Single-molecule FRET efficiency measurement method based on receptor rise time
By using a single-molecule FRET efficiency measurement method based on receptor rise time, the problem of insufficient accuracy in FRET efficiency calculation in traditional methods is solved, achieving high-precision biomolecular distance measurement and expanding the measurement range and anti-interference capability.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- SHENZHEN INST OF ADVANCED TECH CHINESE ACAD OF SCI
- Filing Date
- 2026-01-21
- Publication Date
- 2026-05-01
AI Technical Summary
Existing methods for calculating FRET efficiency are not accurate enough, which affects the precision of biomolecular distance measurements, and limits their detection capabilities at different scales.
A single-molecule FRET efficiency measurement method based on acceptor rise time was adopted. By acquiring fluorescence signals, extracting time-resolved fluorescence decay curves of acceptor channels, and performing function fitting, the acceptor fluorescence rise time and donor fluorescence lifetime were calculated to obtain the fluorescence resonance energy transfer efficiency.
It achieves high-precision and accurate FRET efficiency measurement, can distinguish rise time from photophysical fluctuations in the spectrum, reduces interference such as spectral crosstalk, and expands the measurement range and accuracy.
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Figure CN121963974A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of bioanalytical technology, and in particular to a method for measuring the efficiency of single-molecule FRET based on receptor rise time. Background Technology
[0002] The dynamic conformational changes and interactions of biomolecules (such as proteins and nucleic acids) are fundamental to life activities, and their precise observation is a core challenge in structural biology and molecular biology. Single-molecule fluorescence resonance energy transfer (smFRET) technology has become a key tool for resolving nanoscale conformational dynamics due to its ability to monitor individual biomolecules in real time and dynamically under physiological conditions. This technology labels donor and acceptor fluorophores at specific sites on biomolecules and calculates intramolecular or intermolecular distance changes by measuring changes in fluorescence signals, thereby reflecting their conformational state. Nanoscale distance measurements using smFRET technology rely on FRET efficiency calculations. Theoretical derivation and experimental verification show that the FRET rate and distance satisfy a high-order polynomial function relationship, exhibiting a highly significant distance dependence. However, traditional calculation methods have certain limitations.
[0003] Existing FRET efficiency calculations mainly include: ① Based on the donor-acceptor fluorescence intensity ratio, the calculation formula is as follows: This calculation method involves multiple parameters, the accuracy of which affects the accuracy of the final FRET efficiency calculation, and consequently, the accuracy of the distance measurement. These parameters include the receptor and donor fluorescence intensity values after background removal. and Spectral crosstalk caused by donor fluorescence leakage into the acceptor channel The ratio of receptors directly excited by donor laser Donor-acceptor detector and fluorescent material correction wait.
[0004] ② Based on donor fluorescence lifetime decay, this calculation method has the advantage over methods based on donor-acceptor fluorescence intensity ratios in that the energy transfer time changes exponentially, making it more sensitive; and the parameters are simple in the time dimension, unaffected by fluorescence intensity or spectral crosstalk. However, since both the donor fluorescence lifetime and the donor's own fluorescence lifetime are in the decay curve portion, at low FRET efficiency, it is affected by background interference from the donor's own fluorescence; at high efficiency, the signal is too low to accurately fit, both limiting the accuracy range of FRET. This significantly reduces the detection capability of existing methods when studying the behavior of biomolecules at different scales. Against this backdrop, there is an urgent need to develop a new, high-precision method for calculating FRET efficiency. Summary of the Invention
[0005] In view of this, the present invention provides a method for measuring the efficiency of single-molecule FRET based on receptor rise time to solve the above problems.
[0006] This invention provides a method for measuring single-molecule FRET efficiency based on acceptor rise time, comprising: acquiring a fluorescence signal generated by a fluorescently labeled sample under laser excitation, wherein the sample includes a donor fluorophore and an acceptor fluorophore; extracting a time-resolved fluorescence decay curve of the acceptor channel from the fluorescence signal; performing function fitting on the time-resolved fluorescence decay curve of the acceptor channel to obtain the acceptor fluorescence rise time caused by the fluorescence resonance energy transfer process; and calculating the fluorescence resonance energy transfer efficiency based on the acceptor fluorescence rise time and the fluorescence lifetime of the donor fluorophore itself.
[0007] In another implementation of the present invention, the fluorescence resonance energy transfer system satisfies the following: all donors and acceptors in the system are paired, and there are no unreacted donors or acceptors; the fluorescence lifetimes of the donors and acceptors are single-exponential decays; the excitation wavelength only excites the donor fluorophore, and the acceptor fluorophore is not directly excited by the laser; and there is no donor signal interference in the acceptor fluorescence signal detection channel.
[0008] In another implementation of the present invention, the excited-state fluorescence intensity of the donor fluorophore is expressed as:
[0009] in, It is the fluorescence intensity of the donor molecule at time t; It is the rate constant of the donor molecule in the absence of the acceptor molecule; It is the rate constant of the resonant energy transfer from the donor to the acceptor at time t.
[0010] In another implementation of the present invention, the excited-state fluorescence intensity of the receptor fluorophore is expressed as:
[0011] in, It is the fluorescence intensity of the receptor molecule at time t. It is the rate constant of the acceptor molecule in the absence of a donor molecule.
[0012] In another implementation of the present invention, the formula for calculating the receptor fluorescence rise time is: + = =
[0013] in, FRET energy transfer rate constant, It is the rise time of the acceptor molecule due to fluorescence resonance energy transfer.
[0014] In another implementation of the present invention, the decay time of the donor is expressed as: =
[0015] in, This refers to the decay time of the donor.
[0016] In another implementation of the present invention, the fluorescence resonance energy transfer efficiency is expressed as:
[0017] in, It is the fluorescence lifetime of the donor itself.
[0018] In another aspect, the present invention provides a single-molecule FRET efficiency measurement system based on acceptor rise time, comprising: a fluorescence signal acquisition module for acquiring the fluorescence signal generated by a fluorescently labeled sample under laser excitation, wherein the sample includes a donor fluorophore and an acceptor fluorophore; a fluorescence signal processing module for extracting the time-resolved fluorescence decay curve of the acceptor channel from the fluorescence signal; and a FRET efficiency calculation module for performing function fitting on the time-resolved fluorescence decay curve of the acceptor channel to obtain the acceptor fluorescence rise time caused by the fluorescence resonance energy transfer process; and calculating the fluorescence resonance energy transfer efficiency based on the acceptor fluorescence rise time and the fluorescence lifetime of the donor fluorophore itself.
[0019] In another aspect, the present invention provides an electronic device comprising: a memory, a processor, and a computer program stored in the memory and executable on the processor, wherein the processor, when executing the computer program, implements the steps of a single-molecule FRET efficiency measurement method based on receptor rise time as described in any of the preceding claims. In another aspect, the present invention provides a computer storage medium storing a computer program that, when executed by a processor, implements the steps in a single-molecule FRET efficiency measurement method based on receptor rise time as described in any of the preceding claims.
[0020] The single-molecule FRET efficiency measurement method based on acceptor rise time of the present invention innovatively encodes the FRET rate by using the acceptor rise time, which can cleverly distinguish rise time from photophysical fluctuations in the spectrum, and achieve high precision and accuracy measurement. Attached Figure Description
[0021] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below. By reading the detailed description of the embodiments below, the advantages and benefits of the solutions will become clear to those skilled in the art. The accompanying drawings are only for illustrating preferred embodiments and are not intended to limit the present invention. In the accompanying drawings: Figure 1 This is a schematic diagram of a single-molecule FRET efficiency measurement method based on receptor rise time, according to an embodiment of the present invention.
[0022] Figure 2 This is a schematic diagram illustrating the theoretical relationship between donor fluorescence decay lifetime and acceptor rise lifetime and intermolecular distance, according to an embodiment of the present invention. Detailed Implementation
[0023] To enable those skilled in the art to better understand the technical solutions in the embodiments of the present invention, the technical solutions in the embodiments of the present invention will be clearly and thoroughly described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art should fall within the protection scope of the present invention.
[0024] Figure 1 A schematic flowchart of a single-molecule FRET efficiency measurement method based on receptor rise time is provided for an embodiment of the present invention, as shown below. Figure 1 As shown, this embodiment mainly includes: S101. Obtain the fluorescence signal generated by the fluorescently labeled test sample under laser excitation, wherein the test sample contains a donor fluorophore and an acceptor fluorophore.
[0025] S102. Extract the time-resolved fluorescence decay curve of the receptor channel from the fluorescence signal.
[0026] S103. Perform function fitting on the time-resolved fluorescence decay curve of the receptor channel to obtain the receptor fluorescence rise time caused by the fluorescence resonance energy transfer process.
[0027] S104. The fluorescence resonance energy transfer efficiency is calculated based on the fluorescence rise time of the acceptor and the fluorescence lifetime of the donor fluorophore itself.
[0028] The single-molecule FRET efficiency measurement method based on acceptor rise time of the present invention innovatively encodes the FRET rate by using the acceptor rise time, which can cleverly distinguish rise time from photophysical fluctuations in the spectrum, and achieve high precision and accuracy measurement.
[0029] In another implementation of the present invention, the fluorescence resonance energy transfer system satisfies the following: all donors and acceptors in the system are paired, and there are no unreacted donors or acceptors; the fluorescence lifetimes of the donors and acceptors are single-exponential decays; the excitation wavelength only excites the donor fluorophore, and the acceptor fluorophore is not directly excited by the laser; and there is no donor signal interference in the acceptor fluorescence signal detection channel.
[0030] For example, the fluorescence signal may originate from, but is not limited to, a single-molecule fluorescence detection platform, a fluorescence lifetime imaging microscope, or a time-resolved microplate reading system.
[0031] In another implementation of the present invention, the excited-state fluorescence intensity of the donor fluorophore is expressed as:
[0032] in, It is the fluorescence intensity of the donor molecule at time t; It is the rate constant of the donor molecule in the absence of the acceptor molecule; It is the rate constant of the resonant energy transfer from the donor to the acceptor at time t.
[0033] =
[0034] + = =
[0035] in, It is the FRET energy transfer rate constant.
[0036] In another implementation of the present invention, the excited-state fluorescence intensity of the receptor fluorophore is expressed as:
[0037] in, It is the fluorescence intensity of the receptor molecule at time t. It is the rate constant of the acceptor molecule in the absence of a donor molecule.
[0038] =
[0039] in, It is the fluorescence lifetime of the receptor itself.
[0040] In another implementation of the present invention, the formula for calculating the receptor fluorescence rise time is: + = =
[0041] in, FRET energy transfer rate constant, It is the rise time of the acceptor molecule due to fluorescence resonance energy transfer.
[0042] For example, extract This involves fitting the decay curve using a function model that includes a rise time term. The fitting is performed using the maximum likelihood estimation method. The function model that includes the rise time term is expressed as: I A =-A 1 · + A 2 ·
[0043] Where A1 and A2 are constant coefficients.
[0044] In another implementation of the present invention, theoretically the donor decay time is equal to the acceptor rise time, and the donor decay time is expressed as: =
[0045] in, This refers to the decay time of the donor.
[0046] In another implementation of the present invention, the fluorescence resonance energy transfer efficiency is expressed as:
[0047] in, It is the fluorescence lifetime of the donor itself.
[0048] For example, such as Figure 2 As shown, based on the principles of photophysics, the rise time of the receptor (…) was established. ) and FRET efficiency ( The direct quantitative relationship between them.
[0049] Optionally, in embodiments of the present invention, the research object is not limited to a specific nucleic acid structure. In addition to using a linear double-stranded DNA molecular ruler, DNA origami with precise three-dimensional structure, functionalized RNA structures, or site-labeled stable protein domains can also be used as alternative ruler carriers. These alternative systems can expand the measurable distance range, provide more complex spatial configurations, or better simulate natural biological environments, thereby more comprehensively validating and calibrating the universality and accuracy of receptor rise time-based calculation methods across different spatial scales and biological matrices.
[0050] Alternatively, in terms of data processing algorithms, in addition to maximum likelihood estimation, this invention can also employ alternative analysis methods such as Bayesian inference and machine learning. The Bayesian framework, by introducing prior distributions, can more robustly handle low signal-to-noise ratio data and provide parameter uncertainty estimation; while deep learning-based algorithms can directly extract fluorescence lifetime parameters from raw photon data, improving the automation of the analysis. These alternatives collectively enhance the method's adaptability and analytical efficiency under different data quality conditions.
[0051] Alternatively, in addition to point scanning confocal microscopy systems, the technical solution of this invention can also be adapted to other high time-resolution fluorescence detection platforms. Alternative solutions include using total internal reflection fluorescence microscopy, light-sheet microscopy, or multifocal parallel imaging systems, and being compatible with time-resolution detectors based on different principles, to achieve diverse excitation modes, higher throughput, or instrument configurations more suitable for live-cell imaging.
[0052] This invention is compatible with and makes full use of commonly used fluorophores, overcoming the shortcomings of traditional calculation methods (intensity ratio and donor decay time methods) in terms of measurement accuracy and range, and achieving reliable and accurate measurement of biomolecular structural dynamics under routine experimental conditions. The key points are: Breakthrough in core principles: A high-precision calculation method for single-molecule FRET efficiency based on receptor rise time has been established. The calculation formula is as follows: .
[0053] Compatibility and universality verification: By systematically testing multiple sets of conventional fluorescent FRET pairs with different photophysical properties, the method's broad compatibility with existing commonly used fluorescent labeling systems was verified. This is the core step in its transition from principle to practical application.
[0054] Quantitative proof of superiority: By comparing with the traditional intensity ratio method and donor lifetime method on the same scale, and using molecular dynamics simulations to provide a theoretical benchmark, the significant advantages of the acceptor rise time-based calculation method in terms of measurement accuracy, dynamic range, and anti-interference ability (such as spectral crosstalk) are quantitatively demonstrated.
[0055] Example 1 Using DNA as a "molecular scale" as a model, a high-precision measurement and analysis method based on receptor rise time is constructed through three steps: theoretical derivation, experimental analysis, and simulation verification. This expands the accuracy range of FRET efficiency from 20-80% of traditional methods to 14-95%.
[0056] Specifically, double-stranded DNA sequences were designed, the donor fluorophore positions were fixed, and the acceptor fluorophore was labeled at specific base positions on the complementary strand. A series of donor-acceptor distance models (corresponding to 5, 11, 15, 22, and 24 base pairs, respectively) were constructed to systematically study FRET efficiency at different distances. Based on these five DNA molecular scales covering a range of FRET efficiencies from high to low, measurement standards for the smART FRET method were constructed and validated.
[0057] For example, using Atto532 and Atto647N as donor and acceptor respectively, a DNA "molecular scale" model can be established, and a method based on acceptor rise time can be developed. This method can also be extended to other biomolecular detection applications.
[0058] Example 2 Single-molecule measurements of the FRET process in a double-stranded DNA model were performed using point-scan confocal microscopy. Point-scan confocal microscopy offers sub-millisecond temporal resolution and is equipped with two independent donor and acceptor fluorescence signal detection channels. An avalanche photodiode (APD) with time-correlated single-photon counting was used as the detector to acquire fluorescence signals from donor and acceptor molecules of single double-stranded DNA immobilized on a biotin-functionalized coverslip. Donor-acceptor fluorescence intensity ratio data were directly obtained from the fluorescence intensity in the time trajectory. Donor decay time and acceptor rise time data were obtained by identifying the FRET process region in the time trajectory using a change-point algorithm. Further analysis of this time trajectory region generated fluorescence lifetime curves. The fitting of the fluorescence lifetime curves was calculated using maximum likelihood estimation. The relationships between the donor-acceptor fluorescence intensity ratio, donor decay time, and acceptor rise time with FRET efficiency were investigated.
[0059] The theoretical values of donor-acceptor distance and corresponding FRET efficiency at different positions of the labeled double-stranded DNA were calculated using FPS software simulation. As shown in Table 1, the experimental results were compared with the theoretical values, verifying the accuracy of the calculation method based on acceptor rise time.
[0060] Table 1: FRET efficiency data results obtained based on three calculation methods and FPS simulation.
[0061]
[0062] As can be seen, compared with the prior art, this invention acquires data in the time dimension, the signal changes exponentially, the parameters are simple, and it is not affected by fluorescence intensity, spectral crosstalk, etc.; receptor rise time Compared with its own fluorescence lifetime Completely separated and unaffected by its own fluorescence background; the acceptor fluorescence signal comes only from the FRET process, and is spectrally separated from other photophysical fluctuations, providing a higher single-molecule signal-to-noise ratio.
[0063] The receptor rise time-based calculation method provided by this invention has the application of high-precision calculation of FRET efficiency and has the following extended application scenarios: (1) FRET probe screening and optimization platform: This method serves as a standardized testing platform for systematically evaluating and screening the performance (including brightness, photostability, orientation characteristics, etc.) of novel fluorescent protein variants, synthetic dyes, or quantum dots as FRET pairs, providing optimal probe combinations for biosensor design.
[0064] (2) Absolute distance measurement of biomolecular structure dynamics: The high-precision FRET efficiency distribution measured by the smART method is inverted into the real-time distance distribution of biomolecules, realizing the absolute scale quantification of conformational heterogeneity and transformation dynamics, making up for the shortcomings of static techniques such as cryo-electron microscopy.
[0065] (3) Drug screening and mechanism of action study: Applied to high-throughput drug screening based on FRET, by monitoring the dynamic trajectory of the conformational changes of the target protein before and after the drug molecule binds, not only can active compounds be screened, but their mechanisms of action can also be distinguished (such as allosteric regulation, competitive inhibition, etc.).
[0066] (4) In situ structural biology of cells: Extend the method to single-molecule detection in living cells, and combine it with gene-encoded fluorescent protein labeling to achieve direct observation and quantitative analysis of protein interactions, conformational changes and subcellular organelle dynamics under near-physiological conditions.
[0067] (5) Characterization of nanomaterials and molecular machines: used to characterize the dynamic conformational changes and working cycles of DNA nanomachines, molecular motors or functional nanoparticles, providing key dynamic structural information for their rational design and performance optimization.
[0068] (6) Teaching and Standardized Metrology: Used as a teaching tool for single-molecule biophysics experimental courses for graduate students and senior undergraduates, or as a metrological standard for calibrating FRET measurement modules of commercial fluorescence lifetime imaging microscopes and other equipment.
[0069] These alternative designs and extended uses demonstrate that this invention not only provides an improved method for calculating FRET efficiency, but also serves as a scalable technology platform to promote research and development in multiple interdisciplinary fields such as single-molecule biophysics, chemical biology, and nanotechnology.
[0070] This invention demonstrates exceptional nanoscale sensitivity, successfully elucidating the complex behavioral patterns of biomolecules. This technological breakthrough is expected to open new avenues for biomolecular research, facilitating a deeper and more comprehensive exploration of the mysteries of life processes, and also possesses enormous potential application value in fields such as biomedicine and drug development.
[0071] Another aspect of the present invention provides a single-molecule FRET efficiency measurement system based on receptor rise time, comprising: Fluorescence signal acquisition module: acquires the fluorescence signal generated by the fluorescently labeled test sample under laser excitation, wherein the test sample contains donor fluorophores and acceptor fluorophores.
[0072] Fluorescence signal processing module: Extracts the time-resolved fluorescence decay curve of the receptor channel from the fluorescence signal.
[0073] FRET efficiency calculation module: Performs function fitting on the time-resolved fluorescence decay curve of the receptor channel to obtain the receptor fluorescence rise time caused by the fluorescence resonance energy transfer process; calculates the fluorescence resonance energy transfer efficiency based on the receptor fluorescence rise time and the fluorescence lifetime of the donor fluorophore itself.
[0074] The single-molecule FRET efficiency measurement system based on acceptor rise time of the present invention innovatively encodes the FRET rate by means of acceptor rise time, which can cleverly distinguish rise time from photophysical fluctuations in the spectrum, and achieve high precision and accuracy measurement.
[0075] In another aspect of the present invention, the electronic device includes: a processor, a memory, and a communication bus and a communication interface.
[0076] in: The processor, memory, and communication interface communicate with each other via a communication bus.
[0077] A communication interface is used to communicate with other electronic devices or servers.
[0078] The processor is used to execute programs, specifically, to perform any of the steps of the single-molecule FRET efficiency measurement method based on receptor rise time in the above embodiments.
[0079] Specifically, the program may include program code, which includes computer operation instructions.
[0080] The processor may be a central processing unit (CPU), an application-specific integrated circuit (ASIC), or one or more integrated circuits configured to implement the embodiments of this application. The one or more processors included in the smart device may be processors of the same type, such as one or more CPUs; or they may be processors of different types, such as one or more CPUs and one or more ASICs.
[0081] Memory is used to store programs. Memory may include high-speed RAM, and may also include non-volatile memory, such as at least one disk drive.
[0082] Specifically, the program can be used to cause the processor to execute the steps of any of the single-molecule FRET efficiency measurement methods based on receptor rise time described in the embodiments. The specific implementation of each step in the program can be found in the corresponding descriptions of the steps and units executed in any of the single-molecule FRET efficiency measurement methods based on receptor rise time described above, and will not be repeated here. Those skilled in the art will clearly understand that, for the sake of convenience and brevity, the specific working process of the devices and modules described above can be referred to the corresponding process descriptions in the foregoing method embodiments.
[0083] An exemplary embodiment of this application also provides a non-transitory computer-readable storage medium storing computer instructions, wherein the computer instructions are used to cause a computer to perform the methods of various embodiments of this application.
[0084] The methods described above according to embodiments of the present invention can be implemented in hardware, firmware, or as software or computer code that can be stored in a recording medium (such as a CD-ROM, RAM, floppy disk, hard disk, or magneto-optical disk), or as computer code originally stored on a remote recording medium or a non-transitory machine-readable medium and subsequently stored on a local recording medium, downloaded via a network. Thus, the methods described herein can be processed by software stored on a recording medium using a general-purpose computer, a dedicated processor, or programmable or dedicated hardware (such as an ASIC or FPGA). It is understood that the computer, processor, microprocessor controller, or programmable hardware includes storage components (e.g., RAM, ROM, flash memory, etc.) capable of storing or receiving software or computer code, which, when accessed and executed by the computer, processor, or hardware, implements the methods described herein. Furthermore, when a general-purpose computer accesses code used to implement the methods shown herein, the execution of the code transforms the general-purpose computer into a dedicated computer for executing the methods shown herein.
[0085] Specific embodiments of the present invention have now been described. Other embodiments are within the scope of the appended claims. In some cases, the actions described in the claims can be performed in a different order and still achieve the desired result. Furthermore, the processes depicted in the drawings do not necessarily require a specific or sequential order to achieve the desired result.
[0086] It should be noted that all directional indications (such as up, down, left, right, back, etc.) in the embodiments of the present invention are only used to explain the relative positional relationship between the components in a certain order (as shown in the figure). If the specific order changes, the directional indication will also change accordingly.
[0087] In the description of this invention, the terms "first" and "second" are used only for convenience in describing different components or names, and should not be construed as indicating or implying a sequential relationship, relative importance, or implicitly specifying the number of technical features indicated. Thus, a feature defined with "first" and "second" may explicitly or implicitly include at least one of that feature.
[0088] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.
[0089] It should be noted that although specific embodiments of the present invention have been described in detail with reference to the accompanying drawings, this should not be construed as limiting the scope of protection of the present invention. Various modifications and variations that can be made by those skilled in the art without inventive effort within the scope described in the claims still fall within the scope of protection of the present invention.
[0090] The examples of the embodiments of the present invention are intended to concisely illustrate the technical features of the embodiments of the present invention, so that those skilled in the art can intuitively understand the technical features of the embodiments of the present invention, and are not intended to be an improper limitation of the embodiments of the present invention.
[0091] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.
Claims
1. A method for measuring the efficiency of single-molecule FRET based on receptor rise time, characterized in that, include: The fluorescence signal generated by a fluorescently labeled sample under laser excitation is obtained, wherein the sample contains a donor fluorophore and an acceptor fluorophore; From the fluorescence signal, extract the time-resolved fluorescence decay curve of the receptor channel; The time-resolved fluorescence decay curve of the receptor channel was fitted with a function to analyze the receptor fluorescence rise time caused by the fluorescence resonance energy transfer process. The fluorescence resonance energy transfer efficiency is calculated based on the fluorescence rise time of the acceptor and the fluorescence lifetime of the donor fluorophore itself.
2. The method according to claim 1, characterized in that, The fluorescence resonance energy transfer system satisfies: All donors and acceptors in the system were paired, and there were no unreacted donors and acceptors. The fluorescence lifetimes of both the donor and acceptor exhibit single-exponential decay. The excitation wavelength only excites the donor fluorophore; the acceptor fluorophore will not be directly excited by the laser. The receptor fluorescence signal detection channel is free from donor signal interference.
3. The method according to claim 2, characterized in that, The excited-state fluorescence intensity of the donor fluorophore is expressed as: in, It is the fluorescence intensity of the donor molecule at time t; It is the rate constant of the donor molecule in the absence of the acceptor molecule; It is the rate constant of the resonant energy transfer from the donor to the acceptor at time t.
4. The method according to claim 3, characterized in that, The excited-state fluorescence intensity of the receptor fluorophore is expressed as: in, It is the fluorescence intensity of the receptor molecule at time t. It is the rate constant of the acceptor molecule in the absence of a donor molecule.
5. The method according to claim 4, characterized in that, The formula for calculating the rise time of receptor fluorescence is: + = = in, FRET energy transfer rate constant, It is the rise time of the acceptor molecule due to fluorescence resonance energy transfer.
6. The method according to claim 5, characterized in that, The decay time of the donor is expressed as: = in, This refers to the decay time of the donor.
7. The method according to claim 6, characterized in that, The fluorescence resonance energy transfer efficiency is expressed as: in, It is the fluorescence lifetime of the donor itself.
8. A single-molecule FRET efficiency measurement system based on receptor rise time, characterized in that, include: Fluorescence signal acquisition module: acquires the fluorescence signal generated by the fluorescently labeled test sample under laser excitation, wherein the test sample contains a donor fluorophore and an acceptor fluorophore; Fluorescence signal processing module: Extracts the time-resolved fluorescence decay curve of the receptor channel from the fluorescence signal; FRET efficiency calculation module: Performs function fitting on the time-resolved fluorescence decay curve of the receptor channel to obtain the receptor fluorescence rise time caused by the fluorescence resonance energy transfer process; calculates the fluorescence resonance energy transfer efficiency based on the receptor fluorescence rise time and the fluorescence lifetime of the donor fluorophore itself.
9. An electronic device, characterized in that, include: A memory, a processor, and a computer program stored in the memory and executable on the processor, wherein the processor, when executing the computer program, implements the steps of the single-molecule FRET efficiency measurement method based on receptor rise time as described in any one of claims 1 to 7.
10. A computer storage medium, characterized in that, The computer storage medium stores a computer program that, when executed by a processor, implements the steps in the single-molecule FRET efficiency measurement method based on receptor rise time as described in any one of claims 1 to 7.