CN111979295B - Tyrosine phosphatase biosensor and detection method and application thereof - Google Patents
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Abstract
The invention discloses a tyrosine phosphatase biosensor, a detection method and application thereof, wherein the detection method comprises magnetic beads, peptide-DNA conjugates, DNA templates and signal probes; in the peptide-DNA conjugate, one end of a peptide chain is connected with the 5' end of the first DNA sequence, and the peptide chain contains phosphorylated tyrosine; the other end of the peptide chain is used for connecting magnetic beads; the DNA template comprises two second DNA sequences and a third DNA sequence from the 5 'end to the 3' end, the third DNA sequence can be complementary with the first DNA sequence, double-stranded DNA with two nicking enzyme recognition sites can be formed after hybridization and repolymerization of the DNA template and the first DNA sequence, and the complementary strand of the second DNA sequence is deoxyribozyme; the signal probe is a nucleotide sequence, the two ends of the nucleotide sequence are respectively connected with a fluorophore and a quencher, and the nucleotide sequence can be complementary with the deoxyribozyme; deoxyribozymes are RNA sequences that cleave complementation in a divalent cation-dependent reaction.
Description
Technical Field
The invention belongs to the technical field of analysis and detection, and relates to a tyrosine phosphatase biosensor, a detection method and application thereof.
Background
The disclosure of this background section is only intended to increase the understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art already known to those of ordinary skill in the art.
Tyrosine phosphorylation is regulated precisely by the interaction of tyrosine kinases (PTKs) and tyrosine phosphatases (PTPs) in cells to achieve equilibrium. However, the role of tyrosine phosphatases (PTPs) in regulating tyrosine phosphate balance has been underestimated. Recent studies have shown that protein tyrosine phosphatase 1B (PTP 1B) may play a critical negative regulatory role for insulin/leptin signaling, and that its aberrant activity may lead to type II diabetes and obesity. In addition, the deregulation of other tyrosine phosphatases (PTPs) is closely related to a variety of human diseases and cancers. Therefore, effective and sensitive detection of tyrosine phosphatase (PTP) activity is of great importance for clinical diagnosis, drug discovery and cancer treatment.
To the inventors' knowledge, conventional tyrosine phosphatase (PTP) assays have assays 32 P/ 33 Radiolabelling of the P-labeled phosphorylated peptide/protein substrate released radioactive phosphate and quantitative analysis of the released radioactive phosphate by liquid scintillation analysis are known, but this method is prone to radioactive contamination and is less sensitive. In recent years, non-radioactive methods including colorimetry and fluorescence have been developed, in which the activity of tyrosine phosphatase (PTP) in a phosphorylation reaction is quantitatively analyzed by using absorbance/fluorescence intensity signals using organic phosphates such as p-nitrophenylphosphate (pNPP), 4-methyl-7-hydroxycoumarin phosphate (MUP) and 3, 6-Fluorescein Diphosphate (FDP) as substrates. However, these substrates are simulated natural phosphopeptide/protein substrates, which are poorly specific and may be subject to nonspecific dephosphorylation by various phosphatases, which affects the accurate detection of tyrosine phosphatase (PTP) activity in complex environments. When specific phosphopeptides are used as substrates, the activity of tyrosine phosphatase (PTP) is usually detected by measuring released phosphate ions (Pi) by malachite green colorimetry, which is generally regarded as a standard PTP detection method but has a high background signal in complex samples. Cerium ions (Ce) that can be initiated by dephosphorylation reactions based on the same phosphopeptide substrate 3+ ) Protein tyrosine phosphatase 1B (PTP 1B) activity is determined by a fluorescence method mediated either by a calcein-assisted peptide cleavage in combination with fluorescence quenching based on graphene oxide. However, through the studies of the inventors, it was found that the conventional tyrosine phosphatase (PTP) assayThe method has the defects of low sensitivity, poor specificity in a complex biological system and the like.
Disclosure of Invention
In order to solve the defects of the prior art, the invention aims to provide a tyrosine phosphatase biosensor, a detection method and application thereof, and the detection sensitivity of the tyrosine phosphatase is high.
In order to achieve the above purpose, the technical scheme of the invention is as follows:
in one aspect, a tyrosine phosphatase biosensor comprises magnetic beads, peptide-DNA conjugates, DNA templates, and signaling probes;
the peptide-DNA conjugate comprises a peptide chain and a first DNA sequence, wherein one end of the peptide chain is connected with the 5' end of the first DNA sequence, and the peptide chain contains phosphorylated tyrosine; the other end of the peptide chain is used for connecting magnetic beads;
the DNA template comprises two second DNA sequences and a third DNA sequence from the 5 'end to the 3' end, the third DNA sequence can be complementary with the first DNA sequence, double-stranded DNA with two nicking enzyme recognition sites can be formed after hybridization and repolymerization of the DNA template and the first DNA sequence, and the complementary strand of the second DNA sequence is deoxyribozyme;
the signal probe is a nucleotide sequence, two ends of the nucleotide sequence are respectively connected with a fluorophore and a quencher, and the nucleotide sequence can be complementary with the deoxyribozyme; the deoxyribozymes are RNA sequences that cleave complementation in a divalent cation-dependent reaction.
In another aspect, a method for detecting tyrosine phosphatase provides the above tyrosine phosphatase biosensor;
performing dephosphorylation reaction on magnetic beads of the surface-connected peptide-DNA conjugate and a solution containing tyrosine phosphatase, adding chymotrypsin into a system after the dephosphorylation reaction for performing cleavage reaction, performing magnetic separation to remove the magnetic beads after the cleavage reaction to obtain a cleavage product, performing three-stage DNA amplification reaction on the cleavage product, a DNA template and a signal probe under the action of divalent cations, and performing fluorescence detection on the solution system after the three-stage DNA amplification reaction.
The three-stage DNA amplification reaction is to amplify the cleavage product and the DNA template to produce deoxyribozyme, and to amplify the deoxyribozyme and the DNA template to produce great amount of deoxyribozyme, and to make the deoxyribozyme and the signal probe react circularly under the action of bivalent cation.
In a third aspect, an application of the above-mentioned tyrosine phosphatase biosensor in preparing a reagent for detecting tyrosine phosphatase is provided.
In a fourth aspect, the use of a tyrosine phosphatase biosensor or a method of detecting tyrosine phosphatase as described above for screening for a tyrosine phosphatase inhibitor.
In a fifth aspect, a tyrosine phosphatase detection kit comprises the above tyrosine phosphatase biosensor, chymotrypsin, DNA polymerase, nicking enzyme, magnesium salt and buffer solution.
The beneficial effects of the invention are as follows:
1. the invention has higher sensitivity, which can be attributed to the following three factors: (1) PTP 1B-induced tyrosine dephosphorylation and chymotrypsin-mediated peptide cleavage have high specificity (2) SDA and DNAzyme-mediated signal probe cleavage have high efficiency, and (3) magnetic separation produces low background signals.
2. The invention uses peptide-DNA conjugate as reaction substrate to convert peptide detection into DNA detection. The invention can make the produced DNA with amino acid residue quickly convert into new DNA without amino acid residue, and improves the amplification efficiency.
3. The method has good specificity, so that in the reaction process of protein tyrosine phosphatase 1B (PTP 1B), the acting object of enzyme is very accurate and only acts on tyrosine, so that the method has good specificity and does not respond to non-specific substrates.
Drawings
The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the invention.
FIG. 1 is a schematic diagram of the dephosphorylation-induced tertiary DNA amplification reaction used in a tyrosine phosphatase (PTP) assay in accordance with an embodiment of the present invention. A is a schematic diagram of dephosphorylation cleavage of a peptide-DNA conjugate, and B is a schematic diagram of an assay procedure.
FIG. 2 is a graph showing the results of feasibility experiments in accordance with the embodiments of the invention. (A) Non-denaturing polyacrylamide gel electrophoresis experimental analysis of chymotrypsin cleavage products; channel M, DNA Maker; channel 1, protein tyrosine phosphatase 1B (PTP 1B) and Magnetic Bead (MBs) -linked peptide-DNA conjugates; channel 2, magnetic bead-bound peptide-DNA conjugate; channel 3, protein tyrosine phosphatase 1B (PTP 1B) and peptide-DNA conjugate; channel 4, peptide-DNA conjugate. (B) And (5) non-denaturing polyacrylamide gel electrophoresis experimental analysis of the amplification reaction products. Channel M, DNA Marker; channel 1, reaction product in the presence of protein tyrosine phosphatase 1B (PTP 1B); channel 2, product in the absence of protein tyrosine phosphatase 1B (PTP 1B). (C) The black and red curves represent the fluorescence spectra of dephosphorylation-induced tertiary DNA amplification reactions in the absence and presence of protein tyrosine phosphatase 1B (PTP 1B), respectively. Protein tyrosine phosphatase 1B (PTP 1B) concentration was 4 nanomoles per liter.
FIG. 3 is a graph showing the sensitivity test results of the embodiment of the present invention. (A) Changes in fluorescence spectra corresponding to protein tyrosine phosphatase 1B (PTP 1B) at different concentrations. (B) Changes in fluorescence intensity corresponding to protein tyrosine phosphatase 1B (PTP 1B) at different concentrations. The inset shows the linear relationship between fluorescence intensity and the logarithmic value of protein tyrosine phosphatase 1B (PTP 1B) concentration. Error bars represent standard deviation of three independent experiments.
FIG. 4 is a graph showing the characterization of the results of the specificity experiments of the examples of the present invention. The fluorescence intensity was varied for 4 nanomoles per liter of protein tyrosine phosphatase 1B (PTP 1B), 4 nanomoles per liter of PP2A,40 units per milliliter of PNK,0.005 grams per liter of IgG, and 0.005 grams per liter of BSA, respectively. Error bars represent standard deviation of three independent experiments.
FIG. 5 is a graph showing the characterization of the results of inhibitor analysis experiments in accordance with embodiments of the present invention. (A) Protein tyrosine phosphatase 1B (PTP 1B) relative activity in RK-682 at various concentrations. (B) Na at different concentrations 3 VO 4 Protein tyrosine phosphatase 1B (PTP 1B) relative activity. (C) Na of different concentrations 3 VO 4 The relative activity of the cell extracts in (a). The cell extract was 10000 HepG2 cells. Protein tyrosine phosphatase 1B (PTP 1B) concentration was 4 nanomoles per liter. Error bars represent standard deviation of three independent experiments.
Detailed Description
It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the invention. Unless defined otherwise, 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 belongs.
It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of exemplary embodiments according to the present invention. As used herein, the singular is also intended to include the plural unless the context clearly indicates otherwise, and furthermore, it is to be understood that the terms "comprises" and/or "comprising" when used in this specification are taken to specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof.
In view of the defects of low sensitivity, poor specificity and the like of the existing method for detecting the tyrosine phosphatase, the invention provides a tyrosine phosphatase biosensor, and a detection method and application thereof.
In an exemplary embodiment of the present invention, there is provided a tyrosine phosphatase biosensor comprising a magnetic bead, a peptide-DNA conjugate, a DNA template, and a signaling probe;
the peptide-DNA conjugate comprises a peptide chain and a first DNA sequence, wherein one end of the peptide chain is connected with the 5' end of the first DNA sequence, and the peptide chain contains phosphorylated tyrosine; the other end of the peptide chain is used for connecting magnetic beads;
the DNA template comprises two second DNA sequences and a third DNA sequence from the 5 'end to the 3' end, the third DNA sequence can be complementary with the first DNA sequence, double-stranded DNA with two nicking enzyme recognition sites can be formed after hybridization and repolymerization of the DNA template and the first DNA sequence, and the complementary strand of the second DNA sequence is deoxyribozyme;
the signal probe is a nucleotide sequence, two ends of the nucleotide sequence are respectively connected with a fluorophore and a quencher, and the nucleotide sequence can be complementary with the deoxyribozyme; the deoxyribozymes are RNA sequences that cleave complementation in a divalent cation-dependent reaction.
In the presence of Protein Tyrosine Phosphatases (PTPs), tyrosine in peptide-DNA conjugates can be dephosphorylated and cleaved by chymotrypsin, releasing short single stranded DNA (ssDNA) with amino acid residues. However, the DNA obtained by peptide cleavage contains amino acid residues, and the amplification efficiency of the DNA is limited due to steric hindrance effects. The single-stranded DNA (ssDNA) obtained in the present invention hybridizes with the 3' -end sequence of the DNA template, and the DNA polymerase initiates polymerization to form double-stranded DNA (dsDNA) having two nicking enzyme recognition sites. Nicking enzymes cleave one strand of double-stranded DNA (dsDNA) at a recognition site, creating two new replication sites. Under the catalysis of DNA polymerase and nicking enzyme, strand displacement reactions are continually initiated, producing large numbers of deoxyribose nucleic acid (DNAzyme) sequences. The deoxyribose enzyme (DNAzyme) sequence can hybridize with a new free DNA template, inducing a new round of polymerization, cleavage and release of the deoxyribose enzyme (DNAzyme) sequence, thereby priming DNA exponential amplification, releasing a large number of dnazymes (DNAzyme) continuously. These deoxyribozymes (DNAzyme) can hybridize with the signaling probe moiety in a sequence that is complementary to the sequence of magnesium (Mg 2+ ) With the aid of cleavage of the signaling probe at the intermediate positions of rA and rU, the dissociation of the fluorophore-quencher is induced, thus generating a strong fluorescent signal and releasing the deoxyribose nucleic acid (DNAzyme). The released deoxyribose enzyme (DNAzyme) can be hybridized with a new signaling probe to induce the cyclic cleavage of a large number of signaling probes, and finally the fluorescent signal is obviously amplified. In contrast, tyrosine in peptide-DNA conjugates cannot be cleaved in the absence of Protein Tyrosine Phosphatase (PTP). Therefore, neither release of single-stranded DNA (ssDNA) nor initiation of the tertiary DNA amplification reaction occurs, and thus a fluorescent signal cannot be detected.
In some examples of this embodiment, the peptide-DNA conjugate is attached to the magnetic bead by binding of streptavidin to biotin.
In some examples of this embodiment, the fluorophore is Cy3 and the quencher is BHQ2.
In some examples of this embodiment, the nicking enzyme is nb.
In some examples of this embodiment, the junction between the peptide chain and the first DNA sequence is 2 to 4 amino acids linked to a tyrosine containing phosphorylation.
In some examples of this embodiment, the peptide-DNA conjugate has the sequence:
Lys-Gly-Asp-Gly-Val-pTyr-Ala-Ala-Cys-GGA TCG TCA TGT ACG GCA GCT；
the sequence of the DNA template is: CAT GAG ATT CGT TGT AGC TAG CCT CAA CCC TCA GCC ATG AGA TTC GTT GTA GCT AGC CTC AAC CCT CAG CTG CCG TAC ATG ACG ATC C;
the sequence of the signal probe is as follows: CAT GAG ATA UTC AAC CCT C.
In another embodiment of the present invention, a method for detecting tyrosine phosphatase is provided, and the above tyrosine phosphatase biosensor is provided;
performing dephosphorylation reaction on magnetic beads of the surface-connected peptide-DNA conjugate and a solution containing tyrosine phosphatase, adding chymotrypsin into a system after the dephosphorylation reaction for performing cleavage reaction, performing magnetic separation to remove the magnetic beads after the cleavage reaction to obtain a cleavage product, performing three-stage DNA amplification reaction on the cleavage product, a DNA template and a signal probe under the action of divalent cations, and performing fluorescence detection on the solution system after the three-stage DNA amplification reaction.
The detection method of the present invention is preferably aimed at diagnosis and treatment of non-disease.
In some examples of this embodiment, the reaction temperature of the phosphorylation reaction is 35 to 40℃and the reaction time is 1.5 to 2.5 hours. Inactivating at 80-85 deg.c for 15-25 min after the phosphorylation reaction.
In some examples of this embodiment, the reaction temperature of the cleavage reaction is between 35 and 40℃and the reaction time is between 1.5 and 2.5 hours.
In some examples of this embodiment, the tertiary DNA amplification reaction is performed by first forming double-stranded DNA from the cleavage product and the DNA template, then adding a DNA polymerase and a nicking enzyme to perform a strand displacement amplification reaction, and adding a signaling probe to the system after the strand displacement amplification reaction to incubate.
In one or more embodiments, the DNA polymerase is Vent (exo-) DNA polymerase.
In one or more embodiments, the conditions for double stranded DNA are: heating at 95-100 deg.c for 4-6 min and cooling to room temperature. The room temperature refers to the temperature of the indoor environment, and is generally 15-30 ℃.
In one or more embodiments, the conditions of the strand displacement amplification reaction are: the temperature is 50-60 ℃ and the time is 50-70 min.
In one or more embodiments, the incubation temperature is 35 to 40℃and the incubation time is 1.5 to 2.5 hours.
The third embodiment of the invention provides an application of the tyrosine phosphatase biosensor in preparing a reagent for detecting tyrosine phosphatase.
The fourth embodiment of the invention provides an application of the tyrosine phosphatase biosensor or the detection method of the tyrosine phosphatase in screening of tyrosine phosphatase inhibitors.
In a fifth embodiment of the present invention, there is provided a tyrosine phosphatase detection kit comprising the above tyrosine phosphatase biosensor, chymotrypsin, DNA polymerase, nicking enzyme, magnesium salt, and buffer.
In order to enable those skilled in the art to more clearly understand the technical scheme of the present invention, the technical scheme of the present invention will be described in detail with reference to specific embodiments.
Examples
Ligation of substrates with streptavidin coated magnetic beads: ligation of the substrate to the Magnetic Beads (MBs) was performed according to Invitrogen's instructions. 200 microliter of streptavidin-coated Magnetic Beads (MBs) solution (10 mg/ml) was added to the centrifuge tube and washed twice with 1 Xphosphate buffer (PBS) (pH 7.4). The supernatant was removed by magnetic separation and the beads (MBs) were resuspended to 200. Mu.l. Then, 2. Mu.l of peptide-DNA conjugate (100. Mu.mol/l) and 20. Mu.l of 1 XPBS were mixed with 128. Mu.l of the washed Magnetic Beads (MBs) solution and the mixture was incubated on a mixer for 30 minutes at room temperature. The mixture was magnetically separated to remove unbound substrate and washed 5 times with 1 x Phosphate Buffer (PBS). The mixture was then resuspended to a total volume of 80. Mu.l with 1 XPhosphate buffer (PBS) and the final concentration of magnetic bead-bound peptide-DNA conjugate was 2.5. Mu. Moles per liter.
Protein tyrosine phosphatase 1B (PTP 1B) induced tyrosine dephosphorylation reaction and continuous cleavage reaction of peptide-DNA conjugates: 12 microliter of Magnetic Bead (MBs) linked peptide-DNA conjugate was added to a dephosphorylation reaction system (50 microliters) containing various concentrations of protein tyrosine phosphatase 1B (PTP 1B) and 1 XPP 1B reaction buffer (10 millimoles per liter of 4- (2-hydroxyethyl) -1-piperazine ethanesulfonic acid (HEPES), 1 millimoles per liter of Dithiothreitol (DTT), 2 millimoles per liter of ethylenediamine tetraacetic acid (EDTA)), and incubated for 2 hours at 37 ℃. After the dephosphorylation reaction was completed, the reaction mixture was inactivated at 80℃for 20 minutes. Then, 0.6. Mu.l chymotrypsin (1 mg/ml) was added to the cleavage reaction system (60. Mu.l), followed by incubation at 37℃for 2 hours, and after completion of the cleavage reaction, magnetic separation was performed for 3 minutes, and then the supernatant was collected.
Three-stage DNA amplification reaction based on dephosphorylation product-induced DNAzyme: after the dephosphorylation and cleavage reactions, 30. Mu.l of the supernatant was added to a hybridization reaction system containing 0.7. Mu.l of template at a concentration of 5. Mu.l/l, 3.5. Mu.l of 10 Xannealing buffer (50. Mu.l/l magnesium chloride (MgCl) 2 ) 100 millimoles per liter of Tris-HCl and 0.8 microliter of water, heated at 95 ℃ for 5 minutes, and then cooled slowly to room temperature to form double stranded DNA (dsDNA). After the annealing reaction, the strand displacement amplification reaction was performed in 50. Mu.l of a reaction mixture containing 35. Mu.l of annealed product, 400 mmol dNTPs per l, 8 units per ml of Vent (exo-) DNA polymerase, 0.3 units per. Mu.l of Nb.BbvCI, 5. Mu.l of 10 XCutsmart buffer, and incubated at 55℃for 1 hour. After the strand displacement amplification reaction, 50. Mu.l was usedThe reaction product was mixed with 2.4. Mu.l of signaling probe at a concentration of 10. Mu.mol per liter, 1. Mu.l of 10 XCutsmart buffer and 6.6. Mu.l of water and incubated at 37℃for 1 hour for cyclic cleavage of the signaling probe.
Gel electrophoresis and fluorescence detection: chymotrypsin-catalyzed cleavage reaction products were analyzed by 14% non-denaturing polyacrylamide gel electrophoresis experiments at a constant voltage of 100 volts in 1 XTBE buffer (9 mmol per liter of Tris-HCl), 9 mmol per liter of boric acid (boric acid), 0.2 mmol per liter of ethylenediamine tetraacetic acid (EDTA), pH 8.3) for 90 minutes. The gel was stained with SYBR glass and imaged with the Chemidoc MP imaging system. The products of the tertiary DNA amplification reaction were analyzed by a 12% non-denaturing polyacrylamide gel electrophoresis experiment in 1 XTBE buffer at a constant voltage of 110 volts for 45 minutes. The gel was stained with SYBR glass and imaged with the Chemidoc MP imaging system. For the fluorometry, 60. Mu.l of the amplified product was diluted with ultrapure water to a final volume of 80. Mu.l, and then the fluorometry was performed. Fluorescence intensity at 569 nm emission wavelength was detected with a Hitachi F-7000 spectrometer at an excitation wavelength of 512 nm, and emission spectra were recorded in the wavelength range of 550 to 750 nm, with excitation and emission slits each having a width of 5 nm. Ft-F 0 Concentration of protein tyrosine phosphatase 1B (PTP 1B) was calculated using regression equation, wherein F 0 And Ft represents fluorescence intensity at 569 nm in the absence and presence of protein tyrosine phosphatase 1B (PTP 1B), respectively.
Inhibitor experiments: protein tyrosine phosphatase 1B (PTP 1B) (4 nanomoles per liter) was combined with varying concentrations of (R) -4-hydroxy-5- (hydroxymethyl) -3- (1-oxohexadecyl) -2 (5H) -furanone (RK-682) or sodium vanadate (Na) 3 VO 4 ) After pre-incubation at 37℃for 10 minutes, subsequent experiments were performed following the same procedure as described above for protein tyrosine phosphatase 1B (PTP 1B) analysis, and finally fluorescence detection was performed.
Cell culture and extraction experiments: human embryonic kidney fines were cultured in Dulbecco's Modified Eagle's Medium (DMEM) containing 10% Fetal Bovine Serum (FBS) and 1% penicillin streptomycinCell line (HEK-293 cell), human cervical cancer cell line (HeLa) and human breast cancer cell line (MCF-7). In a mixture containing 10% Fetal Bovine Serum (FBS), 1% penicillin-streptomycin, 0.11 g per liter sodium pyruvate (CH) 3 COCOONa) and 1.5 g per liter sodium bicarbonate (NaHCO) 3 ) Human hepatoma cell line (HepG 2) was cultured in Minimum Eagle Medium (MEM). All cells were at 37℃with 5% CO 2 Is cultured in the environment of (2). In the cell extraction experiments, cells were collected by trypsinization and washed twice with 1 x Phosphate Buffer (PBS) (pH 7.4). The cells were then collected in a centrifuge tube and centrifuged at 800 revolutions per minute (rpm) for 5 minutes. Cell numbers were measured by a Countstar cell counter. Will be about 1X 10 6 Individual cells were suspended in 100 μl RIPA lysis buffer (50 mmol per liter Tris (pH 7.4), 150 mmol per liter sodium chloride (NaCl), 1% polyethylene glycol octylphenyl ether (Triton X-100), 1% sodium deoxycholate (sodium deoxycholate), 0.1% Sodium Dodecyl Sulfate (SDS)) and incubated on ice for 10 minutes, followed by sonication. Lysates were centrifuged at 14000 Xg for 5 min at 4℃and the supernatant was then assayed by measuring absorbance at 280 nm with a Nanodrop 2000C spectrophotometer to quantify total protein concentration.
The sequences employed in the examples are shown in Table 1.
Table 1 sequences used in the examples
pTyr refers to phosphorylated Tyr, and in DNA templates, the underlined regions represent the recognition sequence of the nicking enzyme (nb.bvci), the italicized regions represent the complement to DNAzyme. In the signaling probe, two bases "T" underlined indicate that the base is modified with fluorescein (Cy 3) and quencher 2 (BHQ 2), respectively, and bases rA and rU indicate adenine ribonucleotide and uracil ribonucleotide, respectively.
Wherein the peptide-DNA substrate is formed by joining a polypeptide domain and a DNA domain, the polypeptide domain sequence: lys-Gly-Asp-Gly-Val-pTyr-Ala-Ala-Cys, see SEQ ID NO.1.
DNA domain sequence: GGA TCG TCA TGT ACG GCA GCT, SEQ ID NO.2.
The experimental principle is as shown in fig. 1: three steps are involved: (1) protein tyrosine phosphatase 1B (PTP 1B) -catalyzed tyrosine dephosphorylation reaction induces chymotrypsin cleavage of peptide-DNA conjugate (2) peptide-DNA conjugate cleavage-induced exponential Strand Displacement Amplification (SDA) to produce deoxyribose enzyme (DNAzyme) (3) cycle cleavage of the signaling probe by DNAzyme resulting in amplification of the fluorescent signal. As shown in FIG. 1A, in the presence of protein tyrosine phosphatase 1B (PTP 1B), the tyrosine in the peptide-DNA conjugate can be dephosphorylated and cleaved by chymotrypsin. Detailed assay procedure peptide-DNA conjugates were immobilized on Magnetic Beads (MBs) by biotin-streptavidin interactions, as shown in figure 1B. The presence of protein tyrosine phosphatase 1B (PTP 1B) causes a dephosphorylation reaction on tyrosine, and subsequent cleavage of the peptide-DNA conjugate by chymotrypsin releases short single-stranded DNA (ssDNA) with three amino acid residues. After magnetic separation, the resulting single stranded DNA (ssDNA) can be hybridized to the 3' end sequence of the DNA template to initiate polymerization with Vent (exo-) DNA polymerase to form a double stranded DNA (dsDNA) duplex with two nicking enzyme (nb.bvci) recognition sites. BbvCI can cleave one strand of a double-stranded DNA (dsDNA) duplex to create two new replication sites. Under the catalysis of DNA polymerase and nicking enzyme, strand displacement reaction is continuously initiated, and a large amount of deoxyribozyme (DNAzyme) is produced. Notably, the deoxyribozymes (dnazymes) can hybridize to new free DNA templates to induce a new round of polymerization, cleavage, and release of the deoxyribozymes (dnazymes) resulting in exponential amplification, resulting in a large number of deoxyribozymes (dnazymes). The obtained deoxyribose enzyme (DNAzyme) is a "10-23" type deoxyribose enzyme (DNAzyme) capable of cleaving RNA, and can cleave complementary RNA with high efficiency in a divalent cation-dependent reaction. These deoxyribozymes (DNAzyme) can hybridize with the signaling probe moiety in the presence of the cofactor magnesium ion (Mg 2+ ) With the aid of cleavage of the intermediate position of the signaling probes rA and rU, the dissociation of the fluorophore and quencher is induced, which ultimately leads to an increase in the fluorescence signal andthe deoxyribose enzyme (DNAzyme) is released. The released deoxyribose enzyme (DNAzyme) can be hybridized with a new signaling probe to induce cyclic cleavage of a large number of signaling probes, and finally the fluorescent signal can be amplified significantly. In contrast, in the absence of protein tyrosine phosphatase 1B (PTP 1B), tyrosine in peptide-DNA conjugates cannot be cleaved. Therefore, neither release of single-stranded DNA (ssDNA) nor tertiary DNA amplification reaction occurs, and no fluorescent signal is detected.
Feasibility test for detecting protein tyrosine phosphatase 1B (PTP 1B)
To verify the feasibility of this method, the present example performed an experimental analysis of 14% non-denaturing polyacrylamide gel electrophoresis. As shown in FIG. 2A, a distinct band was observed in the presence of protein tyrosine phosphatase 1B (PTP 1B) and peptide-DNA conjugate (channel 3 of FIG. 2A), and a distinct band was also observed at the same position after magnetic separation in the presence of protein tyrosine phosphatase 1B (PTP 1B) and Magnetic Bead (MBs) -linked peptide-DNA conjugate (channel 1 of FIG. 2A). This suggests that detecting the presence of protein tyrosine phosphatase 1B (PTP 1B) induces dephosphorylation of peptide DNA substrates and is cleaved consecutively by chymotrypsin to produce single stranded DNA (ssDNA) with three amino acid residues. In contrast, after magnetic separation, no bands were observed in the presence of the peptide-DNA conjugate with only Magnetic Beads (MBs) (channel 2 of FIG. 2A), and a more pronounced band corresponding to the intact peptide-DNA conjugate was observed with only the peptide-DNA conjugate. This channel (4 th channel of FIG. 2A) indicates that the peptide-DNA conjugate cannot be cleaved in the absence of protein tyrosine phosphatase 1B (PTP 1B). To verify the peptide cleavage induced exponential DNA amplification reaction, this example uses a 12% non-denaturing polyacrylamide gel electrophoresis experiment to analyze the amplified product after magnetic separation. In the absence of protein tyrosine phosphatase 1B (PTP 1B), two DNA template bands (88 nt) and signaling probe (19 nt) were observed (FIG. 2B, channel 1). In addition to the two DNA template bands (88 nt) and signaling probe (19 nt), three new amplification product bands (68, 33-35 and 9-10 nt) were observed when protein tyrosine phosphatase 1B (PTP 1B) was present (channel 2, FIG. 2B), indicating that the presence of protein tyrosine phosphatase 1B (PTP 1B) prompted the dephosphorylation reaction and chymotrypsin continuous cleavage reaction of the peptide-DNA conjugate to occur, inducing the SDA reaction to occur, thereby generating a polymeric intermediate (68 nt) and an amplification product (33-35 nt) and subsequent DNAzyme induced cyclic cleavage signaling probe reaction to produce shorter cleavage fragments (9-10 nt). The present example further performed fluorescence detection to verify the experiment (fig. 2C). In the absence of detection protein tyrosine phosphatase 1B (PTP 1B), no distinct fluorescent signal was detected (fig. 2C). In contrast, a distinct fluorescent signal was observed in the presence of protein tyrosine phosphatase 1B (PTP 1B) (fig. 2C), indicating that the presence of protein tyrosine phosphatase 1B (PTP 1B) promotes the occurrence of a tyrosine dephosphorylation reaction and a chymotrypsin continuous cleavage peptide-DNA conjugate reaction, the product of which induces an index SDA, yielding a deoxyribose enzyme (DNAzyme) that can catalyze the cyclic cleavage of signaling probes to produce a distinct fluorescent signal. These results indicate that the present protocol is viable for accurate detection of protein tyrosine phosphatase 1B (PTP 1B) activity.
Sensitivity detection
In order to evaluate the sensitivity of detecting the activity of protein tyrosine phosphatase 1B (PTP 1B), the present example carried out analytical measurement of protein tyrosine phosphatase 1B (PTP 1B) at different concentrations, and the results were shown in FIG. 3A, in which fluorescence intensity was enhanced as the concentration of protein tyrosine phosphatase 1B (PTP 1B) was detected from 0.5 picomole per liter to 4 nanomole per liter, and in the range of 0.5 picomole per liter to 100 picomole per liter, the fluorescence intensity exhibited a good linear relationship with the logarithmic value of the concentration of protein tyrosine phosphatase 1B (PTP 1B) (R 2 = 0.9940) (inset of fig. 3B). Regression equation was f=370.22+591.57 log 10 C, wherein F is fluorescence intensity and C is protein tyrosine phosphatase 1B (PTP 1B) concentration. The detection limit was calculated to be 0.24 picomoles per liter (8.97X10) -6 Micrograms per milliliter). The sensitivity of the method was improved by 6 orders of magnitude compared to fluorescent probe-based fluorometry (5.7. Mu.g/ml), by 25-fold compared to chemical sensor-based fluorometry (6. Mu.mol/l), and to calcein-based fluorometry (3X 10) -5 Micrograms per milliliter) And graphene oxide-based fluorometry (1×10 -5 Micrograms per milliliter) was equivalent.
Specificity experiments of PTP
To evaluate the specificity of this protocol, protein phosphatase 2A (PP 2A), polynucleotide kinase (PNK), immunoglobulin G (IgG) and Bovine Serum Albumin (BSA) were used as negative controls. PP2A and PNK are involved in the regulation of intracellular phosphorylation levels. PP2A catalyzes threonine dephosphorylation, but is inactive against tyrosine. PNK can catalyze the transfer of a terminal phosphate group from Adenosine Triphosphate (ATP) to the 5' hydroxyl end of an oligonucleotide. IgG and BSA are two non-specific proteins that have no dephosphorylase activity. As shown in FIG. 4, neither PP2A, PNK, igG nor BSA detected a distinct fluorescent signal, but protein tyrosine phosphatase 1B (PTP 1B) detected a high fluorescent signal, indicating that the presence of protein tyrosine phosphatase 1B (PTP 1B) prompted the tyrosine dephosphorylation reaction and the chymotrypsin continuous cleavage of the peptide-DNA conjugate reaction to occur, the products of which induced a tertiary DNA amplification reaction to generate a stronger fluorescent signal. These results indicate that the proposed method has good specificity for protein tyrosine phosphatase 1B (PTP 1B).
Inhibitor analysis
To examine the feasibility of the method in inhibition experiments, the present example used (R) -4-hydroxy-5- (hydroxymethyl) -3- (1-oxohexadecyl) -2 (5H) -furanone (RK-682) and sodium vanadate (Na) 3 VO 4 ) As inhibitors. As shown in FIG. 5, the relative activities of protein tyrosine phosphatase 1B (PTP 1B) were associated with RK-682 (FIG. 5A) and Na, respectively 3 VO 4 (FIG. 5B) the concentration increases and decreases. IC for calculating RK-682 50 A value of 3.03. Mu. Mol/liter, na 3 VO 4 Is of (2) 50 The value was 2.18 nanomoles per liter, which is consistent with the values obtained for calcein-based and graphene oxide-based fluorometry. Previous studies have shown that protein tyrosine phosphatase 1B (PTP 1B) is involved in insulin signaling regulation. This example evaluates the inhibition ability of inhibitors against human hepatoma cells (HepG 2 cells). When Na is 3 VO 4 From 0 to 1000 nanomoleThe relative activity of protein tyrosine phosphatase 1B (PTP 1B) was correspondingly reduced per liter in HepG2 cells (fig. 5C). IC (integrated circuit) 50 The value calculated was 4.97 nanomoles per liter, consistent with the value obtained with pure protein tyrosine phosphatase 1B (PTP 1B) (2.18 nanomoles per liter) (fig. 5B). These results indicate that the method can be used to screen protein tyrosine phosphatase 1B (PTP 1B) inhibitors.
The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
SEQUENCE LISTING
<110> Shandong university of teachers and students
<120> a tyrosine phosphatase biosensor, and detection method and application thereof
<130>
<160> 4
<170> PatentIn version 3.3
<210> 1
<211> 9
<212> PRT
<213> artificial sequence
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Lys Gly Asp Gly Val Tyr Ala Ala Cys
1 5
<210> 2
<211> 21
<212> DNA
<213> artificial sequence
<400> 2
ggatcgtcat gtacggcagc t 21
<210> 3
<211> 88
<212> DNA
<213> artificial sequence
<400> 3
catgagattc gttgtagcta gcctcaaccc tcagccatga gattcgttgt agctagcctc 60
aaccctcagc tgccgtacat gacgatcc 88
<210> 4
<211> 19
<212> DNA
<213> artificial sequence
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catgagatau tcaaccctc 19
Claims (10)
1. A tyrosine phosphatase biosensor is characterized by comprising magnetic beads, peptide-DNA conjugates, a DNA template, a signal probe, chymotrypsin, vent (exo-) DNA polymerase and nicking enzyme Nb.BbvCI;
the peptide-DNA conjugate comprises a peptide chain and a first DNA sequence, wherein one end of the peptide chain is connected with the 5' end of the first DNA sequence, and the peptide chain contains phosphorylated tyrosine; the other end of the peptide chain is used for connecting magnetic beads;
the DNA template comprises two second DNA sequences and a third DNA sequence from the 5 'end to the 3' end, the third DNA sequence can be complementary with the first DNA sequence, double-stranded DNA with two nicking enzyme recognition sites can be formed after hybridization and repolymerization of the DNA template and the first DNA sequence, and the complementary strand of the second DNA sequence is deoxyribozyme;
the signal probe is a nucleotide sequence which can be complementary to a deoxyribozyme; the deoxyribozyme is an RNA sequence which is complementary by cleavage in a divalent cation dependent reaction;
the tyrosine phosphatase is protein tyrosine phosphatase 1B;
the sequence of the peptide-DNA conjugate is: lys-Gly-Asp-Gly-Val-pTyr-Ala-Ala-Cys-GGA TCG TCA TGT ACG GCA GCT;
the sequence of the DNA template is: CAT GAG ATT CGT TGT AGC TAG CCT CAA CCC TCA GCC ATG AGA TTC GTT GTA GCT AGC CTC AAC CCT CAG CTG CCG TAC ATG ACG ATC C;
the sequence of the signal probe is as follows: CAT GAG A (Cy 3)TrA rUTC AAC CC(BHQ2)TC, performing operation; in the signaling probe, two underlined bases "T" indicate that the base is modified with Cy3 and BHQ2, respectively, and bases rA and rU indicate adenine ribonucleotide, respectivelyAnd uracil ribonucleotides.
2. The tyrosine phosphatase biosensor according to claim 1, wherein the peptide-DNA conjugate is attached to the magnetic bead by binding of streptavidin to biotin.
3. A method for detecting tyrosine phosphatase, which is characterized in that the method does not comprise diagnosis and treatment of diseases, and provides the tyrosine phosphatase biosensor according to any one of claims 1-2;
performing dephosphorylation reaction on magnetic beads of the surface-connected peptide-DNA conjugate and a solution containing tyrosine phosphatase, adding chymotrypsin into a system after the dephosphorylation reaction for performing cleavage reaction, performing magnetic separation to remove the magnetic beads after the cleavage reaction to obtain a cleavage product, performing three-stage DNA amplification reaction on the cleavage product, a DNA template and a signal probe under the action of divalent cations, and performing fluorescence detection on the solution system after the three-stage DNA amplification reaction.
4. The method for detecting tyrosine phosphatase according to claim 3, wherein the reaction temperature of the phosphorylation reaction is 35-40 ℃ and the reaction time is 1.5-2.5 hours;
or the reaction temperature of the cracking reaction is 35-40 ℃ and the reaction time is 1.5-2.5 h;
or, the three-stage DNA amplification reaction is carried out by forming double-chain DNA by the cleavage product and the DNA template, then adding DNA polymerase and nicking enzyme for strand displacement amplification reaction, adding signal probe into the system after strand displacement amplification reaction for incubation, wherein the DNA polymerase is Vent (exo-) DNA polymerase, and the nicking enzyme is Nb.BbvCI.
5. The method for detecting tyrosine phosphatase according to claim 4, wherein the conditions of double-stranded DNA are: heating at 95-100 ℃ for 4-6 minutes, and then cooling to room temperature.
6. The method for detecting tyrosine phosphatase according to claim 4, wherein the conditions for the strand displacement amplification reaction are as follows: the temperature is 50-60 ℃ and the time is 50-70 min.
7. The method for detecting tyrosine phosphatase according to claim 4, wherein the incubation temperature is 35-40 ℃ and the incubation time is 1.5-2.5 h.
8. Use of a tyrosine phosphatase biosensor according to any one of claims 1-2 in the preparation of a reagent for detecting tyrosine phosphatase.
9. Use of the tyrosine phosphatase biosensor according to any one of claims 1-2 or the detection method of tyrosine phosphatase according to any one of claims 3-7 in screening of tyrosine phosphatase inhibitors.
10. A tyrosine phosphatase detection kit is characterized by comprising the tyrosine phosphatase biosensor according to any one of claims 1-2, magnesium salt and buffer solution.
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