The basic principles and application of fluorescent resonant energy transfer technology.

as an efficient optical "molecular ruler", fluorescent resonance energy transfer (FRET) is widely used in biomember molecular interaction, immunoanalytical analysis,
nucleic acid detection
and so on. In
field of
biology, this technique can be used to study the interaction between
proteins and
proteins under the physiological conditions of living cells.
Protein-protein interaction plays an important role in the whole cell life process, because the various components within the cell are extremely complex, so some traditional methods of studying protein-protein interaction, such as yeast double hybridization, immuno precipitation, etc., may lose some important information, can not correctly reflect the dynamic process of protein-protein interaction under the physiological conditions of living cells at that time. FRET technology is a new technology developed recently, which facilitates real-time dynamic research on protein-protein interactions under the physiological conditions of living cells.
I. FreT Technology Basics
Fluorescent Resonance Energy Transfer refers to the transfer of energy to adjacent subject molecules (i.e., energy resonance transfer) by the electrons before the electrons return to the base state when the two fluorescent color groups are close enough to the higher electron energy state when the feeder molecule absorbs a certain frequency of photons.
FRET is a non-radiated energy leap, through the interaction of electrons between molecules, the transfer of the excitation state energy of the supply to the subject excitation state process, so that the fluorescence intensity of the feed is reduced, and the subject can emit stronger than its own characteristic fluorescence (sensitive fluorescence), can also not fluorescence (fluorescence annihilation), but also accompanied by the corresponding shortening or extension of fluorescence life. The efficiency of energy transfer is related to the degree to which the emission
spectral
of the supply overlaps with the absorption spectrum of the receptor, the relative orientation of the jump mate of the supply and the receptor, and the distance between the supply and the receptor. As a resonant energy transfer supply and receptor pair, fluorescent substances must meet the following conditions:
(1) the excitation light of the receptor and the excitation light of the supply should be sufficiently divided, and the light-emitting spectrum of the
(2) supply should overlap with the excitation spectrum of the receptor.
People have successfully applied nucleic acid detection, protein structure, functional analysis, immunoanalytical analysis and cellular structure function detection by using the organism's own fluorescence or marking organic fluorescent dyes on the objects studied. (Traditional organic fluorescent dyes absorb a narrow spectrum, the emission spectrum is often accompanied by a tail, which will affect the degree of overlap between the supply emission spectrum and the subject absorption spectrum, and the supply, the subject emission spectrum interferes with each other.) Recent reports have used luminous quantum dots for resonant energy transfer research, overcoming the shortcomings of organic fluorescent dyes.
Compared to conventional organic fluorescent dye molecules, the emission spectrum of quantum dots is narrow and does not drag, reducing the overlap between the emission spectrum of the feed and the receiver and avoiding interference with each other, and since the quantum dot has a wide spectral excitation range, when it is an energy supply, it is freer to choose the excitation wavelength, which can be maximized. To avoid direct excitation of energy receptors, by changing the composition or size of quantum dots, it can emit light at any wavelength in the visible light region, that is, it can make an energy supply for any lifetime chromosome of the absorption spectrum in the visible region, and ensure the good overlap between the wavelength of the feed and the absorption wavelength of the receptor, increasing the efficiency of resonant energy transfer.take the GFP's two mutants, Cyan fluorescent protein and YFP, as examples: the emission spectrum of the CFP overlaps with the absorption spectrum of YFP, when they When close enough, with the absorption wavelength of CFP, the CFP's hair color group will transfer the energy efficient resonance to the YFP's hair color group, so the CFP emission fluorescence will weaken or disappear, the main emission will be YFP fluorescence. The energy conversion efficiency between the two hair color groups is inversely inversely inversely compared to the six times the spatial distance between them, and the change of space position is very sensitive. For example, to study the interaction between two proteins a and b, a fusion protein can be constructed according to the FRET principle, which consists of three parts: CFP (cyan fluorescent protein), protein b, and YFP (yellow fluorecent protein).
with CFP absorption wavelength 433nm as the excitation wavelength, experimental dexterous design, so that when protein a and b do not interact, CFP and YFP are far away from the fluorescence resonance energy transfer, so it is detected that the CFP emission wavelength is 47 6nm fluorescence, but when protein a interacts with b, the composition changes due to protein b being acted on by protein a, so that CFP and YFP are sufficiently close to the fluorescent resonance energy transfer, at which point the fluorescence of YFP's emission wavelength of 527nm is detected. The genes that encode this fusion protein are genetically modified to express them within the cell, so that protein-protein interactions can be studied under living cell physiological conditions.
, freT technology
With the deepening of life science research, it is particularly important to study the mechanisms of various life phenomena, especially the in-cell protein-protein interaction. Technological progress is essential if major breakthroughs are to be made in these areas of research.
Some traditional research methods continue to develop, providing 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 double hybridization, phosphorylation
antibody
, immunofluorescence, radioactive markers and other methods are applied on the premise of breaking cells or causing damage to cells, can not be real-time under the physiological conditions of living cells on the intracular protein-protein interaction dynamic research. The application of FRET technology combined with genetic engineering and other technologies to make up for this defect, the following is the specific application of FRET technology in the field of related life sciences.
1, detection of enzyme activity changes
(1) in living cells detection protein
kinase
activity
protein phosphatization is an important marker in the process of cell signal transduction, the study of enzyme activity is an important aspect of the study of signaling path. Previously, enzyme activity determination was mainly the use of radioactivity and immunochemical luminescence and other methods, but the premise is to break cells, with cell extract to determine enzyme activity, but also can not do live cells timed, quantitative, positioning of the observation enzyme activity changes.
And the USE method can solve this problem very well: Zhang et al. designed a new probe (a fusion protein) using the FRET principle: the new probe contains a substrate domain specific to known protein kinases, a phosphorylation identification domain combined with the phosphorylated substrate domain. At both ends of this probe protein are GFP derivatives CFP and YFP, which work using the FRET principle.
when the substrate domain is phosphoricized, the internal folding caused by the binding of the phosphoricization recognition domain to the molecule occurs, and energy migration occurs when the two fluorescent proteins are close to each other. If
phosphatase
to dephosphate, the molecule will change reversiblely. The team used several sets of chimacts to study the activity of four known protein kinases: PKA (protein kinase A), Src, Abl, and EGFR (epidermal growth factor receptor).
they transferred the reported probes they had built into cells to detect changes in kinase activity based on FRET. After

treatment of cells, several tyrosine kinases were activated within a few minutes, detecting 25%-35% changes in activity. Using forskolin to activate PKA enhances fret 25%-50% of the changes, and kinases are activated throughout the cytosteal range. If the reported probe is added to the nuclear positioning signal to position it in the core, the FRET change is greatly delayed, which also illustrates the culture of PKA's action. It can be seen that using FRET method can be very good to observe the change of enzyme activity in living cells, and can be timed, quantitative, positioning, is a very effective research method.
.