freyberg, Z. Homogeneous Time-resolved Förster Resonance Energy Transfer-based Assay for Detection of Insulin Secretion.  Vis. Exp.(135), e57531, doi:10.3791/57531 (2018).
  2. 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. Current chemical genomics, 3, 22–32. https://doi.org/10.2174/1875397300903010022
  3. François Degorce (2006) HTRF® : pioneering technology for high-throughput screening, Expert Opinion on Drug Discovery, 1:7, 753-764, DOI: 10.1517/17460441.1.7.753
  4. Jares-Erijman, E. A., & Jovin, T. M. (2003). FRET imaging.Nature Biotechnology, 21(11), 1387-1395. doi:10.1038/nbt896
  5. Miller, T. W., Amason, J. D., Garcin, E. D., Lamy, L., Dranchak, P. K., Macarthur, R., . . . Inglese, J. (2019). Quantitative high-throughput screening assays for the discovery and development of SIRPα-CD47 interaction inhibitors.PloS One, 14(7), e0218897-e0218897. doi:10.1371/journal.pone.0218897
  6. Zeug, A., Woehler, A., Neher, E., & Ponimaskin, E. G. (2012). Quantitative intensity-based FRET approaches–a comparative snapshot. Biophysical Journal, 103(9), 1821–1827. https://doi.org/10.1016/j.bpj.2012.09.031
  7. Creative Diagnostics. (2006, April 4). Polyclonal vs. Monoclonal Antibodies. Retrieved from https://www.creative-diagnostics.com/polyclonal-vs-monoclonal-antibodies.htm

 

Photo References

  1. “Zhendong Smartphone Fertility_08”by SFU – Communications & Marketing is licensed under CC BY 2.0
  2. “File:CSIRO ScienceImage 2628 Multichannel delivery device.jpg”by North Sullivan Photography, CSIRO is licensed under CC BY 3.0
  3. “Spectrophotometer Model 1”by Biology Open Educational Resources is licensed under CC BY-SA 2.0

 

Questions

  1. What is HTRF/FRET screens and how do they work?

HTRF/FRET screens are a target-based screen that works by measuring the fluorescence of the acceptor fluorophore after energy is transferred from the donor fluorophore to the acceptor fluorophores (1,5). This allows the amount of target protein in the sample to be determined by measuring the fluorescence from the sample (1,5).

  1. What are the advantages and disadvantages of using HTRF/FRET?

The advantages of HTRF screens are that these screens are fast, inexpensive compared to many screens, specific, and have a robust fluorescent signal (1). The disadvantages of HTRF/FRET screens are that it utilizes monoclonal antibodies, which are not always available for certain proteins (2).

  1. What are the applications of HTRF/FRET?

The applications of HTRF/FRET include identifying whether drugs bind to the receptor of interest, researching protein interactions in a biological system, and identifying the presence of a protein in a sample (6).

  1. What industries would benefit from this technology?

The pharmaceutical industry often uses this technology for drug screenings. This technology is also used in research and development to identify elements in biological pathways (1).

  1. What are the main components needed to perform a HTRF/FRET screen?

To perform a HTRF/FRET screen you need a donor couple antibody, an acceptor coupled antibody, your sample, and a fluorescence spectrophotometer (1,5).