CN111979295A - Tyrosine phosphatase biosensor and detection method and application thereof - Google Patents
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Abstract
The invention discloses a tyrosine phosphatase biosensor and a detection method and application thereof, wherein the tyrosine phosphatase biosensor 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 a 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 consists of 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, the DNA template can form double-stranded DNA with two nicking enzyme recognition sites after being hybridized and polymerized with 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; dnazymes are complementary RNA sequences that cleave 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 and a detection method and application thereof.
Background
The information in this background section is only for enhancement of 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 that is already known to a person of ordinary skill in the art.
Tyrosine phosphorylation is precisely regulated by the intracellular interactions of tyrosine kinases (PTKs) and tyrosine phosphatases (PTPs) to achieve a balance. However, the role of tyrosine phosphatases (PTPs) has been underestimated in regulating tyrosine phosphate homeostasis. Recent studies have shown that the protein tyrosine phosphatase 1B (PTP1B) may play a key negative regulatory role in insulin/leptin signaling, and that aberrant activity may contribute to type II diabetes and obesity. In addition, dysregulation of other tyrosine phosphatases (PTPs) is closely associated with a variety of human diseases and cancers. Therefore, efficient and sensitive detection of tyrosine phosphatase (PTP) activity is of great importance for clinical diagnosis, drug discovery and cancer treatment.
To the best of the inventors' knowledge, there are assays for the conventional tyrosine phosphatase (PTP) assay32P/33A method of radiolabeling a radioactive phosphate substance released from a P-labeled phosphorylated peptide/protein substrate and a method of quantitative analysis for measuring the released radioactive phosphate substance by a liquid scintillation analyzer, but this method is liable to cause radioactive contamination and has low sensitivity. In recent years, nonradioactive methods including colorimetric methods and fluorescent methods have been developed, in which the activity of tyrosine phosphatase (PTP) in phosphorylation reaction is quantitatively analyzed using absorbance/fluorescence intensity signals when organic phosphates (e.g., p-nitrophenyl phosphate (pNPP), 4-methyl-7-hydroxycoumarin phosphate (MUP), and 3, 6-Fluorescein Diphosphate (FDP)) are used as substrates. However, these substrates are simulated natural phosphopeptide/protein substrates, have poor specificity, and may be affected by non-specific dephosphorylation of various phosphatases in complex environmentsAccurately detecting the activity of tyrosine phosphatase (PTP). The activity of tyrosine phosphatases (PTPs) is usually detected by measuring the released phosphate ion (Pi) by the malachite green colorimetric method when specific phosphopeptides are used as substrates, although this method is generally considered to be a standard PTP detection method, the background signal is high in complex samples. Cerium ion (Ce) initiated by dephosphorylation reaction based on the same phosphopeptide substrate3+) The activity of protein tyrosine phosphatase 1B (PTP1B) is determined by a mediated calcein fluorescence recovery method or a fluorescence method established by combining chymotrypsin-assisted peptide cleavage and graphene oxide fluorescence quenching. However, the inventors have found that the conventional tyrosine phosphatase (PTP) analysis method has the disadvantages 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.
In order to achieve the purpose, the technical scheme of the invention is as follows:
in one aspect, a tyrosine phosphatase biosensor comprises a magnetic bead, a peptide-DNA conjugate, a DNA template, and a signal 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 arranged to be connected with a magnetic bead;
the DNA template consists of 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, the DNA template can form double-stranded DNA with two nicking enzyme recognition sites after being hybridized and polymerized with 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; the dnazymes cleave complementary RNA sequences in a divalent cation-dependent reaction.
On the other hand, a method for detecting tyrosine phosphatase is provided, which comprises providing the above-mentioned tyrosine phosphatase biosensor;
carrying out dephosphorylation reaction on the magnetic beads with the surface connected with the peptide-DNA conjugate and a solution containing tyrosine phosphatase, adding chymotrypsin into a system after the dephosphorylation reaction for carrying out cracking reaction, carrying out magnetic separation after the cracking reaction to remove the magnetic beads so as to obtain a cracking product, carrying out three-stage DNA amplification reaction on the cracking product, a DNA template and a signal probe under the action of divalent cations, and carrying out fluorescence detection on the solution system after the three-stage DNA amplification reaction.
The three-stage DNA amplification reaction is that the cleavage product and a DNA template are subjected to first-stage amplification to generate deoxyribozymes, the deoxyribozymes and the DNA template are continuously subjected to second-stage amplification to generate a large amount of deoxyribozymes, and the deoxyribozymes and the signal probes are subjected to circular cutting signal probe reaction under the action of divalent cations.
In a third aspect, the application of the tyrosine phosphatase biosensor in preparing a reagent for detecting tyrosine phosphatase is provided.
In a fourth aspect, the tyrosine phosphatase biosensor or the method for detecting tyrosine phosphatase is used for screening a tyrosine phosphatase inhibitor.
In a fifth aspect, the tyrosine phosphatase detection kit comprises the above tyrosine phosphatase biosensor, chymotrypsin, DNA polymerase, nickase, magnesium salt, and buffer.
The invention has the beneficial effects that:
1. the invention has higher sensitivity and can be attributed to the following three factors: (1) PTP 1B-induced tyrosine dephosphorylation and chymotrypsin-mediated peptide cleavage had high specificity (2) the index SDA and DNAzyme-mediated cleavage of the signaling probe had high efficiency, and (3) the low background signal generated by magnetic separation.
2. The invention uses peptide-DNA conjugate as reaction substrate to convert peptide detection into DNA detection. The invention can quickly convert the generated DNA with amino acid residues into new DNA without amino acid residues, thereby improving the amplification efficiency.
3. The method has good specificity, so that in the process of protein tyrosine phosphatase 1B (PTP1B), the acting object of the enzyme is very accurate and only acts on tyrosine, so that the method has good specificity and no response to non-specific substrates.
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The accompanying drawings, which are incorporated in and constitute a part of this specification, are included to provide a further understanding of the invention, and are incorporated in and constitute a part of this specification, illustrate exemplary embodiments of the invention and together with the description serve to explain the invention and not to limit the invention.
FIG. 1 is a schematic diagram of the dephosphorylation-induced three-stage DNA amplification reaction used in the tyrosine phosphatase (PTP) assay experiment according to the present invention. A is a schematic representation of dephosphorylation cleavage of a peptide-DNA conjugate, and B is a schematic representation of the assay procedure.
FIG. 2 is a graph representing the results of a feasibility test performed in accordance with an embodiment of the present invention. (A) Performing non-denaturing polyacrylamide gel electrophoresis experimental analysis on the chymotrypsin cleavage product; channel M, DNA marker; channel 1, peptide-DNA conjugates of protein tyrosine phosphatase 1B (PTP1B) and Magnetic Beads (MBs); channel 2, magnetic bead-bound peptide-DNA conjugate; channel 3, protein tyrosine phosphatase 1B (PTP1B) and peptide-DNA conjugates; channel 4, peptide-DNA conjugate. (B) And (3) analyzing the amplification reaction product by using a non-denaturing polyacrylamide gel electrophoresis experiment. Channel M, DNA Marker; channel 1, the reaction product in the presence of protein tyrosine phosphatase 1B (PTP 1B); channel 2, the product in the absence of protein tyrosine phosphatase 1B (PTP 1B). (C) The black and red curves show the fluorescence spectra of the dephosphorylation-induced, tertiary DNA amplification reaction in the absence and presence of protein tyrosine phosphatase 1B (PTP1B), respectively. The protein tyrosine phosphatase 1B (PTP1B) concentration was 4 nmol per liter.
FIG. 3 is a graph representing the results of a sensitivity test according to an embodiment of the present invention. (A) Change in fluorescence spectra corresponding to different concentrations of protein tyrosine phosphatase 1B (PTP 1B). (B) Change in fluorescence intensity corresponding to different concentrations of protein tyrosine phosphatase 1B (PTP 1B). The graph shows the linear relationship between the fluorescence intensity and the logarithmic value of the concentration of protein tyrosine phosphatase 1B (PTP 1B). Error bars represent standard deviations of three independent experiments.
FIG. 4 is a characterization chart of the results of the specificity experiment according to the embodiment of the present invention. The change in fluorescence intensity corresponds to 4 nmol per liter of protein tyrosine phosphatase 1B (PTP1B), 4 nmol 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 deviations of three independent experiments.
FIG. 5 is a graph representing the results of an inhibitor analysis experiment according to an embodiment of the present invention. (A) The relative activity of the protein tyrosine phosphatase 1B (PTP1B) in different concentrations of RK-682. (B) At various concentrations of Na3VO4Protein tyrosine phosphatase 1B (PTP1B) in (1). (C) Different concentrations of Na3VO4The relative activity of the cell extract of (a). The cell extract was 10000 HepG2 cells. The protein tyrosine phosphatase 1B (PTP1B) concentration was 4 nmol per liter. Error bars represent standard deviations of three independent experiments.
Detailed Description
It is to be understood that the following detailed description is exemplary and is intended to provide further explanation of the invention as claimed. 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 invention. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof, unless the context clearly indicates otherwise.
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, a signal 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 arranged to be connected with a magnetic bead;
the DNA template consists of 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, the DNA template can form double-stranded DNA with two nicking enzyme recognition sites after being hybridized and polymerized with 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; the dnazymes cleave complementary RNA sequences in a divalent cation-dependent reaction.
In the presence of Protein Tyrosine Phosphatase (PTP), the tyrosine in peptide-DNA conjugates can be dephosphorylated and cleaved by chymotrypsin, which releases a short single-stranded DNA (ssDNA) with amino acid residues. However, the DNA obtained by peptide cleavage contains amino acid residues, which limit the amplification efficiency of the DNA due to steric hindrance effects. The single-stranded DNA (ssDNA) obtained by the invention is hybridized with the 3' end sequence of the DNA template, and the DNA polymerase starts polymerization reaction to form double-stranded DNA (dsDNA) with 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 nickase, strand displacement reaction is continuously initiated to generate a large amount of deoxyribozyme (DNAzyme) sequences. Deoxyribozyme (DNAzyme) sequences can be hybridized with new free DNA templates to induce a new round of polymerization, cleavage and release of the deoxyribozyme (DNAzyme) sequences, thereby initiating DNA exponential amplification with the continuous release of large amounts of deoxyribozyme (DNAzyme). These deoxyribozymes (DNAzymes) can hybridize to a signaling probe portion, the sequence of which is in magnesium ion (Mg)2+) With the aid of (2), in the middle of rA and rUThe cleavage signal probe is positioned to induce dissociation of the fluorophore-quencher, thereby generating an intense fluorescent signal and releasing the deoxyribozyme (DNAzyme). The released deoxyribozyme (DNAzyme) can be hybridized with a new signal probe to induce the cyclic cleavage of a large number of signal probes, and finally the fluorescence signal is obviously amplified. In contrast, tyrosine in peptide-DNA conjugates cannot be cleaved in the absence of Protein Tyrosine Phosphatases (PTPs). Therefore, neither release of single stranded DNA (ssdna) nor priming of the tertiary DNA amplification reaction can occur, and thus no fluorescence signal can be detected.
In some examples of this embodiment, the peptide-DNA conjugate is attached to the magnetic beads via streptavidin binding to biotin.
In some embodiments of this embodiment, the fluorophore is Cy3 and the quencher is BHQ 2.
In some embodiments of this embodiment, the nicking enzyme is nb.
In some embodiments of this embodiment, the junction of the peptide chain and the first DNA sequence is linked 2 to 4 amino acids to a tyrosine containing phosphorylation.
In some embodiments of this embodiment, the peptide-DNA conjugate has a sequence of:
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, respectively;
the sequence of the signal probe is: CAT GAG ATA UTC AAC CCT C are provided.
In another embodiment of the present invention, there is provided a method for detecting tyrosine phosphatase, comprising providing the above-mentioned tyrosine phosphatase biosensor;
carrying out dephosphorylation reaction on the magnetic beads with the surface connected with the peptide-DNA conjugate and a solution containing tyrosine phosphatase, adding chymotrypsin into a system after the dephosphorylation reaction for carrying out cracking reaction, carrying out magnetic separation after the cracking reaction to remove the magnetic beads so as to obtain a cracking product, carrying out three-stage DNA amplification reaction on the cracking product, a DNA template and a signal probe under the action of divalent cations, and carrying out 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-diseases.
In some examples of this embodiment, the temperature of the phosphorylation reaction is 35-40 ℃ and the reaction time is 1.5-2.5 hours. And inactivating the mixture for 15-25 minutes at 80-85 ℃ after phosphorylation reaction.
In some embodiments of this embodiment, the cracking reaction is performed at a temperature of 35-40 ℃ for 1.5-2.5 hours.
In some examples of this embodiment, the third-stage DNA amplification reaction is performed by forming double-stranded DNA from the cleavage product and the DNA template, then performing a strand displacement amplification reaction by adding DNA polymerase and nicking enzyme, and adding a signal probe to the system after the strand displacement amplification reaction for incubation.
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 for 4-6 minutes at 95-100 ℃, and then cooling to room temperature. The room temperature refers to the temperature of an 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 35-40 ℃ and the incubation time is 1.5-2.5 h.
In a third embodiment of the present invention, there is provided a use of the above-mentioned tyrosine phosphatase biosensor in the preparation of a reagent for detecting tyrosine phosphatase.
In a fourth embodiment of the present invention, there is provided a use of the above-mentioned tyrosine phosphatase biosensor or the method for detecting tyrosine phosphatase for screening a tyrosine phosphatase inhibitor.
In a fifth embodiment of the present invention, a tyrosine phosphatase detection kit is provided, which comprises the above-mentioned tyrosine phosphatase biosensor, chymotrypsin, DNA polymerase, nickase, magnesium salt, and buffer.
In order to make the technical solutions of the present invention more clearly understood by those skilled in the art, the technical solutions of the present invention will be described in detail below with reference to specific embodiments.
Examples
Ligation of the substrate to streptavidin-coated magnetic beads: the ligation of the substrates to Magnetic Beads (MBs) was performed according to the Invitrogen company instructions. 200 μ l of streptavidin-coated Magnetic Beads (MBs) solution (10 mg/ml) was added to the centrifuge tube and washed twice with 1 XPhosphate buffer solution (PBS) (pH 7.4). The supernatant was removed by magnetic separation and the Magnetic 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 Bead (MBs) solution, and the mixture was incubated on a homomixer at room temperature for 30 minutes. The mixture was subjected to magnetic separation, unbound substrate was removed, and washed 5 times with 1 x Phosphate Buffer (PBS). The mixture was then resuspended in 1 x Phosphate Buffered Saline (PBS) to a total volume of 80 μ l and a final concentration of magnetic bead-bound peptide-DNA conjugate of 2.5 μmol per liter.
Tyrosine dephosphorylation reaction induced by protein tyrosine phosphatase 1B (PTP1B) and sequential cleavage reaction of peptide-DNA conjugates: 12 microliters 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 (PTP1B) and 1 XPTP 1B reaction buffer (10 mmoles per liter of 4- (2-hydroxyethyl) -1-piperazineethanesulfonic acid (HEPES), 1 mmoles per liter of Dithiothreitol (DTT), 2 mmoles per liter of ethylenediaminetetraacetic acid (EDTA)), and incubated at 37 ℃ for 2 hours. After the dephosphorylation reaction was completed, it was inactivated at 80 ℃ for 20 minutes. Then, 0.6. mu.l of chymotrypsin (1 mg per ml) was added to the lysis reaction (60. mu.l), followed by incubation at 37 ℃ for 2 hours, magnetic separation was performed for 3 minutes after the completion of the lysis reaction, and then the supernatant was collected.
Three-stage DNA amplification reaction based on dephosphorylated product-induced DNAzyme: after dephosphorylation and cleavage reaction, 30 μmA liter of supernatant was added to the hybridization reaction system containing 0.7. mu.l of template at a concentration of 5. mu. mol/l, 3.5. mu.l of 10 × annealing buffer (50 mmol/l magnesium chloride (MgCl)2) 100 mmoles per liter of Tris-HCl and 0.8 μ l of water, heated at 95 ℃ for 5 minutes and then slowly cooled to room temperature to form double stranded dna (dsdna). After the annealing reaction, a strand displacement amplification reaction was performed in 50. mu.l of a reaction mixture containing 35. mu.l of the annealed product, 400 mmol/l dNTPs, 8 units per ml Vent (exo-) DNA polymerase, 0.3 units per. mu.l 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 of the reaction product was mixed with 2.4. mu.l of a signal probe at a concentration of 10. mu. mol/l, 1. mu.l of 10 XCutsmart buffer and 6.6. mu.l of water, and incubated at 37 ℃ for 1 hour for the cycle-cut signal probe reaction.
Gel electrophoresis and fluorescence detection: the chymotrypsin-catalyzed cleavage reaction products were analyzed by a 14% native polyacrylamide gel electrophoresis experiment in 1 XTBE buffer (9 mmol/l Tris-HCl), 9 mmol/l boric acid (boric acid), 0.2 mmol/l ethylenediaminetetraacetic acid (EDTA), pH 8.3) at constant voltage of 100V, which electrophoresis experiment lasted for 90 min. Gels were stained with SYBR Glod and imaged with a ChemiDoc MP imaging system. The products of the tertiary DNA amplification reaction were analyzed by 12% native polyacrylamide gel electrophoresis experiments in 1 XTBE buffer at constant voltage of 110 volts for 45 minutes. Gels were stained with SYBR Glod and imaged with a ChemiDoc MP imaging system. For the fluorescence measurement, 60. mu.l of the amplification product was diluted with ultrapure water to a final volume of 80. mu.l, and then fluorescence measurement was performed. Fluorescence intensity at an emission wavelength of 569 nm was detected with a Hitachi F-7000 spectrometer at an excitation wavelength of 512 nm and an emission spectrum was recorded over a wavelength range of 550 to 750 nm, with both excitation and emission slits having a width of 5 nm. Ft-F0For the regression equation to calculate the concentration of protein tyrosine phosphatase 1B (PTP1B), wherein F0And Ft is dividedRespectively, the fluorescence intensity at 569 nm in the absence and presence of protein tyrosine phosphatase 1B (PTP 1B).
Inhibitor experiments: protein tyrosine phosphatase 1B (PTP1B) (4 nmol/L) was incubated with varying concentrations of (R) -4-hydroxy-5- (hydroxymethyl) -3- (1-oxycetanyl) -2(5H) -furanone (RK-682) or sodium vanadate (Na)3VO4) After a 10-minute preincubation at 37 ℃, subsequent experiments were performed following the same procedure as the protein tyrosine phosphatase 1B (PTP1B) assay described above, and finally fluorescence detection was performed.
Cell culture and extraction experiments: human embryonic kidney cell line (HEK-293 cells), human cervical cancer cell line (HeLa) and human breast cancer cell line (MCF-7) were cultured in Dulbecco's Modified Eagle Medium (DMEM) containing 10% Fetal Bovine Serum (FBS) and 1% penicillin streptomycin. In the presence of 10% Fetal Bovine Serum (FBS), 1% penicillin-streptomycin, 0.11 g per liter sodium pyruvate (CH)3Coona) and 1.5 g per liter of sodium bicarbonate (NaHCO)3) Human liver cancer cell lines (HepG2) were cultured in Minimum Eagle Medium (MEM). All cells contained 5% CO at 37 deg.C2Is cultured in the environment of (1). In cell extraction experiments, cells were harvested by trypsinization and washed twice with 1 × 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. The cell number was measured by a Countstar cytometer. Will be about 1X 106Individual cells were suspended in 100 microliters of RIPA lysis buffer (50 mmol/l Tris (pH 7.4), 150 mmol/l 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 minutes at 4 ℃ and then supernatants were assayed by measuring absorbance at 280 nm with a Nanodrop 2000C spectrophotometer to quantify total protein concentration.
The sequences used in the examples are shown in Table 1.
Sequences used in the examples of Table 1
pTyr refers to phosphorylated Tyr, the underlined region in the DNA template indicates the recognition sequence for nicking enzyme (nb. In the signaling probe, the underlined two bases "T" indicate that the base is modified with fluorescein (Cy3) and quencher 2(BHQ2), respectively, and the bases rA and rU indicate adenine ribonucleotide and uracil ribonucleotide, respectively.
Wherein the peptide-DNA substrate is formed by connecting a polypeptide domain and a DNA domain, the sequence of the polypeptide domain is as follows: Lys-Gly-Asp-Gly-Val-pTyr-Ala-Ala-Cys, see SEQ ID No. 1.
DNA domain sequence: GGA TCG TCA TGT ACG GCA GCT, see SEQ ID NO. 2.
Experimental principle, as shown in fig. 1: three steps are involved: (1) the tyrosine dephosphorylation reaction catalyzed by protein tyrosine phosphatase 1B (PTP1B) induces chymotrypsin to cleave peptide-DNA conjugate (2) peptide-DNA conjugate cleavage induced exponential Strand Displacement Amplification (SDA) to generate deoxyribozyme (DNAzyme) (3) deoxyribozyme (DNAzyme) cycle cleavage signal probe, which results in amplification of the fluorescent signal. As shown in FIG. 1A, tyrosine in peptide-DNA conjugates can be dephosphorylated and cleaved by chymotrypsin in the presence of protein tyrosine phosphatase 1B (PTP 1B). Detailed assay procedures peptide-DNA conjugates were immobilized on Magnetic Beads (MBs) by biotin-streptavidin interaction, as shown in FIG. 1B. The presence of protein tyrosine phosphatase 1B (PTP1B) leads to dephosphorylation at tyrosine, and subsequent cleavage of the peptide-DNA conjugate by chymotrypsin releases a 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 by Vent (exo-) DNA polymerase to form a double-stranded DNA (dsdna) duplex with two nicking enzyme (nb. Bbvci can cleave one strand of a double-stranded dna (dsdna) duplex to generate two new replication sites. Under the catalysis of DNA polymerase and nickase, the strand displacement reaction is continuously initiatedAnd a large amount of deoxyribozymes (DNAzymes) were produced. Notably, the deoxyribozyme (DNAzyme) can be hybridized with a new free DNA template to induce a new round of polymerization, cleavage and release of the deoxyribozyme (DNAzyme), resulting in exponential amplification, thereby producing a large amount of the deoxyribozyme (DNAzyme). The obtained deoxyribozyme (DNAzyme) is a "10-23" type deoxyribozyme (DNAzyme) which can cleave RNA, and can cleave complementary RNA with high efficiency in a divalent cation-dependent reaction. These deoxyribozymes (DNAzymes) can hybridize to the signaling probe moiety, in the presence of the cofactor magnesium ion (Mg)2+) With the aid of (1), cleavage of the intermediate positions of the signaling probes rA and rU induces dissociation of the fluorophore and the quencher, eventually leading to an increase in the fluorescence signal and release of the deoxyribozyme (DNAzyme). The released deoxyribozyme (DNAzyme) can be hybridized with a new signal probe to induce the cyclic cleavage of a large number of signal probes, and finally the fluorescence signal can be obviously amplified. In contrast, tyrosine in the peptide-DNA conjugate cannot be cleaved in the absence of the protein tyrosine phosphatase 1B (PTP 1B). Therefore, neither release of single stranded DNA (ssDNA) nor tertiary DNA amplification reaction can occur, and no fluorescence signal can be detected.
Feasibility experiment for detecting protein tyrosine phosphatase 1B (PTP1B)
To verify the feasibility of this method, this example was analyzed by 14% native polyacrylamide gel electrophoresis. As shown in FIG. 2A, a distinct band was observed in the coexistence of the protein tyrosine phosphatase 1B (PTP1B) and the peptide-DNA conjugate (channel 3 of FIG. 2A), and a distinct band was also observed at the same position after magnetic separation of the protein tyrosine phosphatase 1B (PTP1B) and the peptide-DNA conjugate (channel 1 of FIG. 2A) linked to Magnetic Beads (MBs). This indicates that detection of the presence of protein tyrosine phosphatase 1B (PTP1B) induces dephosphorylation of the peptide DNA substrate and sequential cleavage by chymotrypsin yields single-stranded DNA (ssDNA) with three amino acid residues. In contrast, after magnetic separation, no band was observed in the presence of the peptide-DNA conjugate linked to Magnetic Beads (MBs) only (lane 2 of FIG. 2A), and a more distinct band corresponding to the intact peptide-DNA conjugate was observed in the presence of the peptide-DNA conjugate alone. This channel (channel 4 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 exponential DNA amplification reaction induced by peptide cleavage, this example used a 12% native polyacrylamide gel electrophoresis experiment to analyze the amplified products after magnetic separation. In the absence of protein tyrosine phosphatase 1B (PTP1B), two DNA template bands (88nt) and a signaling probe (19nt) were observed (FIG. 2B channel 1). When the test protein tyrosine phosphatase 1B (PTP1B) was present, in addition to the two DNA template bands (88nt) and the signal probe (19nt), three new bands of amplification products (68, 33-35, and 9-10nt) were observed (channel 2, FIG. 2B), indicating that the presence of the protein tyrosine phosphatase 1B (PTP1B) prompted the dephosphorylation reaction of the peptide-DNA conjugate and the consecutive cleavage reaction of chymotrypsin, inducing the SDA reaction to occur, thereby generating the polymeric intermediate (68nt) and the amplification products (33-35nt) and the subsequent DNAzyme-induced cyclic cleavage signal probe reaction to produce a shorter cleavage fragment (9-10 nt). This example further performed fluorescence detection to validate the experiment (fig. 2C). In the absence of the test protein tyrosine phosphatase 1B (PTP1B), no significant fluorescent signal was detected (fig. 2C). In contrast, a significant fluorescent signal was observed in the presence of the test protein tyrosine phosphatase 1B (PTP1B) (FIG. 2C), indicating that the presence of the protein tyrosine phosphatase 1B (PTP1B) prompted the occurrence of a tyrosine dephosphorylation reaction and a chymotrypsin consecutive cleavage peptide-DNA conjugate reaction, the product of which induced the index SDA, producing a deoxyribozyme (DNAzyme) that catalyzes the cyclic cleavage of the signaling probe to produce a significant fluorescent signal. These results indicate that the present protocol is feasible for accurate detection of protein tyrosine phosphatase 1B (PTP1B) activity.
Sensitivity detection
To evaluate the sensitivity of detecting the activity of protein tyrosine phosphatase 1B (PTP1B), the assay was carried out for various concentrations of protein tyrosine phosphatase 1B (PTP1B), and the results are shown in FIG. 3A, where the fluorescence intensity increased and was at 0 as the concentration of protein tyrosine phosphatase 1B (PTP1B) was increased from 0.5 pmol per liter to 4 nmol per liter.The fluorescence intensity showed a good linear relationship with the logarithmic value of the protein tyrosine phosphatase 1B (PTP1B) concentration in the range of 5 picomoles per liter to 100 picomoles per liter (R20.9940) (inset of fig. 3B). The regression equation is that F is 370.22+591.57log10C, wherein F is the fluorescence intensity and C is the concentration of protein tyrosine phosphatase 1B (PTP 1B). The detection limit was found by calculation to be 0.24 pmol per liter (8.97X 10)-6Micrograms per milliliter). The sensitivity of the method is improved by 6 orders of magnitude compared to fluorescent probe-based fluorometry (5.7 micrograms per milliliter), by 25-fold compared to chemical sensor-based fluorometry (6 picomoles per liter), and by calcein-based fluorometry (3 × 10-5Microgram per milliliter) and graphene oxide-based fluorometry (1 × 10)-5Micrograms per milliliter) were comparable.
Specificity test of PTP
To assess the specificity of the protocol, protein phosphatase 2A (PP2A), 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 can catalyze threonine dephosphorylation, but is not active against tyrosine. PNK can catalyze the transfer of one terminal phosphate group from Adenosine Triphosphate (ATP) to the 5' hydroxyl group of an oligonucleotide. IgG and BSA are two non-specific proteins with no dephosphorylating activity. As shown in FIG. 4, no significant fluorescent signal was detected for PP2A, PNK, IgG and BSA, while high fluorescent signal was detected for protein tyrosine phosphatase 1B (PTP1B), indicating that the presence of protein tyrosine phosphatase 1B (PTP1B) promotes the occurrence of tyrosine dephosphorylation reaction and chymotrypsin continuous cleavage peptide-DNA conjugate reaction, the product of which induces the occurrence of tertiary DNA amplification reaction, thereby generating a stronger fluorescent signal. These results indicate that the proposed method has good specificity for the protein tyrosine phosphatase 1B (PTP 1B).
Inhibitor assay
To test the feasibility of this approach in inhibition experiments, this example used (R) -4-hydroxy-5- (hydroxymethyl) -3- (1-oxycetanyl) -2(5H) -furanone (RK-682) and sodium vanadate (Na)3VO4) As an inhibitor. As shown in FIG. 5, the relative activities of the protein tyrosine phosphatase 1B (PTP1B) were determined by RK-682 (FIG. 5A) and Na, respectively3VO4(FIG. 5B) the concentration increased and decreased. IC calculated to yield RK-68250A value of 3.03 micromoles per liter, Na3VO4IC of50The value was 2.18 nanomoles per liter, which is consistent with the values obtained for calcein-based fluorometry and graphene oxide-based fluorometry. Previous studies have shown that the protein tyrosine phosphatase 1B (PTP1B) is involved in insulin signaling regulation. This example evaluated the inhibitory ability of the inhibitor against human liver cancer cells (HepG2 cells). When Na is present3VO4Increasing the concentration from 0 to 1000 nmoles per liter, the relative activity of the protein tyrosine phosphatase 1B (PTP1B) in HepG2 cells decreased accordingly (fig. 5C). IC (integrated circuit)50The value was calculated to be 4.97 nmol per liter, consistent with the value obtained using the pure protein tyrosine phosphatase 1B (PTP1B) (2.18 nmol per liter) (fig. 5B). These results indicate that the method can be used to screen for protein tyrosine phosphatase 1B (PTP1B) inhibitors.
The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
SEQUENCE LISTING
<110> university of Shandong Master
<120> 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
<400> 1
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
<400> 4
catgagatau tcaaccctc 19
Claims (10)
1. A tyrosine phosphatase biosensor is characterized by comprising magnetic beads, peptide-DNA conjugates, a DNA template and a signal 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 arranged to be connected with a magnetic bead;
the DNA template consists of 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, the DNA template can form double-stranded DNA with two nicking enzyme recognition sites after being hybridized and polymerized with 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; the dnazymes cleave complementary RNA sequences in a divalent cation-dependent reaction.
2. The tyrosine phosphatase biosensor as set forth in claim 1, wherein the peptide-DNA conjugate is linked to the magnetic beads via streptavidin-biotin binding.
3. The tyrosine phosphatase biosensor as claimed in claim 1, wherein the fluorophore is Cy3 and the quencher is BHQ 2.
4. The tyrosine phosphatase biosensor as claimed in claim 1, wherein the junction of the peptide chain and the first DNA sequence is linked to tyrosine containing phosphorylation by 2 to 4 amino acids.
5. The tyrosine phosphatase biosensor as claimed in claim 1, wherein 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, respectively;
the sequence of the signal probe is: CAT GAG ATA UTC AAC CCT C are provided.
6. A method for detecting tyrosine phosphatase, which comprises providing the tyrosine phosphatase biosensor according to any one of claims 1 to 5;
carrying out dephosphorylation reaction on the magnetic beads with the surface connected with the peptide-DNA conjugate and a solution containing tyrosine phosphatase, adding chymotrypsin into a system after the dephosphorylation reaction for carrying out cracking reaction, carrying out magnetic separation after the cracking reaction to remove the magnetic beads so as to obtain a cracking product, carrying out three-stage DNA amplification reaction on the cracking product, a DNA template and a signal probe under the action of divalent cations, and carrying out fluorescence detection on the solution system after the three-stage DNA amplification reaction.
7. The method for detecting tyrosine phosphatase as claimed in claim 6, wherein the reaction temperature of the phosphorylation reaction is 35 to 40 ℃ and the reaction time is 1.5 to 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 third-level DNA amplification reaction process is that firstly, the cleavage product and the DNA template form double-stranded DNA, then DNA polymerase and nickase are added to carry out the strand displacement amplification reaction, and a signal probe is added to the system after the strand displacement amplification reaction for incubation;
preferably, the conditions for double-stranded DNA are: firstly, heating for 4-6 minutes at 95-100 ℃, and then cooling to room temperature;
preferably, the conditions for the strand displacement amplification reaction are: the temperature is 50-60 ℃, and the time is 50-70 min;
preferably, the incubation temperature is 35-40 ℃, and the incubation time is 1.5-2.5 h.
8. Use of the tyrosine phosphatase biosensor as claimed in any one of claims 1 to 5 in the preparation of a reagent for detecting tyrosine phosphatase.
9. Use of the tyrosine phosphatase biosensor according to any one of claims 1 to 5 or the method for detecting tyrosine phosphatase according to claim 6 or 7 for screening tyrosine phosphatase inhibitors.
10. A tyrosine phosphatase detection kit comprising the tyrosine phosphatase biosensor according to any one of claims 1 to 5, chymotrypsin, DNA polymerase, nickase, magnesium salt, and a buffer solution.
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