Non-natural base DNA aptamer of targeted bacillus cereus 5 / B / 6 metal beta-lactamase and screening method of non-natural base DNA aptamer

Abstract

The invention discloses a non-natural base DNA aptamer targeting bacillus cereus 5 / B / 6 metal beta-lactamase and a screening method of the non-natural base DNA aptamer. The screening method comprises the following steps: respectively taking dNaMTP and dTPT3TP, or dCNMOTP and dTPT3TP as non-natural base nucleoside triphosphate substrates, and amplifying a DNA library containing non-natural bases by utilizing OneTaq DNA polymerase; the method comprises the following steps: carrying out PCR (Polymerase Chain Reaction) reaction on bacillus cereus 5 / B / 6 metal beta-lactamase, degrading 5 '-end phosphorylated DNA chains in a PCR product by virtue of lamda exonuclease to obtain two ssDNA libraries, and screening non-natural base DNA aptamers targeting bacillus cereus 5 / B / 6 metal beta-lactamase on the basis of the libraries to obtain three 70 nt non-natural base DNA aptamers. A biological membrane layer interference analysis result shows that the three non-natural base DNA aptamers have good binding affinity to bacillus cereus 5 / B / 6 metal beta-lactamase.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, and particularly relates to non-natural base DNA aptamer targeting Bacillus cereus 5 / B / 6 metallo-β-lactamase and a screening method thereof. BACKGROUND

[0002] Antibiotic resistance has become a major challenge in the field of global public health, among which β-lactamase-mediated resistance is one of the most important and widespread types of resistance in the clinic. Bacteria can synthesize and secrete β-lactamase, which specifically hydrolyzes the β-lactam ring in the β-lactam antibiotic molecule, destroys the bacteriostatic or bactericidal activity of the antibiotic, and ultimately leads to the failure of antibiotic treatment. According to the Ambler molecular classification, β-lactamase can be divided into A, B, C, and D types, among which A, C, and D types are all serine β-lactamases, which depend on the serine residue in the active center to complete the catalytic reaction, while B type is metallo-β-lactamase (MBLs), which strictly depends on the Zn 2+ ion in the active center to mediate the hydrolysis of antibiotic substrates through Zn 2+ -mediated nucleophilic reaction. The first metallo-β-lactamase was found in a harmless Bacillus cereus strain, and then MBLs-mediated resistance rapidly spread to pathogenic bacteria. Among them, the metallo-β-lactamase from Bacillus cereus 5 / B / 6 has hydrolysis activity to various β-lactam antibiotics including penicillins and cephalosporins, which directly leads to the failure of conventional antibiotic treatment.

[0003] Nucleic acid aptamer has the function of specifically binding to the target similar to antibody, but has more advantages compared with antibody, such as chemical synthesis, low production cost, simple quality control, small molecular weight, easy modification, good thermal stability, and easy transportation. In recent years, the related technologies of nucleic acid aptamer screening and application have developed rapidly, and the application range has been expanded to analytical chemistry, diagnostic detection, targeted therapy, drug development, and other aspects. However, the expansion of the function of natural nucleic acid aptamer is still limited by the limited chemical properties and spatial structure composed of four natural bases. By introducing non-natural bases into nucleic acid aptamer, it is expected to significantly improve the sequence diversity of nucleic acid aptamer, and to expand its chemical structure and functional diversity, providing a new technical path for improving the performance of nucleic acid aptamer.

[0004] At present, the research on nucleic acid aptamer targeting Bacillus cereus 5 / B / 6 metallo-β-lactamase is still limited, and the reported natural DNA aptamer binding affinity (K D ) is mostly between 100-233 nM; although the binding affinity of the 2'-F / OMe modified RNA aptamer is improved, its KD between 70.0-95.9 nM. Therefore, in order to meet the practical application requirements of high sensitivity detection and accurate targeting, it is urgent to obtain a new aptamer with higher affinity to Bacillus cereus 5 / B / 6 metallo-beta-lactamase. SUMMARY

[0005] The primary purpose of the present application is to overcome the defects and deficiencies of the prior art, and provide a non-natural base DNA aptamer targeting Bacillus cereus 5 / B / 6 metallo-beta-lactamase.

[0006] The second purpose of the present application is to provide a screening method for the non-natural base DNA aptamer targeting Bacillus cereus 5 / B / 6 metallo-beta-lactamase.

[0007] The third purpose of the present application is to provide an application of the non-natural base DNA aptamer targeting Bacillus cereus 5 / B / 6 metallo-beta-lactamase.

[0008] The purposes of the present application are achieved by the following technical solutions:

[0009] The non-natural base DNA aptamer targeting Bacillus cereus 5 / B / 6 metallo-beta-lactamase is Apt-BcN, Apt-BcC1 or Apt-BcC2; the nucleic acid sequences of Apt-BcN, Apt-BcC1 and Apt-BcC2 are as follows:

[0010] Apt-BcN: 5'-GGCTTTACACTTTATGACGGGCGGGCAGAATTGGAYTGGGCAGCCTGAAACGGTTTCACACAGTCCCAGT-3';

[0011] Apt-BcC1: 5'-GGCTTTACACTTTATGACGGCACAAGATGGAGAATYTGACACAGACCGACCGGTTTCACACAGTCCCAGT-3';

[0012] Apt-BcC2: 5'-GGCTTTACACTTTATGACGGCAGACCAACCACAAGYTGGATGAAGCGATACGGTTTCACACAGTCCCAGT-3';

[0013] Wherein, Y is dTPT3.

[0014] The screening method for the non-natural base DNA aptamer targeting Bacillus cereus 5 / B / 6 metallo-beta-lactamase comprises the following steps:

[0015] (1) using T70-N30 as a template, using non-natural base nucleotide triphosphates as substrates, using dATP, dTTP, dCTP and dGTP as substrates, and using OneTaq DNA polymerase to perform PCR amplification, and obtaining ssDNA library by degrading the 5' end phosphorylated DNA chain in the PCR product by lambda exonuclease;

[0016] (2) incubating the B. cereus 5 / B / 6 metallo-beta-lactamase with a His tag with Ni magnetic beads, and fixing the B. cereus 5 / B / 6 metallo-beta-lactamase on the Ni magnetic beads;

[0017] (3) incubating the Ni magnetic beads with the B. cereus 5 / B / 6 metallo-beta-lactamase fixed thereon with the ssDNA library in SELEX binding buffer, allowing the ssDNA library to fully bind to the B. cereus 5 / B / 6 metallo-beta-lactamase, washing with SELEX binding buffer, and finally eluting using an imidazole solution and collecting the eluate;

[0018] (4) using the eluate obtained in step (3) as a template, using non-natural base nucleotide triphosphates as substrates, using dATP, dTTP, dCTP and dGTP as substrates, and using OneTaq DNA polymerase to perform PCR amplification, and purifying the amplification product using streptavidin (SA) magnetic beads;

[0019] (5) using the purified product obtained in step (4) as a template and using the same substrates in step (1) to perform PCR amplification, and obtaining the ssDNA library of the next round after being separated into single strands, and repeating steps (2)-(5) until the eluate of the 8th round is obtained;

[0020] (6) performing PCR amplification and sequencing on the eluate of the 8th round, and the complementary sequence of the nucleic acid sequence with the highest frequency of repetition is the non-natural base DNA aptamer targeting the B. cereus 5 / B / 6 metallo-beta-lactamase;

[0021] The sequence of T70-N30 in step (1) is as follows:

[0022] 5'-P-ACTGGGACTGTGTGAAACCGNNNNNNNNNNNNNNXNNNNNNNNNNNNNNNCCGTCATAAAGTGTAAAGCC-3'; wherein: X is dNaM; 5'-P is 5' end phosphorylation;

[0023] The non-natural base nucleotide triphosphates in step (1) are a combination of dNaMTP and dTPT3TP, or a combination of dCNMOTP and dTPT3TP;

[0024] When the non-natural nucleoside triphosphates mentioned in step (1) are a combination of dNaMTP and dTPT3TP; when the biotin-labeled non-natural nucleoside triphosphates mentioned in step (4) are dNaMTP and dTPT3TP Bio TP;

[0025] When the non-natural nucleoside triphosphates mentioned in step (1) are a combination of dCNMOTP and dTPT3TP; and when the biotin-labeled non-natural nucleoside triphosphates mentioned in step (4) are dCNMOTP and dTPT3TP... Bio TP.

[0026] Furthermore, the primers used for PCR amplification in steps (1) and (4) are:

[0027] 5'P-PF-20: 5'-P-ACTGGGACTGTGTGAAACCG-3'; and,

[0028] PR-20:GGCTTTACACTTTATGACGG-3';

[0029] 5'-P is phosphorylated at the 5' end.

[0030] Furthermore, the imidazole solution mentioned in step (3) is an imidazole solution with a concentration of 500 mM or higher.

[0031] Further, the SELEX binding buffer described in step (3) contains the following components: HEPES, NaCl, KCl, MgCl2 and ZnSO4; the pH of the SELEX binding buffer is 7.5; even further, the SELEX binding buffer contains the following components: 20 mM HEPES, 150 mM NaCl, 6 mM KCl, 2 mM MgCl2 and 1 mM ZnSO4.

[0032] The application of the above-mentioned non-natural base DNA aptamers targeting Bacillus cereus 5 / B / 6 metallo-β-lactamases in the detection of Bacillus cereus 5 / B / 6 metallo-β-lactamases.

[0033] The above-mentioned non-natural base DNA aptamers targeting Bacillus cereus 5 / B / 6 metallo-β-lactamases are used in the preparation of Bacillus cereus 5 / B / 6 metallo-β-lactamase detection products.

[0034] Compared with the prior art, the advantages and progress of the present invention are as follows:

[0035] For the first time, two non-natural base pairs, dNaM-dTPT3 and dCNMO-dTPT3, were introduced in parallel into a screening system targeting Bacillus cereus 5 / B / 6 metallo-β-lactamases. Due to the differences in chemical structure between these two non-natural base pairs, they exhibit distinct upstream and downstream sequence preferences during PCR amplification, leading to the enrichment of a population of non-natural DNA aptamers with significantly different sequence compositions during SELEX. This strategy not only effectively overcomes the sequence diversity limitations of the natural four-base system but also achieves high sequence coverage screening of non-natural DNA aptamers by introducing two non-natural base pairs in parallel.

[0036] In each round of SELEX screening, this patent uses dNaMTP and dTPT3TP, or dCNMOTP and dTPT3TP, as non-natural nucleoside triphosphate substrates. OneTaq DNA polymerase is used to amplify and separate the DNA library into single strands, constructing two non-natural DNA libraries. These two libraries are then used to screen for non-natural DNA aptamers targeting Bacillus cereus 5 / B / 6 metallo-β-lactamases, ultimately yielding three 70 nt non-natural DNA aptamers targeting Bacillus cereus 5 / B / 6 metallo-β-lactamases. Electrogel migration assays (EMSA) verified that all three aptamers possess binding activity to metallo-β-lactamases 5 / B / 6. Finally, the binding affinity of these three aptamers to Bacillus cereus 5 / B / 6 metallo-β-lactamases was tested using biofilm layer interferometry (BLI). The results showed that the screened aptamers all had high binding activity to Bacillus cereus 5 / B / 6 metallo-β-lactamases, and have the potential to be developed into detection molecules in the future. Attached Figure Description

[0037] Figure 1 The diagram shows the chemical structures of the non-natural base pairs and natural base pairs used in this patent.

[0038] Figure 2 The image shows the SDS-PAGE gel electrophoresis results of Bacillus cereus 5 / B / 6 metallo-β-lactamase proteins; lane M is the protein marker.

[0039] Figure 3 A schematic diagram of the screening process for non-natural base DNA aptamers targeting Bacillus cereus 5 / B / 6 metallo-β-lactamases.

[0040] Figure 4The images show the gel electrophoresis results of the first round of PCR amplification products, the effectiveness of removing excess primers from the PCR amplification products, and the completeness of lambda exonuclease digestion of the PCR amplification products. In image A, the gel electrophoresis results of the first round of PCR amplification products are shown; lane M represents the low-range ssRNA ladder; lane 1 represents the PCR amplification products using dNaMTP and dTPT3TP; lane 2 represents the PCR amplification products using dCNMOTP and dTPT3TP. Image B shows the gel electrophoresis results of the first round of PCR amplification products after treatment with exonuclease I to remove excess primers; lane M represents the low-range ssRNA ladder. Ladder; Lane 1 shows the product obtained by adding exonuclease I to the PCR amplification products using dNaMTP and dTPT3TP for primer degradation; Lane 2 shows the product obtained by adding exonuclease I to the PCR amplification products using dCNMOTP and dTPT3TP for primer degradation; Lane C shows the gel electrophoresis analysis results verifying whether the PCR amplification products were completely digested with lambda exonuclease; Lane M is the low-range ssRNA ladder; Lane 1 shows the ssDNA obtained by adding lambda exonuclease to the PCR amplification products using dNaMTP and dTPT3TP after removing the primers; Lane 2 shows the product obtained by adding exonuclease I to the obtained Lib-NaM library; Lane 3 shows the ssDNA obtained by adding lambda exonuclease to the PCR amplification products using dCNMOTP and dTPT3TP after removing the primers; Lane 4 shows the product obtained by adding exonuclease I to the obtained Lib-CNMO library.

[0041] Figure 5 This image shows the biotinylated gel electrophoresis migration analysis results of the PCR amplification products eluted from the first round of screening of the Lib-NaM library. Lane M represents the low molecular weight ladder. Lanes 1 and 2, 3 and 4, 5 and 6, and 7 and 8 represent the products from 8, 10, 12, and 14 cycles of PCR amplification of the eluted products from the first round of screening of the Lib-NaM library, respectively. "+" indicates the product after incubation with streptavidin (SA); "-" indicates the product without SA incubation; "P" indicates the amplified product band; and "S" indicates the migration band generated after incubation of the amplified product with SA.

[0042] Figure 6This image shows the biotinylated gel electrophoresis migration analysis results of the PCR amplification products eluted from the first round of screening of the Lib-CNMO library. Lane M represents the low molecular weight ladder. Lanes 1 and 2, 3 and 4, 5 and 6, and 7 and 8 represent the products from 8, 10, 12, and 14 cycles of PCR amplification of the eluted products from the first round of screening of the Lib-CNMO library, respectively. "+" indicates the product after incubation with SA; "-" indicates the product without SA incubation; and "S" indicates the migration band generated after incubation with SA.

[0043] Figure 7 The figures show the real-time quantitative PCR (qPCR) analysis results of each component in each round of screening of the Lib-NaM library; where A~H are the qPCR analysis results of each component in rounds 1-8 respectively; curve E is the final eluted component; U is the component that did not bind to Bacillus cereus 5 / B / 6 metallo-β-lactamase after incubation; W1, W3, W5, W8, and W10 are the solutions obtained from the 1st, 3rd, 5th, 8th, and 10th washes respectively.

[0044] Figure 8 The figures show the real-time quantitative PCR (qPCR) analysis results of each component in each round of screening of the Lib-CNMO library; where A~H are the qPCR analysis results of each component in rounds 1-8 respectively; curve E is the final eluted component; U is the component that did not bind to Bacillus cereus 5 / B / 6 metallo-β-lactamase after incubation; W1, W3, W5, W8, and W10 are the solutions obtained from the 1st, 3rd, 5th, 8th, and 10th washes respectively.

[0045] Figure 9 The images show the predicted secondary structures of three non-natural base DNA aptamers: A is the predicted secondary structure of aptamer Apt-BcN screened from the Lib-NaM library; B is the predicted secondary structure of aptamer Apt-BcC1 screened from the Lib-CNMO library; and C is the predicted secondary structure of aptamer Apt-BcC2 screened from the Lib-CNMO library.

[0046] Figure 10The figure shows the EMSA validation analysis results of the binding activity of three non-natural DNA aptamers with Bacillus cereus 5 / B / 6 metallo-β-lactamases; in the figure, lane M is the low molecular weight ladder; lane 1 is aptamer Apt-BcN; lane 2 is the product of aptamer Apt-BcN incubated with metallo-β-lactamase; lane 3 is aptamer Apt-BcC1; lane 4 is the product of aptamer Apt-BcC1 incubated with metallo-β-lactamase; lane 5 is aptamer Apt-BcC2; and lane 6 is the product of aptamer Apt-BcC2 incubated with metallo-β-lactamase.

[0047] Figure 11 The diagram shows the biofilm layer interference (BLI) analysis results of the binding affinity of three non-natural DNA aptamers and Bacillus cereus 5 / B / 6 metallo-β-lactamases; where A, B, and C are the BLI analysis results of the binding affinity of aptamers Apt-BcN, Apt-BcC1, and Apt-BcC2 to metallo-β-lactamases, respectively. Detailed Implementation

[0048] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. It should be noted that any aspects not specifically described in detail are implementable or understandable by those skilled in the art upon reference to existing technology. Reagents or instruments whose manufacturers are not specified are considered to be commercially available products.

[0049] The primers, ssDNA containing non-natural bases, plasmids pET28a(+)-5 / B / 6, and BL21(DE3) used in the following examples were purchased or synthesized by Shanghai Sangon Biotech Co., Ltd.; the dNTPs used were purchased by Shanghai Baisai Biotechnology Co., Ltd.; and the deoxyribonucleoside triphosphates dNaMTP, dTPT3TP, and dCNMOTP containing non-natural bases used were purchased by WuXi AppTec, and their structural formulas are shown below. Figure 1 As shown; the dTPT3 used Bio TP was prepared in our laboratory (dTPT3) BioThe TP preparation method has been disclosed in the article "Wang, Guangyuan, et al. "Enzymatic synthesis of DNA with an expanded genetic alphabet using terminal deoxynuceotidyltransferase." ACS Synthetic Biology 11.12 (2022): 4142-4155."; the OneTaq DNA polymerase, 5× OneTaq buffer, exonuclease I, 10× exonuclease I buffer, lambda exonuclease, and 10× lambda exonuclease buffer used were purchased from New England Biolabs, USA.

[0050] The sequences involved in the following embodiments are shown in Table 1.

[0051] Table 1. Sequences involved in the embodiments

[0052]

[0053] The protein sequences of the His-tagged Bacillus cereus 5 / B / 6 metallo-β-lactamases used in the following examples are shown below:

[0054] MHKVIKNETGTISISQLNKNVWVHTELGYFSGEAVPSNGLVLNTSKGLVLVDSSWDDKLTKELIEMVEKKFKKRVTDVIITHAHADRIGGMKTLKERGIKAHSTALTAELAKKNGYEEPLGDLQSVT NLKFGNMKVETFYPGKGHTEDNIVVWLPQYQILAGGCLVKSASSKDLGNVADAYVNEWSTSIENVLKRYGNINLVVPGHGEVGDRGLLLHTLDLLKKLAAALEHHHHHH*; where: HHHHHH is the His tag.

[0055] The expression and purification steps of the His-tagged Bacillus cereus 5 / B / 6 metallo-β-lactamase are as follows:

[0056] (1) Transform plasmid pET28a(+)-5 / B / 6 into Escherichia coli BL21(DE3) competent cells, spread the transformation product on LB solid medium containing kanamycin, and incubate overnight at 37 ℃ with the medium inverted.

[0057] (2) Pick a single colony from the solid culture medium above and inoculate it into 2× YT medium (10 g / L yeast extract, 16 g / L tryptone, 5 g / L NaCl) with 50 μg / mL kanamycin added, and culture overnight at 37 °C with shaking.

[0058] (3) Transfer the overnight culture to fresh culture medium of the same formula at a ratio of 1:100, and continue to culture at 37 °C until the bacterial OD reaches the target value. 600 The concentration was increased to 0.6–0.8; then 1 mM IPTG and 1 mM ZnSO4 were added to induce protein expression, and the mixture was incubated at 25 °C for 18 h.

[0059] (4) The bacterial culture was centrifuged at 4 ℃ and 6000 rpm to collect the bacterial cells. The cells were resuspended in 1× buffer A (50 mM Tris-HCl, 5 mM imidazole, 150 mM NaCl, pH 8.0) and then homogenized using a high-pressure homogenizer. The homogenized liquid was centrifuged at 4 ℃ and 10000 rpm for 40 min, and the supernatant was filtered through a 0.45 μm filter membrane. The supernatant was then added to a nickel column containing Ni-NTA resin and incubated for 2 h to allow for complete binding.

[0060] (5) Rinse the nickel column with 10 column volumes of 1× buffer A, then perform gradient elution with elution buffer (50 mM Tris-HCl, 50–500 mM imidazole, 300 mM NaCl, pH 8.0) and collect the target protein.

[0061] (6) Collect the elution fractions with imidazole concentrations of 200 mM and 300 mM, and concentrate them using Amicon ultrafiltration centrifuge tubes (MWCO: 10 kDa). After concentrating to a certain volume, add protein dialysis buffer (50 mM Tris-HCl, 150 mM NaCl, 2 mM DTT, 0.1% (v / v) Tween 20, 1 mM ZnSO4, pH 7.5) to the ultrafiltration tube, centrifuge at 4500 rpm for 30 min, and repeat this operation 3 times. Collect the target protein in the ultrafiltration tube, measure the volume, add an equal volume of 80% glycerol, mix thoroughly, and store at -20 ℃ for later use.

[0062] The purified and concentrated protein was verified by 10% SDS-PAGE gel electrophoresis, and the results are as follows: Figure 2 As shown, the theoretical molecular weight of Bacillus cereus 5 / B / 6 metallo-β-lactamase protein is 26.1 kDa. The SDS-PAGE gel results show that the band size is consistent with the molecular weight, indicating that the target protein was successfully expressed.

[0063] The following examples illustrate the procedure for screening non-natural DNA aptamers targeting Bacillus cereus 5 / B / 6 metallo-β-lactamases. Figure 3 As shown.

[0064] Example 1: Design and synthesis of ssDNA library and primers containing non-natural bases

[0065] A 70 nt ssDNA library was constructed, consisting of 20 nt fixed sequence regions (essential for primer binding) at both the 5' and 3' ends, and a 30 nt random sequence region in the middle, with non-natural bases located at position 15 of the 30 nt random sequence region; the 70 nt ssDNA library is shown below:

[0066] T70-N30: 5'-P-ACTGGGACTGTGTGAAACCGNNNNNNNNNNNNNNXNNNNNNNNNNNNNCCGTCATAAAGTGTAAAGCC-3'.

[0067] The primers 5'P-PF-20 and PR-20 for amplifying the ssDNA library are shown below.

[0068] 5'P-PF-20: 5'-P-ACTGGGACTGTGTGAAACCG-3';

[0069] PR-20:GGCTTTACACTTTATGACGG-3';

[0070] Where: X is dNaM; 5'-P is 5'-terminal phosphorylation.

[0071] Example 2: Preparation of two ssDNA libraries containing non-natural bases

[0072] Using T70-N30 as a template, initial libraries were constructed by PCR using dNaMTP and dTPT3TP, or dCNMOTP and dTPT3TP, as non-natural nucleoside triphosphate substrates. The PCR reaction system is shown in Table 2. The reaction program was 94 ℃, 2 min; (96 ℃, 30 s; 60 ℃, 30 s; 68 ℃, 4 min) × 6; 68 ℃, 10 min. The library constructed using dNaMTP and dTPT3TP as non-natural nucleoside triphosphate substrates was named Lib-NaM, and the library constructed using dCNMOTP and dTPT3TP as non-natural nucleoside triphosphate substrates was named Lib-CNMO.

[0073] Table 2 PCR amplification reaction system

[0074]

[0075] The two PCR amplification products were purified using a Zymo kit, and then incubated with exonuclease I at 37 °C for 16 h to remove excess primers. The reaction system is shown in Table 3. The PCR amplification products and the products obtained after degrading excess primers with exonuclease I were analyzed by 15% denaturing polyacrylamide gel electrophoresis. The results are shown in Table 3. Figure 4 A and Figure 4 As shown in B in the diagram.

[0076] Table 3. Nucleotide exonuclease I degradation primer reaction system

[0077]

[0078] The PCR amplification products, after primer removal, were purified using the Zymo kit. Lambda exonuclease was then added and incubated at 37 °C for 1.5 h to degrade and remove the 5' phosphorylated DNA strands. The reaction system is shown in Table 4. The lambda exonuclease degradation products were purified using the Zymo kit to obtain two non-natural ssDNA libraries: Lib-NaM and Lib-CNMO. 5 μL of each Lib-NaM and Lib-CNMO library were added, and 1 μL of exonuclease I was added to each library. The libraries were incubated at 37 °C for 1 h to verify the complete removal of the 5' phosphorylated DNA strands from the PCR amplification products by lambda exonuclease. The exonuclease I degradation products were analyzed by 15% denaturing polyacrylamide gel electrophoresis. The results are shown in Table 4. Figure 4 As shown in C in the figure. The 5' phosphorylated DNA strands in both groups of samples were completely degraded by lambda exonuclease, and the obtained Lib-NaM and Lib-CNMO ssDNA libraries had high purity.

[0079] Table 4 Lambda exonuclease degradation reaction system

[0080]

[0081] Example 3: Screening process for non-natural DNA aptamers targeting Bacillus cereus 5 / B / 6 metallo-β-lactamases

[0082] (1) Pretreatment of magnetic beads: Take 20 μL of Ni magnetic beads and wash them three times with SELEX binding buffer (containing the following components: 20 mM HEPES, 150 mM NaCl, 6 mM KCl, 2 mM MgCl2, 1 mM ZnSO4; pH 7.5; the remainder is nuclease-free water) to remove impurities and ensure the surface of the magnetic beads is clean. Mix the Ni magnetic beads with His-tagged Bacillus cereus 5 / B / 6 metallo-β-lactamase at a final concentration of 1 μM and incubate at room temperature for 12 h to allow the protein to fully bind to the magnetic beads. Then, wash three more times with SELEX binding buffer to remove unbound protein and impurities to obtain pretreated Ni magnetic beads.

[0083] (2) Library screening: Two non-natural ssDNA libraries were mixed with SELEX binding buffer and incubated at 95 °C for 10 min, then slowly cooled to room temperature to obtain a cooled mixture. The cooled mixture was then added to pretreated Ni magnetic beads and incubated at 37 °C for 2 h to allow the library to fully bind to the target protein. After incubation, the supernatant was directly aspirated; this was the unbound component U. The Ni magnetic beads were then washed with 200 μL of SELEX binding buffer each time to remove non-specifically bound DNA. This was repeated 10 times, and the wash buffers W1-W10 were collected. Finally, 100 μL of 500 mM imidazole solution was added, and the DNA specifically bound to the target was eluted by rinsing. The eluent E was collected and purified using a Zymo kit to enrich the ssDNA library.

[0084] Example 4: Template preparation for the next round of ssDNA library construction after each round of screening

[0085] PCR amplification was performed using elution buffer E as a template, employing dTPT3. Bio TP and dNaMTP or dTPT3 Bio TP and dCNMO were used as substrates for non-natural nucleoside triphosphates. The PCR cycle number was optimized, selecting a cycle number with high non-natural base retention and few specific contaminants for PCR amplification. The PCR reaction system is shown in Table 5. The reaction program was 94 ℃, 2 min; (96 ℃, 30 s; 60 ℃, 30 s; 68 ℃, 4 min) × (8-14); 68 ℃, 10 min. The PCR amplification products were analyzed by 6% polyacrylamide gel electrophoresis, and the optimal PCR cycle number was determined. The results are shown below. Figure 5 and Figure 6As shown, PCR amplification was performed for 8 cycles. DNA strands containing non-natural base pairs in the PCR product were purified and enriched using SA magnetic beads. The final purified product was used as a template for the next round of ssDNA library construction to ensure that DNA strands containing non-natural base pairs were not lost in large quantities due to non-specific amplification during the screening process.

[0086] The SA magnetic bead purification steps are as follows:

[0087] (1) Take 50 μL of SA magnetic beads with a concentration of 10 mg / mL and place them in a centrifuge tube. Wash them three times with 200 μL of BWBS buffer (containing the following components: 10 mM Tris–HCl, 1 mM EDTA, 1 M NaCl, 0.01% Tween 20; pH 7.5; the remainder is nuclease-free water).

[0088] (2) The PCR product was diluted to 300 μl with BWBS buffer and mixed with the washed SA magnetic beads. The mixture was then incubated at 37 °C with slow shaking for 2 h. After incubation, the SA magnetic beads were washed 8 times with BWBS buffer.

[0089] (3) Add 100 μL of 95% formamide to the SA magnetic beads, incubate in a metal bath at 95 °C for 10 min, collect the supernatant, repeat the above operation 3 times, combine the supernatants and purify using the Zymo kit.

[0090] The method for constructing the ssDNA library in the next round is basically the same as in Example 2, except that the purified product obtained in Example 4 is used as the template for PCR amplification. After constructing the library, the steps of Example 3 and Example 4 are performed again. The steps of Example 2-4 are repeated for a total of 8 rounds of screening. In each round of screening, unbound component U, washing buffer W1-W10 and elution buffer E are obtained.

[0091] Table 5 Template preparation for the next round of ssDNA libraries

[0092]

[0093] Example 5: qPCR analysis of each component obtained from screening the Lib-NaM and Lib-CNMO libraries.

[0094] The unbound component U, washing buffers W1-W10, and elution buffer E obtained from each of the eight rounds of screening were analyzed by qPCR. The specific steps are as follows: using unbound component U, washing buffers W1-W10, and elution buffer E as templates, and 5'P-PF-20 and PR-20 as primers, qPCR was performed. The reaction system is shown in Table 6, and the reaction program is: 94 ℃, 2 min; (94 ℃, 30 s; 60 ℃, 30 s; 72 ℃, 1 min) × 40; 72 ℃, 5 min. The qPCR results are shown in Table 6. Figure 7 and Figure 8 As shown in the figure, compared with the curves of washing buffers W5-W10, the curve of elution buffer E has a smaller cycle number of peaks and a faster peak formation time. This indicates that non-natural base DNA aptamers were enriched in both the Lib-NaM and Lib-CNMO libraries during the 1st to 8th rounds of screening. Moreover, in the 7th and 8th rounds of screening, the cycle number of peak formation for the curve of elution buffer E was significantly smaller than that for the unbound component U, indicating that non-natural base DNA aptamers targeting Bacillus cereus 5 / B / 6 metallo-β-lactamases were enriched in large quantities during the 7th and 8th rounds of screening.

[0095] Table 6. qPCR of each component in the screening process for non-natural base DNA aptamers.

[0096]

[0097] Example 6: High-throughput sequencing of the library obtained from the final round of SELEX screening.

[0098] Using the libraries selected in the 8th round of screening as templates, Lib-NaM and Lib-CNMO libraries were amplified by PCR using upstream primers Seq-F1 and Seq-F2 with different barcode sequences and the same downstream primer PR-20, respectively. The reaction system is shown in Table 7. The reaction program was 94 ℃, 2 min; (96 ℃, 30 s; 55 ℃, 30 s; 68 ℃, 4 min) × 10; 68 ℃, 10 min. After purification of the PCR amplification products using the Zymo kit, they were analyzed by 15% denaturing polyacrylamide gel electrophoresis. A single band of the correct size was excised and purified using a polyacrylamide gel DNA recovery kit to obtain a high-purity library. The final library was sent to Shanghai Sangon Biotech Co., Ltd. for high-throughput sequencing using the Illumina XPus sequencing platform in PE150 mode.

[0099] Table 7. PCR amplification system for preparing high-throughput sequencing libraries

[0100]

[0101] Sequencing results were compared, and all sequences were sorted from highest to lowest frequency. The complementary sequences of the most frequently occurring sequences were likely non-natural DNA aptamers for Bacillus cereus 5 / B / 6 metallo-β-lactamase. The sequence ranked first in the Lib-NaM library was found to have a complementary sequence of Apt-BcN, while the sequences tied for first place in the Lib-CNMO library were found to have complementary sequences of Apt-BcC1 and Apt-BcC2, respectively. The secondary structures of these three sequences were predicted using M-fold sequencing, and the results are as follows: Figure 9 As shown.

[0102] Example 7: Electrophoretic migration assay (EMSA) to verify the binding activity of DNA aptamers containing non-natural bases to Bacillus cereus 5 / B / 6 metallo-β-lactamase.

[0103] The three sequences Apt-BcN, Apt-BcC1, and Apt-BcC2 were placed in SELEX binding buffer and incubated at 95 °C for 10 min, then slowly cooled to room temperature and placed on ice for 10 min to fold into secondary structures. The folded aptamers were then co-incubated with Bacillus cereus 5 / B / 6 metallo-β-lactamases for 1 h, and the binding activity of each DNA aptamer to the enzyme was verified by 5% non-denaturing polyacrylamide gel electrophoresis. EMSA analysis results are shown below. Figure 10 As shown, all three aptamers have the activity of binding to Bacillus cereus 5 / B / 6 metallo-β-lactamases.

[0104] Example 8. Determination of K+ binding between three non-natural DNA aptamers and Bacillus cereus 5 / B / 6 metallo-β-lactamase using biomembrane layer interferometry (BLI). D value

[0105] This example uses a Sartorius Octet R4 biomolecular interaction analyzer to detect the binding affinity of the aptamer to Bacillus cereus 5 / B / 6 metallo-β-lactamase using biolayer interferometry (BLI). In this example, bovine serum albumin (BSA) was added to the SELEX binding buffer at a final concentration of 1 mg / mL to reduce the influence of nonspecific binding. A SELEX binding buffer with a concentration of 0 for Bacillus cereus 5 / B / 6 metallo-β-lactamase was used as a control.

[0106] First, the 5' biotinylated aptamer was placed in binding buffer containing 5 mM MgCl2 and incubated at 95 °C for 10 min, then slowly cooled to room temperature and incubated on ice for 10 min to complete folding. The BLI assay procedure is as follows:

[0107] (1) Sensor equilibration: The probe with streptavidin pre-fixed on its surface was immersed in SELEX binding buffer and equilibrated for 60 s.

[0108] (2) Aptamer immobilization: The probe was immersed in a 5'-terminal biotinylated aptamer solution with a final concentration of 50 nM for 300 s.

[0109] (3) Sensor secondary balancing: Immerse the probe with the aptamer fixed in SELEX binding buffer for 60 s to complete baseline zeroing.

[0110] (4) Sample detection: The probes were immersed in different concentrations of Bacillus cereus 5 / B / 6 metallo-β-lactamase solution and incubated for 300 s to allow the aptamers to bind to Bacillus cereus 5 / B / 6 metallo-β-lactamase, and then dissociated in SELEX binding buffer for 600 s.

[0111] (5) Probe regeneration: Treat the probe with 5 M sodium chloride solution for 30 s to achieve probe regeneration.

[0112] The results of the BLI experiment are as follows: Figure 11 As shown, the K of the aptamer Apt-BcN was thus measured. D It is 2.367 × 10 -8 M, K of aptamer Apt-BcC1 D 3.209 × 10 -8 M; K of aptamer Apt-BcC2 D 3.939 × 10 -8 M. Experimental results showed that the three non-natural base DNA aptamers had good binding affinity to Bacillus cereus 5 / B / 6 metallo-β-lactamase.

Claims

1. A non-natural DNA aptamer targeting Bacillus cereus 5 / B / 6 metallo-β-lactamase, characterized in that, The non-natural base DNA aptamer is Apt-BcN, Apt-BcC1, or Apt-BcC2; the nucleic acid sequences of Apt-BcN, Apt-BcC1, and Apt-BcC2 are shown below: Apt-BcN: 5'-GGCTTTACACTTTATGACGGGCGGGCAGAATTGGAYTGGGCAGCCTGAAACGGTTTCACACAGTCCCAGT-3'; Apt-BcC1: 5'-GGCTTTACACTTTATGACGGCACAAGATGGAGAATYTGACACAGACCGACCGGTTTCACACAGTCCCAGT-3'; Apt-BcC2: 5'-GGCTTTACACTTTATGACGGCAGACCAACCACAAGYTGGATGAAGCGATACGGTTTCACACAGTCCCAGT-3'; Where Y is dTPT3.

2. The method for screening non-natural DNA aptamers targeting Bacillus cereus 5 / B / 6 metallo-β-lactamase as described in claim 1, characterized in that, Includes the following steps: (1) Using T70-N30 as a template, non-natural nucleoside triphosphates as substrates, and dATP, dTTP, dCTP and dGTP as substrates, PCR amplification was performed using OneTaq DNA polymerase, and ssDNA library was obtained by degrading the 5' phosphorylated DNA strand in the PCR product by lambda exonuclease. (2) Bacillus cereus 5 / B / 6 metallo-β-lactamase with His tag was co-incubated with Ni magnetic beads to immobilize Bacillus cereus 5 / B / 6 metallo-β-lactamase on Ni magnetic beads; (3) Incubate the Ni magnetic beads immobilized with Bacillus cereus 5 / B / 6 metallo-β-lactamase and the ssDNA library in SELEX binding buffer to fully bind the ssDNA library with Bacillus cereus 5 / B / 6 metallo-β-lactamase, wash with SELEX binding buffer, and finally elute with imidazole solution and collect the eluent. (4) Using the elution buffer obtained in step (3) as a template, non-natural nucleoside triphosphates as substrates, and dATP, dTTP, dCTP and dGTP as substrates, PCR amplification was performed using OneTaq DNA polymerase. The amplification products were purified using streptavidin magnetic beads. (5) Using the purified product obtained in step (4) as a template, perform PCR amplification with the same substrate as in step (1), then separate the single strands to obtain the next round of ssDNA library, and repeat steps (2)-(5) until 8 rounds of elution buffer are obtained. (6) The elution buffer from the 8th round was subjected to PCR amplification and sequencing. The complementary sequence of the nucleic acid sequence with the highest repetition frequency was a non-natural base DNA aptamer targeting Bacillus cereus 5 / B / 6 metallo-β-lactamase. The sequence of T70-N30 mentioned in step (1) is as follows: 5'-P-ACTGGGACTGTGTGAAACCGNNNNNNNNNNNNNNXNNNNNNNNNNNNNNNCCGTCATAAAGTGTAAAGCC-3'; where: X is dNaM; 5'-P is 5'-terminal phosphorylation; The non-natural nucleoside triphosphates mentioned in step (1) are a combination of dNaMTP and dTPT3TP, or a combination of dCNMOTP and dTPT3TP; When the non-natural nucleoside triphosphates mentioned in step (1) are a combination of dNaMTP and dTPT3TP; when the non-natural nucleoside triphosphates mentioned in step (4) are dNaMTP and dTPT3TP Bio TP; When the non-natural nucleoside triphosphates mentioned in step (1) are a combination of dCNMOTP and dTPT3TP; when the non-natural nucleoside triphosphates mentioned in step (4) are dCNMOTP and dTPT3TP Bio TP.

3. The screening method according to claim 2, characterized in that, The primers used for PCR amplification in steps (1) and (5) are: 5'P-PF-20: 5'-P-ACTGGGACTGTGTGAAACCG-3'; and, PR-20:GGCTTTACACTTTATGACGG-3'; 5'-P is phosphorylated at the 5' end.

4. The screening method according to claim 2, characterized in that, The SELEX binding buffer described in step (3) contains the following components: HEPES, NaCl, KCl, MgCl2 and ZnSO4; The pH of the SELEX binding buffer is 7.

5.

5. The screening method according to claim 4, characterized in that, SELEX binding buffer contains the following components: 20 mM HEPES, 150 mM NaCl, 6 mM KCl, 2 mM MgCl2 and 1 mM ZnSO4.

6. The screening method according to claim 2, characterized in that, The imidazole solution mentioned in step (3) is an imidazole solution with a concentration of 500 mM or higher.

7. The use of the non-natural base DNA aptamer targeting Bacillus cereus 5 / B / 6 metallo-β-lactamase as described in claim 1 in the detection of Bacillus cereus 5 / B / 6 metallo-β-lactamase.

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