A Measuring Method of Donor-Acceptor Distance Distribution Based on Fluorescence Correlation Spectrum

A technique of fluorescence correlation spectrum and measurement method, which is applied in the field of measurement of fluorescence resonance energy transfer distance distribution, and can solve the problems of lack of technical effect, measurement, and inability to accurately calculate the influence of receptor concentration.

Active Publication Date: 2016-08-24
SOUTH CHINA NORMAL UNIVERSITY
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Problems solved by technology

However, when the concentration ratio of the donor and acceptor is not 1:1, the fluorescence signal of the acceptor channel includes the contribution from the fluorescence resonance energy transfer and the influence of the concentration of the acceptor molecule, because neither the spectroscopy nor the fluorescence lifetime method can measure the concentration of the system. Donor-acceptor concentration was measured, so the effect of acceptor concentration on acceptor fluorescence signaling pathways cannot be accurately calculated
Although some correction schemes try to solve the problem of donor-receptor non-pairing fluorescence resonance energy transfer measurement, most of them are based on experience or fitting parameters, and cannot obtain good technical results.

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  • A Measuring Method of Donor-Acceptor Distance Distribution Based on Fluorescence Correlation Spectrum
  • A Measuring Method of Donor-Acceptor Distance Distribution Based on Fluorescence Correlation Spectrum
  • A Measuring Method of Donor-Acceptor Distance Distribution Based on Fluorescence Correlation Spectrum

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Embodiment 1

[0112] The distance between the donor-acceptor pair ECFP-Venus was varied to construct 3 independent FRET systems, 10%, 35%, 75%. The triplet weight distribution of each system exhibits a narrow peak distribution, such as image 3 shown in the curve. The triplet weight distribution of each system is obtained by inversion calculation using the maximum entropy algorithm, such as image 3 shown by the dotted solid line. The maximum entropy inversion results show that the calculated results in the single-distance FRET system are in good agreement with the actual distribution.

Embodiment 2

[0114] Placing three donor-acceptor pairs at different distances in the same system, the same maximum entropy inversion can also obtain ideal results, such as image 3 The solid line in the inversion results of a mixture of three donor-acceptor distances in equal proportions.

[0115] In calculating the transfer rate constant k ET , the triplet relaxation time τ is implied tri A to the transfer rate constant k ET There is a one-to-one corresponding functional relationship between them. Although the function cannot be expressed explicitly, the functional relationship can be represented graphically, such as Figure 4a , Figure 4b shown.

[0116] transfer rate constant k ET The function of the distance r to the donor acceptor is given by The energy transfer rate constant formula is given by:

[0117] k ET ( r ) = Q D ...

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Abstract

The invention discloses a donor-acceptor distance distribution measuring method based on fluorescence correlation spectroscopy. Fluorescent labeled living cells of donors and acceptors are subjected to a selective donor excitation operation through a fluorescence correlation spectroscopy module of a laser scanning confocal microscope in different laser power. A correlation card acquires photon number fluctuation signals of a donor channel and an acceptor channel to form a self-correlation curve. Energy transfer rate and energy transfer efficiency can be calculated through triplet state analysis of the self-correlation curve of the acceptors. In addition, triplet state distribution of the energy transfer rate and the energy transfer efficiency can be inverted from the self-correlation curve of the triplet state of the acceptors through a maximum entropy algorithm so that donor-to-acceptor distance distribution can be calculated. A fluorescence correlation spectrum analysis method itself is an excellent technology of measuring concentration of a dilute solution, so that the measuring method can be used for measuring donor-acceptor non-paired fluorescence resonance energy transfer efficiency and donor-acceptor non-paired distance distribution after integration items of overlap items of the spectrum being corrected with respect to the donor-acceptor concentration proportion. The measuring method is convenient to use and high in measuring precision.

Description

technical field [0001] The invention relates to a method for measuring the distance distribution of fluorescence resonance energy transfer, in particular to a method for measuring the distance distribution of a donor and an acceptor based on a fluorescence correlation spectrum. Background technique [0002] Fluorescence resonance energy transfer is a photophysical process in which an excited donor fluorescent molecule transfers excited state energy to a neighboring ground state acceptor molecule through dipole-dipole long-range Coulomb interactions. The occurrence of fluorescence resonance energy transfer requires the distance between the donor and acceptor molecules to be between 1 and 10 nm. This spatial scale is exactly the linearity of various molecular interactions in living cells, so FRET technology is especially suitable for the study of intracellular molecular interactions and molecular conformational changes. [0003] When fluorescence resonance energy transfer occ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N21/64
Inventor 郭周义陈同生陈秉灵
Owner SOUTH CHINA NORMAL UNIVERSITY
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