A method for detecting polynucleotide kinase activity based on CRISPR / Cas12a

Abstract

The present invention discloses a method for detecting polynucleotide kinase activity based on CRISPR / Cas12a, and belongs to the field of biotechnology. The detection method uses CRISPR / Cas12a as the signal output platform of the detection system, and integrates the signal amplification characteristics of TdT primer extension technology and Klenow fragment amplification technology, and realizes the quantitative detection of polynucleotide kinase enzyme activity with this strategy. The present invention further improves the detection sensitivity, realizes highly sensitive, specific, rapid, economical and efficient multiple detection of target nucleic acids, does not require professional operators and complex equipment, and is suitable for on-site deployment and instant detection.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and in particular relates to a method for detecting polynucleotide kinase activity based on CRISPR / Cas12a. Background Art

[0002] Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated proteins (Cas), namely the CRISPR-Cas system, have been widely and effectively used in gene editing and expression regulation. In recent years, the CRISPR-Cas system has been widely developed as a highly sensitive and selective tool for nucleic acid detection, including CRISPR / Cas9, CRISPR / Cas12a, CRISPR / Cas13a and CRISPR / Cas14a systems. The CRISPR / Cas12 system is composed of a crRNA and a Cas12 protein. The target DNA is complementary to the spacer sequence of the crRNA in CRISPR / Cas12a, which can stimulate CRISPR / Cas12 to trans-cleave the single ssDNA signal probe, thereby releasing a fluorescent signal. The target that CRISPR / Cas12 can recognize can be either dsDNA or ssDNA. The recognition of dsDNA requires a PAM site (TTTN, N is an arbitrary base) to be carried in the target sequence, while the recognition of ssDNA does not require a PAM sequence. The biggest advantage of CRISPR / Cas12 lies in its programmability. That is, by simply replacing the spacer sequence of crRNA, CRISPR / Cas12 can be transformed into a tool for recognizing any DNA sequence, and perform specific target shearing and non-specific trans-cutting. Compared with the existing probes, CRISPR / Cas12 can recognize multiple target sequences by changing the crRNA sequence. CRISPR / Cas12 recognition has sequence-dependent characteristics. Only when the target and crRNA are complementary can the activity of Cas12 be activated. Compared with traditional enzyme cleavage, CRISPR / Cas12 shows stronger specificity. CRISPR / Cas12 itself is a signal amplification system. Once the trans-cutting activity is activated by the target, the signal probe is continuously cut, and the detection signal will be continuously amplified, thus ensuring the sensitivity of the detection. CRISPR / Cas12 has been applied to the detection of nucleic acids or nucleic acid-related samples, with high specificity and sensitivity.

[0003] Terminal deoxyribonucleotidyl transferase (TdT, referred to as terminal transferase) can add deoxynucleotides to the 3'-OH end of single-stranded DNA (ssDNA) without a template. Terminal transferase can attach oligo dA or dC to the 3'-OH end of one group of DNA molecules, and oligo dT or dG to the 3'-OH end of another group of DNA molecules. The two groups of molecules are mixed to anneal the homopolymer tails to form circular molecules.

[0004] Klenow fragment is the DNA polymerase I of Escherichia coli treated with subtilisin (or trypsin), and the original enzyme molecule is cut into two fragments, wherein the large fragment has a molecular weight of 76kD and the small fragment has a molecular weight of 34kD. The large fragment is usually called Klenow fragment, and Klenow fragment can also be obtained by cloning technology. The fragment retains the 5'-3' polymerase and 3'-5' exonuclease activities of DNA polymerase I, but lacks the 5'-3' exonuclease activity of the complete enzyme. After the DNA polymerase I (DNA-pol I) is disconnected, there is another 323 amino acid residue fragment, which retains the 5'-3' exonuclease activity.

[0005] Polynucleotide kinase (PNK) is a bifunctional DNA / RNA end-processing enzyme that catalyzes the transfer of γ-phosphate groups from adenosine triphosphate (ATP) to the 5'-hydroxyl group of oligonucleotides or nucleic acids, and is essential for repairing DNA strand breaks induced by endogenous or exogenous factors. PNK can catalyze the phosphorylation of the 5'-OH end of DNA molecules and the dephosphorylation of the 3'-phosphate group. Phosphorylation of nucleic acids plays an important role in normal cellular activities, such as DNA recombination, DNA replication, and DNA repair during strand breaks. The catalytic activity of PNK can directly affect the phosphorylation process of nucleic acids, thereby affecting a series of metabolic processes. Moreover, human PNK is involved in maintaining the integrity of the genome, and abnormal expression of PNK activity is associated with a variety of diseases, such as neurodegenerative diseases, spinocerebellar ataxia, Werner syndrome, etc. In addition, PNK is also considered a potential target in cancer treatment and can be used as a biomarker for disease diagnosis and prognosis. Sensitive detection of its activity is of great significance in biochemical research, drug development, clinical diagnosis and treatment.

[0006] Traditional detection methods for PNK include polyacrylamide gel electrophoresis, radioisotope 32P labeling, radioautography, but the widespread application of traditional methods is limited by their own shortcomings, including radiation pollution, time-consuming, laborious operation and expensive instruments. In order to overcome the above shortcomings, many new PNK detection methods have been developed in recent years, including colorimetry, fluorescence analysis, electrochemical method and electrochemiluminescence method, but most of these methods rely on the auxiliary cutting of λ nuclease (λ-exo), but λ-exo may also cut non-phosphorylated probes, resulting in non-specific enhancement and false positive samples, so they cannot be used for accurate quantitative detection of PNK. Therefore, it is imperative to develop a PNK quantitative detection method that does not rely on λ-exo and has high sensitivity and good specificity. Summary of the invention

[0007] In view of the actual needs of high sensitivity and high specificity in the detection of polynucleotide kinase activity, the present invention considers using CRISPR / Cas12a as the signal output platform of the detection system, and integrates the signal amplification characteristics of TdT primer extension technology and Klenow fragment amplification technology, so as to realize the quantitative detection of polynucleotide kinase enzyme activity with this strategy.

[0008] In order to construct an ultrasensitive detection system for polynucleotide kinases, the present invention combines CRISPR-Cas12a with terminal deoxynucleotidyl transferase (TdT) to specifically and sensitively detect PNK activity in cell lysates. First, we designed an Oligo dT primer probe, which was phosphorylated at its 3' end to avoid nonspecific amplification. In the absence of target PNK, the Oligo dT primer probe cannot activate the CRISPR-Cas12a unit, resulting in a very low background signal. In the presence of target polynucleotide kinases, PNK can convert the Oligo dT primer probe 3'-phosphate group (3'-PO 4 ) dephosphorylated to 3'-OH.

[0009] As a special DNA polymerase, TdT directly catalyzes the sequential addition of deoxynucleotide triphosphates (dNTPs) to the 3'-OH of DNA molecules without the need for a specific template strand. Therefore, in the presence of TdT and dATP, the free 3'-OH end DNA probe can be amplified by TdT to produce a polyadenine (poly A) tailed single-stranded DNA (named T-An, where n represents the number of A bases).

[0010] After heating to remove TdT activity, the remaining primer fragment will complement the T-An single-stranded base to form a double-stranded DNA. After adding the Klenow fragment and dTTP, the Klenow fragment will use T-An as a template and the primer as a primer to produce a large amount of P-Tm-At single-stranded DNA (m is the number of T bases, t is the number of T bases in the primer); the At region will fold back and hybridize in the Tm region, and then the Klenow fragment will use the Tm region as a template and the At region as a primer to amplify, and finally form a double-stranded product (TA dsDNA) with completely complementary pairing of T bases and A bases.

[0011] TA dsDNA can be used as an activator to supplement the recognition region (poly U) of crRNA, which can activate multiple CRISPR-Cas12a units and reverse cut a large number of reporter probes (ssDNA modified with fluorescent group-quencher group pairs at both ends), thereby releasing fluorescent signals. DNA amplifies the output fluorescent signal through the extension of TdT enzyme, Klenow fragment and reverse cutting activity of Cas12a, which can be used for the detection of PNK targets.

[0012] In order to achieve the above technical purpose, the present invention adopts the following technical solutions:

[0013] A method for detecting the activity of a polynucleotide kinase based on CRISPR / Cas12a, wherein the method utilizes CRISPR-Cas12a, terminal deoxynucleotidyl transferase TdT, and a Klenow fragment to jointly detect the activity of the polynucleotide kinase.

[0014] Preferably, the detection method comprises the following steps:

[0015] (1) TdT primer extension reaction: Construct a 20 μL reaction system containing the following components at the final concentration: 400 nM oligodT primer probe, 3 μL 1×T4 polynucleotide kinase reaction buffer, 3 μL 1×TdT reaction buffer, 5 mM dATP, 5 U TdT, 3 μL ddH 2 O, 5 μL sample extract, PNK dephosphorylation and TdT primer extension reaction are incubated at 35-38°C for 15-90 minutes, and then TdT is inactivated to obtain solution I;

[0016] (2) Klenow polymerase reaction: A 20 μL reaction system was constructed, which included the following components at the final concentrations: 5 mM dTTP, 5 U Klenow fragment, 8 μL 1×NEBuffer 2, 8 μL ddH 2O, mixed with solution I and incubated at 35-38°C for 15-90 minutes to obtain solution II;

[0017] (3) CRISPR / Cas 12a reverse cleavage reaction: Add 1-3 μL 1 μM Cas12a, 2-4 μL 400 nM crRNA, 0.5-1.5 μL 50 μM reporter probe, and 12-16 μL 1× reaction buffer to solution II, incubate at 35-38°C for 15-45 minutes, and add ddH 2 After adding 200 μL of HO, the fluorescence was measured.

[0018] More preferably, the OligodT primer probe in step (1) is 5'-TTT TTT TTT TTT TTT TTTTT-PO 4 -3'.

[0019] More preferably, the composition of NEBuffer2 in step (2) is: 50mM NaCl, 10mM Tris-HCl, 10mM MgCl 2 ,1mM DTT,pH 7.9@25℃.

[0020] More preferably, the reaction temperature in steps (1), (2) and (3) is 35-38°C.

[0021] More preferably, in step (3), the reporter probe is 5'-FAM-TTATT-BHQ1-3'.

[0022] More preferably, the composition of the reaction buffer in step (3) is: 50 mM NaCl, 10 mM Tris-HCl, 10 mM MgCl 2 ,0.1mg / mL BSA,Ph=7.9.

[0023] More preferably, the crRNA in step (3) is 5'-UAAUUU CUACUAAGU GUG UGUAUU UUU UUU UUUUUU UUU UUU U-3.

[0024] A polynucleotide kinase activity detection product, comprising CRISPR-Cas12a, terminal deoxynucleotidyl transferase TdT and a Klenow fragment.

[0025] Preferably, the product is a kit.

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] (1) The present invention uses a dual enzyme combined amplification technology based on terminal transferase TdT and Klenow fragment as a signal amplification method to maximize the amplification of trace target signals;

[0028] (2) The present invention uses CRISPR / Cas12a as the signal output platform of the detection system and double-stranded DNA as the activation chain. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 The schematic diagram is a schematic diagram of the polynucleotide kinase activity detection of the present invention.

[0030] Figure 2 The fluorescence spectra of the reaction systems measured in the emission range of 505nm-650nm in different wavelengths in Example 1, the concentration of PNK added to the reaction group was 1.0U / mL; Curve 1: no enzyme was present; Curve 2: PNK alone; Curve 3: TdT alone; Curve 4: Klenow fragment alone; Curve 5: TdT and Klenow fragment were present; Curve 6: PNK and Klenow fragment were present; Curve 7: PNK and TdT were present; Curve 8: PNK, TdT, Klenow fragment were present, Figure 2 From top to bottom, the curves are 8-7-3-5-2-4-6-1.

[0031] Figure 3 It is a fluorescence spectrum diagram of the fluorescence emission intensity at 520nm in Example 1, wherein Curve 1: no enzyme is present; Curve 2: PNK is present alone; Curve 3: TdT is present alone; Curve 4: Klenow fragment is present alone; Curve 5: TdT and Klenow fragment are present; Curve 6: PNK and Klenow fragment are present; Curve 7: PNK and TdT are present; Curve 8: PNK, TdT, Klenow fragment are present.

[0032] Figure 4 This is a diagram for verifying the specific fluorescence experiment of the system in Example 2, wherein DNA adenine methyltransferase (DAM), 80 U / mL; phi29 DNA polymerase, 100 U / mL; T4 Ligase, 200 U / mL; DpnI restriction endonuclease, 200 U / mL; bovine serum albumin (BSA), 5 mg / mL; and polynucleotide kinase (PNK), 1 U / mL.

[0033] Figure 5 The fluorescence detection results after adding different cell extracts in Example 1.

[0034] Figure 6 These are the detection results of different concentrations of PNK in Example 3.

[0035] Figure 7 This is a linear relationship diagram between fluorescence intensity and PNK concentration in Example 3. DETAILED DESCRIPTION

[0036] Example 1

[0037] This embodiment provides a method for detecting polynucleotide kinase activity based on CRISPR / Cas12a, and the specific steps are as follows:

[0038] (1) Design of probe sequence

[0039] Oligo dT primer (SEQ ID NO.1): 5'-TTT TTT TTT TTT TTT TTT TT-PO 4 -3';

[0040] Reporterprobe:5'-FAM-TTATT-BHQ1-3';

[0041] crRNA(SEQ ID NO.2):5'-UAA UUU CUA CUA AGU GUG UGU AU U UUU UUU UUU UUU UUU UUU U -3' (the underlined part is the recognition sequence of TA dsDNA).

[0042] (2) Experimental plan

[0043] 2.1 PNK dephosphorylation reaction

[0044] (1) TdT primer extension reaction: a 20 μL reaction system was constructed, containing 400 nM OligodT primer probe (3'-PO 4 ), 3μL 1×T4 polynucleotide kinase reactionbuffer, 3μL 1×TdTreactionbuffer, 5mM dATP, 5U TdT, 3μL ddH 2 O, different concentrations of PNK, PNK dephosphorylation and TdT primer extension reaction were incubated at 35-38°C for 15-90 minutes, and then TdT was inactivated by heating the mixture at 80°C for 5-10 minutes to obtain solution I;

[0045] (2) Klenow polymerase reaction: Construct a 20 μL reaction system containing 5 mM dTTP, 5 U Klenow fragment, 8 μL 1× NEBuffer 2 (50 mM NaCl, 10 mM Tris-HCl, 10 mM MgCl2 ,1mM DTT, pH 7.9@25℃), 8μL ddH 2 O, mixed with solution I and incubated at 35-38°C for 15-90 minutes to obtain solution II;

[0046] (3) CRISPR / Cas 12a reverse cleavage reaction: Add 2 μL 1 μM Cas12a, 3 μL 400 nm crRNA, 1 μL 50 μM reporter probe, and 14 μL 1× reaction buffer (50 mM NaCl, 10 mM Tris-HCl, 10 mM MgCl 2 , 0.1 mg / mL BSA, pH = 7.9, 25 ° C), incubate at 35-38 ° C for 15-45 minutes, add ddH 2 After adding 200 μL of HO, the fluorescence was measured.

[0047] 2.2 Feasibility Verification

[0048] The feasibility of the system was verified by polyacrylamide gel electrophoresis and fluorescence spectroscopy experiments:

[0049] (1) Polyacrylamide gel electrophoresis: 12% native polyacrylamide gel (native-PAGE) was prepared with 5×TBE buffer, and 15μL reaction solution containing 3μL 6×loading buffer was mixed to prepare the electrophoresis sample. The gel was electrophoresed at a constant voltage of 100V for 60-90 minutes in 1×TBE buffer (9mM Tris-HCl, pH 7.9, 9mM boric acid, 0.2mM EDTA), and then stained with 1×Gel Red nucleic acid dye for imaging.

[0050] (2) Fluorescence spectrum experiment: The fluorescence spectrum of the prepared samples was measured by a fluorescence spectrophotometer (F4500, Hitachi) with an excitation wavelength of 492 nm and the fluorescence spectrum of the reaction system measured in the emission range of 505 nm-650 nm. The results are shown in Figure 2 , (the pictures are curves 8-7-3-5-2-4-6-1 from top to bottom); a strong fluorescence response can only be observed when PNK is present and the system components are complete, and there is no obvious difference in the peak fluorescence values ​​of curves 1 to 6.

[0051] In the fluorescence test, the excitation and emission slits were set to 5nm, the PMT voltage was set to 700V, and the fluorescence emission intensity was measured at 520nm. The results are as follows: Figure 3 As shown ( Figure 3 and Figure 2 The test samples in are exactly the same. Figure 3 The histogram in the figure is the statistics of fluorescence peaks).

[0052] 2.3 Determination of PNK activity in cell samples

[0053] HepG2 cells (liver cancer cells) and HL-7702 (normal liver cells) were selected as model cells for intracellular PNK activity detection. Both HepG2 and HL-7702 cells were cultured in DMEM (10% fetal bovine serum, 100U / mL penicillin, 100U / mL streptomycin) medium. The cultured cells were washed with ice PBS and separated by trypsin digestion. After washing again with ice PBS, the cell density was counted using a hemocytometer. Cell extracts were obtained using a nuclear protein extraction kit and can be stored at -80°C for future use.

[0054] like Figure 5 As shown in the figure, the fluorescence was significantly enhanced after adding HepG2 cell extract, which was similar to the fluorescence result of pure PNK. However, the fluorescence intensity after adding HL-7702 cell extract was significantly lower than that of HepG2 cell extract, which also showed that the expression of PNK in cancer cell lines was higher than that in normal cells.

[0055] Example 2

[0056] This example verifies the specificity of the polynucleotide kinase activity detection method in Example 1, and the specific steps are as follows:

[0057] When determining the specificity of the detection method, different enzymes or proteins are selected to replace the PNK in Example 1, and the PNK activity detection method in Example 1 is followed.

[0058] The concentration of PNK was 1 U / mL; the concentration of DAM was 80 U / mL; the concentration of phi29 was 100 U / mL; the concentration of T4 Ligase was 200 U / mL; the concentration of DpnI restriction endonuclease was 200 U / mL; the concentration of BSA was 5 mg / mL. For the results, see Figure 4 .

[0059] Example 3

[0060] This example verifies the sensitivity of the polynucleotide kinase activity detection method in Example 1, and the specific steps are as follows:

[0061] Different concentrations of PNK (0, 0.0001, 0.001, 0.01, 0.1, 0.25, 0.5, 0.75, 1.0 U / mL) were added to the detection system, and the corresponding fluorescence response at 520 nm was measured. For experimental steps, see the PNK activity detection method steps, and for experimental results, see Figure 6As the PNK concentration (0-1U / mL) increases, the fluorescence signal of the sensing system gradually increases. There is a good linear correlation between the fluorescence intensity at 520nm and the PNK concentration (0-1U / mL), and the corresponding equation is Y=836.9*X+115.9, R 2 =0.9962, where F is the fluorescence intensity of the sensor system at 520 nm, X is the concentration of PNK (U / mL), and the direct detection limit is 1×10 - 4 U / mL. Figure 6 The values ​​from the middle curve from bottom to top are 0, 0.0001, 0.001, 0.01, 0.1, 0.25, 0.5, 0.75, 1.0 U / mL, Figure 7 It is a linear relationship graph.

[0062] High sensitivity and specificity are the two most critical factors in the construction of biosensors. CRISPR / Cas has sequence-dependent recognition characteristics, which ensures the specificity of target detection. In addition, after CRISPR / Cas12a recognizes the target nucleic acid, the trans-cleavage triggered by it continuously shears the probe of the exogenous signal, making CRISPR / Cas a signal amplification and readout system, thereby ensuring detection sensitivity. The combination of CRISPR / Cas and bio-amplification technology will further improve detection sensitivity, achieve highly sensitive, specific, rapid, and cost-effective multiple detection of target nucleic acids, without the need for professional operators and complex equipment, and is suitable for on-site deployment and instant detection.

[0063] Terminal deoxynucleotidyl transferase (TdT) is a unique template-free DNA polymerase that can randomly add deoxynucleoside triphosphates (dNTPs) to the 3'-OH end of single-stranded DNA (ssDNA) to extend the ssDNA chain. Therefore, when only dATP is added, TdT can amplify a long single strand with a polyA tail without a template.

[0064] It can provide long template chains for subsequent amplification steps to further amplify the target signal.

[0065] The introduction of Klnew fragments amplifies the long single strand into a double strand, activating CRISPR / Cas12a. At the same time, the detection system in the present invention does not rely on the auxiliary cutting of λ-exo, which can reduce background signals, reduce false positives, and improve detection specificity; at the same time, the sequence design is simple, the amplification method is single but the amplification cycle is diverse, and trace target signals can be amplified.

[0066] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A polynucleotide kinase activity detection product, characterized in that: The product components are as follows: OligodT primer probe for TdT primer extension reaction, 1×T4 polynucleotide kinasereactionbuffer, 1×TdTreaction buffer, dATP, terminal deoxynucleotidyl transferase TdT, ddH2O; and dTTP, Klenow fragment, 1×NEBuffer2 for Klenow polymerase reaction; and Cas12a, crRNA, reporter probe, and 1×reaction buffer for CRISPR / Cas 12a reverse cleavage reaction; The terminal deoxynucleotidyl transferase TdT is used for primer extension reaction, and the specific steps include: constructing a 20 μL reaction system, the system is composed of the following components with final concentrations: 400 nM OligodT primer probe, 3 μL 1×T4 polynucleotide kinase reaction buffer, 3 μL 1×TdT reaction buffer, 5 mM dATP, 5 U dT, 3 μL ddH2O, 5 μL sample extract, incubating PNK dephosphorylation and TdT primer extension reaction at 35-38° C. for 15-90 minutes, and then inactivating TdT to obtain solution I; The Klenow fragment is used for Klenow polymerase reaction, and the specific steps include: constructing a 20 μL reaction system, the system is composed of the following components with final concentrations: 5 mM dTTP, 5 U Klenow fragment, 8 μL 1×NEBuffer2, 8 μL ddH2O, mixing with solution I, and incubating at 35-38° C. for 15-90 minutes to obtain solution II; The Cas12a is used for CRISPR / Cas 12a reverse cutting reaction, and the specific steps include: adding 1-3 μL 1 μM Cas12a, 2-4 μL 400nM crRNA, 0.5-1.5 μL 50 μM reporter probe, and 12-16 μL 1×reaction buffer to solution II, incubating at 35-38° C. for 15-45 minutes, adding ddH2O to supplement to 200 μL, and measuring fluorescence; The OligodT primer probe in the primer extension reaction is 5'-TTT TTT TTT TTT TTT TTT TTT TT-PO4 -3'; The composition of NEBuffer2 in the Klenow polymerase reaction is: 50mM NaCl, 10mM Tris-HCl, 10mM MgCl2, 1mM DTT, pH 7.9; The reporter probe in the CRISPR / Cas 12a reverse cleavage reaction is 5'-FAM-TTATT-BHQ1-3'; The crRNA in the CRISPR / Cas 12a reverse cleavage reaction is 5'-UAAUUU CUACUAAGU GUG UGUAUU UUU UUUUUUU UUU UUUU-3'; The composition of 1×reaction buffer in the CRISPR / Cas 12a reverse cleavage reaction is: 50 mM NaCl, 10 mM Tris-HCl, 10 mM MgCl2, 0.1 mg / mL BSA, pH=7.

9.

2. The polynucleotide kinase activity detection product according to claim 1, characterized in that: The reaction temperatures of the primer extension reaction, Klenow polymerase reaction, and CRISPR / Cas 12a reverse cleavage reaction are all 35-38°C.

3. The polynucleotide kinase activity detection product according to claim 1, characterized in that: The product is a test kit.

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