Digital single-molecule enzyme multi-activity characteristic cross-scale heterogeneity dynamic evaluation method

Abstract

The invention discloses a digital single-molecule enzyme multi-activity characteristic cross-scale heterogeneity dynamic evaluation method, which comprises the following steps: diluting a pre-combined enzyme-template-primer ternary complex to a single-molecule level so as to load the enzyme-template-primer ternary complex into a reaction chamber in a micropore array chip for rolling circle amplification, a molecular beacon is combined with an amplification product to release a fluorescence signal; monitoring and acquiring the fluorescence signal in real time to generate a time-resolved fluorescence intensity curve; extracting a slope parameter of the fluorescence intensity curve so as to obtain a catalytic synthesis rate through conversion calculation for evaluating enzyme activity; the distribution of catalytic synthesis rate values is fitted using an n-fold Gaussian mixture model to obtain a standard deviation for assessing population homogeneity and a coefficient of variation for assessing stability. According to the invention, cross-scale analysis of single-molecule resolution, multi-parameter dynamic association and environmental interference processing can be realized at the same time, and resolution limitation of a traditional analysis technology is broken through by integrating single-molecule fluorescence tracking and population statistics.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biomolecule detection, and in particular to a method for dynamically evaluating the cross-scale heterogeneity of single-molecule enzyme multi-activity characteristics. BACKGROUND

[0002] In the field of biotechnology, the catalytic performance of enzymes directly determines the efficiency and accuracy of core technologies such as DNA sequencing and biosynthesis. In particular, the development of cutting-edge platforms such as single-molecule real-time sequencing (SMRT) has placed unprecedented demands on the catalytic rate, reaction uniformity, and environmental stability of DNA polymerase. However, current enzyme activity analysis methods have fundamental limitations. The paradigm of relying on population average measurements cannot reveal the heterogeneity characteristics of single enzyme molecules, which has become a key bottleneck restricting the rational design and screening of high-performance enzymes.

[0003] Traditional enzyme activity detection techniques such as fluorescence substrate hydrolysis and gel electrophoresis analysis measure the collective behavior of millions to hundreds of millions of enzyme molecules to output average activity values. This method based on the assumption of population uniformity essentially masks the significant differences in kinetic parameters among single enzyme molecules. In a population of DNA polymerases, there may be a high-rate subgroup and a low-rate subgroup, but traditional detection can only report a statistical average value. In addition, the sensitivity differences of enzyme molecules to environmental disturbances, laser irradiation, and ion concentration fluctuations are hidden, which may lead to the failure to identify key performance defects. The absence of such single-molecule heterogeneity makes directed evolution engineering lose its dominance, and may result in over-reliance on rate improvement in mutant screening, while ignoring the coordinated optimization of uniformity and stability, ultimately leading to the inability of high-rate mutants to be practically applied due to hidden defects.

[0004] To overcome the limitations of population analysis, single-molecule detection techniques have been introduced into enzyme research, such as single-molecule fluorescence resonance energy transfer (smFRET) and optical tweezer manipulation. However, these techniques still have certain limitations. smFRET can observe enzyme conformation changes in real time, but it is difficult to simultaneously correlate catalytic rate and functional stability. Optical tweezer technology is good at analyzing enzyme-substrate interactions under mechanical force, but it cannot simulate real reaction conditions in solution environment. Emerging nanopore sequencing platforms can obtain DNA translocation speed, but it is difficult to distinguish between enzyme heterogeneity and environmental noise contributions. In addition, the throughput limitations and statistical requirements of these techniques cannot fully adapt, and the high cost of equipment such as super-resolution microscopes and complex fluorescence labeling processes further hinder their large-scale application. Therefore, the shortcomings of existing single-molecule platforms in terms of throughput, multi-parameter integration, and environmental simulation capabilities have hindered the research of enzyme heterogeneity from the phenomenon observation stage to the establishment of a quantifiable evaluation standard.

[0005] Therefore, how to develop a cross-scale analysis method capable of realizing single molecule resolution, multi-parameter dynamic correlation and environmental interference processing at the same time has become an urgent need to break through the development bottleneck of enzyme screening evaluation for single molecule sequencing. SUMMARY

[0006] In order to overcome the shortcomings of the prior art, the purpose of the present application is to provide a digital single molecule enzyme multi-activity characteristic cross-scale heterogeneity dynamic evaluation method, which has the advantages of being capable of realizing single molecule resolution, multi-parameter dynamic correlation and environmental interference processing at the same time.

[0007] The purpose of the present application is achieved by the following technical solutions: According to the present application, a digital single molecule enzyme multi-activity characteristic cross-scale heterogeneity dynamic evaluation method is provided, which comprises: diluting the pre-bound enzyme-template-primer ternary complex to a single molecule level to load the reaction chamber in the microwell array chip for rolling circle amplification, and using molecular beacons to combine with the amplification product to release a fluorescence signal; real-time monitoring and acquiring the fluorescence signal to generate a time-resolved fluorescence intensity curve; extracting the slope parameter of the fluorescence intensity curve to calculate the catalytic synthesis rate by conversion for evaluating enzyme activity; using an n-Gaussian mixture model to fit the catalytic synthesis rate value distribution to obtain the standard deviation for evaluating population homogeneity and the coefficient of variation for evaluating stability.

[0008] In some exemplary embodiments, the size of the microwell array chip is (10-20) x (10-20) mm, and has more than 10,000 reaction chambers, the reaction chamber size is (65-90) pm x (50-80) pm x (80-120) pm, the pitch is 80-120 pm, and the microwell array chip is treated by surface activation, hydrophobic coating deposition, and selective hydrophilization to enhance the hydrophilicity inside the reaction chamber and the hydrophobicity outside the reaction chamber.

[0009] In some exemplary embodiments, the dilution of the pre-bound enzyme-template-primer ternary complex to a single molecule level to load the reaction chamber in the microwell array chip for rolling circle amplification specifically comprises: obtaining a target dilution factor based on the volume of the reaction chamber according to the Poisson distribution formula to dilute the pre-bound enzyme-template-primer ternary complex to a predetermined concentration; loading the diluted sample into the microwell array chip and distributing it to each reaction chamber by capillary action or centrifugal force, so that at least part of the reaction chambers contain only one ternary complex; The catalytic reaction is started to perform rolling circle amplification, and the molecular beacon is specifically combined with the corresponding matching sequence during the amplification process to release the fluorescence signal.

[0010] In some exemplary embodiments, when the enzyme-template-primer ternary complex is prepared, the template DNA is connected to the first end of the primer by the T4 ligase to form a circular template as a substrate for the polymerase.

[0011] In some exemplary embodiments, when the enzyme-template-primer ternary complex is prepared, the circular DNA template and the primer are also mixed in a volume ratio of 1: (2-3) in the reaction buffer for pre-hybridization, and the concentration of dNTPs is optimized to balance the amplification efficiency and background noise.

[0012] In some exemplary embodiments, the reaction buffer includes 50 mM Tris-HCl, 10 mM MgCl2, 100 mM KCl, and 1 mM dithiothreitol, the pH of the reaction buffer is maintained at 7.5-8.5, and the concentration of dNTPs is optimized to 0.4 mM.

[0013] In some exemplary embodiments, the real-time monitoring and acquisition of the fluorescence signal to generate a time-resolved fluorescence intensity curve specifically includes: The fluorescence intensity of each reaction chamber is monitored in real time by a high-frame-rate camera to generate a raw fluorescence image to extract the fluorescence signal, and a time-resolved fluorescence intensity curve is generated based on the real-time acquired fluorescence signal intensity.

[0014] In some exemplary embodiments, the slope parameter of the fluorescence intensity curve is extracted to calculate the catalytic synthesis rate by conversion for evaluating enzyme activity specifically includes: After suppressing optical noise by using image enhancement technology, feature hole positioning is performed based on a pre-defined hole site template to determine the reaction hole site in the microhole array chip; The time sequence fluorescence intensity data of each reaction hole site is acquired, and the slope parameter of the corresponding fluorescence intensity curve is extracted; The catalytic synthesis rate is calculated by conversion according to the calibration formula μ=k / (α·β), and the catalytic synthesis rate is used to evaluate enzyme activity, wherein μ is the catalytic synthesis rate, k is the slope parameter, α is the background fluorescence gain coefficient, and β is the beacon binding efficiency correction factor.

[0015] In some exemplary embodiments, the n-order Gaussian mixture model is used to fit the catalytic synthesis rate value distribution to obtain the standard deviation for evaluating population homogeneity and the coefficient of variation for evaluating stability specifically includes: A statistical histogram is generated based on the obtained catalytic synthesis rate, and the distribution characteristics are modeled using an n-fold Gaussian function fitting method according to the modeling formula, which is: Where n is the Gaussian multiplicity, y is the derivative of the fluorescence value, and x is the temperature sequence. The fitting function of the scipy library in Python is used to obtain the parameters that minimize the equation error. Population homogeneity was assessed by calculating the standard deviation of the catalytic synthesis rate at 1000 reaction sites based on the obtained n-fold Gaussian mixture model. The stability was assessed by calculating the coefficient of variation of the expected catalytic synthesis rate sequence over 900 minutes at 1-minute intervals based on the obtained n-fold Gaussian mixture model.

[0016] In some exemplary embodiments, when evaluating uniformity and stability, the enzyme synthesis rate at different concentrations is also monitored in real time to establish a double reciprocal curve, and the results are calculated according to the formula... The dynamic Michaelis constant of a single enzyme is obtained, where V0 is the reaction rate and dV is the constant. max The maximum reaction rate is given by [S], where [S] is the substrate concentration and dK is the denominator. m This is the dynamic Michaelis constant.

[0017] In some exemplary embodiments, the method further includes: The enzyme population was irradiated with a 532 nm laser, and the formula was used to... Calculate the photodamage coefficient to quantify enzyme stability; Constructing 50-200 mM Na + Gradient, 5-20 mM Cl - Gradient, 50-200 mM K + Gradient and 5-20 mMMg 2+ The gradient ion interference test system, through the formula Ion sensitivity index was calculated to assess the environmental sensitivity of the enzyme.

[0018] In summary, compared with the prior art, the present invention has the following beneficial effects: This invention provides a dynamic evaluation method for the cross-scale heterogeneity of multiple activity characteristics of digital single-molecule enzymes, enabling real-time cross-scale analysis of the multiple activity characteristics of single enzyme molecules. By integrating single-molecule fluorescence tracking and population statistics, it breaks through the resolution limitations of traditional analytical techniques, providing a dynamic and multi-dimensional evaluation tool for enzyme heterogeneity research. It can also reveal the complex trade-offs between rate, uniformity, and stability of single enzyme molecules, promoting the transformation of enzyme analysis from "static averaging" to "dynamic individualization". Attached Figure Description

[0019] Figure 1A flowchart of the method for dynamically evaluating the cross-scale heterogeneity of the multi-activity characteristics of the digital single-molecule enzyme in the embodiments of the present application is shown.

[0020] Figure 2 A basic principle diagram of the method for dynamically evaluating the cross-scale heterogeneity of the multi-activity characteristics of the digital single-molecule enzyme in the embodiments of the present application is shown.

[0021] Figure 3 A principle diagram of the method for performing rolling circle amplification and generating a fluorescence intensity curve in the embodiments of the present application is shown.

[0022] Figure 4 A principle diagram of the method for loading the ternary complex into a microwell array chip in the embodiments of the present application is shown.

[0023] Figure 5 A result diagram of the verification using wild-type phi 29 DNA polymerase in the embodiments of the present application is shown.

[0024] Figure 6 A result diagram of the evaluation of the multi-activity characteristics of mutant phi 29 DNA polymerases M1 / M3 / M5 in the embodiments of the present application is shown.

[0025] Figure 7 A result diagram of the multi-activity characteristics of different phi 29 DNA polymerases in the embodiments of the present application is shown.

[0026] Figure 8 A result diagram of the evaluation of the multi-activity characteristics of different phi 29 DNA polymerases after laser treatment in the embodiments of the present application is shown. DETAILED DESCRIPTION

[0027] The technical solutions in the embodiments of the present application will be described clearly and completely below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments of the present application. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application.

[0028] As shown in Figures 1 to 8 The present application provides a method for dynamically evaluating the cross-scale heterogeneity of the multi-activity characteristics of a digital single-molecule enzyme (dMACE), which comprises: S100, diluting the enzyme-template-primer ternary complex after pre-combination to a single-molecule level to load the reaction chamber in a microwell array chip for rolling circle amplification, and using a molecular beacon to combine with the amplification product to release a fluorescence signal.

[0029] Wherein, when the enzyme-template-primer ternary complex is prepared, the template DNA is connected to the first by the primer to form a circular template as the substrate of the polymerase by T4 ligase, and the rolling circle amplification reaction is started after the circular template is obtained to perform amplification. In the amplification process, deoxyribonucleotide triphosphates (dATP, dTTP, dCTP, dGTP) are used to synthesize new DNA chains. Rolling circle amplification (RCA) is a constant temperature nucleic acid amplification technology based on circular DNA template, and linear DNA products containing hundreds of repeated units can be generated after amplification.

[0030] In one specific example, when the enzyme-template-primer ternary complex is prepared, a ligation reaction system with a total volume of 20 μL is first configured, which contains: T4 DNA ligase buffer, primer DNA, template DNA, nuclease-free water, and T4 DNA ligase. The initial concentration of the primer DNA is 10 μM, and the volume added is 2 μL, so that the final concentration reaches 2 μM; the initial concentration of the template DNA is 10 μM, and 4 μL is added, and the final concentration is 0.5 μM; nuclease-free water is used to make up the reaction system to a total volume of 20 μL; finally, 2 μL of T4 DNA ligase (400 U / μL) is added to start the ligation reaction.

[0031] When preparing, the template DNA, primer DNA and water are mixed in a microcentrifuge tube according to the proportion, and after centrifugation, the centrifuge tube is taken out and placed in a 4°C refrigerator for cooling for 2 minutes, then the complete ligation reaction system is prepared according to the above proportion, shaken and mixed, and centrifuged, then reacted at 16°C overnight, in some embodiments, it can also be selected to react at 25°C for 2 hours or at 65°C for 10 minutes, and the ligation reaction process is completed.

[0032] After the ligation reaction is completed, the digestion step is entered, and a digestion system containing Exo III buffer (buffer), Exo I buffer (buffer), Exo III enzyme (400 U / μL), Exo I enzyme (400 U / μL), and ddH2O (Double Distilled Water, double distilled water) is prepared. After adding the digestion system to the ligation reaction product, it is reacted at 37°C for 45 minutes, then heated at 80°C for 15 minutes to inactivate the enzyme activity, and finally stored at 4°C.

[0033] The sample after the digestion step can be directly used for characterization, or diluted by one time, or purified by a purification kit, and the purified sample needs to be diluted by 20 times with a dilution buffer before the final effect characterization. The re-characterization can be analyzed by using a Thermo NanoDrop series ultraviolet spectrophotometer, a fragment analyzer or an agarose gel electrophoresis method.

[0034] Meanwhile, when the enzyme-template-primer ternary complex is prepared, the circular DNA template and the primer are mixed in a reaction buffer according to a volume ratio of 1: (2-3) for pre-hybridization, and the concentration of dNTPs is optimized to balance the amplification efficiency and background noise; wherein the reaction buffer includes 50 mM Tris-HCl, 10 mM MgCl2, 100 mM KCl and 1 mM dithiothreitol (DTT), Tris-HCl (Tris (Hydroxymethyl) Aminomethane Hydrochloride) is a commonly used biochemical buffer reagent, the PH of the reaction buffer is maintained at 7.5-8.5, and the concentration of dNTPs is optimized to 0.4 mM.

[0035] In some embodiments, Tris-HCl can also be replaced by HEPES (4-hydroxyethyl piperazine ethanesulfonic acid), and the PH is maintained at 7.5-8.5, the purpose of which is to provide the most suitable PH reaction environment for the enzyme; magnesium ions are necessary cofactors for enzyme catalytic activity, which participate in the binding and hydrolysis of dNTP (deoxyribonucleotide triphosphate); and the addition of reducing agents such as DTT can maintain the stability of the enzyme, and protein stabilizers such as bovine serum albumin (BSA) can also be added to reduce the adsorption loss and non-specific inhibition of the enzyme on the pipe wall.

[0036] In one embodiment, the reaction system is optimized by mixing the circular DNA template and the primer in a volume ratio of 1:2.5 in a reaction buffer, wherein the reaction buffer comprises 50 mM Tris-HCl, 10 mM MgCl2, 100 mM KCl, 1 mM DTT, 0.1 mg / mL BSA, and 5 μM molecular beacon; the pH of Tris-HCl is 7.5, MgCl2 is used to activate catalytic activity, KCl is used to maintain ionic strength, DTT is used to prevent enzyme oxidation inactivation, BSA is used to reduce surface adsorption loss, the molecular beacon is designed as a stem-loop structure, the 5 bp stem is modified with a BHQ1 quenching group, the 15 nt loop is completely complementary to the template repeat unit, and the 5' end is labeled with a FAM fluorescent group; the optimal dNTP concentration is determined by testing a gradient of 0.1-1.0 mM, and the optimal value is 0.4 mM, at which the rolling circle amplification efficiency and background noise ratio reach a peak; the molecular beacon is used at a concentration of 50 nM, at which the amplification product binding efficiency is the highest, ensuring that the fluorescence signal triggers specificity.

[0037] It can be understood that, when the ternary complex is configured, the rolling circle amplification has theoretically started due to the presence of the initiator reagent in the reaction system, and the amplification reaction is continuously performed. On this basis, the ternary complex is diluted to a single molecule level, and then loaded into a microwell array chip for continuous amplification, and then the subsequent evaluation and calculation are performed according to the fluorescence signal intensity in the microwell array chip after amplification.

[0038] Specifically, S100 specifically comprises: S101, obtaining a target dilution multiple based on a Poisson distribution formula according to the volume of the reaction chamber, so as to dilute the enzyme-template-primer ternary complex after pre-binding to a predetermined concentration.

[0039] Wherein, the Poisson distribution formula is λ=C*V*N, wherein λ represents the average number of events occurring in a unit of time or space, and the target value of λ is usually set to 0.1-0.2; C represents the sample concentration (Concentration), with a unit of mol / L (moles / liter), which is used to describe the density of entities (such as molecules, particles) that can occur in the system; V represents the sample volume (Volume), with a unit of L (liter), which is used to define the physical space range observed; N represents Avogadro's constant (Avogadro's constant), which is 6.022×10 23 mol −1 , which is a bridge for converting the number of moles into the absolute number of particles.

[0040] The target dilution factor is calculated according to the formula, to avoid too large single-step dilution error, a multi-step dilution method is usually adopted, and the dilution solution is preferably PBS containing BSA or other specific buffer, so as to maintain the stability of the ternary complex and prevent the dissociation or inactivation of the ternary complex. Each step of dilution adopts a high-precision pipette and a low-adsorption suction head, and is fully mixed to ensure uniformity.

[0041] S102, loading the diluted sample into the microwell array chip and distributing it to each reaction chamber by capillary action or centrifugal force, so that at least part of the reaction chambers contain only one ternary complex.

[0042] The microwell array chip preferably adopts a silicon-based microwell array chip prepared by a photolithography process, which contains a large number of reaction chambers in the form of microwells to realize high-density integration of reaction units, and each reaction chamber is physically isolated, and the volume of the reaction chamber can be determined by the pore size and the pore depth; the diluted sample is the ternary complex dilution solution diluted to the target dilution factor, after the diluted sample is loaded into the microwell array chip, it can be distributed to each reaction chamber by capillary action or centrifugal force, in a large number of reaction chambers, most of the reaction chambers are microwells, a part of the reaction chambers contain exactly one ternary complex, and a very small part of the reaction chambers contain two or more ternary complexes, and since each reaction chamber is physically isolated, a single enzyme molecule can be observed independently.

[0043] The size of the microwell array chip is (10-20) x (10-20) mm, and has more than 10,000 reaction chambers, the size of the reaction chamber is (65-90) μm x (50-80) μm x (80-120) μm, the pitch is 80-120 μm, and the microwell array chip is treated by surface activation, hydrophobic coating deposition, and selective hydrophilization to enhance the hydrophilicity inside the reaction chamber and the hydrophobicity outside the reaction chamber.

[0044] In one specific example, the microwell array chip is prepared by a high-precision photolithography process, specifically, a regular array with a hole length of 82 μm, a hole width of 65 μm, a hole depth of 97 μm, and a hole pitch of 100 μm is constructed on a silicon-based substrate to realize high-density integration of more than 10,000 reaction chambers per square centimeter, and the surface of the microwell array chip is treated by oxygen plasma hydrophilization to ensure uniform filling of the reaction chamber with the reaction solution. The enzyme-template-primer ternary complex is diluted to 0.7 molecules per reaction chamber.

[0045] Further, to verify the dilution effect, the proportion of whether it conforms to the Poisson distribution can also be judged by observing the fluorescence signal points through a microscope tube, or the initial concentration can be evaluated and optimized according to the final number of fluorescence signals.

[0046] S103, start catalytic reaction to perform rolling circle amplification, and make the molecular beacon specifically bind to the corresponding matching sequence to release the fluorescence signal during the amplification process.

[0047] During the rolling circle replication process, the molecular beacon in the system specifically and complementarily binds to the corresponding matching sequence of the DNA product. The molecular beacon is a stem-loop structure with a stem length of 5 bp and a loop length of 15 nt. The repetitive sequence targeting region is completely complementary to the repetitive unit of the DNA product to ensure the characteristic of signal triggering. During the binding process, the stem-loop structure of the molecular beacon unfolds, causing the fluorescence group and the quenching group to separate, thereby releasing a high-intensity fluorescence signal. The fluorescence intensity increases over time, and the change slope is proportional to the enzyme activity rate, as shown in Figure 3 Figure 3 The principle diagram of the rolling circle amplification process is shown in

[0048] S200, real-time monitoring and acquiring the fluorescence signal to generate a time-resolved fluorescence intensity curve, specifically: real-time monitoring the fluorescence intensity change of each reaction chamber through a high-frame-rate camera and generating a raw fluorescence image to extract the fluorescence signal, and generating a time-resolved fluorescence intensity curve based on the real-time acquired fluorescence signal intensity. In actual operation, only the fluorescence signal of the positive well can be detected, that is, the reaction chamber containing a single ternary complex, as shown in Figure 4 Figure 4 The principle diagram of the fluorescence signal intensity change in the microwell array chip is shown in

[0049] S300, extracting the slope parameter of the fluorescence intensity curve to calculate the catalytic synthesis rate through conversion for evaluating the enzyme activity.

[0050] Specifically, step S300 specifically includes: S301, after suppressing optical noise by using image enhancement technology, performing feature hole positioning based on a pre-defined well site template to determine the reaction well site in the microwell array chip.

[0051] When obtaining the raw fluorescence image, adaptive threshold processing is first used to enhance the image contrast, and then Canny operator is used for edge detection to extract significant edge features, and then the target region in the image is accurately positioned through contour finding operation, for example, mutual correlation algorithm or machine learning segmentation model can be used for accurate positioning. The target region is the reaction well site, thereby excluding non-specific region interference.

[0052] S302, acquiring the time sequence fluorescence intensity data of each reaction well site, and extracting the slope parameter of the corresponding fluorescence intensity curve.

[0053] ​​After the reaction well sites are located, the fluorescence signal extraction and processing phase is entered, raw fluorescence intensity data is extracted from the located reaction well sites, and the raw fluorescence intensity data is sequentially subjected to normalization processing to eliminate system errors, baseline correction to remove background signal interference, and smoothing filtering using a Butterworth filter to suppress high-frequency noise. After normalization processing, the fluorescence intensity is mapped to the [0, 1] interval to eliminate inter-well optical differences. According to the obtained fluorescence intensity and time relationship, a time-resolved fluorescence intensity curve is generated, and then the slope parameter of the fluorescence intensity curve is extracted, and the instantaneous growth rate of the fluorescence intensity is calculated to capture the dynamic characteristics of the molecular binding / dissociation event.

[0054] S303, a catalytic synthesis rate is calculated by conversion according to a calibration formula μ=k / (α·β), the catalytic synthesis rate is used to evaluate enzyme activity, wherein μ is the catalytic synthesis rate, k is the slope parameter, α is the background fluorescence gain coefficient, and β is the beacon binding efficiency correction factor, the background fluorescence gain coefficient α and the beacon binding efficiency β are calibrated by a standard curve, and the catalytic synthesis rate can be used to evaluate the absolute enzyme activity of the reaction process.

[0055] S400, the catalytic synthesis rate value distribution is fitted using an n-order Gaussian mixture model to obtain a standard deviation for evaluating population uniformity and a coefficient of variation for evaluating stability.

[0056] Specifically, step S400 specifically includes: S401, generating a statistical histogram based on the obtained catalytic synthesis rate, and fitting the distribution characteristics according to a modeling formula using an n-order Gaussian function fitting method, the modeling formula being: wherein n is the Gaussian order, y is the derivative of the fluorescence value, x is the temperature sequence, and the parameters that minimize the equation error are obtained using the fitting function of the scipy library in Python, a1-a n is the number of samples conforming to the Gaussian distribution, σ1-σ n is the standard deviation of the function, used to represent the dispersion degree of the sample distribution; wherein μ1 to μ n are mean parameters of each Gaussian component, comprehensively representing the distribution characteristics of the fluorescence signal, the window size of the adaptive threshold processing can be adaptively adjusted according to the image resolution, the cutoff frequency and order of the Butterworth filter can be optimally set according to the signal characteristics, and the number n of Gaussian components can be determined according to the actual data distribution characteristics by a model selection criterion. This implementation not only guarantees the systematicness and integrity of the processing flow, but also has the advantages of strong adaptability and high configurability, realizing efficient and automated processing and precise quantitative analysis of fluorescence image data.

[0057] S402, evaluating population homogeneity by calculating the standard deviation of catalytic synthesis rate of 1000 reaction wells according to the obtained n-Gaussian mixture model; S403, calculating the coefficient of variation of the expected value sequence of catalytic synthesis rate at 1-minute intervals within 900 minutes according to the obtained n-Gaussian mixture model to evaluate stability.

[0058] Generally, the expectation maximization algorithm can be used to optimize the mixed model to represent the characteristic values of each substate, and the model robustness is verified by residual analysis and Bayesian information criterion during the process, which provides direct evidence at the molecular mechanism level for nanoscale processes such as enzyme kinetics and protein folding.

[0059] Further, in evaluating homogeneity and stability, it also includes: S404, real-time monitoring of enzyme synthesis rate at different concentrations to establish a double-reciprocal curve, and according to the formula to obtain the dynamic Michaelis constant of single molecule enzyme, wherein V0 is the reaction rate, dV max is the maximum reaction rate, [S] is the substrate concentration, and dK m is the dynamic Michaelis constant. Through the above process, dynamic monitoring from a single time point to a long period of time can be covered.

[0060] Further, in some embodiments, the method further comprises: S501, using a 532 nm laser to irradiate the enzyme population, and calculating the light damage coefficient to quantify the stability of the enzyme according to the formula , wherein γ is the light damage coefficient, which is a quantitative index for measuring the sensitivity of enzyme molecules to light damage. The larger the γ value, the faster the enzyme activity decays with the increase of light intensity, indicating that the enzyme has poorer light stability and is more easily damaged by light. Conversely, the smaller the γ value, the better the light stability of the enzyme, and the more resistant to light. μ represents the activity of the enzyme, which will gradually decrease during the experiment with the increase of light time or light intensity. The natural logarithm form lnμ is used in the formula to more clearly reveal the kinetic characteristics of activity decay (which usually conforms to first-order reaction kinetics), that is, the relative change rate of activity. P represents the light pressure, which can be more broadly understood as the dose or intensity of light, which can refer to the light power density (such as W / cm²), light energy dose (such as J / cm²), or irradiation time, and dP represents a small change in light intensity.

[0061] The whole derivative term d (ln μ) / dP represents the rate of change of the natural logarithm of enzyme activity with light pressure, and the value itself is negative because when the light intensity (P) increases, the enzyme activity (μ) is always decreasing, therefore, the negative sign (-) before the formula plays a key role: it converts this negative rate into a positive value, so that the final calculation of the light damage coefficient γ is an intuitive positive number, which is convenient for comparison and data analysis between different enzyme molecules or experimental conditions.

[0062] S502, constructing 50-200 mM Na + gradient, 5-20 mM Cl - gradient, 50-200 mM K + gradient and 5-20 mM Mg 2+ ion interference test system, through the formula The ion sensitivity index is calculated to evaluate the environmental sensitivity of the enzyme, wherein the formula S ion is the environmental sensitivity of the enzyme, Δµ is the change amount of the enzyme catalytic synthesis rate before and after the ion concentration change, µ0 is the initial catalytic synthesis rate of the enzyme, ΔC is the change amount of the ion concentration, and C0 is the initial concentration.

[0063] The test results are output as a three-dimensional activity map (μ, δ, θ) and an environmental sensitivity matrix. The standard deviation θ of the catalytic synthesis rate detected under different conditions increases or decreases, which indicates that the ion concentration fluctuation significantly affects the enzyme reaction uniformity. At the same time, a negative control group is set to ensure data validity. By introducing laser irradiation and different ion concentration gradient treatment, the influence of environmental factors on enzyme activity, efficiency and stability can be systematically judged.

[0064] The present application provides a kind of digital single molecule enzyme multi-activity characteristic cross-scale heterogeneity dynamic evaluation method, realizes the real-time analysis of single enzyme molecule multi-activity characteristic cross-scale, breaks through the resolution limit of traditional analysis technology by integrating single molecule fluorescence tracking and population statistics, provides dynamic, multi-dimensional evaluation tool for enzyme heterogeneity research, and can reveal the complex trade-off relationship of single enzyme molecule in rate, uniformity and stability, promote enzyme analysis from "static average" to "dynamic individualization".

[0065] The present application realizes single molecule resolution by physical isolation of micropore array chip, converts enzyme activity into quantifiable fluorescence track by RCA signal amplification, and analyzes the dynamic correlation of dynamic catalytic rate, uniformity and stability by combining intelligent algorithm. In a typical case, dK m <50 nM and δ 2The high-performance mutant with a theta value of 18% or less in 900 minutes of continuous synthesis meets the stringent requirements of SMRT sequencing on enzyme long-term work. The method breaks through the limitations of traditional population average analysis and provides a molecular level multi-dimensional evaluation benchmark for enzyme rational design, especially suitable for application in SMRT sequencing enzyme mutant screening.

[0066] Taking the analysis of the functional heterogeneity of a single phi29 DNA polymerase as an example, the comparative test of wild type and different mutants shows that DNA polymerases with more appropriate speed and better stability can be screened and evaluated within a certain range, which reveals the hidden stability defects caused by the mutation, and the specific test results are as shown in Figures 5 to 8 Figure 5 The results of using wild-type phi 29 DNA polymerase for verification are shown in the figure, wherein A is a statistical distribution graph of the slope of the negative control group without enzyme changing with time, used for calibrating the data of the experimental group; B is a statistical distribution graph of the slope of the M0 single molecule enzyme effective positive hole changing with time, the expected value µ represents the enzyme activity rate, the standard deviation θ represents the enzyme uniformity, and the coefficient of variation δ reflects the enzyme stability; C is a multi-activity characteristic heat map of M0 single molecule enzyme; and D is a multi-activity characteristic distribution graph of M0 single molecule enzyme. Figure 5 In the method, the performance evaluation of M0 enzyme is more accurate through calibration by the negative control group, and the monitoring of the change with time can better reflect the stability characteristics of the enzyme, Figure 5 In the method, the core purpose of the experiment of the negative control group (µ = 2.90, δ 2 = 0.20, θ = 1.4) shown in A is to quantify and calibrate the accuracy, uniformity and stability of the method through positive and negative calibration and long-term monitoring; Figure 5 In the method, the wild-type M0 (µ = 4.72, δ 2 = 0.61, θ = 25.8) shown in B, the abscissa in the figure represents the slope of the fluorescence intensity changing with time, and the ordinate represents the frequency of occurrence.

[0067] Figure 6 ​Figure results of evaluating the multiple activity characteristics of mutant phi29 DNA polymerase M1 / M3 / M5 in the embodiments of the present application. Among them, A figure, the first row is the statistical distribution of the effective positive hole slope of M1 single molecule enzyme changing with time, the expected value µ represents the activity rate, the standard deviation θ represents the uniformity, the coefficient of variation δ represents the stability, the second row is the statistical distribution result figure of the effective positive hole slope, and the third row is the clustering scatter plot of the effective positive hole slope; B figure, the first row is the statistical distribution figure of the effective positive hole slope of M3 single molecule enzyme changing with time, the second row is the statistical distribution result figure of the effective positive hole slope, and the third row is the clustering scatter plot of the effective positive hole slope; C figure, the first row is the statistical distribution figure of the effective positive hole slope of M5 single molecule enzyme changing with time, the second row is the statistical distribution result figure of the effective positive hole slope, and the third row is the clustering scatter plot of the effective positive hole slope; D figure, the first row is the statistical distribution figure of the effective positive hole slope of commercial enzyme C1 changing with time, the second row is the statistical distribution result figure of the effective positive hole slope, and the third row is the clustering scatter plot of the effective positive hole slope; E figure, the first row is the statistical distribution figure of the effective positive hole slope of commercial enzyme C2 changing with time, the second row is the statistical distribution result figure of the effective positive hole slope, and the third row is the clustering scatter plot of the effective positive hole slope; F figure, the first row is the statistical distribution figure of the effective positive hole slope of commercial enzyme C3 changing with time, the second row is the statistical distribution result figure of the effective positive hole slope, and the third row is the clustering scatter plot of the effective positive hole slope; Figure 6 The dMACE platform was used to simultaneously quantify the catalytic synthesis rate (µ, expected value of slope distribution), uniformity (δ, standard deviation) and stability (θ, coefficient of variation of µ changing with time) of three mutant (M1, M3, M5) and three commercial (C1, C2, C3) phi29 DNA polymerase variants at the single molecule level, and each column shows three sets of analysis results of one enzyme; (a-c) Characterization of mutant polymerases: (a) mutant M1 (µ = 1.86, δ = 0.16, θ = 10.4), (b) mutant M3 (µ = 5.58, δ = 0.47, θ = 27.3), (c) mutant M5 (µ = 0.99, δ = 0.22, θ = 12.5); (d-f) Characterization of commercial enzymes: (d) commercial enzyme C1 (µ = 6.53, δ = 0.79, θ = 21.1), (e) commercial enzyme C2 (µ = 5.07, δ = 0.64, θ = 19.71), (f) commercial enzyme C3 (µ = 0.80, δ = 0.34, θ = 17.5); 2 = 0.16, θ = 10.4), (b) mutant M3 (µ = 5.58, δ 2 = 0.47, θ = 27.3), (c) mutant M5 (µ = 0.99, δ 2 = 0.22, θ = 12.5); (d-f) Characterization of commercial enzymes: (d) commercial enzyme C1 (µ = 6.53, δ 2 = 0.79, θ = 21.1), (e) commercial enzyme C2 (µ = 5.07, δ 2 = 0.64, θ = 19.71), (f) commercial enzyme C3 (µ = 0.80, δ 2 = 0.34, θ = 17.5); For each enzyme (a to f): top row (time-dependent distribution): the statistical distribution of catalytic slopes (k) in all positive microwells is constantly changing throughout the 900-minute experiment, the solid line traces the time trajectory of the mean value (μ n ) in each time interval; middle row (final histogram): the frequency distribution of the final calibrated slope values (k) in all single-molecule reactions, the curve represents a Gaussian fit to the distribution, the center defines μ, and the width (standard deviation) defines δ; bottom row (clustered scatter plot): a scatter plot of each individual slope value obtained, illustrating the dispersion and clustering of single-molecule activities, each point represents the catalytic rate of a single enzyme molecule; These data reveal significant differences in catalytic rates and molecular uniformity among different enzyme variants, for example, mutant M5 exhibits higher uniformity (low δ) and stability (low θ) despite a slower catalytic rate, while commercial preparation C1 has a higher catalytic rate but greater molecular heterogeneity (high δ).

[0068] Figure 7 The results of the multi-activity characteristics of different phi29 DNA polymerases in the embodiments of the present application are shown in the figures, where the expected value µ represents the activity rate, the standard deviation θ represents the uniformity, and the coefficient of variation δ represents the stability. In the figures, the first row of A shows the statistical distribution of the effective positive slopes of C1 and M5 mixed enzymes over time, the second row shows the statistical distribution of the effective positive hole slopes, the third row shows the clustered scatter plot of the effective positive hole slopes, and the fourth row shows the violin plot of the effective positive hole slopes; the first row of B shows the statistical distribution of the effective positive slopes of C2 and C3 mixed enzymes over time, the second row shows the statistical distribution of the effective positive hole slopes, the third row shows the clustered scatter plot of the effective positive hole slopes, and the fourth row shows the violin plot of the effective positive hole slopes; the first row of C shows the statistical distribution of the effective positive slopes of M3 and M5 mixed enzymes over time, the second row shows the statistical distribution of the effective positive hole slopes, the third row shows the clustered scatter plot of the effective positive hole slopes, and the fourth row shows the violin plot of the effective positive hole slopes; the first row of D shows the statistical distribution of the effective positive slopes of M0 and M1 mixed enzymes over time after mixing, the second row shows the statistical distribution of the effective positive hole slopes, the third row shows the clustered scatter plot of the effective positive hole slopes, and the fourth row shows the violin plot of the effective positive hole slopes; Figure 7 In the middle, dMACE platform was used to analyze the multi-activity characteristics of the heterogeneous mixture containing two different phi29 DNA polymerase variants in each sample, and the method successfully identified and quantified the enzyme subpopulations according to their different catalytic characteristics; For each mixture (a-d), four perspectives are analyzed: Top row (slope distribution over time): Evolution of the slope (k) distribution in all active single-enzyme micro-wells over the course of the 900-minute experiment, bimodal distribution indicates the presence of two enzyme populations with different catalytic synthesis rates (μ1and μ2); Second row (final slope histogram): Frequency distribution of the final slope values at the end of the experiment, solid lines represent Gaussian fits to each sub-population, from which the average catalytic rate (μ, center of the peak) and homogeneity (δ, standard deviation of the fit) can be derived; Third row (cluster scatter plot): Scatter plot of each individual slope value, clearly showing the data points clustering into two distinct groups, corresponding to the two enzyme variants in the mixture; Bottom row (violin plot): Illustration of the distribution (shape), statistical quartiles (inner box plot), and median (white dot) of the slope values in the population; this plot highlights the differences in density and distribution of the two sub-populations.

[0069] Figure 8 For the results of evaluating the multi-activity characteristics of different phi 29 DNA polymerases after laser treatment in the embodiments of the application, the expected value µ represents the activity rate, the standard deviation θ represents the homogeneity, and the coefficient of variation δ represents the stability. Among them, A is a schematic diagram of the enzyme activity distribution after laser treatment; B is the first row of the multi-activity characteristic heat map of C1 single molecule enzyme before laser treatment, and the second row is the multi-activity characteristic heat map of C1 single molecule enzyme after laser treatment; C is the first row of the multi-activity characteristic heat map of C2 single molecule enzyme before laser treatment, and the second row is the multi-activity characteristic heat map of C2 single molecule enzyme after laser treatment; D is the first row of the multi-activity characteristic heat map of C3 single molecule enzyme before laser treatment, and the second row is the multi-activity characteristic heat map of C3 single molecule enzyme after laser treatment; E is the first row of the multi-activity characteristic heat map of M0 single molecule enzyme before laser treatment, and the second row is the multi-activity characteristic heat map of M0 single molecule enzyme after laser treatment; F is the first row of the multi-activity characteristic heat map of M1 single molecule enzyme before laser treatment, and the second row is the multi-activity characteristic heat map of M1 single molecule enzyme after laser treatment; G is the first row of the multi-activity characteristic heat map of M3 single molecule enzyme before laser treatment, and the second row is the multi-activity characteristic heat map of M3 single molecule enzyme after laser treatment; H is the first row of the multi-activity characteristic heat map of M5 single molecule enzyme before laser treatment, and the second row is the multi-activity characteristic heat map of M5 single molecule enzyme after laser treatment; Figure 8 Quantifying the impact of laser-induced on phi29 DNA polymerase multi-activity characteristics, enzyme samples from six polymerase variants (commercial: C1, C2, C3; wild type: M0; mutant: M1, M3, M5) were irradiated with 30 mW laser for 30 minutes, and then their functional parameters were evaluated in real time using the dMACE platform for quantitative analysis of the multi-activity spectrum to evaluate the light stability, which is a key indicator in SMRT sequencing applications; (a) The enzyme complex was first subjected to laser treatment, then encapsulated in the dMACE platform and real-time activity monitoring was performed to quantify the changes in catalytic performance; (b-h) Multidimensional analysis of each polymerase variant before and after laser irradiation, top row (heat map): two-dimensional density plot of catalytic slope (k, x-axis) versus observation time (time interval, y-axis), color intensity corresponds to the number of individual enzyme molecules exhibiting a specific slope at a given time, intuitively demonstrating the dynamic activity distribution and its evolution.

[0070] Quantified multi-activity parameters after laser treatment were: (b) C1: μ = 4.37, δ 2 = 1.35, θ = 30.8; (c) C2: μ = 3.79, δ 2 = 0.76, θ = 26.4; (d) C3: μ = 0.78, δ 2 = 0.39, θ = 21.3; (e) M0: μ = 3.43, δ 2 = 1.02, θ = 43.1; (f) M1: μ = 1.44, δ 2 = 0.23, θ = 26.5; (g) M3: μ = 4.21, δ 2 = 0.84, θ = 63.7; (h) M5: μ = 0.82, δ 2 = 0.26, θ = 20.7; These data clearly indicate that laser irradiation leads to specific attenuation of enzyme activity and a clear pattern of functional decay, with mutant M5 exhibiting excellent photostability, having the lowest coefficient of variation (θ = 20.7), indicating minimal loss of activity consistency over time, which is a highly desirable property for long-read sequencing applications.

[0071] The above embodiments only express several embodiments of the present application, and the description is more specific and detailed, but it cannot be understood as limiting the scope of the patent. It should be noted that for ordinary skilled in the art, without departing from the concept of the present application, a number of modifications and improvements can be made, which are equivalent modifications and evolution of the above embodiments according to the essential technology of the present application, and these all belong to the protection scope of the present application.

Claims

1. A method for dynamic evaluation of cross-scale heterogeneity of multi-activity characteristics of digital single-molecule enzymes, characterized in that, include: The pre-bound enzyme-template-primer ternary complex was diluted to the single-molecule level and loaded into the reaction chamber of a microporous array chip for rolling circle amplification. Molecular beacons were used to bind to the amplification products and release fluorescent signals. The fluorescence signal is monitored and acquired in real time to generate a time-resolved fluorescence intensity curve; The slope parameter of the fluorescence intensity curve is extracted to calculate the catalytic synthesis rate through conversion, which is used to evaluate enzyme activity; The distribution of catalytic synthesis rate values ​​was fitted using an n-fold Gaussian mixture model to obtain the standard deviation for assessing population homogeneity and the coefficient of variation for assessing stability.

2. The method for dynamic evaluation of cross-scale heterogeneity of multi-activity characteristics of digital single-molecule enzymes according to claim 1, characterized in that, The microporous array chip has a size of (10-20) × (10-20) mm and has more than 10,000 reaction chambers. The reaction chambers have a size of (65-90) μm × (50-80) μm × (80-120) μm and a spacing of 80-120 μm. The microporous array chip is subjected to surface activation, hydrophobic coating deposition, and selective hydrophilication treatment to enhance the hydrophilicity inside the reaction chambers and the hydrophobicity outside.

3. The method for dynamic evaluation of cross-scale heterogeneity of multi-activity characteristics of digital single-molecule enzymes according to claim 1, characterized in that, The pre-bound enzyme-template-primer ternary complex is diluted to a single-molecule level and loaded into the reaction chamber of a microwell array chip for rolling circle amplification. This process specifically includes: The target dilution factor is obtained based on the Poisson distribution formula according to the volume of the reaction chamber, so as to dilute the pre-bound enzyme-template-primer ternary complex to the predetermined concentration. The diluted sample is loaded into a microporous array chip and distributed to each reaction chamber by capillary action or centrifugal force, so that at least some of the reaction chambers contain only one ternary complex. The catalytic reaction is initiated to perform rolling circle amplification, and during the amplification process, the molecular beacon specifically binds to the corresponding matching sequence to release a fluorescent signal.

4. The method for dynamic evaluation of cross-scale heterogeneity of multi-activity characteristics of digital single-molecule enzymes according to claim 1, characterized in that, When preparing the enzyme-template-primer ternary complex, T4 ligase uses primers to ligate the first and last parts of the template DNA into a circular template, which serves as the substrate for the polymerase.

5. The method for dynamic evaluation of cross-scale heterogeneity of multi-activity characteristics of digital single-molecule enzymes according to claim 1, 2, or 3, characterized in that, When preparing the enzyme-template-primer ternary complex, the circular DNA template and primers are mixed in the reaction buffer at a volume ratio of 1:(2-3) for pre-hybridization, and the concentration of dNTPs is optimized to balance amplification efficiency and background noise.

6. The method for dynamic evaluation of cross-scale heterogeneity of multi-activity characteristics of digital single-molecule enzymes according to claim 5, characterized in that, The reaction buffer comprises 50 mM Tris-HCl, 10 mM MgCl2, 100 mM KCl, and 1 mM dithiothreitol. The pH of the reaction buffer is maintained at 7.5-8.5, and the concentration of the dNTPs is optimized to 0.4 mM.

7. The method for dynamic evaluation of cross-scale heterogeneity of multi-activity characteristics of digital single-molecule enzymes according to claim 1, characterized in that, The real-time monitoring and acquisition of the fluorescence signal to generate a time-resolved fluorescence intensity curve specifically involves: The fluorescence intensity changes of each reaction chamber are monitored in real time by a high frame rate camera to generate raw fluorescence images for extracting fluorescence signals, and time-resolved fluorescence intensity curves are generated based on the real-time acquired fluorescence signal intensity.

8. The method for dynamic evaluation of cross-scale heterogeneity of multi-activity characteristics of digital single-molecule enzymes according to claim 1, characterized in that, The extraction of the slope parameter of the fluorescence intensity curve to calculate the catalytic synthesis rate for evaluating enzyme activity specifically includes: After suppressing optical noise using image enhancement technology, feature hole positioning is performed based on a predefined hole location template to determine the reaction hole locations in the micro-hole array chip; Obtain the time-series fluorescence intensity data for each reaction site and extract the slope parameter of the corresponding fluorescence intensity curve; The catalytic synthesis rate is calculated using the calibration formula μ=k / (α·β), which is used to evaluate enzyme activity. Here, μ is the catalytic synthesis rate, k is the slope parameter, α is the background fluorescence gain coefficient, and β is the beacon binding efficiency correction factor.

9. The method for dynamic evaluation of cross-scale heterogeneity of multi-activity characteristics of digital single-molecule enzymes according to claim 8, characterized in that, The specific steps of fitting the catalytic synthesis rate distribution using an n-fold Gaussian mixture model to obtain the standard deviation for assessing population homogeneity and the coefficient of variation for assessing stability include: A statistical histogram is generated based on the obtained catalytic synthesis rate, and the distribution characteristics are modeled using an n-fold Gaussian function fitting method according to the modeling formula, which is: Where n is the Gaussian multiplicity, y is the derivative of the fluorescence value, and x is the temperature sequence. The fitting function of the scipy library in Python is used to obtain the parameters that minimize the equation error. Population homogeneity was assessed by calculating the standard deviation of the catalytic synthesis rate at 1000 reaction sites based on the obtained n-fold Gaussian mixture model. The stability was assessed by calculating the coefficient of variation of the expected catalytic synthesis rate sequence over 900 minutes at 1-minute intervals based on the obtained n-fold Gaussian mixture model.

10. The method for dynamic evaluation of cross-scale heterogeneity of multi-activity characteristics of digital single-molecule enzymes according to claim 9, characterized in that, To assess homogeneity and stability, the enzyme synthesis rate at different concentrations was monitored in real time to establish a double reciprocal curve, and the results were analyzed according to the formula... The dynamic Michaelis constant of a single enzyme is obtained, where V0 is the reaction rate and dV is the constant. max The maximum reaction rate is given by [S], where [S] is the substrate concentration and dK is the denominator. m This is the dynamic Michaelis constant.

11. The method for dynamic evaluation of cross-scale heterogeneity of multi-activity characteristics of digital single-molecule enzymes according to claim 1, characterized in that, The method further includes: The enzyme population was irradiated with a 532 nm laser, and the formula was used to... Calculate the photodamage coefficient to quantify enzyme stability; Constructing 50-200 mM Na + Gradient, 5-20 mM Cl - Gradient, 50-200 mM K + Gradient and 5-20 mM Mg 2+ The gradient ion interference test system, through the formula Ion sensitivity index was calculated to assess the environmental sensitivity of the enzyme.

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