Fully genetically encoded nad + Probes, dna fragments, expression vectors and cells and uses

Abstract

This invention discloses the fully genetically encoded NAD + This invention relates to the field of bioprobe technology, encompassing probes, DNA fragments, expression vectors, cells, and applications. Specifically, it provides a fully genetically encoded NAD... + The probe, which genetically encodes NAD + The amino acid sequence of the probe is any one of the sequences shown in SEQ ID NO: 1 to SEQ ID NO: 9. This invention provides a total of 9 probes that can be used to detect NAD. + Concentration of NAD based on fluorescence resonance energy transfer + The probe possesses high spatiotemporal resolution, high specificity, resistance to physiological concentration pH interference, and anti-NAD properties. + Analogous interference properties can reflect NAD in living cells and their subcellular organelles. + The actual fluctuations in concentration enrich the methods for detecting NAD. + A concentration-based probe library based on fluorescence resonance energy transfer for NAD + Concentration detection offers more options.

Description

Technical Field

[0001] This invention relates to the field of biological probe technology, and more particularly to the genetically encoded NAD. + Probes, DNA fragments, expression vectors, and cells and their applications. Background Technology

[0002] Oxidized nicotinamide adenine dinucleotide (NAD) + NAD plays a central role in various processes such as energy metabolism, DNA repair, and gene expression regulation, and has therefore been the subject of extensive and in-depth research. + Its classic role is as a cofactor of oxidoreductases in metabolic processes, but recent extensive basic and clinical research has revealed its crucial role in signaling pathways, epigenetic regulation, and energy balance. More importantly, NAD+... + Imbalances in NAD+ levels can lead to metabolic disorders, inflammation, neurodegeneration, and aging. Therefore, NAD+... + Metabolism is becoming a target for clinical intervention. Achieving NAD+ in living cells and subcellular structures. + High-fidelity real-time measurement of dynamic concentration changes is crucial for studying NAD. + It is of great help in regulating metabolic pathways, disease occurrence, and drug action mechanisms.

[0003] Currently, NAD in biological samples + The content can be detected by the following methods: (1) Enzyme-linked colorimetric method: using an enzyme-catalyzed reaction to convert NAD+ into color. + All of it is converted to NADH (reduced NAD), and then the generated NADH is used to reduce a dye molecule to change its color. Based on NAD... + A standard curve was established using the absorbance (or fluorescence value) of the standards and the corresponding generated dyes, and the NAD content in the actual sample was then calculated by reverse inference. + concentration.

[0004] (2) High-performance liquid chromatography-mass spectrometry: In the high-performance liquid chromatography-mass spectrometry method, different components in the sample to be tested are separated by high-performance liquid chromatography, and the absorbance of different components is detected at the same time. During high-performance liquid chromatography, different components are ionized and enter the mass spectrometer. The nucleus-mass ratio is used to identify different components, and the ion intensity is used to quantify different components.

[0005] (3) NAD based on fluorescence intensity changes + Fluorescent probes: designed using protein engineering to sense NAD + A fluorescent protein probe. This method typically uses NAD+ as a probe. + The sensing protein fuses with a cyclically arranged fluorescent protein (typically cpYFP) and utilizes NAD+. +Sensory proteins and NAD + The conformational change following specific binding modulates the fluorescent protein's conformation, further affecting its fluorescence intensity, thus indicating the presence of NAD+ in the system based on the intensity of the fluorescence signal. + concentration.

[0006] (4) Semi-synthetic or total synthetic NAD based on the principle of resonant energy transfer + Bioluminescent protein probes: This method utilizes the principle of fluorescence or bioluminescence resonance energy transfer to detect NAD. + The concentration is determined, and semi-synthetic or fully synthetic protein probes are constructed using recombinant proteins (protein moiety) and synthetic fluorescent ligand molecules (synthetic moiety) or another recombinant protein to achieve NAD. + Detection. These probes consist of luciferase (or fluorescent protein), NAD+, etc. + It is formed by the fusion of a binding protein and a self-labeled protein or fluorescent protein, wherein the self-labeled protein is covalently linked to a synthetic molecule containing a red fluorescent group and an NAD+. + Ligands that bind to proteins. NAD + NAD+ affinity receptor system for binding proteins and ligands + Concentration regulation, therefore, under natural conditions, the NAD in the probe + When the binding protein and ligand do not bind, the probe remains in an open state. The fluorophore and luciferase (or fluorescent protein) are distant, resulting in weak energy resonance transfer. The probe as a whole emits the original emission light of the luciferase (or fluorescent protein). When NAD+ is present in the system... + When the concentration increases, NAD + The binding protein and ligand bring the fluorophore and luciferase (or fluorescent protein) closer together, causing energy resonance transfer and making the probe emit the color of the fluorophore. The luminescence color of this probe varies with the NAD content in the sample. + The concentration of NAD+ changes, therefore, measuring the ratio of the luminescence intensity of luciferase (or fluorescent protein) to that of the fluorophore can quantify the NAD+ content in the system. + concentration.

[0007] NAD + Neither colorimetric kits nor high-performance liquid chromatography-mass spectrometry (HPLC-MS) could measure NAD in live cells. + Concentration. This type of method requires lysing a large number of cells (approximately one million), and this lysis process results in the loss of cellular NAD5. + Temporal and spatial information on concentration; simultaneously, the sample lysis process alters intracellular NAD+. + Concentration can lead to inaccurate results; furthermore, analyzing a single sample takes 10 to 30 minutes and relies on large instruments, severely limiting the analysis of large batches of samples. + One of the rate-limiting steps in metabolic research and drug development.

[0008] Live-cell NAD based on fluorescence intensity changes has been developed + The probe cannot accurately reflect NAD in living cells and subcellular structures due to interference from factors such as analogues, ATP (adenosine triphosphate), ADP (adenosine diphosphate), and pH. + Dynamic changes. Currently available whole-genome synthetic fluorescent probes (such as cpVenus-LigA and FiNAD) achieve NAD based on changes in fluorescence intensity (rather than changes in fluorescence resonance energy transfer efficiency). + Sensing. cpVenus-LigA is interfered with by nucleotides (ATP and ADP), NMN (nicotinamide mononucleotide), and NR (nicotinamide riboside), and under physiological ATP concentrations (above 500 μM), it inhibits NAD. + Its affinity for NAD decreases sharply; NMN and NR can also interfere with its effect on NAD. + The response to NAD was completely lost in the presence of 500 μM NMN or NR. + The response limits its use for assessing NAD. + The application of supplements is necessary; furthermore, pH significantly affects the probe's response, requiring additional pH correction steps during application. FiNAD is another type of fluorescent NAD. + The probe is a modified version of the NADH fluorescent probe. Although FiNAD responds to NAD... + However, it still responds to the original NADH ligand, and when the NADH level is higher than 10 μM, it can cause a significant change in fluorescence ratio, making the probe unable to accurately reflect NAD in mitochondria. + Horizontal fluctuations. In addition, similar to cpVenus-LigA, FINAD is also drastically affected by nucleotides (ATP and ADP) and physiological concentrations such as pH.

[0009] Developed semi-synthetic fluorescent or bioluminescent NADs based on resonance energy transfer + Although the probe is tolerant to physiological pH, it is still affected by ATP and ADP. Furthermore, because the semi-synthetic probe is composed of recombinant proteins and synthetic fluorescent ligand molecules, it is difficult to detect NAD+ in live cells. + During detection, cells can only autonomously express the fusion protein portion of the probe, while the synthesized molecule portion needs to be provided extracellularly. This dependence on extracellular synthesis limits the application of this type of probe in long-term observation of large numbers of live cell samples. In some live cellular processes such as autophagy and endoplasmic reticulum stress, pH or energy states (such as ATP) fluctuate significantly, thus preventing the aforementioned probes from reflecting true NAD. + The level of understanding, or even the opposite conclusion.

[0010] In the inventors' earlier work, they developed NAD based on bioluminescent resonance energy transfer. + Probe (CN116063546A), this series of probes is suitable for high-throughput screening of NAD. + However, the bioluminescent signals (such as luciferase) in these probes are weak, photon scattering is severe, and long exposure times lead to reduced signal-to-noise ratio and spatial resolution, making them unsuitable for fluorescence microscopy. Furthermore, this series of probes is also affected by ATP and ADP, and cannot accurately measure NAD in their presence. + Concentration. And currently, NAD based on fluorescence resonance energy transfer... + The number of probes is still relatively limited, and there is an urgent need to develop NAD based on fluorescence resonance energy transfer. + Probe.

[0011] Therefore, existing technologies still need to be improved and developed. Summary of the Invention

[0012] In view of the shortcomings of the prior art, the purpose of this invention is to provide a fully genetically encoded NAD. + Probes, DNA fragments, expression vectors, and cells and applications are designed to provide tools for detecting NAD. + Concentration of NAD based on fluorescence resonance energy transfer + Probes to enrich the detection of NAD + A concentration-based probe library based on fluorescence resonance energy transfer for NAD + Concentration testing offers more options.

[0013] The technical solution of the present invention is as follows: In a first aspect, the present invention provides a genetically encoded NAD... + The probe, wherein the fully genetically encoded NAD + The amino acid sequence of the probe is any one of the sequences shown in SEQ ID NO: 1 to SEQ ID NO: 9.

[0014] A second aspect of the present invention provides a DNA fragment, wherein the DNA fragment encodes the fully genetically encoded NAD as described above. + Probe.

[0015] A third aspect of the present invention provides an expression vector, wherein the expression vector comprises the DNA fragment of the present invention as described above.

[0016] In a fourth aspect, the present invention provides a cell, wherein the cell comprises the expression vector of the present invention as described above.

[0017] A fifth aspect of the present invention provides a genetically encoded NAD as described above.+ The application of probes, wherein the fully genetically encoded NAD... + Probes are used to prepare NAD + Concentration detection reagent or NAD + Concentration detection kit.

[0018] A sixth aspect of the present invention provides a genetically encoded NAD as described above. + Applications of probes not for disease diagnosis, wherein the fully genetically encoded NAD... + The probe is used to detect NAD in solution or living cells. + concentration.

[0019] A seventh aspect of the present invention provides a genetically encoded NAD as described above. + The application of probes, wherein the fully genetically encoded NAD... + Probes are used to screen NAD + Regulator.

[0020] Optionally, the NAD + Regulators include NAD + agonists or NAD + Inhibitors.

[0021] An eighth aspect of the present invention provides a NAD + The concentration detection method includes the following steps: The present invention, as described above, fully genetically encodes NAD. + Probes with different NAD content + Solutions of different concentrations are mixed to obtain a mixture; The luminescence intensity of the mixed solution was detected at wavelengths of 520 nm and 595 nm. The ratio of the luminescence intensity at the two wavelengths was calculated, and the relationship between the luminescence intensity ratio and the NAD of the solution was used. + A standard curve was obtained by plotting the concentration. Provide the test solution, and the fully genetically encoded NAD + The probe is mixed with the test solution to obtain the test mixture; The luminescence intensity of the test mixture was detected at wavelengths of 520 nm and 595 nm, and the ratio of the luminescence intensity of the test mixture at the two wavelengths was calculated. The NAD content in the test solution is obtained by comparing the ratio of the luminescence intensity of the test mixture at the two wavelengths with the standard curve. + The concentration; or, Provide the test solution, and use the fully genetically encoded NAD as described above in this invention. + The probe is mixed with the test solution to obtain the test mixture; The luminescence intensity of the test mixture was detected at wavelengths of 520 nm and 595 nm. The ratio of the luminescence intensity at the two wavelengths was calculated. Based on the ratio of the luminescence intensity at the two wavelengths, the NAD content in the test mixture was qualitatively detected. + concentration.

[0022] A ninth aspect of the present invention provides intracellular NAD+. + The concentration detection method includes the following steps: The expression vector of the present invention, as described above, was transformed into a vector containing different NAD+ cells using a lentiviral infection method. + In live cells at a concentration of [specific concentration], the luminescence intensity of live cells at wavelengths of 520 nm and 595 nm was measured, and the ratio of luminescence intensity at the two wavelengths was calculated. This ratio was then used in conjunction with the NAD[value] of the live cells. + A standard curve was obtained by plotting the concentration. Provide live cells to be tested, and transfect the expression vector into the live cells to be tested using a lentiviral infection method. Then, detect the luminescence intensity of the live cells to be tested at wavelengths of 520 nm and 595 nm, and calculate the ratio of the luminescence intensity of the live cells to be tested at the two wavelengths. The NAD of the live cells under test was obtained by comparing the ratio of the luminescence intensity of the two wavelengths with the standard curve. + Concentration; or, Provide live cells to be tested, and transform the expression vector of the present invention as described above into the live cells to be tested by lentivirus infection. Then, detect the luminescence intensity of the live cells to be tested at wavelengths of 520 nm and 595 nm, and calculate the ratio of the luminescence intensity of the live cells to be tested at the two wavelengths. The NAD content in the live cells was qualitatively measured based on the ratio of the luminescence intensity of the live cells at the two wavelengths. + concentration.

[0023] Beneficial effects: This invention provides a total of 9 methods for detecting NAD. + Concentration of NAD based on fluorescence resonance energy transfer + Probes have enriched the methods for detecting NAD. + A concentration-based probe library based on fluorescence resonance energy transfer for NAD + Concentration detection offers more options.

[0024] The probe provided by this invention is a fluorescence ratiometric probe based on resonance energy transfer that encodes the entire NAD genome. + Protein probes that utilize the principle of fluorescence resonance energy transfer to detect NAD. + The concentration has achieved self-calibration, no longer relying on a single wavelength, but instead using the intensity ratio of dual wavelengths to detect NAD. +Concentration. This invention achieves full-genome encoding of the probe structure and completes resonance energy transfer NAD independent of synthetic fluorescent ligand molecules. + Concentration detection. The probe provided by this invention has high spatiotemporal resolution, high specificity, resistance to physiological concentration pH interference, and anti-NAD properties. + Analogous substances interfere with the expression of NAD+ in living cells and their subcellular organelles (cytoplasm, nucleus, mitochondria, peroxisomes, and endoplasmic reticulum). + The actual fluctuation of concentration was realized in live cells and subcompartments of NAD. + Fluorescence microscopy and NAD + Concentration detection can be applied to NAD + Metabolic studies can also be used as a tool to target NAD. + Intervention compound screening.

[0025] Furthermore, the probe with the amino acid sequence shown in SEQ ID NO: 9 not only possesses the aforementioned characteristics, but it is also resistant to ATP and ADP interference, making it a probe that combines high spatiotemporal resolution, high specificity, and resistance to physiological concentrations of pH and NAD. + The fully genetically encoded NAD analogues and their ATP and ADP interference properties + Probe. Attached Figure Description

[0026] Figure 1 It is genetically encoded as NAD + A schematic diagram illustrating the working principle of the probe.

[0027] Figure 2 The probe FrNADS in Example 2 is used to target different NADs. + Titration chart of concentration solution.

[0028] Figure 3 Example 2: Detection of NAD by probe FrNADS under different pH conditions + The concentration results are shown in the graph.

[0029] Figure 4 For example, the probe FrNADS detects NAD in Example 2. + A graph showing the results of analogue concentrations.

[0030] Figure 5 For example, in Example 2, the probe FrNADS was used to detect NAD under ATP interference. + The concentration results are shown in the graph.

[0031] Figure 6 In Example 3, the probe FrNADS was used to locate NAD in different subcellular regions for in vitro monitoring. + The graph shows the results of concentration changes, where A represents the cytoplasm, B represents the nucleus, and C represents the mitochondria.

[0032] Figure 7 In Example 4, FrNADS revealed that SLC25A17 transports NAD. + The results for peroxisome analysis are shown in Figure A, where knockdown of SLC25A17 significantly reduces peroxisome NAD. + The results are shown in Figure B, where overexpression of SLC25A17 significantly increases peroxisome NAD50. + The result graph is horizontal. Detailed Implementation

[0033] This invention provides a fully genetically encoded NAD + Probes, DNA fragments, expression vectors, cells, and applications: To make the objectives, technical solutions, and effects of this invention clearer and more explicit, the invention is further described in detail below. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

[0034] 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 in this specification is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

[0035] This invention provides a fully genetically encoded NAD + The probe (which can also be called a fully genetically encoded NAD) + (Biosensor), wherein the fully genetically encoded NAD + The amino acid sequence of the probe is any one of the sequences shown in SEQ ID NO: 1 to SEQ ID NO: 9.

[0036] This invention is based on rational design guided by the three-dimensional structure of proteins. First, it designs conformational receptors influenced by NAD. + The highly regulated protein domain, namely NAD + The response protein is a mutant of the DNA ligase (LigA) (containing an amino acid mutation in the ligand-binding region); then NAD... + The N-terminus and C-terminus of the response protein are fused to a receptor and a donor, respectively, to form a fluorescence resonance energy transfer (FRET) phenomenon. The receptor and donor are a red fluorescent protein (mScarlet-I3, the receptor) and a green fluorescent protein (mStayGold-E138D, the donor), respectively. Finally, the inventors discovered NAD... + Whether the junction between the response protein and the receptor can undergo fluorescence resonance energy transfer to the final probe, and whether NAD can be achieved. + Concentration detection is crucial; therefore, this invention optimizes NAD.+ The linkage mechanism between the response protein and the fluorescence resonance energy transfer (FRET) receptor (by mutating the N-terminal or C-terminal amino acids, i.e., introducing different mutations in the linker region, because probes obtained by directly fusing the three parts cannot detect NAD). + (Concentration) to maximize the dynamic range of the probe and achieve the required functions of the probe.

[0037] The working principle of the probe provided by this invention is as follows: Figure 1 As shown, when NAD + The response protein does not bind to NAD + When the probe structure is in an "open" state, the distance between the fluorescent resonance energy transfer acceptor (mScarlet-I3 in the figure) and the donor (mStayGold-E138D in the figure) is relatively large, resulting in low resonance energy transfer efficiency. The probe as a whole emits light from the fluorescent resonance energy transfer donor. When NAD... + Response protein binds to NAD + After molecule formation, its conformation changes from "open" to "closed," prompting the fluorescent resonance energy transfer (FRET) probe to approach the acceptor, resulting in a high FRET efficiency. The probe then emits light from the FRET acceptor. NAD + Changes in the resonance energy transfer efficiency induced by molecules ultimately manifest as changes in the luminescence intensity of the donor and acceptor in the probe's fluorescence resonance energy transfer. The ratio of these two luminescence intensities can then be used to indicate the NAD content in the system. + The concentration.

[0038] This invention provides a total of 9 methods for detecting NAD. + Concentration of NAD based on fluorescence resonance energy transfer + Probes have enriched the methods for detecting NAD. + A concentration-based probe library based on fluorescence resonance energy transfer for NAD + Concentration detection offers more options.

[0039] The probe provided by this invention is a robust, resonance energy transfer-based, fluorescence ratiometric probe encoding the entire NAD genome. + Protein probes that utilize the principle of fluorescence resonance energy transfer to detect NAD. + The concentration has achieved self-calibration, no longer relying on a single wavelength, but instead using the intensity ratio of dual wavelengths to detect NAD. + Concentration. This invention achieves full-genome encoding of the probe structure and completes resonance energy transfer NAD independent of synthetic fluorescent ligand molecules. + Concentration detection. This invention, through a special design, provides a probe with high spatiotemporal resolution, high specificity, resistance to physiological concentration pH interference, and resistance to NAD. +Analogous substances interfere with the expression of NAD+ in living cells and their subcellular organelles (cytoplasm, nucleus, mitochondria, peroxisomes, and endoplasmic reticulum). + The actual fluctuation of concentration was realized in live cells and subcompartments of NAD. + Fluorescence microscopy and NAD + Concentration detection can be applied to NAD + Metabolic studies can also be used as a tool to target NAD. + Intervention compound screening.

[0040] ATP and ADP molecules are NAD + NAD is a component of molecules (composed of NAM and ADP), therefore NAD + The probe's receptive domain naturally binds to ATP and ADP, meaning that ATP and ADP bind to NAD. + Competitive binding to the probe, thus interfering with the probe response. This invention further improves upon the amino acid sequence shown in SEDID NO: 1, specifically for NAD... + For the response protein component, the R202A mutation (based on the amino acid sequence of natural EfLigA) was first introduced to reduce the affinity of the probe for ADP or ATP. Further introduction of the D91L mutation (based on the amino acid sequence of natural EfLigA) further reduced ATP and ADP interference. Neither the R202A nor the D91L mutation affected the affinity of the probe for NAD+. + The probe exhibits high affinity for ATP and ADP, thus mitigating interference from ATP and ADP. Therefore, the probe with the amino acid sequence shown in SEQ ID NO: 9 not only possesses the aforementioned characteristics but also resists ATP and ADP interference, making it a probe with high spatiotemporal resolution (suitable for fluorescence microscopy), high specificity, and resistance to physiological concentrations of pH and NAD. + The fully genetically encoded NAD analogues and their ATP and ADP interference properties + The probe effectively solved the problem of NAD obtained in the inventor's previous research. + The probe (CN116063546A) suffers from interference from ATP and ADP, has low spatial resolution, and is unsuitable for fluorescence microscopy.

[0041] This invention also provides a DNA fragment, wherein the DNA fragment encodes the fully genetically encoded NAD as described above in this invention. + Probe. This invention provides a novel NAD probe with improved functionality. + The amino acid sequence of the probe and its encoding gene sequence.

[0042] This invention also provides an expression vector, wherein the expression vector comprises the DNA fragment described above in this invention.

[0043] This invention also provides a cell, wherein the cell includes the expression vector described above in this invention.

[0044] This invention also provides a fully genetically encoded NAD as described above in this embodiment. + The application of probes, wherein the fully genetically encoded NAD... + Probes are used to prepare NAD + Concentration detection reagent or NAD + Concentration detection kit.

[0045] This invention also provides a fully genetically encoded NAD as described above in this embodiment. + Applications of probes not for disease diagnosis, wherein the fully genetically encoded NAD... + The probe is used to detect NAD in solution or living cells. + concentration.

[0046] This invention also provides a fully genetically encoded NAD as described above in this embodiment. + The application of probes, wherein the fully genetically encoded NAD... + Probes are used to screen NAD + Regulator.

[0047] In some implementations, the NAD + Regulators include NAD + agonists or NAD + Inhibitors.

[0048] This invention also provides an NAD embodiment. + The concentration detection method includes the following steps: The fully genetically encoded NAD as described in the embodiments of the present invention is as follows. + Probes with different NAD content + Solutions of different concentrations are mixed to obtain a mixture; The luminescence intensity of the mixed solution was detected at wavelengths of 520 nm and 595 nm. The ratio of the luminescence intensity at the two wavelengths was calculated, and the relationship between the luminescence intensity ratio and the NAD of the solution was used. + A standard curve was obtained by plotting the concentration. Provide the test solution, and the fully genetically encoded NAD + The probe is mixed with the test solution to obtain the test mixture; The luminescence intensity of the test mixture was detected at wavelengths of 520 nm and 595 nm, and the ratio of the luminescence intensity of the test mixture at the two wavelengths was calculated. The NAD content in the test solution is obtained by comparing the ratio of the luminescence intensity of the test mixture at the two wavelengths with the standard curve.+ The concentration; or, Provide the test solution, and use the fully genetically encoded NAD as described in the embodiments of the present invention. + The probe is mixed with the test solution to obtain the test mixture; The luminescence intensity of the test mixture was detected at wavelengths of 520 nm and 595 nm. The ratio of the luminescence intensity at the two wavelengths was calculated. Based on the ratio of the luminescence intensity at the two wavelengths, the NAD content in the test mixture was qualitatively detected. + concentration.

[0049] This invention uses a fully genetically encoded NAD. + The probe can achieve NAD + Qualitative and quantitative measurements of NAD concentration can be performed by comparing the ratio of luminescence intensity at wavelengths of 520 nm and 595 nm in different test solutions. + The concentration level, i.e., the realization of NAD + Qualitative measurement of concentration. Alternatively, a standard curve can be plotted first, and then the NAD content of the test solution can be obtained based on the ratio of the emission intensity at 520 nm and 595 nm wavelengths of the standard curve to that of the test solution. + Concentration value, i.e., achieving NAD + Quantitative measurement of concentration.

[0050] This invention also provides an intracellular NAD+. + The concentration detection method includes the following steps: The expression vector described above in the embodiments of the present invention was transformed into a vector containing different NAD values ​​using a lentiviral infection method. + In live cells at a concentration of [specific concentration], the luminescence intensity of live cells at wavelengths of 520 nm and 595 nm was measured, and the ratio of luminescence intensity at the two wavelengths was calculated. This ratio was then used in conjunction with the NAD[value] of the live cells. + A standard curve was obtained by plotting the concentration. Provide live cells to be tested, and transfect the expression vector into the live cells to be tested using a lentiviral infection method. Then, detect the luminescence intensity of the live cells to be tested at wavelengths of 520 nm and 595 nm, and calculate the ratio of the luminescence intensity of the live cells to be tested at the two wavelengths. The NAD of the live cells under test was obtained by comparing the ratio of the luminescence intensity of the two wavelengths with the standard curve. + Concentration; or, Provide live cells to be tested, and transform the expression vector of the present invention as described above into the live cells to be tested by lentivirus infection. Then, detect the luminescence intensity of the live cells to be tested at wavelengths of 520 nm and 595 nm, and calculate the ratio of the luminescence intensity of the live cells to be tested at the two wavelengths. The NAD content in the live cells was qualitatively measured based on the ratio of the luminescence intensity of the live cells at the two wavelengths. + concentration.

[0051] This invention uses a fully genetically encoded NAD. + The probe can achieve NAD + Qualitative and quantitative measurements of NAD concentration can be performed by comparing the ratio of luminescence intensity at 520 nm and 595 nm wavelengths in different cells, allowing for qualitative comparison of NAD concentration in different living cells. + The concentration level, i.e., the realization of NAD + Qualitative measurement of concentration. Alternatively, a standard curve can be plotted first, and then the concentration of NAD+ in live cells can be obtained based on the ratio of the emission intensity of the standard curve to that of live cells at wavelengths of 520 nm and 595 nm. + Concentration value, i.e., achieving NAD + Quantitative measurement of concentration.

[0052] The present invention will be further described below through specific embodiments.

[0053] Example 1 This embodiment provides eight fully genetically encoded NADs as shown in Table 1. + Probes are used to verify the genetically encoded NAD. + The probe can respond to NAD with high specificity + The titration experiment includes the following steps: (1) Preparation of probe solution and NAD + solution Probe solutions: Dilute each purified probe to 2 μM with HEPES buffer (50 mM HEPES, 120 mM NaCl, 2 mM CaCl2, 2 mM MgCl2, pH 7.2) and temporarily store in an ice box for later use. NAD + Solutions: Different concentrations of NAD were prepared using a 3-fold serial dilution method with HEPES buffer (50 mM HEPES, 120 mM NaCl, 2 mM CaCl2, 2 mM MgCl2, pH 7.2). + Solution (NAD + The solutions, with concentrations of 10 mM, 3.3 mM, 1.1 mM, 370 μM, 123 μM, 41.2 μM, 13.7 μM and 4.5 μM, were stored in an icebox.

[0054] (2) Add 90 μL of probe solution and 10 μL of NAD to the black 96-well microplate. +Immediately mix the solution by gently pipetting and aspirating multiple times (at least 8 times) using a multichannel pipette; using a Flex Station3 multi-channel microplate reader in fluorescence mode, with the excitation wavelength set to 480 nm, measure the luminescence intensity at 595 nm and 520 nm, continuously monitoring for 10 min, and calculate the average value of the ratio of luminescence intensity at 595 nm to 520 nm during this time period. The probe is used with different NAD... + The emission spectrum at a given concentration was obtained by monitoring the emission intensity within the wavelength range of 500 to 600 nm.

[0055] Among them, the maximum value of the average ratio of the luminous intensity at 595 nm to 520 nm for probes 1 to 8 (R) max The minimum value of the average ratio of the luminous intensity at 595 nm and 520 nm (R) min The ratio of (R) max / R min The values ​​were 2.3, 1.7, 1.8, 1.6, 1.7, 1.8, 1.5, and 1.2 respectively (Table 1). It can be seen that these probes are effective against NAD+. + It has high responsiveness and can be used to detect NAD. + concentration.

[0056] Table 1. Eight NADs + Probe information and titration results

[0057] Example 2 In this embodiment, probe 1 in Table 1 was further optimized to obtain probe FrNADS (whose amino acid sequence is shown in SEQ ID NO: 9), and the following tests were performed.

[0058] 1. To verify that the probe FrNADS can respond to NAD with high specificity. + The titration experiment includes the following steps: (1) Preparation of probe solution and NAD + solution Probe solution: Dilute the purified probe FrNADS to 2 μM with HEPES buffer (50 mM HEPES, 120 mM NaCl, 2 mM CaCl2, 2 mM MgCl2, pH 7.2) and temporarily store in an ice box for later use. NAD + Solutions: Different concentrations of NAD were prepared using a 3-fold serial dilution method with HEPES buffer (50 mM HEPES, 120 mM NaCl, 2 mM CaCl2, 2 mM MgCl2, pH 7.2). + Solution (NAD+ The solutions were prepared at concentrations of 50 mM, 16.5 mM, 5.5 mM, 1850 μM, 615 μM, 206 μM, 68.5 μM, 22.5 μM, and 0 μM, and were stored in an icebox.

[0059] (2) Add 90 μL of probe solution and 10 μL of NAD to the black 96-well microplate. + The final NAD+ concentrations in the solution were 5 mM, 1.65 mM, 0.55 mM, 185 μM, 61.5 μM, 20.6 μM, 6.85 μM, 2.25 μM, and 0 μM, respectively. The solution was immediately mixed by gently pipetting and aspirating multiple times (at least 8 times) using a multichannel pipette. Using a Flex Station3 multi-mode microplate reader in fluorescence mode, with the excitation wavelength set to 480 nm, the luminescence intensity at 520 nm and 595 nm was measured. This was monitored continuously for 10 min, and the average ratio of the luminescence intensity at 595 nm to 520 nm during this time period was calculated (referred to as the 595 / 520 intensity ratio). The FrNADS probe was used in different NAD+ concentrations... + The emission spectrum at a given concentration was obtained by monitoring the emission intensity within the wavelength range of 500 to 600 nm.

[0060] The results are as follows Figure 2 As shown, it can be seen that in different NAD... + Concentration (NAD) + In 90 μL probe solution and 10 μL NAD + At different final concentrations in the solution mixture, the 595 / 520 light intensity ratio of the probe FrNADS varies, and within a certain NAD... + With NAD concentration range + The increase in concentration with increasing concentration indicates that the probe FrNADS can be used for NAD. + Quantitative determination of concentration, with suitable C 50 Value (the concentration of the analyte that causes a 50% change in probe conformation).

[0061] 2. The effect of pH on the probe FrNADS was tested, including the following steps: The probe FrNADS was diluted to 2 μM using HEPES buffer (50 mM HEPES, 120 mM NaCl, 2 mM CaCl2, 2 mM MgCl2) at different pH values ​​(6.8, 7.0, 7.2, 7.4, 7.6, 7.8, and 8.0, respectively). The remaining steps were the same as the titration experiment described above.

[0062] The results are as follows Figure 3As shown, the FrNADS probe is not affected by the physiological pH range, meaning that the FrNADS probe can be used to detect NAD+. + The concentration is not affected by the physiological pH range.

[0063] (3) Probe FrNADS on NAD + Analog responsiveness testing includes the following steps: The NAD in the above titration experiment + Replace them respectively with NMN (nicotinamide mononucleotide), NaMN (nicotinic acid mononucleotide), NAM (nicotinamide), NR (nicotinamide riboside), NRH (dihydronicotinamide riboside), NADH (reduced NAD), and NADP. + (Nicotinamide adenine dinucleotide phosphate) and NADPH (reduced NADP) + The remaining steps are the same as those in the titration experiment.

[0064] The results are as follows Figure 4 As shown, the probe FrNADS does not respond to the aforementioned NAD. + Analogous, namely, using the probe FrNADS to detect NAD + At concentrations unaffected by the above NAD + Interference from analogues indicates that the probe FrNADS is affected by NAD. + It has a high degree of selectivity.

[0065] (4) ATP interference experiment, including the following steps: The NAD used in the above titration experiment + Add an additional 1 mM ATP to the solution, and the remaining steps are the same as the titration experiment described above.

[0066] The results are as follows Figure 5 As shown, the probe FrNADS is less affected by ATP and can resist ATP interference.

[0067] Example 3 This embodiment utilizes a mammalian cell line that stably expresses the probe FrNADS to measure intracellular NAD in living cells. + The dynamic changes in concentration include the following steps: (1) The coding gene of the probe FrNADS was cloned into the pCDH vector, and a stable HEK293 cell line was prepared using the lentiviral method. The cells were seeded at an appropriate amount in 3.5 cm glass-bottomed cell culture dishes, and the culture medium was DMEM medium (high glucose, phenol red-free) containing 10% FBS (fetal bovine serum). The cells were cultured at 37°C and 5% CO2 for 24 h.

[0068] (2) After culture, FK866 (a NAMPT inhibitor, which can effectively reduce intracellular NAD) was used to further reduce intracellular NAD.+ (Level) and NR (NAD) + Precursor, which can effectively increase intracellular NAD + Stable cell lines were treated with horizontal concentrations of 10 nM and 500 μM.

[0069] After treating cells with compounds (FK866 and NR, respectively) for 24 h, the culture medium was removed, the cells were washed with PBS, and finally, live-cell imaging solution was added. The light intensities of the GFP and RFP channels were acquired using an inverted fluorescence microscope. Based on the detection principle of the probe FrNADS, the ratio of the emission intensity of the RFP and GFP channels indicates the NAD content in live cells. + concentration.

[0070] The results are as follows Figure 6 As shown ( This indicates that p is less than 0.0001. (Indicating p < 0.001), it can be seen that in cells treated with FK866, the ratio of intracellular light emission intensity decreased, indicating increased intracellular NAD+. + The concentration decreased. In NR-treated cells, the ratio of intracytoplasmic luminescence intensity increased, indicating intracytoplasmic NAD+ concentration decreased. + Concentration increases.

[0071] Cells treated with FK866 showed a decrease in the ratio of intranuclear light emission intensity, indicating intranuclear NAD3. + The concentration decreased. In NR-treated cells, the ratio of intranuclear luminescence intensity increased, indicating intranuclear NAD+. + Concentration increases.

[0072] FK866-treated cells showed a decrease in the ratio of intramitochondrial light emission intensity, indicating intramitochondrial NAD+. + The concentration decreased. In NR-treated cells, the ratio of intramitochondrial luminescence intensity increased, indicating increased NAD+ concentration in mitochondria. + Concentration increases.

[0073] The changes described above are consistent with expectations, indicating that the probe FrNADS is able to respond to intracellular NAD+. + The concentration changes can be observed, and NAD+ levels can be achieved in living cells and subcompartments (cytoplasm, nucleus, mitochondria). + Fluorescence microscopy.

[0074] Example 4 The coding gene for the probe FrNADS was cloned into the pCDH vector, and a stable HEPG2 cell line was prepared using lentiviral technology. Cells were seeded at an appropriate amount in 3.5 cm glass-bottomed cell culture dishes in DMEM medium (high glucose, phenol red-free) containing 10% FBS. The cells were cultured at 37°C and 5% CO2 for 24 h.

[0075] Using RNA interference technology, shRNA and expression plasmids (constructed on the pLV3-CMV-Puro vector) of target genes (such as PXMP2, NUDT12, and SLC25A17) were added to cultured HEPG2 cells for transient transfection to knock down and upregulate the expression levels of the target genes. Forty-eight hours after transient transfection, the light intensity of the GFP and RFP channels in the probe-stabilized cells was collected using fluorescence microscopy, and the light intensity ratio was calculated to indicate the intracellular NAD+ levels. + concentration.

[0076] The results are as follows Figure 7 As shown (ns indicates no significant difference), (Indicates p < 0.05), the results show that the probe FrNADS can reveal the NAD transport of SLC25A17. + To peroxisomes, knockdown of SLC25A17 significantly reduced peroxisome NAD. + Levels of SLC25A17 overexpression significantly increased peroxisome NAD50 levels. + The levels demonstrated that the peroxisome transporter SLC25A17 can regulate NAD in peroxisomes. + level.

[0077] As can be seen from the above embodiments, the NAD provided by the present invention + The probe simultaneously achieved NAD based on fluorescence resonance energy transfer. + Quantification and probe whole-genome encoding enable self-calibration of the probe signal, i.e., NAD based on the ratio of dual-wavelength light intensity. + Quantitative analysis was achieved, enabling live-cell NAD50 analysis without relying on synthetic fluorescent ligand molecules. + Monitoring. Of particular importance, NAD... + The probe avoided NAD + Analogs (NADH and NMN) and physiological pH range interference, and tolerance to ATP and ADP.

[0078] It should be understood that the application of the present invention is not limited to the examples above. Those skilled in the art can make improvements or modifications based on the above description, and all such improvements and modifications should fall within the protection scope of the appended claims.

Claims

1. A genetically encoded NAD + The probe is characterized by, The fully genetically encoded NAD + The amino acid sequence of the probe is shown in SEQ ID NO:

9.

2. A DNA fragment, characterized in that, The DNA fragment encodes the fully genetically encoded NAD as described in claim 1. + Probe.

3. An expression carrier, characterized in that, The expression vector comprises the DNA fragment as described in claim 2.

4. A cell, characterized in that, The cell comprises the expression vector of claim 3.

5. A fully genetically encoded NAD as described in claim 1 + The application of probes is characterized by, The fully genetically encoded NAD + Probes are used to prepare NAD + Concentration detection reagent or NAD + Concentration detection kit.

6. A fully genetically encoded NAD as described in claim 1 + The application of probes for purposes other than disease diagnosis is characterized by, The fully genetically encoded NAD + The probe is used to detect NAD in solution or living cells. + concentration.

7. A fully genetically encoded NAD as described in claim 1 + The application of probes is characterized by, The fully genetically encoded NAD + Probes are used to screen NAD + Regulator.

8. The application according to claim 7, characterized in that, The NAD + Regulators include NAD + agonists or NAD + Inhibitors.

9. A non-disease diagnostic NAD + A method for detecting concentration, characterized in that, Includes the following steps: The fully genetically encoded NAD as described in claim 1 + Probes with different NAD content + Solutions of different concentrations are mixed to obtain a mixture; The luminescence intensity of the mixed solution was detected at wavelengths of 520 nm and 595 nm. The ratio of the luminescence intensity at the two wavelengths was calculated, and the relationship between the luminescence intensity ratio and the NAD of the solution was used. + A standard curve was obtained by plotting the concentration. Provide the test solution, and use the fully genetically encoded NAD as described in claim 1. + The probe is mixed with the test solution to obtain the test mixture; The luminescence intensity of the test mixture was detected at wavelengths of 520 nm and 595 nm, and the ratio of the luminescence intensity of the test mixture at the two wavelengths was calculated. The NAD content in the test solution is obtained by comparing the ratio of the luminescence intensity of the test mixture at the two wavelengths with the standard curve. + The concentration; or, Provide the test solution, and use the fully genetically encoded NAD as described in claim 1. + The probe is mixed with the test solution to obtain the test mixture; The luminescence intensity of the test mixture was detected at wavelengths of 520 nm and 595 nm. The ratio of the luminescence intensity at the two wavelengths was calculated. Based on the ratio of the luminescence intensity at the two wavelengths, the NAD content in the test mixture was qualitatively detected. + concentration.

10. A live-cell NAD for non-disease diagnostic purposes + A method for detecting concentration, characterized in that, Includes the following steps: The expression vector described in claim 3 was transferred into cells containing different NAD values ​​via lentivirus infection. + In live cells at a concentration of [specific concentration], the luminescence intensity of live cells at wavelengths of 520 nm and 595 nm was measured, and the ratio of luminescence intensity at the two wavelengths was calculated. This ratio was then used in conjunction with the NAD[value] of the live cells. + A standard curve was obtained by plotting the concentration. Provide live cells to be tested, and transfer the expression vector described in claim 3 into the live cells to be tested by lentivirus infection. Then, detect the luminescence intensity of the live cells to be tested at wavelengths of 520 nm and 595 nm, and calculate the ratio of the luminescence intensity of the live cells to be tested at the two wavelengths. The NAD of the live cells under test was obtained by comparing the ratio of the luminescence intensity of the two wavelengths with the standard curve. + Concentration; or, Provide live cells to be tested, and transfer the expression vector described in claim 3 into the live cells to be tested by lentivirus infection. Then, detect the luminescence intensity of the live cells to be tested at wavelengths of 520 nm and 595 nm, and calculate the ratio of the luminescence intensity of the live cells to be tested at the two wavelengths. The NAD content in the live cells was qualitatively measured based on the ratio of the luminescence intensity of the live cells at the two wavelengths. + concentration.

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