A fully genetically encoded NMN protein probe based on resonance energy transfer and its application

Abstract

The present invention discloses a fully genetically encoded NMN protein probe based on resonance energy transfer and its application, specifically discloses a fully genetically encoded NMN protein probe, which is composed of a resonance energy transfer receptor, an NMN response protein and a resonance energy transfer donor in series; wherein the NMN response protein is a mutant of DNA ligase, and the mutant sequence of the DNA ligase is shown in SEQ ID NO.1, SEQ ID NO.2 or SEQ ID NO.3; the resonance energy transfer donor is selected from luciferase or fluorescent protein; the resonance energy transfer receptor is selected from fluorescent protein and the fluorescent protein used as the resonance energy transfer receptor is different from the fluorescent protein of the resonance energy transfer donor. The fully genetically encoded NMN molecular probe based on resonance energy transfer of the present invention can respond to NMN molecules in vitro and in cells.

Description

Technical Field

[0001] The patent of this invention belongs to the field of biological probes, involving the fields of aging medicine, cell biology, molecular biology, drug development, etc., and specifically relates to a fully genetically encoded NMN protein probe based on resonance energy transfer and its application. Background Art

[0002] NAD + It is an important coenzyme molecule that participates in many important processes such as cell energy metabolism, DNA repair, protein post-transcriptional modification, gene regulation and circadian rhythm. + A consensus in the field of metabolic research is that in some tissues or cells, NAD + The concentration decreases with aging. For example, the model animal mouse shows NAD in skeletal muscle, adipose tissue and hippocampus as aging progresses. + The concentration of NAD + The decrease in concentration affects the activity of SIRT1 and PARPs, changes calcium ion homeostasis, and reduces mitochondrial function. + Lower levels could help prevent or treat age-related diseases.

[0003] NMN is NAD + As a direct precursor of the salvage synthesis pathway, supplementing with NMN is a quick way to increase NAD + Many studies have shown that supplementing NMN can effectively increase intracellular NAD levels in a variety of pathological conditions such as aging-related diseases, obesity-related metabolic disorders, diabetes, and ischemia-reperfusion injury. + Several recent clinical trials of NMN have also demonstrated its benefits in humans: first, taking NMN (tens of milligrams to thousands of milligrams per day) is safe; second, taking NMN can improve the aerobic capacity of amateur runners, and it is dose-dependent; finally, for overweight women with prediabetes, oral NMN can improve the insulin sensitivity of muscle tissue without obvious side effects.

[0004] Therefore, in-depth research and characterization of the metabolic rules of NMN in living cells and the realization of real-time measurement of the dynamic changes of NMN concentration are of great significance for studying NMN or NAD + It is of great help in the regulation of metabolic pathways, disease occurrence and drug action mechanisms, and is expected to provide new solutions for improving aging and metabolic-related diseases.

[0005] Currently, the NMN content in biological samples can be detected by the following methods:

[0006] 1) High performance liquid chromatography-mass spectrometry:

[0007] In the HPLC-MS method, the different components in the sample to be tested are separated by HPLC chromatography, and the absorbance intensity of the different components is detected at the same time. During the HPLC chromatography, the different components enter the mass spectrometer through ionization, and the different components are qualitatively determined by the nucleus-mass ratio, and the different components are quantitatively determined by the ion intensity.

[0008] 2) Fluorescence detection method:

[0009] The alkylpyridine group of NMN is derivatized with acetophenone to enable it to produce fluorescence. Finally, the fluorescence intensity is measured by a fluorescence photometer. A standard curve is drawn based on the standard sample and the corresponding fluorescence intensity, and the NMN concentration in the sample is reversed.

[0010] However, both HPLC-MS and NMN fluorescence detection methods cannot measure the concentration of NMN in living cells. This type of method requires the lysis of a large number of cells (about one million), and this lysis process loses the temporal and spatial information of the cell NMN concentration; at the same time, the sample lysis process will change the intracellular NMN concentration, resulting in inaccurate results; in addition, the derivatization of NMN requires a high temperature of 100°C, which may degrade NMN; finally, the detection of a single sample takes 10 to 30 minutes and relies on large instruments, which severely limits the detection of large batches of samples and is one of the rate-limiting steps in NMN metabolism research and drug development. Summary of the invention

[0011] In view of the deficiencies of the prior art, the present invention realizes a fully gene-encoded NMN protein probe based on resonance energy transfer.

[0012] In terms of probe function, the present invention 1) realizes the full gene encoding of the probe structure, which can be expressed in living cells; 2) realizes the self-calibration of the probe signal, that is, NMN quantification based on the dual-wavelength light intensity ratio.

[0013] In terms of probe structure, the present invention 1) designs a protein domain whose conformation is drastically regulated by NMN, namely, NMN response protein; 2) fuses the N-terminus and C-terminus of the NMN response protein with a receptor and a donor that can form a resonance energy transfer phenomenon. The resonance energy transfer donor and receptor are a luciferase and a fluorescent protein, or two fluorescent proteins of different colors, respectively; 3) optimizes the linking mode between the NMN response protein and the resonance energy transfer donor and receptor to maximize the dynamic range of the probe. Specifically, P312 is mutated to G312 in LigA mutant 3, V1 is mutated to I in cpNLuc mutant 2, and S223 is mutated to L in mScarlet-I mutant 2.

[0014] In terms of probe application, the present invention constructed a cell line that stably expresses the NMN resonance energy transfer protein probe, and achieved the characterization of NMN concentration in living cells and their subcellular structures.

[0015] The specific technical solutions of the present invention are as follows:

[0016] In one aspect, the present invention provides a fully genetically encoded NMN protein probe, which is composed of a resonance energy transfer receptor, an NMN response protein, and a resonance energy transfer donor connected in series;

[0017] The NMN response protein is a mutant of DNA ligase, and the sequence of the mutant of DNA ligase is shown in SEQ ID NO.1, SEQ ID NO.2 or SEQ ID NO.3;

[0018] The resonance energy transfer donor is selected from luciferase or fluorescent protein; the resonance energy transfer acceptor is selected from fluorescent protein, and the fluorescent protein used as the resonance energy transfer acceptor is different from the fluorescent protein of the resonance energy transfer donor.

[0019] Further, the resonance energy transfer donor is selected from a ring-arranged bioluminescent protein cpNLuc or a green fluorescent protein mNeoGreen;

[0020] Preferably, the sequence of the cpNLuc is SEQ ID NO.4 or SEQ ID NO.5; the sequence of the green fluorescent protein mNeoGreen is SEQ ID NO.8.

[0021] Further, the resonance energy transfer acceptor is selected from green fluorescent protein mNeoGreen or red fluorescent protein mScarlet-I;

[0022] Preferably, the sequence of the mScarlet-I is SEQ ID NO.6 or SEQ ID NO.7; the sequence of the green fluorescent protein mNeoGreen is SEQ ID NO.8.

[0023] Further, the NMN protein probe is formed by connecting mScarlet-I, NMN response protein and cpNLuc in series; or

[0024] The NMN protein probe is formed by connecting mScarlet-I, NMN response protein and mNeoGreen in series;

[0025] Preferably, the amino acid sequence of the NMN protein probe is shown as SEQ ID NO.9, SEQ ID NO.10 or SEQ ID NO.11.

[0026] In another aspect, the present invention provides the use of the fully genetically encoded NMN protein probe in the preparation of a reagent for detecting NMN concentration.

[0027] In another aspect, the present invention provides a composition for detecting NMN concentration, wherein the composition comprises the fully genetically encoded NMN protein probe;

[0028] Preferably, the composition further comprises a bioluminescent substrate.

[0029] In another aspect, the present invention provides a nucleotide sequence encoding the fully genetically encoded NMN protein probe.

[0030] In another aspect, the present invention provides a vector, comprising a nucleotide sequence encoding the fully genetically encoded NMN protein probe;

[0031] Preferably, the vector is a lentiviral expression vector.

[0032] Furthermore, the nucleotide coding sequence of the cytoplasmic localization protein, the nucleotide coding sequence of the nuclear localization protein and the nucleotide coding sequence of the mitochondrial localization protein are added to the amino terminus of the nucleotide sequence of the fully genetically encoded NMN protein probe.

[0033] In another aspect, the present invention provides a cell expressing the fully genetically encoded NMN protein probe;

[0034] Preferably, the cell is obtained by transferring the vector into a living cell, and the vector can be translated in the cell to obtain the NMN protein probe.

[0035] In another aspect, the present invention provides the use of the cells in preparing an experimental model for studying NMN;

[0036] Preferably, the experimental model is used to study the cellular transport mechanism of NMN, or to study agonists or inhibitors of NMN synthesis and metabolism, or to study NAD + Agonists or inhibitors of synthetic and metabolic processes.

[0037] In another aspect, the present invention provides a method for detecting NMN concentration, the method comprising the following steps:

[0038] S11) mixing any of the protein probes described above with a reagent to detect the concentration of NMN,

[0039] S12) detecting the luminescence intensity at the maximum luminescence wavelength of the resonance energy transfer donor and the resonance energy transfer acceptor in the probe respectively, and calculating the ratio of the luminescence intensity of the two;

[0040] S13) regressing on the standard curve to obtain the corresponding NMN concentration; or

[0041] S13) detecting the ratio of luminescence intensity at different time points, and obtaining the trend of NMN concentration change at different time points; or

[0042] S13) detecting the change in the ratio of luminescence intensity after adding different active ingredients to obtain the effect of different active ingredients on the change in NMN concentration;

[0043] Preferably, in step S11), when the resonance energy transfer donor in the protein probe is luciferase, a bioluminescent substrate needs to be added before detecting the luminescence intensity;

[0044] Preferably, the method for preparing the standard curve in step S13) is to use NMN with different standard concentrations, and respectively use steps S11-S12 to detect the luminescence intensity ratios corresponding to the different concentrations of NMN, and use the logarithm of the NMN concentration as the horizontal axis and the luminescence intensity ratio as the vertical axis to make a standard curve.

[0045] Furthermore, the method further comprises adjusting the pH of the mixed solution in step S11) to 6.8-7.2.

[0046] In another aspect, the present invention provides a method for detecting the concentration of NMN in living cells, the method comprising the following steps:

[0047] S21) transferring the vector into the cells to be tested by lentiviral infection, and using the fluorescent signal of the fluorescent protein as a marker to screen and obtain a stable cell line to be tested;

[0048] S22) detecting the luminescence intensity of the resonance energy transfer donor and the resonance energy transfer acceptor in the NMN protein probe encoded by the nucleotide in the vector at the maximum luminescence wavelength, respectively, and calculating the ratio of the luminescence intensity of the two;

[0049] S23) regressing the standard curve to obtain the corresponding intracellular NMN concentration; or

[0050] S23) detecting the ratio of luminescence intensity at different time points, and obtaining the trend of changes in intracellular NMN concentration at different time points; or

[0051] S23) Detect the change in the ratio of luminescence intensity after adding different active ingredients to obtain the effect of different active ingredients on the change in intracellular NMN concentration.

[0052] Furthermore, the method is used to detect the concentration of NMN in the cytoplasm, nucleus or mitochondria in living cells.

[0053] The beneficial effects of the present invention are:

[0054] The present invention innovatively realizes a full-gene encoding NMN protein probe based on resonance energy transfer. In the background technology, both the high-performance liquid chromatography-mass spectrometry method and the NMN fluorescence detection method cannot measure the NMN concentration in living cells. This type of method requires the lysis of a large number of cells (about one million), and this lysis process loses the temporal and spatial information of the cell NMN concentration; at the same time, the sample lysis process will change the intracellular NMN concentration, resulting in inaccurate results; in addition, a high temperature of 100°C is required for the derivatization of NMN, which may degrade NMN; finally, it takes 10 to 30 minutes to detect a single sample, and it relies on large instruments, which severely limits the detection of large batches of samples, and is one of the rate-limiting steps in NMN metabolism research and drug development. The NMN protein probe provided by the present invention not only realizes the NMN quantification based on resonance energy transfer and the full-gene encoding of the probe, that is, it realizes the self-calibration of the probe signal, that is, the NMN quantification based on the dual-wavelength light intensity ratio; it also realizes the monitoring of NMN in living cells that does not rely on exogenous molecules. The full-gene-encoded NMN molecular probe based on resonance energy transfer of the present invention can respond to NMN molecules in vitro and in cells, and has the advantages of high specificity, a suitable C50 value (the concentration of the analyte that causes 50% probe conformational change) and a high dynamic range. BRIEF DESCRIPTION OF THE DRAWINGS

[0055] Figure 1 This is a schematic diagram of the principle of the present invention based on the resonance energy transfer NMN molecular probe to detect the concentration of NMN molecules. When the probe is not bound to the NMN molecule, the NMN sensing protein is in an "open" state, and when it is bound to NMN, the NMN sensing protein conformation changes to a "closed" state. The resonance energy transfer efficiency in the "open" and "closed" states is different, which can indicate the NMN level.

[0056] Figure 2 A is the purified NMN molecular probe NMNS 1.0 and NMNS 1.1 Titration curve in response to NMN molecules. The horizontal axis is the NMN concentration, and the vertical axis is the corresponding ratio of light intensity at 590nm and 440nm. Figure 2 B is the purified molecular probe NMNS 1.1 For the titration curve of NMN analogs, the horizontal axis is the NMN concentration, and the vertical axis is the corresponding light intensity ratio at 590nm and 440nm. Figure 2 C is the molecular probe NMNS 1.1 The titration curve in response to AXP, with the horizontal axis representing the NMN concentration and the vertical axis representing the corresponding light intensity ratio at 590 nm and 440 nm. Figure 2 D is molecular probe NMNS 1.1The titration curve of NMN response under physiological pH range conditions, the horizontal axis is the NMN concentration, and the vertical axis is the corresponding light intensity ratio at 590nm and 440nm. Figure 2 E is the molecular probe NMNS 1.1 Bioluminescence spectrum in response to NMN, with the horizontal axis being the wavelength (nm) and the vertical axis being the normalized bioluminescence intensity.

[0057] Figure 3 A is the purified NMN molecular probe NMNS 2.0 Titration curve in response to NMN molecules. The horizontal axis is the NMN concentration, and the vertical axis is the corresponding ratio of light intensity at 590nm and 515nm. Figure 3 B is the purified molecular probe NMNS 2.0 For the titration curve of NMN analogs, the horizontal axis is the NMN concentration, and the vertical axis is the corresponding light intensity ratio at 590nm and 515nm. Figure 3 C is the molecular probe NMNS 2.0 The titration curve in response to AXP, with the horizontal axis representing the NMN concentration and the vertical axis representing the corresponding light intensity ratio at 590 nm and 515 nm. Figure 3 D is molecular probe NMNS 2.0 The titration curve of NMN response under physiological pH range conditions, the horizontal axis is the NMN concentration, and the vertical axis is the corresponding light intensity ratio at 590nm and 515nm. Figure 3 E is the molecular probe NMNS 2.0 Bioluminescence spectrum in response to NMN, with the horizontal axis being the wavelength (nm) and the vertical axis being the normalized bioluminescence intensity.

[0058] Figure 4 A is a fluorescence micrograph of stably transfected cells with the nuclear localization probe NMNS1.1. From left to right, they are the DAPI channel, the 561nm channel, and the overlap of the DAPI channel and the 561nm channel. The better the overlap between the 561nm channel and the DAPI channel, the more rigorous the localization of the probe NMNS1.1 in the cell nucleus. Figure 4 B is a fluorescence micrograph of cells stably transfected with the mitochondrial localization probe NMNS1.1. From left to right, they are the Mitotracker dye channel, the 561nm channel, and the overlay of the Mitotracker dye channel and the 561nm channel. The better the overlay of the 561nm channel and the Mitotracker dye channel, the more rigorous the localization of the probe NMNS1.1 in the mitochondria.

[0059] Figure 5A is the cytoplasmic NMN level after HEK 293T cells were treated with 500 μM NAD+ precursor molecules NAM, NMN, NR, NRH and 10 nM NAMPT inhibitor FK866 for 6 hours. Figure 5 B is the mitochondrial NMN level after HEK 293T cells were treated with 500 μM NAD+ precursor molecules NAM, NMN, NR, NRH and 10 nM NAMPT inhibitor FK866 for 6 hours. Figure 5 C is the nuclear NMN level of HEK 293T cells treated with 500 μM NAD+ precursor molecules NAM, NMN, NR, NRH and 10 nM NAMPT inhibitor FK866 for 6 h. All P values ​​were calculated using one-way ANOVA.

[0060] Figure 6 A shows the changes in intracellular NMN levels within 10 min after PBS treatment or addition of 100 μM NMN to HEK 293T (Ctrl Vector) or HEK293T cells overexpressing Slc12a8 protein (Slc12a8-OE). Figure 6 B is the statistical data of the changes in intracellular NMN levels in HEK 293T cells expressing Ctrl Vector or overexpressing Slc12a8 protein (Slc12a8-OE) after treatment with PBS or addition of 100 μM NMN. DETAILED DESCRIPTION

[0061] In order to understand the present invention more clearly, the present invention is further described with reference to the following examples and accompanying drawings. The examples are only used for explanation and are not intended to limit the present invention in any way. In the examples, each raw material reagent is commercially available, and the experimental methods without specifying specific conditions are conventional methods and conventional conditions well known in the art, or according to the conditions recommended by the instrument manufacturer.

[0062] Example 1

[0063] The fully genetically encoded NMN protein probe based on resonance energy transfer provided by the present invention is composed of three parts: an NMN response protein whose conformation is drastically regulated by NMN, a donor and an acceptor that can form a resonance energy transfer phenomenon, which is composed of a resonance energy transfer acceptor, an NMN response protein and a resonance energy transfer donor in series. Wherein the NMN response protein is a mutant of a DNA ligase (LigA), and the LigA mutant can be selected from LigA mutant 1, LigA mutant 2 or LigA mutant 3. The resonance energy transfer donor and the acceptor are a luciferase and a fluorescent protein, or two fluorescent proteins of different colors, such as a resonance energy transfer donor can be a circularly permuted NanoLuciferase (cpNLuc) or a green fluorescent protein mNeoGreen, and the resonance energy transfer acceptor is a red fluorescent protein mScarlet-I, cpNLuc can be selected from cpNLuc mutant 1 or cpNLuc mutant 2, and mScarlet-I can be selected from mScarlet-I mutant 1 or mScarlet-I mutant 2.

[0064] The principle of the resonance energy transfer-based fully genetically encoded NMN protein probe for detecting NMN molecule concentration is as follows Figure 1 When the NMN response protein does not bind to the NMN molecule, the probe structure is in an "open" state, resulting in a long distance between the resonance energy transfer donor and the receptor, a low resonance energy transfer efficiency, and the probe as a whole emits the light of the resonance energy transfer donor. When the NMN response protein binds to the NMN molecule, its conformation changes from an "open" state to a "closed" state, causing the resonance energy transfer donor and the receptor to approach, forming a higher resonance energy transfer efficiency, and the probe emits the light of the resonance energy transfer receptor. The change in resonance energy transfer efficiency caused by the NMN molecule is ultimately manifested as a change in the intensity of the emission wavelength of the resonance energy transfer donor and the receptor in the probe. The ratio of the emission light intensity can then indicate the concentration of NMN in the system.

[0065] The sequence of LigA mutant 1 is shown in SEQ ID NO.1:

[0066] LTLTAATTRAQELRKQLNQYSHEYYVKDQPSVEDYVYDRLYKELVDIETEFPDLITPDSPTQNVGGKVLSGFEKAPHDIPMYSLNKGFSKEDIFAFDERVRKAIGKPVAYCCELLIDGLAISLRYENGVFVRGATRGDGTVGENITENLRTVRSVPMDLTEPISVEVRGECYMPKQSFVALNEEREENGQDIFANPRNAAAGSLRQLDTKIVAKRNLNTFLATVADFGPMKAKTQFEALEELSAIGFRTNPERQLCQSIDEVWAYIEEYHEKRSTLPYEINGIVIKVNEFALQDELGFTVKAPRWAIAYKFP

[0067] The sequence of LigA mutant 2 is shown in SEQ ID NO.2:

[0068] LTLTAATTRAQELRKQLNQYDHEYYVKDQPSVEDYVYDRLYKELVDIETEFPDLITPDSPTQNVGGKVLSGFEKAPHDIPMYSMNRGFSKEDIFAFDERVRKAIGKPVAYCCELLIDGLDISLRYENGVFVRGATRGDGTVGENITENLRTVRSVPMDLTEPISVEVRGECYMPKQSFVALNEEREENGQDIFANPRNAAAGSLRQLDTKIVAKRNLNTFLKQVADFGPMKAKTQFEALEELSAIGFRTNPERQLCQSIDEVWAYIEEYHEKRSTLPYEINGIVIKVNEFALQDELGFTVKAPRWAIAYKFP

[0069] The sequence of LigA mutant 3 is shown in SEQ ID NO.3:

[0070] LTLTAATTRAQELRKQLNQYDHEYYVKDQPSVEDYVYDRLYKELVDIETEFPDLITPDSPTQNVGGKVLSGFEKAPHDIPMYSMNRGFSKEDIFAFDERVRKAIGKPVAYCCELLIDGLDISLRYENGVFVRGATRGDGTVGENITENLRTVRSVPMDLTEPISVEVRGECYMPKQSFVALNEEREENGQDIFANPRNAAAGSLRQLDTKIVAKRNLNTFLKQVADFGPMKAKTQFEALEELSAIGFRTNPERQLCQSIDEVWAYIEEYHEKRSTLPYEINGIVIKVNEFALQDELGFTVKAPRWAIAYKFG

[0071] The sequence of cpNLuc mutant 1 is shown in SEQ ID NO.4:

[0072] VDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILAGGTGGSGGTGGSMVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYE

[0073] The sequence of cpNLuc mutant 2 is shown in SEQ ID NO.5:

[0074] IDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILAGGTGGSGGTGGSMVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYE

[0075] The sequence of mScarlet-I mutant 1 is shown in SEQ ID NO.6:

[0076] MVSKGEAVIKEFMRFKVHMEGSMNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFSWDILSPQFMYGSRAFIKHPADIPDYYKQSFPEGFKWERVMNFEDGGAVTVTQDTSLEDGTLIYKVKLRGTNFPPDGPVMQKKTMGWEASTERLYPEDGVLKGDIKMALRLKDGGRYLADFKTTYKAKKPVQMPGAYNVDRKLDITSHNEDYTVVEQYERSEGRHST

[0077] The sequence of mScarlet-I mutant 2 is shown in SEQ ID NO.7:

[0078] MVSKGEAVIKEFMRFKVHMEGSMNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFSWDILSPQFMYGSRAFIKHPADIPDYYKQSFPEGFKWERVMNFEDGGAVTVTQDTSLEDGTLIYKVKLRGTNFPPDGPVMQKKTMGWEASTERLYPEDGVLKGDIKMALRLKDGGRYLADFKTTYKAKKPVQMPGAYNVDRKLDITSHNEDYTVVEQYERSEGRHLT

[0079] The sequence of mNeoGreen is shown in SEQ ID NO.8:

[0080] LAATHELHIFGSINGVDFDMVGQGTGNPNDGYEELNLKSTKGDLQFSPWILVPHIGYGFHQYLPYPDGMSPFQAAMVDGSGYQVHRTMQFEDGASLTVNYRYTYEGSHIKGEAQVKGTGFPADGPVMTNSLTAADWCRSKKTYPNDKTIISTFKWSYTTGNGKRYRSTARTTYTFAKPMAANYLKNQPMYVFRKTELKHSKTELNFKEWQKAFTDVMGMDELYK

[0081] This example prepares NMNS 1.0 probe, NMNS 1.1 probe and NMNS 2.0 probe, and the probe sequence is as follows:

[0082] NMNS 1.0The probe, mScarlet I mutant 1-LigA mutant 1-cpNluc mutant 1, has an amino acid sequence as shown in SEQ ID NO.9:

[0083] MVSKGEAVIKEFMRFKVHMEGSMNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFSWDILSPQFMYGSRAFIKHPADIPDYYKQSFPEGFKWERVMNFEDGGAVTVTQDTSLEDGTLIYKVKLRGTNFPPDGPVMQKKTMGWEASTERLYPEDGVLKGDIKMALRLKDGGRYLADFK TTYKAKKPVQMPGAYNVDRKLDITSHNEDYTVVEQYERSEGRHSTLTLTAATTRAQELRKQLNQYSHEYYVKDQPSVEDYVYDRLYKELVDIETEFPDLITPDSPTQNVGGKVLSGFEKAPHDIPMYSLNKGFSKEDIFAFDERVRKAIGKPVAYCCELLIDGLAISLRYENGVFVRGA TRGDGTVGENITENLRTVRSVPMDLTEPISVEVRGECYMPKQSFVALNEEREENGQDIFANPRNAAAGSLRQLDTKIVAKRNLNTFLATVADFGPMKAKTQFEALEELSAIGFRTNPERQLCQSIDEVWAYIEEYHEKRSTLPYEINGIVIKVNEFALQDELGFTVKAPRWAIAYKFPV DQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILAGGTGGSGGTGGSMVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYE

[0084] NMNS 1.1 The probe, mScarlet I mutant 1-LigA mutant 2-cpNluc mutant 2, has an amino acid sequence as shown in SEQ ID NO.10:

[0085] MVSKGEAVIKEFMRFKVHMEGSMNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFSWDILSPQFMYGSRAFIKHPADIPDYYKQSFPEGFKWERVMNFEDGGAVTVTQDTSLEDGTLIYKVKLRGTNFPPDGPVMQKKTMGWEASTERLYPEDGVLKGDIKMALRLKDGGRYLADFKTTYKAKKPVQMPGAYNVDRKLDITSHNEDYTVVEQYERSEGRHSTLTLTAATTRAQELRKQLNQYDHEYYVKDQPSVEDYVYDRLYKELVDIETEFPDLITPDSPTQNVGGKVLSGFEKAPHDIPMYSMNRGFSKEDIFAFDERVRKAIGKPVAYCCELLIDGLDISLRYENGVFVRGATRGDGTVGENITENLRTVRSVPMDLTEPISVEVRGECYMPKQSFVALNEEREENGQDIFANPRNAAAGSLRQLDTKIVAKRNLNTFLKQVADFGPMKAKTQFEALEELSAIGFRTNPERQLCQSIDEVWAYIEEYHEKRSTLPYEINGIVIKVNEFALQDELGFTVKAPRWAIAYKFPIDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILAGGTGGSGGTGGSMVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYE

[0086] NMNS 2.0 Probe, mScarlet I Mutant 2 - LigA Mutant 3 - mNeoGreen, the amino acid sequence of the probe is shown in SEQ ID NO.11:

[0087] MVSKGEAVIKEFMRFKVHMEGSMNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFSWDILSPQFMYGSRAFIKHPADIPDYYKQSFPEGFKWERVMNFEDGGAVTVTQDTSLEDGTLIYKVKLRGTNFPPPDGPVMQKKTMGWEASTERLYPEDGVLKGDIKMALRLKDGGRYLADFKTTYKAKKPVQM PGAYNVDRKLDITSHNEDYTVVEQYERSEGRHLTLTLTAATTRAQELRKQLNQYDHEYYVKDQPSVEDYVYDRLYKELVDIETEFPDLITPDSPTQNVGGKVLSGFEKAPHDIPMYSMNRGFSKEDIFAFDERVRKAIGKPVAYCCELLIDGLDISLRYENGVFVRGATRGDGTVGENITENLRTVRSVP MDLTEPISVEVRGECYMPKQSFVALNEEREENGQDIFANPRNAAAGSLRQLDTKIVAKRNLNTFLKQVADFGPMKAKTQFEALEELSAIGFRTNPERQLCQSIDEVWAYIEEYHEKRSTLPYEINGIVIKVNEFALQDELGFTVKAPRWAIAYKFGLAATHELHIFGSINGVDFDMVGQGTGNPNDGYEE LNLKSTKGDLQFSPWILVPHIGYGFHQYLPYPDGMSPFQAAMVDGSGYQVHRTMQFEDGASLTVNYRYTYEGSHIKGEAQVKGTGFPADGPVMTNSLTAADWCRSKKTYPNDKTIISTFKWSYTTGNGKRYRSTARTTYTFAKPMAANYLKNQPMYVFRKTELKHSKTELNFKEWQKAFTDVMGMDELYK

[0088] In this example, by introducing mutations into the LigA mutant, probes NMNS with different affinities can be designed. 1.0 and NMNS 1.1 , and their C50 values ​​were 1.34μM and 7.56μM ( Figure 2 A), can be used for NMN detection scenarios in different concentration ranges. The probe has high selectivity for NMN molecules ( Figure 2 B, C). The probe did not respond to NAM and NADPH. Although the probe responded to NADH and NRH, the C50 value was higher (>1mM).+ The response dynamic range of NMN molecules is small. The titration curves under different pH conditions show that the probe is stable under the pH conditions in the physiological range ( Figure 2 D).

[0089] Preparation of NMNS 2.0 The probe replaced cpNLuc with the fluorescent protein mNeoGreen, and then developed an NMN molecular probe based on fluorescence resonance energy transfer, with a signal response change of 1.2 times and a C50 value of 30.2μM ( Figure 3 ).

[0090] The following is a detailed description of NMNS 1.0 Probe, NMNS 1.1 Probes and NMNs 2.0 The probe is described in detail.

[0091] Example 2: Determination of NMN content in samples using a fully genetically encoded NMN molecular probe based on bioluminescence resonance energy transfer (BRET)

[0092] Verification of the NMN molecular probe NMNS provided by the present invention 1.0 and NMNS 1.1 It can be used to determine the NMN content in the sample by performing a titration experiment as shown in the following steps:

[0093] (1) Prepare the following samples

[0094] Probe solution: Dilute the purified NMN molecular probe to 2 nM with HEPES buffer (50 mM NaCl, 50 mM HEPES, pH 7.2) and temporarily place in an ice box for later use;

[0095] NMN solution: NMN solutions of different concentrations (3 mM, 1 mM, 0.33 mM, 0.11 mM, 37.0 μM, 12.3 μM, 4.12 μM and 1.37 μM) or (500 μM, 167 μM, 55.6 μM, 18.5 μM, 6.1 μM, 2.1 μM, 0.68 μM and 0.23 μM) were prepared by 3-fold serial gradient dilution method. The buffer used was HEPES buffer, which was stored in an ice box.

[0096] NMN analogs NMNH and NaMN were prepared at concentrations of 3mM, 1mM, 0.33mM, 0.11mM, 37.0μM, 12.3μM, 4.12μM and 1.37μM; NMN analogs NAM, NR, NRH, NAD + , NADH, NADP +The concentrations of NADPH prepared were 50mM, 16.7mM, 5.56mM, 1.85mM, 617μM, 206μM, 68.6μM and 22.9μM; the concentrations of AMP, ADP and ATP prepared were 50mM, 16.7mM, 5.56mM, 1.85mM, 617μM, 206μM, 68.6μM and 22.9μM.

[0097] Bioluminescent substrate solution: Dilute the Furimazine (purchased from Promega) solution 100-fold with water and store in an ice box away from light.

[0098] (2) Add 80 μL of probe solution, 10 μL of NMN solution, and 10 μL of bioluminescent substrate solution to a white 96-well ELISA plate, and immediately mix by gently pipetting several times (at least 8 times) with a multichannel pipette; read the luminescence mode at wavelengths of 440 nm and 590 nm (NMNS) using a Flex Station3 multifunctional ELISA reader. 1.0 、NMNS 1.1 ) and continuously monitored for 5 minutes, and calculated the average acceptor-to-donor luminescence ratio during this period. The luminescence spectrum of the probe at different NMN concentrations was obtained by monitoring the light intensity in the wavelength range of 360-650nm.

[0099] Depend on Figure 2 As shown in A, at different NMN concentrations, the probe NMNS 1.0 、NMNS 1.1 The 590 / 440 light intensity ratio is different and within a certain NMN concentration range (NMNS 1.0 :23nM-50μM;NMNS 1.1 :137nM-300μM) increases with the increase of concentration. 1.0 、NMNS 1.1 Can be used to determine the content of NMN. 1.0 C 50 The value of 1.34 μM is suitable for high-sensitivity NMN level detection in vitro, while NMN 1.1 C 50 The value of 7.56 μM is suitable for physiological NMN level detection.

[0100] Depend on Figure 2 B shows that the probe NMN 1.1 The response to NMN analogs was low, and under physiological conditions, these compounds (NMNH, NaMN, NAM, NAM, NR, NRH, NAD + , NADH, NADP + and NADPH) will not affect the probe's response to NMN. Figure 2 C shows that ATP has an effect on the probe at physiological concentrations, but the intracellular ATP content generally maintains a steady state, so the NMN probe can still respond to NMN at a stable ATP level. Figure 2 D shows that pH probe NMNS 1.1 The response to NMN is affected, so it is necessary to correct for pH levels when measuring intracellular NMN levels. Figure 2 E is the probe NMNS 1.1 The luminescence intensity scanning spectrum clearly shows that NMNS 1.1 There are significant luminescence peaks at 590nm and 440nm.

[0101] Example 3: Determination of NMN content in samples using a fully genetically encoded NMN molecular probe based on fluorescence resonance energy transfer (FRET)

[0102] Verification of the NMN molecular probe NMNS provided by the present invention 2.0 It can be used to determine the NMN content in the sample by performing a titration experiment as shown in the following steps:

[0103] (1) Prepare the following samples

[0104] Probe solution: Dilute the purified NMN molecular probe to 1 μM with HEPES buffer (50 mM NaCl, 50 mM HEPES, pH 7.2) and temporarily place in an ice box for later use;

[0105] NMN solution: NMN solutions of different concentrations (10 mM, 3.33 mM, 1.11 mM, 0.37 mM, 123 μM, 41.2 μM, 13.7 μM and 4.57 μM) were prepared by 3-fold serial gradient dilution method. The buffer used was HEPES buffer and stored in an ice box.

[0106] NMN analogs NMNH and NaMN were prepared at concentrations of 10mM, 3.33mM, 1.11mM, 0.37mM, 123μM, 41.2μM, 13.7μM and 4.57μM; NMN analogs NAM, NR, NRH, NAD + , NADH, NADP + The concentrations of NADPH prepared were 50mM, 16.7mM, 5.56mM, 1.85mM, 617μM, 206μM, 68.6μM and 22.9μM; the concentrations of AMP, ADP and ATP prepared were 50mM, 16.7mM, 5.56mM, 1.85mM, 617μM, 206μM, 68.6μM and 22.9μM.

[0107] (2) Add 90 μL of probe solution and 10 μL of NMN solution to a black 96-well ELISA plate, and immediately mix by gently pipetting several times (at least 8 times) with a multichannel pipette; use a Flex Station3 multifunctional ELISA reader in fluorescence mode, set the excitation wavelength to 470 nm, measure the emission wavelengths at 515 nm and 590 nm, monitor continuously for 5 minutes, and calculate the average 590 nm to 515 nm luminescence ratio during this period. The luminescence spectrum of the probe at different NMN concentrations was obtained by monitoring the emission intensity value in the wavelength range of 500-600 nm.

[0108] Depend on Figure 3 As shown in A, at different NMN concentrations, the probe NMNS 2.0 The 590 / 515 light intensity ratio is different, and increases with the increase of NMN concentration within a certain NMN concentration range (0.45μM-1mM). 2.0 Can be used to determine the content of NMN. 2.0 C 50 The value is 30.2 μM. Although it cannot be used to detect physiological NMN levels, it can be used to study the cellular transport of NMN.

[0109] Depend on Figure 3 B shows that the probe NMN 2.0 The response to NMN analogs was low, and under physiological conditions, these compounds (NMNH, NaMN, NAM, NAM, NAD + , NADH, NADP + and NADPH) will not affect the probe's response to NMN. Figure 3 C shows that ATP has an effect on the probe at physiological concentrations, but the intracellular ATP content generally maintains a steady state, so the NMN probe can still respond to NMN at a stable ATP level. Figure 3 D shows that pH probe NMNS 2.0 The response to NMN is affected, so it is necessary to correct for pH levels when measuring intracellular NMN levels. Figure 3 E is the probe NMNS 2.0 The luminescence intensity scanning spectrum clearly shows that NMNS 2.0 There are significant luminescence peaks at 590nm and 515nm.

[0110] Example 4 Mammalian cell lines stably expressing NMN molecular probes are used to measure the dynamic changes of NMN concentration in living cells

[0111] The NMN molecular probe provided by the present invention can detect changes in the concentration of NMN molecules in living cells and their substructures. 1.1) were cloned into the pCDH-CMV-MCS-EF1-Neo vector, and NES (MLQNELALKLAGLDINKT), 2x CoXVIII (SVLTPLLLRGLTGSARRLPVPRAKIHSLGDPMSVLTPLLLRGLTGSARRLPVPRAKIHSLGDPK) or NTS (DPKKKRKV) sequence tags were added to the amino terminus of the probe encoding gene to achieve cytoplasmic localization (Cyto-NMNS 1.1 ), nuclear localization (Nuc-NMNS 1.1 ) and mitochondrial localization (Mito-NMNS 1.1 ) probe plasmid. Then, HEK 293T stable cell line was prepared by lentiviral method. Figure 4 It can be seen that the probe can be well localized in the cell nucleus or mitochondria. HEK 293T cells were plated in a 96-well white cell culture plate with an inoculation of 10,000 cells per well, and the amount of DMEM medium (high glucose, no phenol red) containing 10% (v / v) fetal bovine serum (FBS) was 100 μL. After culturing for 24 hours at 37°C and 5% CO2, 10 nM FK866 (NAMPT inhibitor, which can effectively reduce intracellular NAD + horizontal), or 500 μM NAD + The stable cell lines were treated with precursor compounds (NAM, NMN, NR, NRH). After the cells were treated with the compounds for 6 hours, the culture medium was replaced with fresh DMEM culture medium containing Furimazine substrate (high glucose, no phenol red, Furimazine substrate diluted 1000-fold). The emission intensity at wavelengths of 590nm and 440nm was detected using the Flex Station3 multifunctional microplate reader. According to the detection principle of the probe, the ratio of the emission intensity at wavelengths of 590nm and 440nm indicates the NMN concentration in living cells. The test found that the ratio decreased in cells treated with FK866, indicating a decrease in the intracellular NMN concentration. While NAD + The ratio of cells treated with precursor compounds increased to varying degrees, indicating an increase in intracellular NMN concentration. This change was consistent with expectations, indicating that the NMN probe can respond to changes in intracellular NMN concentration ( Figure 5 ).

[0112] Example 4 NMNS 1.1 Detection of NMN transport kinetics in stably transfected HEK 293T cells

[0113] The stable NMNS provided by the present invention 1.1The HEK 293T cell line can be used to study the mechanism of NMN transport in living cells. HEK 293T cells were plated in a 96-well white cell culture plate at a seeding volume of 10,000 cells per well. The culture medium was DMEM medium (high glucose, no phenol red) containing 10% (v / v) FBS and cultured at 37°C, 5% CO2 for 24 hours. Take out the cell culture plate, remove the culture medium, and clean each well three times with 100 μL of Hank's balanced salt solution (HBSS). Then, add 100 μL of HBSS buffer containing Furimazine substrate (diluted 1000-fold) and the corresponding concentration of NMN to each well, and immediately place it on the FlexStation3 multi-function microplate reader to read the light intensity values ​​at 590nm and 440nm wavelengths. According to the detection principle of this probe, the ratio of the emission light intensity at wavelengths of 590nm and 440nm indicates the NMN concentration in living cells. By Figure 6 It can be seen that after adding NMN, the probe has a significant response, indicating that NMN enters living cells very quickly. In addition, cells overexpressing Slc12a8 protein have faster NMN transport than cells expressing empty vector, supporting the view that Slc12a8 is an NMN transporter.

[0114] Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the invention.

Claims

1. A fully genetically encoded NMN protein probe, characterized in that: It is composed of a resonance energy transfer receptor, an NMN response protein and a resonance energy transfer donor connected in series, and the amino acid sequence of the NMN protein probe is shown in SEQ ID NO.9, SEQ ID NO.10 or SEQ ID NO.

11.

2. Use of the fully genetically encoded NMN protein probe according to claim 1 in the preparation of a reagent for detecting NMN concentration.

3. A composition for detecting NMN concentration, characterized in that: The composition comprises the fully genetically encoded NMN protein probe according to claim 1.

4. The composition according to claim 3, characterized in that The composition also includes a bioluminescent substrate.

5. A nucleic acid molecule encoding the fully genetically encoded NMN protein probe of claim 1.

6. A carrier, characterized in that The vector comprises the nucleic acid molecule of claim 5.

7. The carrier according to claim 6, characterized in that The vector is a lentiviral expression vector.

8. The carrier according to claim 6, characterized in that A nucleotide coding sequence for a cytoplasmic localization protein, a nucleotide coding sequence for a nuclear localization protein, and a nucleotide coding sequence for a mitochondrial localization protein are added to the amino terminus of the nucleic acid molecule according to claim 5.

9. A cell expressing the fully genetically encoded NMN protein probe of claim 1.

10. The cell according to claim 9, characterized in that The cell is obtained by transferring the vector according to claim 6 into a living cell, and the vector can be translated in the cell to obtain the NMN protein probe according to claim 1.

11. Use of the cell according to claim 9 in preparing an experimental model for studying NMN.

12. The use according to claim 11, characterized in that The experimental model is used to study the cellular transport mechanism of NMN, or to study agonists or inhibitors of NMN synthesis and metabolism, or to study NAD + Agonists or inhibitors of synthetic and metabolic processes.

13. A method for detecting NMN concentration, characterized in that: The method comprises the following steps: S11) mixing the protein probe according to claim 1 with a reagent to detect the concentration of NMN, S12) detecting the luminescence intensity at the maximum luminescence wavelength of the resonance energy transfer donor and the resonance energy transfer acceptor in the probe respectively, and calculating the ratio of the luminescence intensity of the two; S13) regressing on the standard curve to obtain the corresponding NMN concentration; or S13) detecting the ratio of luminescence intensity at different time points, and obtaining the trend of NMN concentration change at different time points; or S13) Detect the change in the ratio of luminescence intensity after adding different active ingredients to obtain the effect of different active ingredients on the change in NMN concentration.

14. The method according to claim 13, characterized in that The method for preparing the standard curve in step S13) is to use NMN of different standard concentrations, and use steps S11-S12 to detect the luminescence intensity ratios corresponding to the different concentrations of NMN, and use the logarithm of the NMN concentration as the horizontal axis and the luminescence intensity ratio as the vertical axis to make a standard curve.

15. The method according to claim 13, characterized in that The method further comprises adjusting the pH of the mixed solution in step S11) to 6.8-7.

2.

16. A method for detecting NMN concentration in living cells, characterized in that: The method comprises the following steps: S21) transferring the vector described in claim 6 into the cells to be tested by lentiviral infection, and using the fluorescent signal of the fluorescent protein as a marker for screening to obtain a stable cell line to be tested; S22) detecting the luminescence intensity of the resonance energy transfer donor and the resonance energy transfer acceptor in the NMN protein probe encoded by the nucleotide in the vector at the maximum luminescence wavelength, respectively, and calculating the ratio of the luminescence intensity of the two; S23) regressing the standard curve to obtain the corresponding intracellular NMN concentration; or S23) detecting the ratio of luminescence intensity at different time points, and obtaining the trend of changes in intracellular NMN concentration at different time points; or S23) Detect the change in the ratio of luminescence intensity after adding different active ingredients to obtain the effect of different active ingredients on the change in intracellular NMN concentration.

17. The method according to claim 16, characterized in that The method is used to detect the concentration of NMN in the cytoplasm, nucleus or mitochondria in living cells.

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