Fluorescence Resonance Energy Transfer (FRET) for Molecular Analysis

What is Fluorescence Resonance Energy Transfer?

Fluorescence Resonance Energy Transfer (FRET) has brought about a seismic shift in the realm of biological research over the past few years. This powerful and sophisticated technique is a non-radiative energy transfer mechanism, which occurs between two chromophores – typically a donor and an acceptor – when they come into close proximity. The energy transfer takes place owing to the dipole-dipole interaction between the molecules of the donor and acceptor. The technique of FRET has an extensive and widespread usage in various domains of biology, such as protein-protein interactions, DNA-protein interactions, and signaling pathways.

Fluorescence Resonance Energy Transfer Principle

The principle of energy transfer between two fluorescent molecules forms the basis of FRET. The donor fluorophore, which has a specific wavelength, absorbs light and then emits it at a longer wavelength. When the donor fluorophore approaches the acceptor fluorophore, it transfers energy to the excited acceptor, which then emits light at a longer wavelength than the donor.The efficiency of FRET depends on the distance between the donor and acceptor fluorophores, as well as their relative orientations. This intricate and fascinating phenomenon of FRET has revolutionized the way researchers study various biological processes.

How Does Fluorescence Resonance Energy Transfer Work?

FRET can be used to detect changes in distance or conformation of molecules in a sample. To perform FRET, two fluorescent molecules, the donor and acceptor fluorophores, are chosen based on their spectral properties. The donor fluorophore is excited with light of a specific wavelength, which causes it to emit light at a longer wavelength. If the acceptor fluorophore is within a certain distance of the donor fluorophore, the energy from the excited donor fluorophore is transferred to the acceptor fluorophore, which then emits light at a longer wavelength than the donor.

We can measure the efficiency of FRET by calculating the ratio of the emission intensity of the acceptor fluorophore to the emission intensity of the donor fluorophore. This ratio, known as the FRET efficiency, is proportional to the distance between the donor and acceptor fluorophores. Measuring FRET efficiency reveals the distance between two fluorophores and any changes in distance or conformation of the molecules.

Schematic diagrams depicting the three conditions (Broussard et al., 2013).

Single Molecule Fluorescence Resonance Energy Transfer

Single-molecule fluorescence resonance energy transfer (smFRET) is a variant of FRET. In smFRET, individual molecules are immobilized on a surface and their fluorescence is measured using TIRF microscopy. This technique allows the observation of individual molecules in real time, providing unprecedented insights into the dynamics of molecular interactions.

Single-molecule FRET event (Wallace et al., 2017)

What Can We Use Fluorescence Resonance Energy Transfer For?

FRET has an expansive array of applications in biological research. One of its pivotal roles is in drug discovery, where we can utilize it to scrutinize the interactions between drugs and their targets. By endowing drugs and their targets with donor and acceptor fluorophores, FRET can facilitate the measurement of the binding affinity of the drug to its target, as well as decipher the intricate mechanism of action of the drug.

Another significant application of FRET is in studying protein-protein interactions, which is of paramount importance in comprehending various biological processes. By affixing donor and acceptor fluorophores to two proteins of interest, FRET can precisely measure the strength and dynamics of their interaction. Researchers can harness this critical information to create new drugs that specifically target protein-protein interactions.

FRET also offers immense potential in cell biology, allowing researchers to explore the movement and localization of molecules in living cells. By grafting donor and acceptor fluorophores onto molecules, FRET can trail the real-time movement of these molecules and determine how they are transported within cells. Insights from these investigations could lead to novel therapies for diseases like cancer that involve abnormal cell movement and migration.

FRET

Moreover, FRET is a valuable tool in studying enzyme activity and signaling pathways in cells. By attaching donor and acceptor fluorophores to substrates and enzymes, FRET can effectively monitor the activity of the enzyme in real-time and investigate the dynamics of enzymatic reactions. Researchers can leverage the data obtained from these studies to design biosensors that detect enzyme activity and develop new drugs that specifically target the enzymes involved in various disease processes.

Finally, We also can use FRET to study gene expression and regulation in living cells. By grafting donor and acceptor fluorophores onto DNA or RNA molecules, FRET can track the binding of transcription factors or other regulatory molecules to specific DNA sequences in real-time. This information can help understand gene expression mechanisms and aid in developing new therapies for genetic diseases.