CN115724998A - Resonance energy transfer-based total genetic coding NMN protein probe and application thereof - Google Patents
Resonance energy transfer-based total genetic coding NMN protein probe and application thereof Download PDFInfo
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Abstract
The invention discloses a resonance energy transfer-based total genetic coding NMN protein probe and application thereof, and particularly discloses a total genetic coding NMN protein probe, which is formed by connecting 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 sequence of the mutant of the DNA ligase is shown as SEQ ID NO.1, SEQ ID NO.2 or SEQ ID NO. 3; the resonance energy transfer donor is selected from luciferase or a fluorescent protein; the resonance energy transfer acceptor is selected from fluorescent proteins and the fluorescent protein as the resonance energy transfer acceptor is different from the fluorescent protein as the resonance energy transfer donor. The whole gene coding NMN molecular probe based on resonance energy transfer can respond to NMN molecules in vitro and in cells.
Description
Technical Field
The invention belongs to the field of biological probes, relates to the fields of aging medicine, cell biology, molecular biology, drug development and the like, and particularly relates to a resonance energy transfer-based total genetic coding NMN protein probe and application thereof.
Background
NAD + Is an important coenzyme molecule and participates in a plurality of important processes such as cell energy metabolism, DNA repair, protein post-transcriptional modification, gene regulation, circadian rhythm and the like. NAD (nicotinamide adenine dinucleotide) + One consensus in the field of metabolic research is that: NAD in some tissues or cells + The concentration will decrease with aging. For example, model animal mice will develop skeletal muscle, adipose tissue and brain hippocampal NAD as aging progresses + The concentration is reduced. And NAD + A decrease in concentration may affect the activity of SIRT1 and PARPs, alter calcium homeostasis, and decrease mitochondrial function. Thus, intracellular NAD is reversed + The reduced levels are useful in preventing or treating aging-related disorders.
NMN is NAD + The direct precursor of the salvage synthesis pathway, NMN supplementation, is a rapid increase of NAD + A horizontally effective means. Many researches show that NMN supplementation can effectively promote intracellular NAD (nicotinamide adenine dinucleotide) under various pathological conditions such as aging-related diseases, obesity-related metabolic disorders, diabetes, ischemia-reperfusion injury and the like + Level, improve cognitive function, and improve depressive behavior. Several recent clinical trials of NMN have also demonstrated their benefits in humans: first, NMN (tens of milligrams to thousands of milligrams per day) is safe to administer; secondly, the aerobic exercise capacity of amateur runners can be improved by taking NMN, and the dosage dependence characteristic is presented; finally, for overweight women with pre-diabetes, oral NMN can increase insulin sensitivity in muscle tissue without significant side effects.
Therefore, the metabolism rule of NMN in living cells is deeply researched and characterized, the real-time measurement of the dynamic change of the NMN concentration is realized, and the research on NMN or NAD + The metabolic pathway regulation, the disease generation and the drug action mechanism are greatly helpful, and a new scheme is expected to be provided for improving aging and metabolic related diseases.
At present, the NMN content in biological samples can be detected as follows:
1) High performance liquid chromatography-mass spectrometry combination:
in the high performance liquid chromatography-mass spectrometry combined method, different components in a sample to be detected are separated by using the high performance liquid chromatography method, and the light absorption intensities of the different components are detected at the same time. And (3) during high performance liquid chromatography, different components enter a mass spectrum through ionization, the different components are qualitative through nuclear mass ratio, and the different components are quantitative through ion strength.
2) Fluorescence detection method:
performing acetophenone derivatization aiming at an alkyl pyridine group of the NMN, further enabling the alkyl pyridine group to generate fluorescence, finally measuring fluorescence intensity through a fluorescence photometer, drawing a standard curve according to a standard product and the corresponding fluorescence intensity, and further performing reverse-extrapolation to obtain the concentration of the NMN in the sample.
However, neither hplc-ms and NMN fluorescence detection methods are able to measure NMN concentrations in living cells. This type of method requires lysis of a large number of cells (about one million), a process that loses temporal and spatial information of the concentration of cellular NMN; meanwhile, the intracellular NMN concentration can be changed in the sample cracking process, so that the result is inaccurate; in addition, the derivatization treatment of the NMN requires a high temperature condition of 100 ℃, which may degrade the 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.
Disclosure of Invention
Aiming at the defects of the prior art, the invention realizes the whole gene coding NMN protein probe based on resonance energy transfer.
In terms of probe functions, the invention 1) realizes the whole gene coding of the probe structure and can express in living cells; 2) Self-calibration of probe signals, i.e. NMN quantification based on dual wavelength light intensity ratio, is achieved.
On the structure of a probe, the invention 1) designs a protein structural domain with conformation which is severely regulated and controlled by NMN, namely NMN response protein; 2) The N end and the C end of the NMN response protein are fused with an acceptor and a donor which can form a resonance energy transfer phenomenon respectively. The resonance energy transfer donor and the acceptor are respectively luciferase and fluorescent protein or two fluorescent proteins with different colors; 3) The linkage between the NMN responsive protein and the resonance energy transfer donor and acceptor is optimized to maximize the dynamic range of the probe. The concrete expression is as follows: the mutation P312 was G312 in the LigA mutant 3, the mutation V1 was I in the cpNLuc mutant 2, and the mutation S223 was L in the mScarlet-I mutant 2.
In the aspect of probe application, the invention constructs a cell line for stably expressing the NMN resonance energy transfer protein probe and realizes the characterization of the NMN concentration of the living cells and the subcellular structures thereof.
The specific technical scheme of the invention is as follows:
the invention provides a total genetic code NMN protein probe, which is formed by connecting 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 sequence of the mutant of the DNA ligase is shown as SEQ ID NO.1, SEQ ID NO.2 or SEQ ID NO. 3;
the resonance energy transfer donor is selected from luciferase or a fluorescent protein; the resonance energy transfer acceptor is selected from fluorescent proteins and the fluorescent protein as the resonance energy transfer acceptor is different from the fluorescent protein as the resonance energy transfer donor.
Further, the resonance energy transfer donor is selected from the group consisting of circularly arranged bioluminescent protein cpNLuc or green fluorescent protein meneogreen;
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.
Further, the resonance energy transfer acceptor is selected from green fluorescent protein mNeoGreen or red fluorescent protein mScarlet-I;
preferably, the sequence of 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.
Further, the NMN protein probe is formed by tandem of mScarlet-I, NMN response protein and cpNLuc; or
The NMN protein probe is formed by connecting mSacrlet-I, NMN response protein and mNeoGreen in series;
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.
In another aspect, the invention provides the use of the whole genetic code NMN protein probe in the preparation of a reagent for detecting the concentration of NMN.
In yet another aspect, the present invention provides a composition for detecting NMN concentration, wherein said composition comprises said whole genetic code NMN protein probe;
preferably, a bioluminescent substrate is also included in the composition.
In yet another aspect, the invention provides a nucleotide sequence encoding said complete genetic code NMN protein probe.
In yet another aspect, the present invention provides a vector comprising a nucleotide sequence encoding said complete genetic code NMN protein probe;
preferably, the vector is a lentiviral expression vector.
Furthermore, the nucleotide coding sequence of cytoplasmic localization protein, the nucleotide coding sequence of nuclear localization protein and the nucleotide coding sequence of mitochondrial localization protein are added at the amino terminal of the nucleotide sequence of the complete genetic coding NMN protein probe.
In yet another aspect, the invention provides a cell expressing said complete genetic code NMN protein probe;
preferably, the cells are obtained by transferring said vector in living cells, said vector being capable of obtaining said NMN protein probe in translation in the cells.
In another aspect, the invention provides the use of said cell in the preparation of an experimental model for studying NMN;
preferably, the experimental model is used for studying the cellular transport mechanism of NMN, or for studying agonists or inhibitors of NMN synthesis and metabolic processes, or for studying NAD + Of synthetic and metabolic processesAn agonist or an inhibitor.
In another aspect, the present invention provides a method for detecting NMN concentration, the method comprising the steps of:
s11) mixing the protein probe with a reagent for detecting the NMN concentration,
s12) detecting the luminous intensity under the maximum luminous wavelength of the resonance energy transfer donor and the resonance energy transfer acceptor in the probe respectively, and calculating the ratio of the luminous intensity of the resonance energy transfer donor and the resonance energy transfer acceptor;
s13) regressing on a standard curve to obtain the corresponding NMN concentration; or
S13) detecting the ratio of the light intensity at different time points, and obtaining the variation trend of the NMN concentration at different time points; or
S13) detecting the change of the ratio of the luminous intensity after different active ingredients are added, and obtaining the influence of the different active ingredients on the NMN concentration change;
preferably, in step S11), when the resonance energy transfer donor in the protein probe is luciferase, a bioluminescent substrate is added before detecting the luminescence intensity;
preferably, the preparation method of the standard curve in step S13) is to adopt NMNs with different standard concentrations, respectively adopt steps S11 to S12 to detect the corresponding luminous intensity ratios of the NMNs with different concentrations, and use the logarithmic value of the NMN concentration as the abscissa and the luminous intensity ratio as the ordinate to make the standard curve.
Further, the method further comprises adjusting the pH of the mixed solution in the step S11) to 6.8-7.2.
In yet another aspect, the present invention provides a method for detecting NMN concentration in living cells, the method comprising the steps of:
s21) transferring the vector into a cell to be tested by a lentivirus infection method, and screening by taking a fluorescent signal of a fluorescent protein as a marker to obtain a stable transfer cell line to be tested;
s22) detecting the luminous intensity under the maximum luminous wavelength of a resonance energy transfer donor and a resonance energy transfer acceptor in the NMN protein probe coded by the nucleotide in the carrier respectively, and calculating the ratio of the luminous intensity of the two;
s23) regressing on a standard curve to obtain the NMN concentration in the corresponding cells; or
S23) detecting the ratio of the light intensity at different time points, and obtaining the change trend of the concentration of the NMN in the cells at different time points; or alternatively
S23) detecting the change of the ratio of the luminous intensity after adding different active ingredients to obtain the influence of the different active ingredients on the change of the concentration of the NMN in the cells.
Further, the method is used for detecting the concentration of NMN in the cytoplasm, nucleus or mitochondria within living cells.
The invention has the beneficial effects that:
the invention innovatively realizes the whole gene coding NMN protein probe based on resonance energy transfer. In the background art, the NMN concentration in living cells cannot be measured by the combined high performance liquid chromatography-mass spectrometry and the NMN fluorescence detection method, and the NMN concentration in living cells cannot be measured by the combined high performance liquid chromatography-mass spectrometry and the NMN fluorescence detection method. This type of method requires lysis of a large number of cells (about one million), a process that loses temporal and spatial information of the concentration of cellular NMN; meanwhile, the intracellular NMN concentration can be changed in the sample cracking process, so that the result is inaccurate; in addition, the derivatization treatment of the NMN requires a high temperature condition of 100 ℃, which may degrade the NMN; finally, the detection of a single sample takes 10 to 30 minutes, and depends on a large-scale instrument, which severely limits the detection of a large number of samples, and is one of the rate-limiting steps in NMN metabolism research and drug development. The NMN protein probe provided by the invention not only realizes the NMN quantification based on resonance energy transfer and the whole gene coding of the probe, namely, the self-calibration of the probe signal is realized, namely, the NMN quantification based on the dual-wavelength light intensity ratio is realized; and realizes the NMN monitoring of living cells independent of exogenous molecules. The whole gene coding NMN molecular probe based on resonance energy transfer can respond to NMN molecules in vitro and in cells, and has the advantages of high specificity, proper C50 value (concentration of a substance to be detected when 50% of probe conformation changes is caused), high dynamic range and the like.
Drawings
Fig. 1 is a schematic diagram of the principle of detecting the concentration of the NMN molecule based on the resonance energy transfer NMN molecular probe of the present invention. The NMN-sensing protein is in an "open" state when the probe does not bind the NMN molecule, and the NMN-sensing protein conformation changes to a "closed" state when NMN is bound. The efficiency of the resonance energy transfer differs between the "open" and "closed" states, which in turn can indicate the NMN level.
FIG. 2A shows purified NMN molecular probe NMNS 1.0 And NMNS 1.1 Titration curves for NMN molecular response. The abscissa is the concentration of NMN and the ordinate is the ratio of the light intensities at 590nm and 440nm, respectively. FIG. 2B shows purified molecular probe NMNS 1.1 For titration curves of NMN analogs, the abscissa is NMN concentration and the ordinate is the corresponding light intensity ratio at 590nm and 440 nm. FIG. 2C shows molecular probe NMNS 1.1 The response to AXP is a titration curve with NMN concentration on the abscissa and the corresponding ratio of light intensity at 590nm to 440nm on the ordinate. FIG. 2D shows molecular probe NMNS 1.1 Titration curves for NMN response at physiological pH range with NMN concentration on the abscissa and light intensity ratio at 590nm and 440nm on the ordinate. FIG. 2E shows molecular probe NMNS 1.1 Bioluminescence spectra in response to NMN, wavelength (nm) on the abscissa and normalized bioluminescence intensity on the ordinate.
FIG. 3A shows purified NMN molecular probe NMNS 2.0 Titration curves for NMN molecular response. The abscissa is the NMN concentration and the ordinate is the ratio of the light intensity at 590nm and 515nm, respectively. FIG. 3B shows purified molecular probe NMNS 2.0 For titration curves of NMN analogue, the abscissa is NMN concentration and the ordinate is the corresponding ratio of light intensity at 590nm and 515 nm. FIG. 3C shows the molecular probe NMNS 2.0 The titration curve in response to AXP has NMN concentration on the abscissa and the ratio of light intensity at 590nm to 515nm on the ordinate. FIG. 3D shows molecular probe NMNS 2.0 Titration curves for NMN response at physiological pH range with NMN concentration on the abscissa and light intensity ratio at corresponding 590nm and 515nm on the ordinate. FIG. 3E shows the molecular probe NMNS 2.0 Bioluminescence spectra in response to NMN, wavelength (nm) on the abscissa and normalized bioluminescence intensity on the ordinate.
FIG. 4A is a fluorescent micrograph of a nuclear localization probe NMNS1.1 stably transfected cell, from left to right, of the DAPI channel, the 561nm channel, and the superposition of the DAPI and 561nm channels, respectively. The better the overlap of the 561nm channel with the DAPI channel, indicating a more stringent localization of probe NMNS1.1 in the nucleus. FIG. 4B is a fluorescence micrograph of a mitochondrion localization probe NMNS1.1 stably transfected cell, from left to right, showing the Mitotracker dye channel, the 561nm channel, and the superposition of the Mitotracker dye channel and the 561nm channel, respectively. The better the overlap of the 561nm channel with the Mitotracker dye channel, indicating the more stringent the localization of probe NMNS1.1 at mitochondria.
FIG. 5A is the cytoplasmic NMN levels of HEK 293T cells treated with 500. Mu.M of the NAD + precursor molecules NAM, NMN, NR, NRH and 10nM of the NAMPT inhibitor FK866 for 6 h. FIG. 5B is the mitochondrial NMN levels after HEK 293T cells were treated with 500. Mu.M of the NAD + precursor molecules NAM, NMN, NR, NRH and 10nM of the NAMPT inhibitor FK866 for 6 h. FIG. 5C is nuclear NMN levels in HEK 293T cells treated with 500. Mu.M of the NAD + precursor molecules NAM, NMN, NR, NRH and 10nM of the NAMPT inhibitor FK866 for 6 h. All P values were calculated using One-way analysis of variance (One-way anova).
FIG. 6A shows the change in intracellular NMN levels within 10min after PBS treatment or addition of 100. Mu.M NMN to HEK 293T (Ctrl Vector) or HEK 293T cells overexpressing Slc12a8 protein (Slc 12a 8-OE). FIG. 6B is a statistical plot of the change in intracellular NMN levels following PBS treatment of Ctrl Vector or HEK 293T cells overexpressing Slc12a8 protein (Slc 12a 8-OE) or addition of 100. Mu.M NMN.
Detailed Description
In order that the invention may be more clearly understood, it will now be further described with reference to the following examples and the accompanying drawings. The examples are for illustration only and do not limit the invention in any way. In the examples, each raw reagent material is commercially available, and the experimental method not specifying the specific conditions is a conventional method and a conventional condition well known in the art, or a condition recommended by an instrument manufacturer.
Example 1
The invention provides a resonance energy transfer-based total genetic coding NMN protein probe, which consists of three parts, namely an NMN response protein, a donor and an acceptor, wherein the conformation of the NMN response protein is severely regulated and controlled by NMN, and the donor and the acceptor can form a resonance energy transfer phenomenon, and the resonance energy transfer acceptor, the NMN response protein and the resonance energy transfer donor are connected in series. Wherein the NMN responsive protein is a mutant of DNA ligase (LigA), and the LigA mutant can be selected from the group consisting of LigA mutant 1, ligA mutant 2 and LigA mutant 3. The resonance energy transfer donor and the acceptor are respectively a Luciferase and a fluorescent protein, or two fluorescent proteins with different colors, for example, the resonance energy transfer donor can be a circularly arranged bioluminescent protein (cpNLuc) or a green fluorescent protein mNeoGreen, the resonance energy transfer acceptor is a red fluorescent protein mReclet-I, the cpNLuc can be selected from cpNLuc mutant 1 or cpNLuc mutant 2, the mReclet-I can be selected from mReclet-I mutant 1 or mReclet-I mutant 2.
The principle of detecting the concentration of the NMN molecule by the resonance energy transfer-based full genetic coding NMN protein probe is shown in figure 1, when the NMN response protein is not combined with the NMN molecule, the probe structure is in an open state, so that the distance between a resonance energy transfer donor and a receptor is long, the resonance energy transfer efficiency is low, and the probe integrally emits light of the resonance energy transfer donor. When the NMN response protein is combined with the NMN molecule, the conformation of the NMN response protein is changed from an open state to a closed state, the resonance energy transfer donor and the receptor are enabled to be close to each other, higher resonance energy transfer efficiency is formed, and the probe emits light of the resonance energy transfer receptor. The change of the resonance energy transfer efficiency caused by the NMN molecule is finally shown as the change of the emission wavelength intensity of the resonance energy transfer donor and acceptor in the probe. This ratio of emitted light intensities in turn indicates the concentration of NMN in the system.
The sequence of the LigA mutant 1 is shown as SEQ ID NO. 1:
LTLTAATTRAQELRKQLNQYSHEYYVKDQPSVEDYVYDRLYKELVDIETEFPDLITPDSPTQNVGGKVLSGFEKAPHDIPMYSLNKGFSKEDIFAFDERVRKAIGKPVAYCCELLIDGLAISLRYENGVFVRGATRGDGTVGENITENLRTVRSVPMDLTEPISVEVRGECYMPKQSFVALNEEREENGQDIFANPRNAAAGSLRQLDTKIVAKRNLNTFLATVADFGPMKAKTQFEALEELSAIGFRTNPERQLCQSIDEVWAYIEEYHEKRSTLPYEINGIVIKVNEFALQDELGFTVKAPRWAIAYKFP
the sequence of the LigA mutant 2 is shown as SEQ ID NO. 2:
LTLTAATTRAQELRKQLNQYDHEYYVKDQPSVEDYVYDRLYKELVDIETEFPDLITPDSPTQNVGGKVLSGFEKAPHDIPMYSMNRGFSKEDIFAFDERVRKAIGKPVAYCCELLIDGLDISLRYENGVFVRGATRGDGTVGENITENLRTVRSVPMDLTEPISVEVRGECYMPKQSFVALNEEREENGQDIFANPRNAAAGSLRQLDTKIVAKRNLNTFLKQVADFGPMKAKTQFEALEELSAIGFRTNPERQLCQSIDEVWAYIEEYHEKRSTLPYEINGIVIKVNEFALQDELGFTVKAPRWAIAYKFP
the sequence of the LigA mutant 3 is shown in SEQ ID NO. 3:
LTLTAATTRAQELRKQLNQYDHEYYVKDQPSVEDYVYDRLYKELVDIETEFPDLITPDSPTQNVGGKVLSGFEKAPHDIPMYSMNRGFSKEDIFAFDERVRKAIGKPVAYCCELLIDGLDISLRYENGVFVRGATRGDGTVGENITENLRTVRSVPMDLTEPISVEVRGECYMPKQSFVALNEEREENGQDIFANPRNAAAGSLRQLDTKIVAKRNLNTFLKQVADFGPMKAKTQFEALEELSAIGFRTNPERQLCQSIDEVWAYIEEYHEKRSTLPYEINGIVIKVNEFALQDELGFTVKAPRWAIAYKFG
the sequence of the cpNLuc mutant 1 is shown in SEQ ID NO. 4:
VDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILAGGTGGSGGTGGSMVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYE
the sequence of the cpNLuc mutant 2 is shown as SEQ ID NO. 5:
IDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILAGGTGGSGGTGGSMVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYE
the sequence of the mScarlet-I mutant 1 is shown as SEQ ID NO. 6:
MVSKGEAVIKEFMRFKVHMEGSMNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFSWDILSPQFMYGSRAFIKHPADIPDYYKQSFPEGFKWERVMNFEDGGAVTVTQDTSLEDGTLIYKVKLRGTNFPPDGPVMQKKTMGWEASTERLYPEDGVLKGDIKMALRLKDGGRYLADFKTTYKAKKPVQMPGAYNVDRKLDITSHNEDYTVVEQYERSEGRHST
the sequence of the mScarlet-I mutant 2 is shown in SEQ ID NO. 7:
MVSKGEAVIKEFMRFKVHMEGSMNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFSWDILSPQFMYGSRAFIKHPADIPDYYKQSFPEGFKWERVMNFEDGGAVTVTQDTSLEDGTLIYKVKLRGTNFPPDGPVMQKKTMGWEASTERLYPEDGVLKGDIKMALRLKDGGRYLADFKTTYKAKKPVQMPGAYNVDRKLDITSHNEDYTVVEQYERSEGRHLT
the sequence of mNeoGreen is shown in SEQ ID NO. 8:
LAATHELHIFGSINGVDFDMVGQGTGNPNDGYEELNLKSTKGDLQFSPWILVPHIGYGFHQYLPYPDGMSPFQAAMVDGSGYQVHRTMQFEDGASLTVNYRYTYEGSHIKGEAQVKGTGFPADGPVMTNSLTAADWCRSKKTYPNDKTIISTFKWSYTTGNGKRYRSTARTTYTFAKPMAANYLKNQPMYVFRKTELKHSKTELNFKEWQKAFTDVMGMDELYK
this example prepares NMNS 1.0 Probe, NMNS 1.1 Probe and NMNS 2.0 The probe has the following sequence:
NMNS 1.0 a probe, namely mScarlet I mutant 1-LigA mutant 1-cpNluc mutant 1, wherein the amino acid sequence of the probe is shown as SEQ ID NO. 9:
MVSKGEAVIKEFMRFKVHMEGSMNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFSWDILSPQFMYGSRAFIKHPADIPDYYKQSFPEGFKWERVMNFEDGGAVTVTQDTSLEDGTLIYKVKLRGTNFPPDGPVMQKKTMGWEASTERLYPEDGVLKGDIKMALRLKDGGRYLADFKTTYKAKKPVQMPGAYNVDRKLDITSHNEDYTVVEQYERSEGRHSTLTLTAATTRAQELRKQLNQYSHEYYVKDQPSVEDYVYDRLYKELVDIETEFPDLITPDSPTQNVGGKVLSGFEKAPHDIPMYSLNKGFSKEDIFAFDERVRKAIGKPVAYCCELLIDGLAISLRYENGVFVRGATRGDGTVGENITENLRTVRSVPMDLTEPISVEVRGECYMPKQSFVALNEEREENGQDIFANPRNAAAGSLRQLDTKIVAKRNLNTFLATVADFGPMKAKTQFEALEELSAIGFRTNPERQLCQSIDEVWAYIEEYHEKRSTLPYEINGIVIKVNEFALQDELGFTVKAPRWAIAYKFPVDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILAGGTGGSGGTGGSMVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYE
NMNS 1.1 a probe, namely mScarlet I mutant 1-LigA mutant 2-cpNluc mutant 2, wherein the amino acid sequence of the probe is shown as SEQ ID NO. 10:
MVSKGEAVIKEFMRFKVHMEGSMNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFSWDILSPQFMYGSRAFIKHPADIPDYYKQSFPEGFKWERVMNFEDGGAVTVTQDTSLEDGTLIYKVKLRGTNFPPDGPVMQKKTMGWEASTERLYPEDGVLKGDIKMALRLKDGGRYLADFKTTYKAKKPVQMPGAYNVDRKLDITSHNEDYTVVEQYERSEGRHSTLTLTAATTRAQELRKQLNQYDHEYYVKDQPSVEDYVYDRLYKELVDIETEFPDLITPDSPTQNVGGKVLSGFEKAPHDIPMYSMNRGFSKEDIFAFDERVRKAIGKPVAYCCELLIDGLDISLRYENGVFVRGATRGDGTVGENITENLRTVRSVPMDLTEPISVEVRGECYMPKQSFVALNEEREENGQDIFANPRNAAAGSLRQLDTKIVAKRNLNTFLKQVADFGPMKAKTQFEALEELSAIGFRTNPERQLCQSIDEVWAYIEEYHEKRSTLPYEINGIVIKVNEFALQDELGFTVKAPRWAIAYKFPIDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILAGGTGGSGGTGGSMVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYE
NMNS 2.0 the probe, mSCarlet I mutant 2-LigA mutant 3-mNeoGreen, has an amino acid sequence shown in SEQ ID NO. 11:
MVSKGEAVIKEFMRFKVHMEGSMNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFSWDILSPQFMYGSRAFIKHPADIPDYYKQSFPEGFKWERVMNFEDGGAVTVTQDTSLEDGTLIYKVKLRGTNFPPDGPVMQKKTMGWEASTERLYPEDGVLKGDIKMALRLKDGGRYLADFKTTYKAKKPVQMPGAYNVDRKLDITSHNEDYTVVEQYERSEGRHLTLTLTAATTRAQELRKQLNQYDHEYYVKDQPSVEDYVYDRLYKELVDIETEFPDLITPDSPTQNVGGKVLSGFEKAPHDIPMYSMNRGFSKEDIFAFDERVRKAIGKPVAYCCELLIDGLDISLRYENGVFVRGATRGDGTVGENITENLRTVRSVPMDLTEPISVEVRGECYMPKQSFVALNEEREENGQDIFANPRNAAAGSLRQLDTKIVAKRNLNTFLKQVADFGPMKAKTQFEALEELSAIGFRTNPERQLCQSIDEVWAYIEEYHEKRSTLPYEINGIVIKVNEFALQDELGFTVKAPRWAIAYKFGLAATHELHIFGSINGVDFDMVGQGTGNPNDGYEELNLKSTKGDLQFSPWILVPHIGYGFHQYLPYPDGMSPFQAAMVDGSGYQVHRTMQFEDGASLTVNYRYTYEGSHIKGEAQVKGTGFPADGPVMTNSLTAADWCRSKKTYPNDKTIISTFKWSYTTGNGKRYRSTARTTYTFAKPMAANYLKNQPMYVFRKTELKHSKTELNFKEWQKAFTDVMGMDELYK
this example allows the design of probes NMNS generating different affinities by introducing mutations in the LigA mutant 1.0 And NMNS 1.1 The C50 values are 1.34 μ M and 7.56 μ M respectively (FIG. 2A), and the method can be used for NMN detection scenes with different concentration ranges. The probe has high selectivity to NMN molecules (FIG. 2B, C). Probes were not responsive to NAM and NADPH; the probe responds to NADH and NRH, but has a high C50 value (>1 mM); and probe pair NADP + And NMN molecules have a smaller dynamic range of response. Titration curves at different pH conditions indicated that the probe appeared stable at pH conditions in the physiological range (fig. 2D).
Preparation of NMNS 2.0 The probe replaces cpNLuc with fluorescent protein mNeoGreen, and then an NMN molecular probe based on fluorescence resonance energy transfer is developed, wherein the signal response strain is 1.2 times, and the C50 value is 30.2 mu M (figure 3).
Specific examples of NMNS are described below 1.0 Probe, NMNS 1.1 Probe and NMNS 2.0 The probe will be described in detail.
Example 2 full genetic code NMN molecular probes based on Bioluminescence Resonance Energy Transfer (BRET) for determination of NMN content in a sample
Verification of the NMN molecular probe NMNS provided by the invention 1.0 And NMNS 1.1 Can be used for NMN content determination in a sample, and a titration experiment is carried out according to the following steps:
(1) The following samples were prepared
Probe solution: the purified NMN molecular probe was diluted to 2nM with HEPES buffer (50mM NaCl,50mM HEPES, pH 7.2) and temporarily placed in an ice box for use;
NMN solution: NMN solutions of different concentrations (3 mM,1mM,0.33mM,0.11mM, 37.0. Mu.M, 12.3. Mu.M, 4.12. Mu.M and 1.37. Mu.M) or (500. Mu.M, 167. Mu.M, 55.6. Mu.M, 18.5. Mu.M, 6.1. Mu.M, 2.1. Mu.M, 0.68. Mu.M and 0.23. Mu.M) were prepared by 3-fold serial gradient dilution using HEPES buffer and stored in ice boxes;
NMN analogues NMNH and NaMN were formulated at concentrations of 3mM,1mM,0.33mM,0.11mM, 37.0. Mu.M, 12.3. Mu.M, 4.12. Mu.M and 1.37. Mu.M; NMN analogs NAM, NR, NRH, NAD + ，NADH，NADP + And NADPH at a concentration of 50mM,16.7mM,5.56mM,1.85mM, 617. Mu.M, 206. Mu.M, 68.6. Mu.M and 22.9. Mu.M; AMP, ADP and ATP are prepared at concentrations of 50mM,16.7mM,5.56mM,1.85mM, 617. Mu.M, 206. Mu.M, 68.6. Mu.M and 22.9. Mu.M.
Bioluminescent substrate solution: furimazine (from Promega) solution was diluted 100-fold with water and stored in ice-box protected from light.
(2) Respectively adding 80 mu L of probe solution, 10 mu L of LNMN solution and 10 mu L of bioluminescence substrate solution into a white 96-hole enzyme label plate, and immediately and gently blowing and sucking for multiple times (at least 8 times) by using a multi-channel pipettor for uniformly mixing; reading the wavelength 440nm and the wavelength 590nm (NMNS) by a Flex Station3 multifunctional microplate reader in a luminescence mode 1.0 、NMNS 1.1 ) Processing the light intensity value, continuously monitoring for 5min, and calculating the average receptor ratio of the time periodDonor luminescence ratio. The luminescence spectra of the probe at different NMN concentrations were obtained by monitoring the intensity of light in the wavelength range 360-650 nm.
As can be seen from FIG. 2A, probe NMNS was present at different NMN concentrations 1.0 、NMNS 1.1 The 590/440 light intensity ratios are different and within a certain NMN concentration range (NMNS) 1.0 ：23nM-50μM；NMNS 1.1 :137nM-300 μ M) increased with increasing concentration. Illustrating the NMN molecular probe NMNS of the invention 1.0 、NMNS 1.1 Can be used for measuring the NMN content. NMN 1.0 C of (A) 50 A value of 1.34. Mu.M is suitable for the in vitro detection of highly sensitive NMN levels 1.1 C of (A) 50 A value of 7.56 μ M is suitable for physiological NMN level detection.
As can be seen from FIG. 2B, probe NMN 1.1 The response to NMN analogues is low, and under physiological conditions, these compounds (NMNH, naMN, NAM, NAM, NR, NRH, NAD) + ，NADH，NADP + And NADPH) does not affect the probe's response to NMN. From fig. 2C, it can be seen that ATP has an effect on the probe at physiological concentrations, but intracellular ATP content generally remains steady, so the NMN probe can still respond to NMN at a steady ATP level. As can be seen from FIG. 2D, pH vs. Probe NMNS 1.1 In response to NMN effects, pH levels need to be corrected when making intracellular NMN level determinations. FIG. 2E is probe NMNS 1.1 The luminous intensity scans the map, so that the NMNS can be clearly seen 1.1 There are significant luminescence peaks at 590nm and 440 nm.
Example 3 Fluorescence Resonance Energy Transfer (FRET) -based fully genetically encoded NMN molecular probes for determination of NMN content in a sample
Verification of the NMN molecular probe NMNS provided by the invention 2.0 Can be used for NMN content determination in a sample, and a titration experiment is carried out according to the following steps:
(1) The following samples were prepared
Probe solution: the purified NMN molecular probe was diluted to 1. Mu.M with HEPES buffer (50mM NaCl,50mM HEPES, pH 7.2) and temporarily placed in an ice box for use;
NMN solution: NMN solutions of different concentrations (10mM, 3.33mM,1.11mM,0.37mM, 123. Mu.M, 41.2. Mu.M, 13.7. Mu.M and 4.57. Mu.M) were prepared by 3-fold serial gradient dilution, using HEPES buffer, and stored in an ice box;
NMN analogues NMNH and NaMN were formulated at concentrations of 10mM,3.33mM,1.11mM,0.37mM, 123. Mu.M, 41.2. Mu.M, 13.7. Mu.M and 4.57. Mu.M; NMN analogs NAM, NR, NRH, NAD + ，NADH，NADP + And NADPH at a concentration of 50mM,16.7mM,5.56mM,1.85mM, 617. Mu.M, 206. Mu.M, 68.6. Mu.M and 22.9. Mu.M; AMP, ADP and ATP are prepared at concentrations of 50mM,16.7mM,5.56mM,1.85mM, 617. Mu.M, 206. Mu.M, 68.6. Mu.M and 22.9. Mu.M.
(2) Respectively adding 90 mu L of probe solution and 10 mu L of MN solution into a black 96-hole enzyme label plate, and immediately and gently blowing and sucking for multiple times (at least 8 times) by using a multi-channel pipettor for uniformly mixing; and (3) setting the excitation wavelength to 470nm by using a Flex Station3 multifunctional microplate reader in a fluorescence mode, measuring the emission wavelengths at 515nm and 590nm, continuously monitoring for 5min, and calculating the average 590nm to 515nm luminescence ratio of the time period. The luminescence spectra of the probe at different NMN concentrations were obtained by monitoring the emission intensity values in the wavelength range 500-600 nm.
As can be seen in FIG. 3A, probe NMNS was present at different NMN concentrations 2.0 The 590/515 light intensity ratio of (A) was different and increased with increasing concentration over a range of NMN concentrations (0.45. Mu.M-1 mM). Illustrative NMN molecular Probe NMNS of the invention 2.0 Can be used for measuring the NMN content. NMN 2.0 C of (A) 50 The value was 30.2. Mu.M, and although not useful for the detection of physiological NMN levels, it was useful for the study of cellular transport of NMN.
As can be seen from FIG. 3B, probe NMN 2.0 The response to NMN analogues is lower, under physiological conditions, these compounds (NMNH, naMN, NAM, NAM, NAD) + ，NADH，NADP + And NADPH) does not affect the probe response to NMN. From fig. 3C, it can be seen that ATP has an effect on the probe at physiological concentrations, but intracellular ATP content generally maintains a steady state, so the NMN probe can still respond to NMN at a steady ATP level. From FIG. 3D, it can be seen that the pH vs probe NMNS 2.0 In response to NMN effects, pH levels need to be corrected when making intracellular NMN level determinations. FIG. 3E is probe NMNS 2.0 Luminous intensityScanning the map, the NMNS can be clearly seen 2.0 There are significant luminescence peaks at 590nm and 515 nm.
Example 4 mammalian cell lines stably expressing NMN molecular probes for measuring the dynamic change in NMN concentration in living cells
The NMN molecular probe provided by the invention can detect the concentration change of NMN molecules in living cells and substructures thereof. The probe (NMNS) 1.1 ) The coding gene is cloned to a pCDH-CMV-MCS-EF1-Neo vector, NES (MLQNELALKLGLGLLDINKT), 2x CoXVIII (SVLTPLLLRGLTGSARRLPVPRAKIHSLGDPMSVLTPLLLRGLTGSARRLPVPRAKIHSLGDPK) or NTS (DPKKKRKV) sequence labels are respectively added at the amino end of the coding gene of the probe, and cytoplasmic localization (Cyto-NMNS) is respectively realized 1.1 ) Nuclear localization (Nuc-NMNS) 1.1 ) And mitochondrial localization (Mito-NMNS) 1.1 ) And (5) constructing a probe plasmid. Subsequently, a HEK 293T stable cell line was prepared using a lentivirus method. As can be seen from FIG. 4, the probe was well localized to the nucleus or mitochondria. HEK 293T cells were plated in 96-well white cell culture plates at an inoculum size of 10,000 cells per well in 100 μ L DMEM medium (high sugar, phenol red free) containing 10% (v/v) Fetal Bovine Serum (FBS). At 37 ℃,5% CO 2 After 24h of incubation under these conditions, using 10nM FK866 (NAMPT inhibitor, it was effective in reducing intracellular NAD + Level), or 500. Mu.M NAD + Precursor compounds (NAM, NMN, NR, NRH) treated stable cell lines. After 6h of compound treatment, the medium was changed to fresh DMEM medium containing Furimazine substrate (high sugar, no phenol red, 1000-fold dilution of Furimazine substrate). The emission light intensity of 590nm and 440nm wavelength is detected by a Flex Station3 multifunctional microplate reader. According to the detection principle of the probe, the ratio of the emission light intensity of 590nm and 440nm wavelength indicates the NMN concentration in the living cells. This ratio decreased in FK 866-treated cells as detected, indicating a decrease in intracellular NMN concentration. And NAD + The ratio increased to various degrees in cells treated with the precursor compound, indicating an increase in intracellular NMN concentration. This change was consistent with expectations, indicating that the NMN probe was able to respond to changes in intracellular NMN concentration (fig. 5).
Example 4NMNS 1.1 Detection of NMN transport kinetics by stably-transformed HEK 293T cells
The invention provides a stable NMNS 1.1 The HEK 293T cell line of (a) can be used to study the mechanism of NMN transport by living cells. HEK 293T cells plated at an inoculum size of 10,000 cells per well in 96-well white cell culture plates in DMEM medium (high-sugar, phenol red-free) containing 10% (v/v) FBS, at 37 ℃ with a 5% CO 2 Culturing under the condition for 24h. The cell culture plates were removed and the medium removed and cleared 3 times with 100 μ L Hank's Balanced Salt Solution (HBSS) per well. Then, 100. Mu.L of HBSS buffer containing Furimazine substrate (diluted 1000-fold) and NMN at the corresponding concentration was added to each well and immediately placed in a Flex Station3 multifunctional microplate reader to read the light intensity at 590nm and 440 nm. According to the detection principle of the probe, the ratio of the emission light intensities of 590nm and 440nm wavelength indicates the NMN concentration in the living cells. As can be seen in fig. 6, the probe had a clear response after NMN addition, indicating that the rate of NMN entry into living cells was rapid. Furthermore, cells over-expressing the Slc12a8 protein had more rapid NMN transport than cells expressing the empty vector, supporting the idea that Slc12a8 is an NMN transporter.
It should be understood that the above examples are only for clarity of illustration and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications therefrom are within the scope of the invention.
Claims (15)
1. A total genetic code NMN protein probe is characterized in that the probe is formed by connecting 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 sequence of the mutant of the DNA ligase is shown as SEQ ID NO.1, SEQ ID NO.2 or SEQ ID NO. 3;
the resonance energy transfer donor is selected from luciferase or a fluorescent protein; the resonance energy transfer acceptor is selected from fluorescent proteins and the fluorescent protein as the resonance energy transfer acceptor is different from the fluorescent protein as the resonance energy transfer donor.
2. A complete genetically encoded NMN protein probe according to claim 1, characterized in that the resonance energy transfer donor is selected from the group consisting of circularly permuted bioluminescent protein cpNLuc or green fluorescent protein meneogreen;
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.
3. The whole genetically encoded NMN protein probe according to claim 1, wherein the resonance energy transfer receptor is selected from the group consisting of green fluorescent protein mNeoGreen or red fluorescent protein mScarlet-I;
preferably, the sequence of 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.
4. The fully genetically encoded NMN protein probe of claim 1, wherein the NMN protein probe is formed from a mScarlet-I, NMN response protein and cpNLuc in tandem; or
The NMN protein probe is formed by connecting mScplet-I, NMN response protein and mNeoGreen in series;
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.
5. Use of a whole-genetic-encoded NMN protein probe according to any one of claims 1 to 4 in the preparation of a reagent for detecting NMN concentration.
6. A composition for detecting NMN concentration, comprising a whole-gene-encoded NMN protein probe according to any one of claims 1 to 4;
preferably, a bioluminescent substrate is also included in the composition.
7. A nucleotide sequence encoding the complete genetic code NMN protein probe of any of claims 1-4.
8. A vector comprising the nucleotide sequence of claim 7;
preferably, the vector is a lentiviral expression vector.
9. The vector of claim 8, wherein the nucleotide coding sequence for cytoplasmic localization protein, the nucleotide coding sequence for nuclear localization protein and the nucleotide coding sequence for mitochondrial localization protein are added at the amino terminus of the nucleotide sequence of claim 7.
10. A cell expressing a whole gene encoding an NMN protein probe according to any one of claims 1 to 4;
preferably, the cell is obtained by transferring the vector of claim 8 or 9 into a living cell, said vector being capable of obtaining the NMN protein probe of any one of claims 1 to 4 in translation in the cell.
11. Use of the cell of claim 10 for the preparation of an experimental model for studying NMN;
preferably, the experimental model is used for studying the cellular transport mechanism of NMN, or for studying agonists or inhibitors of NMN synthesis and metabolic processes, or for studying NAD + Agonists or inhibitors of synthetic and metabolic processes.
12. A method of detecting NMN concentration, comprising the steps of:
s11) mixing the protein probe of any one of claims 1 to 4 with a reagent for detecting NMN concentration,
s12) detecting the luminous intensity under the maximum luminous wavelength of the resonance energy transfer donor and the resonance energy transfer acceptor in the probe respectively, and calculating the ratio of the luminous intensity of the resonance energy transfer donor and the resonance energy transfer acceptor;
s13) regressing on a standard curve to obtain the corresponding NMN concentration; or alternatively
S13) detecting the ratio of the light intensity at different time points, and obtaining the variation trend of the NMN concentration at different time points; or alternatively
S13) detecting the change of the ratio of the luminous intensity after different active ingredients are added, and obtaining the influence of the different active ingredients on the NMN concentration change;
preferably, in step S11), when the resonance energy transfer donor in the protein probe is luciferase, a bioluminescent substrate is added before detecting the luminescence intensity;
preferably, the preparation method of the standard curve in step S13) is to adopt NMNs with different standard concentrations, respectively adopt steps S11 to S12 to detect the corresponding luminous intensity ratios of the NMNs with different concentrations, and use the logarithmic value of the NMN concentration as the abscissa and the luminous intensity ratio as the ordinate to detect and make the standard curve.
13. The method as claimed in claim 12, further comprising adjusting the pH of the mixed solution in the step S11) to 6.8-7.2.
14. A method for detecting NMN concentration in living cells, comprising the steps of:
s21) transferring the vector of claim 8 or 9 into a cell to be tested by a lentivirus infection method, and screening by taking a fluorescent signal of a fluorescent protein as a marker to obtain a stable transfer cell line to be tested;
s22) detecting the luminous intensity under the maximum luminous wavelength of a resonance energy transfer donor and a resonance energy transfer acceptor in the NMN protein probe coded by the nucleotide in the carrier respectively, and calculating the ratio of the luminous intensity of the two;
s23) regressing on a standard curve to obtain the NMN concentration in the corresponding cell; or
S23) detecting the ratio of the light intensity at different time points, and obtaining the change trend of the concentration of the NMN in the cells at different time points; or
S23) detecting the change of the ratio of the luminous intensity after adding different active ingredients to obtain the influence of the different active ingredients on the change of the concentration of the NMN in the cells.
15. The method according to claim 14, wherein the method is used to detect NMN concentration in cytoplasm, nucleus or mitochondria within living cells.
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| PCT/CN2022/138161 WO2024087336A1 (en) | 2022-10-28 | 2022-12-09 | All-genetically encoded nmn protein probe based on resonance energy transfer, and use thereof |
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