Basic Principles and Applications of Fluorescence Resonance Energy Transfer Technology
As an efficient optical "molecular ruler", fluorescence resonance energy transfer (FRET) has a wide range of applications in biological macromolecular interactions, immunoassays, and nucleic acid detection. In the field of molecular biology, this technique can be used to study protein-protein interactions under physiological conditions in living cells.
Protein-protein interactions play an important role in the whole process of cell life. Because various components in cells are extremely complex, some traditional methods to study protein-protein interactions such as yeast two-hybrid and immunoprecipitation may lose some important information and cannot correctly reflect the dynamic changes of protein-protein interactions under the physiological conditions of living cells at that time. FRET technology is a new technology recently developed, which provides convenience for real-time dynamic study of protein-protein interactions under physiological conditions in living cells.
Basic principles of FRET technology
Fluorescence resonance energy transfer refers to that when two fluorescent chromophores are close enough, the donor molecule is excited to a higher electronic energy state after absorbing a certain frequency of photons, and the energy transfer to the adjacent acceptor molecule is realized through dipole interaction before this electron returns to the ground state (i.e., energy resonance transfer occurs).
FRET is a non-radiative energy transition that transfers the energy of the donor excited state to the acceptor excited state through the dipole interaction between molecules, so that the donor fluorescence intensity is reduced, while the acceptor can emit a characteristic fluorescence stronger than itself (sensitized fluorescence) or no fluorescence (fluorescence burst), which is also accompanied by a corresponding shortening or prolongation of the fluorescence lifetime. The efficiency of energy transfer is related to the degree of overlap between the emission spectrum of the donor and the absorption spectrum of the acceptor, the relative orientation of the transition dipole between the donor and the acceptor, and the distance between the donor and the acceptor. As the donor and acceptor pair for resonance energy transfer, the fluorescent substance shall meet the following conditions:
The excitation light of the acceptor and the donor should be sufficiently separated;
The emission spectrum of the donor and the excitation spectrum of the acceptor should overlap.
It has been successfully applied to nucleic acid detection, protein structure, functional analysis, immunoassay and organelle structure and function detection by using the fluorescence of the organism itself or labeling organic fluorescent dyes to the studied objects. The absorption spectrum of traditional organic fluorescent dyes is narrow, and the emission spectrum is often accompanied by tailing, which will affect the overlap degree of donor emission spectrum and acceptor absorption spectrum, and the donor and acceptor emission spectra interfere with each other. Some recent reports have used emitting quantum dots for resonance energy transfer studies, which overcomes the shortcomings of organic fluorescent dyes.
Compared with traditional organic fluorescent dye molecules, the emission spectrum of QDs is very narrow and not trailing, reducing the overlap of donor and acceptor emission spectra and avoiding mutual interference; because QDs have a wide spectral excitation range, when they are used as energy donors, they can more freely select the excitation wavelength and can minimize the direct excitation of energy acceptors; by changing the composition or size of QDs, they can emit light at any wavelength in the visible region, that is to say, they can act as energy donors for any lifetime of absorption spectra in the visible region, and ensure the good overlap of donor emission wavelength and acceptor absorption wavelength, increasing the resonance energy transfer efficiency.
Application of FRET technology
With the deepening of life science research, the research on the mechanism of various life phenomena, especially the protein-protein interaction in cells, has become particularly important. In order to achieve major breakthroughs in research in these areas, technological progress is essential.
The continuous development of some traditional research methods provides extremely favorable conditions for the study of protein-protein interactions, but at the same time, these research methods also have many defects: such as yeast two-hybrid, phosphorylated antibodies, immunofluorescence, radiolabeling, etc. The premise of the application of the method is to break cells or cause damage to cells, and it is impossible to conduct dynamic research on intracellular protein-protein interactions in real time under the physiological conditions of living cells. The application of FRET technology combined with genetic engineering and other technologies just makes up for this defect.