A hairpin probe for detecting DNA glycosylase, a biosensor, a detection method and application

Abstract

The application discloses a hairpin probe for detecting DNA glycosylase, a biosensor and a detection method. The hairpin probe is a stem-loop structure, the stem region of the hairpin probe is a T7 promoter sequence, and the third base A left to the starting site GGG is replaced by a deoxyinosine base I. The amino acid sequence of the hairpin probe is shown as SEQ ID NO. 1. The sensor comprises the hairpin probe, human deoxyuracil / deoxythymine endonuclease, deoxyribonucleotide premix, ribonucleotide premix, KF polymerase, T7 RNA polymerase, RNAase inhibitor and CRISPR-Cas12a reaction solution. The biosensor generates a large amount of crRNA in the presence of hAAG, so that the CRISPR-Cas12a signal produces a two-stage signal amplification reaction. The detection method realizes low-background and high-sensitivity hAAG detection, is simple to operate, and can accurately measure endogenous hAAG activity in cancer cells at a single cell level.

Description

Technical Field

[0001] This invention belongs to the field of bioanalytical technology, specifically relating to a biosensor for detecting DNA glycosidases and a detection method thereof. Background Technology

[0002] Minor DNA damage (such as oxidative damage, small alkylation products, and various types of single-strand breaks) can be removed and corrected via the base excision repair (BER) pathway. The key to the BER pathway is DNA glycosylase, as it initiates the entire pathway. Different types of DNA glycosidases specialize in different types of chemical damage. Human alkyladenine DNA glycosidase (hAAG) is a monofunctional DNA glycosidase that acts on alkylated bases and has very broad substrate specificity, capable of cleaving various alkylated purines, including 3-methyladenine, 7-methylguanine, and 1,N-methyladenine. 6 -Adenine, deoxyinosine (I), and N1-methylguanine and 1,N 2 - The oxidative deamination product of deoxyguanine. By hydrolyzing the N-glycosidic bond between the target base and the deoxyribose, hAAG releases free bases and leaves apurinol / pyrimidine-free (AP) sites in DNA. Balanced expression of hAAG is important for alkylation damage repair, and overexpression of hAAG may lead to an increased risk of colon cancer, lung cancer, and gastric cancer.

[0003] Currently, methods for detecting hAAG include high-performance liquid chromatography (HPLC), mass spectrometry (MS), radioisotope labeling, paramagnetic bead trapping, and gel electrophoresis. These methods suffer from drawbacks such as time consumption, harmful radiation, and poor sensitivity. Recent biosensors based on electrochemical and fluorescence strategies for detecting hAAG activity require complex nanomaterial preparation, cleaning, and separation steps. Furthermore, the high background noise inherent in these methods, coupled with a lack of appropriate signal amplification strategies, results in relatively low sensitivity for these biosensors. Therefore, the development of simple and sensitive biosensors for glycosylation enzyme detection remains an urgent need. Summary of the Invention

[0004] Objectives of the Invention: The first objective of this invention is to provide a hairpin probe for efficient detection of hAAG activity; the second objective is to provide a biosensor for efficient detection of hAAG activity; the third objective is to provide a detection method for the above-mentioned biosensor to detect hAAG activity; and the fourth objective is to provide the application of the above-mentioned hairpin probe, biosensor, and detection method in screening hAAG inhibitors / activators.

[0005] Technical solution: The present invention provides a hairpin probe, wherein the hairpin probe has a stem-loop structure, the stem region of the hairpin probe is a T7 promoter sequence, and the third base A to the left of the start site GGG is replaced by a deoxyinosine base I; the amino acid sequence of the hairpin probe is shown in SEQ ID NO.1.

[0006] The present invention discloses a biosensor for detecting DNA glycosidase, the sensor comprising the aforementioned hairpin probe, human depurinyl / depyrimidine endonuclease, deoxyribonucleotide premix, ribonucleotide premix, KF polymerase, T7 RNA polymerase, RNase inhibitor, and CRISPR-Cas12a reaction solution; the DNA sequence of the loop region of the hairpin probe is complementary to crRNA, serving as a template for post-cutting transcriptional amplification to synthesize crRNA; the amino acid sequence of the crRNA is shown in SEQ ID NO. 2; the CRISPR-Cas12a reaction solution comprises double-stranded activating DNA, a reporter probe, and Cas12a protein; the double-stranded activating DNA consists of a TS portion and an NTS portion, the TS portion being complementary to the crRNA.

[0007] The hairpin probe designed in this invention consists of two regions: a stem and a loop. The stem region comprises a functionalized T7 promoter sequence, in which the third base (A) to the left of the start site (GGG) is replaced by a deoxyinosine base (I). The loop region comprises the complementary DNA sequence of crRNA, which is the transcription product and a component of the CRISPR-Cas12a system. Double-stranded activating DNA (dsDNA activator) is another component of the CRISPR-Cas12a system, and its TS portion is complementary to the crRNA. The reporter probe is labeled with FAM at the 5' end and BHQ at the 3' end; the FAM is quenched by BHQ.

[0008] In the presence of hAAG, damaged deoxyinosine bases in the hairpin probe stem are specifically recognized by hAAG and effectively cleaved from the I:T pair, leaving AP sites. After creating a single nucleotide gap, APE1 completely cleaves the base linkage and simultaneously unfolds the hairpin probe loop. Subsequently, the unfolded hairpin probe serves as a template and primer, initiating polymerization in the presence of KF polymerase and dNTPs. This produces two stable dsDNA duplexes (Case 1: complete polymerization forming a perfectly complementary duplex structure; Case 2: complete polymerization of the T7 polymerase recognition region but incomplete polymerization of the entire DNA strand), initiating a T7 RNA polymerase-catalyzed transcription reaction. This generates a large amount of crRNA, providing signal amplification fuel for the CRISPR-Cas12a system. The generated crRNA is designed to contain a guide sequence and repeat sequences. The repeat sequences are essential for crRNA anchoring to the Cas12a protein, and the guide sequence is complementary to the dsDNA activator to activate cis-cleavage of the CRISPR-Cas12a system. When the ternary complex of Cas12a / crRNA / dsDNA activator is formed, the trans-cleavage activity of the CRISPR-Cas12a system is activated, exhibiting high trans-cleavage efficiency (conversion rate of approximately 1250 / s), cleaving multiple reporter probes and generating a strong recovery fluorescent signal. Conversely, the deletion of hAAG fails to induce the removal of deoxyinosine, and neither the initiation of transcription nor the generation of crRNA occurs. The CRISPR-Cas12a system cannot be activated, and therefore the FAM signal cannot be observed.

[0009] Furthermore, the amino acid sequence of the reporting probe is shown in SEQ ID NO.3.

[0010] Furthermore, the reporting probe is labeled with a fluorescent group and a quenching group.

[0011] The detection method for DNA glycosidase using the above-mentioned biosensor according to the present invention includes the following steps:

[0012] (1) Mix the hairpin probe, human depurinase / depyrimidine endonuclease and DNA glycosidase hAAG standard samples or test samples containing hAAG, and incubate at a constant temperature at a conventional temperature to obtain the product.

[0013] The hairpin probe has a stem-loop structure. The stem region of the hairpin probe is the T7 promoter sequence, and the third base A to the left of the start site GGG is replaced by a deoxyinosine base I. The amino acid sequence of the hairpin probe is shown in SEQ ID NO.1. The DNA sequence of the loop region of the hairpin probe is complementary to the crRNA and serves as a template for post-cleavage transcriptional amplification to synthesize crRNA. The amino acid sequence of the crRNA is shown in SEQ ID NO.2.

[0014] (2) The product obtained in step (1) is mixed with deoxyribonucleotide premix, ribonucleotide premix, KF polymerase, RNase inhibitor and T7 RNA polymerase, and incubated at a constant temperature at a normal temperature. The reaction is then terminated by high temperature incubation to obtain the product.

[0015] (3) The product obtained in step (2) is mixed with the CRISPR-Cas12a reaction solution and incubated at a constant temperature. Fluorescence detection is then performed. A standard curve is plotted based on the fluorescence values ​​of the hAAG standard samples of different concentrations. The FAM fluorescence value of the test sample containing hAAG is substituted into the standard curve to obtain the concentration of DNA glycosidase hAAG. The CRISPR-Cas12a reaction solution includes double-stranded activated DNA, a reporter probe, and Cas12a protein. The double-stranded activated DNA consists of a TS part and an NTS part. The TS part is complementary to crRNA. The reporter probe is labeled with a fluorescent group and a quenching group.

[0016] Further, the hairpin probe concentration in step (1) is 0.5-100 nM; the hAAG concentration in the DNA glycosidase hAAG standard sample is 1.0 × 10⁻⁶. -10 -1.0×10 -2 U / μL.

[0017] Furthermore, the amount of KF polymerase used in step (2) is 0.5-20U; the amount of T7 RNA polymerase used is 5-25U; step (2) also includes NEBuffer 2 and RNAPol reaction buffer.

[0018] Further, the concentration of Cas12a protein in step (3) is 0.08-3.2 μM; the concentration of the reporter probe is 0.032-0.32 μM.

[0019] The application of the hairpin probe, biosensor, and detection method described in this invention in screening hAAG inhibitors / activators.

[0020] Furthermore, the detection wavelength for fluorescence detection in step (3) is 492 nm.

[0021] Beneficial Effects: Compared with existing technologies, the present invention has the following significant advantages: The biosensor described in this application, through a designed hairpin probe, generates a large amount of crRNA in the presence of hAAG, resulting in a two-stage signal amplification reaction of the CRISPR-Cas12a signal: transcriptional amplification catalyzed by T7 RNA polymerase and loop cleavage catalyzed by the CRISPR-Cas12a system. The detection method of this application achieves low background and high sensitivity hAAG detection, with a detection limit of 9.25 × 10⁻⁶. - 11U / μL, dynamic range from 1.0×10 -10 The concentration is up to 0.01 U / μL, the operation is simple, and it can accurately measure the endogenous hAAG activity in cancer cells at the single-cell level. Attached Figure Description

[0022] Figure 1 This is a schematic diagram illustrating the principle of the biosensor of the present invention.

[0023] Figure 2 The image shows the results of 12% non-denaturing polyacrylamide gel electrophoresis (PAGE). Lane A represents the detection target of hairpin probes at various stages; lane M is the DNA standard molecular weight control; lane 1 is the product after the synthesized hairpin probe is cleaved; lane 2 is hairpin probe + hAAG + APE1 + 10×NEBuffer 4 + 10×NEBuffer 2; lane 3 is hairpin probe + APE1 + 10×NEBuffer 4 + 10×NEBuffer 2; B is the detection target of polymerization and transcription reaction products. Lane M is the DNA standard molecular weight control; lane 1 is the synthesized crRNA; lane 2 is hairpin probe + hAAG + APE1 + KF polymerase; lane 3 is hairpin probe + APE1 + KF polymerase; lane 4 is hairpin probe + hAAG + APE1 + KF polymerase + T7 RNA polymerase; lane 5 is hairpin probe + APE1 + KF polymerase + T7 RNA polymerase, and lanes 2-5 in B are all added with +10×NEBuffer 4 + 10×NEBuffer 2 + 10×RNAPol reaction buffer + dNTPs + NTPs).

[0024] Figure 3 The FAM fluorescence emission spectra were measured with and without hAAG.

[0025] Figure 4 A represents the fluorescence signal response of the biosensor of this invention to different concentrations of hAAG (a→k: 1.0×10⁻⁶). -10 1.0×10 –9 1.0×10 –8 1.0×10 –7 1.0×10 -6 1.0×10 -5 1.0×10 -4 1.0×10 -3 A represents the concentrations of 0.01, 0.1, and 1 U / μL; B represents the fluorescence intensity at 524 nm as a function of hAAG concentration; C represents the linear relationship between fluorescence intensity and the logarithm of hAAG concentration (range 1.0 × 10⁻⁶). -10 Up to 0.01 U / μL, the error bar represents the standard deviation of the three experiments.

[0026] Figure 5 The fluorescence intensity responses of hAAG, uracil DNA glycosylase (UDG), Dam methyltransferase (Dam), polynucleotide kinase, protein kinase A (PKA), and bovine serum albumin (BSA), as well as the control group containing only reaction buffer, were measured using the biosensor of this invention (all detection concentrations were 0.1 U / μL, and error bars represent the standard deviation of the three experiments).

[0027] Figure 6 The graph shows the detection results of the initial velocity when the hairpin probe concentration is 0-200 nM (where the hAAG concentration is 0.1 U / μL, and the error bars represent the standard deviation of the three experiments).

[0028] Figure 7 For the optimized hairpin probe concentration (error bars represent the standard deviation of the three experiments).

[0029] Figure 8 To optimize the activities of KF polymerase and T7 RNA polymerase (error bars represent the standard deviation of three experiments).

[0030] Figure 9 To optimize the concentration of Cas12a protein and reporter probe (error bars represent the standard deviation of three experiments).

[0031] Figure 10 1-500μM Cd 2+ The results of the effect on the relative activity of hAAG are shown in the figure (where the hAAG concentration is 0.1 U / μL, and the error bars represent the standard deviation of the three experiments).

[0032] Figure 11 The results of using the biosensor of this invention to detect reagent samples are shown in the figure. (A represents the response fluorescence intensity of cell extracts, and the detection objects are: cervical cancer cells (HeLa), human non-small cell lung cancer cells (A549), human normal liver cells (HL-7702), heat-inactivated HeLa cell extracts, and a control group containing only lysis buffer, with a cell count of 10,000 in each case; B represents the linear relationship between fluorescence intensity at 524 nm and the logarithm of the number of HeLa cells; C represents the linear relationship between fluorescence intensity at 524 nm and the logarithm of the number of A549 cells; and the error bars represent the standard deviation of the three experiments.) Detailed Implementation

[0033] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0034] The reagents and instruments used in this experiment are as follows:

[0035] Human alkyl adenine DNA glycosidase standard (hAAG), human purine / pyrimidine endonuclease 1 (APE1), Klenow fragment DNA polymerase (3'→5'exo-, KF polymerase), T7 RNA polymerase, Cas12a protein (Cpf1), deoxyribonucleotide (dNTPs) premix (i.e., deoxyguanosine, deoxyadenosine, deoxycytidine, and deoxythymidine), ribonucleotide (NTPs) premix (i.e., adenosine triphosphate, uridine triphosphate, guanine trinucleotide phosphate, and cytosine triphosphate), 10×NEBuffer 4 (10 mM DTT, 100 mM magnesium acetate, 500 mM potassium acetate, 200 mM Tris acetate, pH 7.9), 1, 0×NEBuffer 2 (composition: 100 mM MgCl2, 500 mM NaCl, 10 mM DTT, 100 mM Tris-HCl (pH 7.9) 10×RNAPol reaction buffer (400 mmol / L Tris-HCl, 60 mmol / L MgCl2, 20 mmol / L spermine, 100 mmol / L DTT, pH 7.9), uracil DNA glycosidase (UDG), DNA adenine methyltransferase (Dam-MT), T4 polynucleotide kinase (T4 PNK), protein kinase (PKA), and bovine serum albumin (BSA) were purchased from New England Biolabs (Ipswich, MA, USA). RNase-free TE buffer and chromium chloride (II) were purchased from Sigma-Aldrich (St. Louis, Missouri, USA). SYBR Gold nucleic acid dye was purchased from Thermo Fisher Scientific (Waltham, Massachusetts, USA). RNase inhibitor (Recombinant RNase Inhibitor, 2313Q, 500U concentration, 40U / μL) was purchased from TaKaRa Bio Inc. (Dalian, China). Human cervical cancer cell lines (HeLa cells), human lung adenocarcinoma cell lines, and human liver cell lines (HL-7702 cells) were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). All other reagents were analytical grade and used as is without further purification. Ultrapure water obtained from the Millipore filtration system (Temecula, CA, USA) was used in all experiments.

[0036] Fluorescence spectra were measured using a micro-quartz dish on a Hitachi F-7000 fluorescence spectrophotometer (Tokyo, Japan) at an excitation wavelength of 492 nm.

[0037] Example 1: Preliminary Preparation

[0038] Hairpin probe (SEQ ID NO: 1: (Bold text indicates the cleavage site; underlined text indicates the complementary sequence to crRNA.)

[0039] crRNA: (SEQ ID NO: 2: The underlined part is the sequence complementary to the hairpin probe, and the bolded part is the sequence complementary to the dsDNA activator TS.

[0040] Double-stranded activated DNA (dsDNA activator, dsDNA activator TS SEQ ID NO.4): The bolded portion is the sequence complementary to crRNA; dsDNA activator NTS SEQ ID NO.5: 5'-GAC AGA CAT ACT TTA TGA CACATG CTG GTC CCT ATA ACT GTC TGT GGA ATG TCA-3');

[0041] Report probe (SEQ ID NO.3: 5'-FAM-TTA TT-BHQ-3');

[0042] The product after the hairpin probe is cut (long chain SEQ ID NO.6: 5'-TAG GGA CCA GCA TGT GTC AATCTA CAC TTA GTA GAA ATT ACC CTA TAG TGA GTC GTA TTA-3', short chain SEQ ID NO.7: 5'-TAATAC GAC TCA CT-3');

[0043] The above sequence was synthesized by TaKaRa Bio Inc. (Dalian, China).

[0044] All the above oligonucleotides were dissolved in 1× RNase-free TE buffer (10 mM HCl, 1 mM EDTA, pH 7.5) to a final concentration of 10 μM to prepare a stock solution.

[0045] Before use, the hairpin probe was incubated in 1× annealing buffer (1.5 mM MgCl2 and 10 mM Tris-HCl, pH 8.0) at 95°C for 5 minutes, and then cooled to room temperature to fold into a perfect hairpin structure. dsDNA activator TS and dsDNA activator NTS were mixed in a 1:1 ratio and incubated in 1× annealing buffer at 95°C for 5 minutes, followed by cooling to room temperature to form double-stranded activated DNA.

[0046] Example 2: Detection of DNA glycosidase

[0047] (1) Mix 1 nanomolar hairpin probe per liter, 0.5 U / μL human depurinyl / depyrimidine endonuclease (APE 1), 1 μL 10×NEBuffer 2 and 1 μL 10×NEBuffer 4, and reagents with concentrations of 1.0×10⁻⁶. -10 1.0×10 –9 1.0×10 –8 1.0×10 –7 1.0×10 -6 1.0×10 -5 1.0×10 -4 1.0×10 -3 Mix with 0.01 U / μL of hAAG standard sample and incubate at 37°C for 20 minutes;

[0048] Mix 1 nanomolar hairpin probe per liter, 0.5 U / μL human depurinyl / depyrimidine endonuclease (APE 1), 1 μL 1×NEBuffer 2 and 1 μL 10×NEBuffer 4 with the test sample containing hAAG, and incubate at 37°C for 20 minutes.

[0049] (2) Mix 6 μL of each of the two reaction products obtained in step (1) with 1 μL of 1×RNAPol reaction buffer, 100 nanomoles per liter of deoxyribonucleotide premix, 250 micromoles per liter of ribonucleotide premix, 0.25 U / μL KF polymerase, 1 μL of RNase inhibitor, and 1 U / μL of T7 RNA polymerase to form a 20 μL system. React at 37°C for 30 minutes, and then incubate at 65°C for 10 minutes to terminate the reaction.

[0050] (3) Mix 15 μL of the polymerized transcription reaction product from step (2) with 15 μL of CRISPR-Cas12a reaction solution (containing 3 μL of 10×NEBuffer 2, 0.32 μmol / L double-stranded activated DNA (dsDNA activator), 0.16 μmol / L reporter probe, and 0.32 μmol / L Cas12a protein), and incubate at 37°C for 30 minutes. Perform fluorescence detection and plot a standard curve based on the FAM fluorescence values ​​of different concentrations of hAAG standard samples. Figure 4 C) Substitute the FAM fluorescence value of the test sample containing hAAG into the standard curve to obtain the concentration of DNA glycosidase hAAG.

[0051] Example 3: 12% non-denaturing polyacrylamide gel electrophoresis (PAGE) test of probe and product

[0052] 3.1 The product of the hairpin probe synthesized in Example 1 after cleavage (long chain + short chain after annealing) was added to water (control) as lane 1. The hairpin probe was added to hAAG, APE1, 10×NEBuffer 4, and 10×NEBuffer 2 as lane 2. The hairpin probe was added to APE1, 10×NEBuffer 4, and 10×NEBuffer 2 as lane 3. 12% non-denaturing polyacrylamide gel electrophoresis was performed on each lane. The results are as follows: Figure 2 As shown in Figure A, in the presence of hAAG, the running speed of the hairpin probe is consistent with that of the synthesized hairpin probe after cleavage. Furthermore, due to the unwinding of the ring region, its running speed is faster than that of the intact hairpin probe without hAAG. These results indicate that, with the assistance of APE1, hAAG can accurately cleave damaged deoxyinosine and destroy the hairpin probe.

[0053] 3.2 To further investigate whether the production of crRNA for transcription initiation can proceed, the polymer and transcription products generated in this method were analyzed by 12% non-denaturing PAGE. The crRNA synthesized in Example 1 (control group) was used as lane 1; the hairpin probe, hAAG, APE1, and KF polymerase were used as lane 2; the hairpin probe, APE1, and KF polymerase were used as lane 3; the hairpin probe, hAAG, APE1, KF polymerase, and T7 RNA polymerase were used as lane 4; and the hairpin probe, APE1, KF polymerase, and T7 RNA polymerase were used as lane 5. The results are as follows: Figure 2 As shown in Figure B, in the presence of hAAG, APE1, and KF polymerase, a 60 bp polymerized dsDNA product band appeared, moving more slowly than the hairpin probe, indicating that KF polymerase-catalyzed polymerization occurred. Finally, in the presence of T7 RNA polymerase, a 43 nt band identical to the synthesized crRNA appeared, indicating crRNA production. Conversely, when hAAG was absent, only the hairpin probe band was detected, indicating that no polymerization or transcription reaction occurred. These results clearly demonstrate the initiation of transcription-initiated crRNA generation. Fluorescence spectroscopy was also used in the experiment to verify the feasibility of the entire assay. Figure 3 The presence of hAAG resulted in a higher FAM signal, but no significant FAM was detected in the absence of hAAG. These results suggest that the presence of hAAG can induce CRISPR-Cas12a signal amplification during transcription initiation.

[0054] In this embodiment, each lane was filled with loading buffer, electrophoresis sample loading buffer, and the nucleic acid dye SYBRGold.

[0055] Example 4: Sensitivity Analysis of Biosensors

[0056] Based on the detection method of Example 2, this example measures the fluorescence intensity of the biosensor in response to different concentrations of hAAG. The concentration of the hAAG standard sample in step (1) of Example 2 is changed to 1.0 × 10⁻⁶. -10 1.0×10 –9 1.0×10 –8 1.0×10 –7 1.0×10 -6 1.0×10 -5 1.0×10 -4 1.0×10 -3 0.01, 0.1 and 1 U / μL, the results are as follows Figure 4 As shown, the fluorescence intensity at 524 nm increases with hAAG concentration from 1.0 × 10⁻⁶. -10 The effect is enhanced when U / μL is increased to 1 U / μL. Figure 4 (A and B). Furthermore, on a logarithmic scale, the fluorescence intensity is at 1.0 × 10⁻⁶. -10 Linear correlation with hAAG concentration in the range of U / μL to 0.01 U / μL ( Figure 4 C). The corresponding standard curve equation is F = 9886.56 + 932.61log 10 C (R 2 =0.9989), where F is the fluorescence intensity at 524 nm and C is the concentration of hAAG (U / μL). Based on the rule of adding three times the standard deviation to the mean control signal, the limit of detection is estimated to be 9.25 × 10⁻⁶. -11 U / μL. Compared with fluorescence assay based on rolling circle amplification (detection limit: 2.0 × 10⁻⁶). -7 (U / μL) and dephosphorylation-mediated chemiluminescent biosensor (detection limit: 1.53 × 10⁻⁶). -9 Compared to (U / μL), the sensitivity of the biosensor constructed in this application is improved by 2162 (9.25×10⁻⁶ U / μL). -11 / 2.0×10 -7 (U / μL) times and 17 (9.25×10) times. -11 / 1.53×10 -9 (U / μL) times.

[0057] Example 5: Specificity Analysis of Biosensors

[0058] Based on the detection method of Example 2, this example measures the fluorescence intensity of the biosensor to various biomarkers. The different concentrations of hAAG standard samples in step (1) of Example 2 are replaced with uracil DNA glycosidase (UDG), DNA adenine methyltransferase (Dam MTase), polynucleotide kinase (T4 PNK), protein kinase (PKA), and bovine serum albumin (BSA), respectively. Figure 5 As shown, only hAAG showed a high FAM signal. Conversely, no significant fluorescence signal was detected in the presence of UDG, Dam MTase, T4 PNK, PKA, or BSA, similar to the control group with only reaction buffer. This indicates that the proposed method has excellent specificity in distinguishing unrelated biomolecules and other DNA glycosidase members.

[0059] Example 6: Feasibility of the kinetic analysis method

[0060] This embodiment measures the initial velocity (V) of the hairpin probe concentration change, and changes the hairpin probe concentration in step (1) of embodiment 2 to 1, 5, 15, 25, 50, 100, 150, and 200 nM respectively. Figure 6 As shown, in the presence of 0.1 U / μL hAAG, the initial velocity gradually increased with the hairpin probe concentration from 5 nmol / L to 200 nmol / L, after incubation at 37 °C for 5 min. Vmax was determined to be 238.87 / s, and Km was calculated to be 15.35 nmol / L. The Km value is consistent with the values ​​obtained from radiometric assays (13–42 nmol / L). The results indicate that the proposed method can be used for hAAG kinetic analysis.

[0061] The results are as follows Figure 7 As shown, the optimal concentration range was ultimately selected as 0.5-100 nanomoles per liter.

[0062] Example 7: Optimization of KF polymerase and T7 RNA polymerase activities

[0063] Referring to the method of Example 2, the amount of KF polymerase added in step 2 was changed to 0.5, 2.5, 5, 10, and 20 U, respectively. Only a 0.1 U / μL hAAG standard sample was added. The remaining steps and conditions were the same as in Example 2. The results are as follows: Figure 8 As shown in Figure A, the F / F0 value increases as the amount of KF polymerase increases from 1U to 5U, and then decreases. Therefore, 5U of KF polymerase was selected as the preferred amount in this study.

[0064] Referring to the method of Example 2, the amount of T7 RNA polymerase added in step 2 was changed to 5, 10, 15, 20, and 25 U, respectively, and the hAAG concentration was 0.1 U / μL of hAAG standard sample. The remaining steps and conditions were the same as in Example 2. The results are as follows: Figure 8 As shown in Figure B, the F / F0 ratio increased significantly with the increase of T7 RNA polymerase from 5 U to 15 U, and then stabilized after exceeding 15 U. Therefore, the optimal amount of T7 RNA polymerase was determined to be 15 U in this study.

[0065] Example 8: Optimization of Cas12a protein and reporter probe dosage

[0066] Referring to the method of Example 2, the amount of Cas12a protein added in step 2 was changed to 0.02, 0.04, 0.08, 0.16, 0.32, and 3.20 μM, respectively, and the hAAG concentration was 0.1 U / μL of hAAG standard sample. The remaining steps and conditions were the same as in Example 2. The results are as follows: Figure 9 As shown in Figure A, the F / F0 value increased as the Cas12a protein concentration increased from 0.02 μM to 0.32 μM, and then decreased beyond 0.32 μM.

[0067] Referring to the method of Example 2, the amount of the reporting probe added in step 2 was changed to 0.016, 0.032, 0.08, 0.16, and 0.32 μM, respectively. The test sample containing hAAG was replaced with an hAAG standard sample with a concentration of 0.1 U / μL. The remaining steps and conditions were the same as in Example 2. The results are as follows: Figure 9 As shown in B, the F / F0 value increases as the reporter probe concentration increases from 0.016 μM to 0.16 μM, and then tends to stabilize when the concentration exceeds 0.16 μM.

[0068] The Cas12a protein can assemble with the transcribed crRNA and dsDNA activator to form a ternary complex, which sequentially trans-cleaves the reporter probe to generate a strong FAM fluorescence signal. Background interference signals will be introduced by excess Cas12a protein and reporter probe. Therefore, a concentration of 0.32 μM Cas12a protein and 0.16 μM reporter probe was selected as the optimal concentration for the method.

[0069] Example 9 Inhibitor Experiment

[0070] Chromium(II) is a common broad-spectrum inhibitor of DNA glycosidases. It can inhibit DNA substrate cleavage by binding to the substrate and directly inactivate the catalytic activity of DNA glycosidases by binding to its active site.

[0071] Referring to the method of Example 2, the test sample containing hAAG in step (1) was replaced with an hAAG standard sample with a concentration of 0.1 U / μL, and additionally Cd was added at concentrations of 1, 20, 40, 45, 60, 80, 110, 150, 180, 210, 250, 300, and 500 μM. 2+ The remaining steps and conditions are the same as in Example 2. The results are as follows: Figure 10 As shown, Cd 2+ The relative activity of hAAG was induced in a concentration-dependent manner. A 50% reduction in hAAG activity (IC50) was achieved. 50 The required inhibitor concentration was determined to be 51.93 μmol / L, comparable to the value obtained from radiometric assays (approximately 100 μmol / L). This result demonstrates that our method can be used for screening hAAG inhibitors, showing great potential in drug discovery and disease treatment.

[0072] Example 10: Actual Sample Testing

[0073] To test the feasibility of the proposed biosensor system for real-world sample analysis, this embodiment measured the endogenous hAAG concentration in human cervical cancer cell line (HeLa cells), human lung adenocarcinoma cell line (A549 cells), and human liver cell line (HL-7702 cells).

[0074] Human cervical cancer cell line (HeLa cells), human lung adenocarcinoma cell line (A549 cells), and human hepatocyte cell line (HL-7702 cells) were cultured in Dulbecco modified Eagle's medium (DMEM, Life Technologies, USA) containing 10% FBS (Life Technologies, USA) and 1% penicillin-streptomycin (Gibco, USA). All cells were cultured at 37°C in a humidified atmosphere containing 5% CO2. Nuclear extracts were collected at the growth index stage using a nuclear extraction kit (ActiveMotif, Carlsbad, CA, USA).

[0075] Referring to the method of Example 2, the test samples containing hAAG in step (1) were replaced with nuclear extracts and lysis buffers of 10,000 human cervical cancer cell lines (HeLa cells), human lung adenocarcinoma cell lines (A549 cells), human liver cell lines (HL-7702 cells), and inactivated hAAG (HeLa cells heated in a 95°C water bath for 10 min) cells. The remaining steps and conditions were the same as in Example 2.

[0076] The results are as follows Figure 11As shown in Figure A, low background signals were detected in the control group containing only lysis buffer and in the heat-treated (inactivated) HeLa cell extract. Low fluorescence signals were detected in the nuclear extract of HL-7702 cells, only slightly higher than those measured in the control group, indicating a lack of hAAG in normal cells. Conversely, high fluorescence signals were detected in the presence of nuclear extracts from HeLa cells and A549 cells, consistent with the overexpression of hAAG in human cancer cells.

[0077] This embodiment also investigated the relationship between fluorescence intensity and the number of HeLa and A549 cells. The total number of cells in the culture dish was determined by counting the HeLa and A549 cells cultured in the dish using a cell counter. Nuclear extracts from cells at the growth index stage were collected using a nuclear extraction kit (ActiveMotif, Carlsbad, CA, USA). The number of cells per microliter of extract was calculated by dividing the total number of cells by the volume of the nuclear extract. Volumes of HeLa cell extracts corresponding to 10,000, 1,000, 10,000, and 100,000 cells, and volumes of A549 cell extracts corresponding to 10, 100, 1,000, and 10,000 cells were taken. The test samples containing hAAG in step (1) were replaced with the above-mentioned extracts, while the remaining steps and conditions were the same as in Example 2. The linear relationship between cell number and fluorescence intensity was obtained by detecting the intensity of the fluorescence signal.

[0078] like Figure 11 As shown in B and C, fluorescence intensity was linearly correlated on a logarithmic scale with the number of HeLa cells in the range of 10,000–100,000 cells and the number of A549 cells in the range of 50,000–100,000 cells, respectively. The regression equation for the nuclear extract of HeLa cells was F = 2308.10 + 986.20 log 10 N (R 2 =0.9922), the regression equation for the nuclear extract of A549 cells is F = 232.85 + 802.19log 10 N Where F is the fluorescence intensity at 524 nm and N is the cell number. The detection limit reaches a single cell, demonstrating the high accuracy of the proposed method for detecting endogenous hAAG in cell extracts.

Claims

1. A biosensor for detecting DNA glycosidase, characterized in that, The sensor comprises a hairpin probe, a human depurinyl / depyrimidine endonuclease, a deoxyribonucleotide premix, a ribonucleotide premix, a KF polymerase, a T7 RNA polymerase, an RNase inhibitor, and a CRISPR-Cas12a reaction solution. The DNA sequence of the loop region of the hairpin probe is complementary to the crRNA, serving as a template for transcriptional amplification and synthesis of crRNA after cleavage. The hairpin probe has a stem-loop structure, with the stem region being the T7 promoter sequence, and the third base A to the left of the start site GGG being replaced by a deoxyinosine base I. The nucleotide sequence of the hairpin probe is shown in SEQ ID NO.1; the nucleotide sequence of the crRNA is shown in SEQ ID NO.

2. The CRISPR-Cas12a reaction solution comprises double-stranded activating DNA, a reporter probe, and Cas12a protein; the double-stranded activating DNA consists of a TS portion and an NTS portion; the nucleotide sequence of the reporter probe is shown in SEQ ID NO.3; The nucleotide sequence of the TS portion of the double-stranded activated DNA is shown in SEQ ID NO.4, and the nucleotide sequence of the NTS portion is shown in SEQ ID NO.

5.

2. The biosensor for detecting DNA glycosidase according to claim 1, characterized in that, The reporting probe is labeled with fluorescent and quenching groups.

3. A non-diagnostic detection method for DNA glycosidase using the biosensor according to any one of claims 1-2, characterized in that, Includes the following steps: (1) Mix the hairpin probe, human depurinase / depyrimidine endonuclease and DNA glycosidase hAAG standard samples or test samples containing hAAG, and incubate at a constant temperature to obtain the product. The hairpin probe has a stem-loop structure. The stem region of the hairpin probe is a T7 promoter sequence, and the third base A to the left of the start site GGG is replaced by a deoxyinosine base I. The nucleotide sequence of the hairpin probe is shown in SEQ ID NO.

1. The DNA sequence of the loop region of the hairpin probe is complementary to the crRNA and serves as a template for transcriptional amplification of crRNA after cleavage. The nucleotide sequence of the crRNA is shown in SEQ ID NO.

2. (2) The product obtained in step (1) is mixed with deoxyribonucleotide premix, ribonucleotide premix, KF polymerase, RNase inhibitor and T7 RNA polymerase, and incubated at a constant temperature at a normal temperature. The reaction is then terminated by high temperature incubation to obtain the product. (3) Mix the product obtained in step (2) with the CRISPR-Cas12a reaction solution, incubate at a constant temperature, and then perform fluorescence detection. Plot a standard curve based on the fluorescence values ​​of the different concentrations of hAAG standard samples. Substitute the FAM fluorescence value of the test sample containing hAAG into the standard curve to obtain the concentration of DNA glycosidase hAAG. The CRISPR-Cas12a reaction solution includes double-stranded activated DNA, a reporter probe, and Cas12a protein. The double-stranded activated DNA consists of a TS part and an NTS part, and the TS part is complementary to crRNA. The report probe is labeled with fluorescent and quenching groups.

4. The detection method according to claim 3, characterized in that, The hairpin probe concentration in step (1) is 0.5-100 nM; the hAAG concentration in the DNA glycosidase hAAG standard sample is 1.0 × 10⁻⁶. −10 -1.0×10 -2 U / µL.

5. The detection method according to claim 3, characterized in that, The amount of KF polymerase used in step (2) is 0.5-20U; the amount of T7 RNA polymerase used is 5-25U; step (2) also includes NEBuffer 2 and RNAPol reaction buffer.

6. The detection method according to claim 3, characterized in that, The concentration of Cas12a protein in step (3) is 0.08-3.2 µM; the concentration of the reporter probe is 0.032-0.32 µM.

7. The application of a biosensor according to any one of claims 1 to 2 and a detection method according to any one of claims 3 to 6 in screening hAAG inhibitors / activators.

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