WO2020014883A1 - Aptamère d'adn simple brin reconnaissant spécifiquement la tobramycine et application associée - Google Patents

Aptamère d'adn simple brin reconnaissant spécifiquement la tobramycine et application associée Download PDF

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WO2020014883A1
WO2020014883A1 PCT/CN2018/096048 CN2018096048W WO2020014883A1 WO 2020014883 A1 WO2020014883 A1 WO 2020014883A1 CN 2018096048 W CN2018096048 W CN 2018096048W WO 2020014883 A1 WO2020014883 A1 WO 2020014883A1
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tobramycin
aptamer
stranded dna
detection
sequence
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PCT/CN2018/096048
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Chinese (zh)
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周楠迪
聂晶晶
张玉红
韩旭艳
田亚平
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江南大学
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/115—Aptamers, i.e. nucleic acids binding a target molecule specifically and with high affinity without hybridising therewith ; Nucleic acids binding to non-nucleic acids, e.g. aptamers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/16—Aptamers

Definitions

  • the invention relates to a single-stranded DNA aptamer that specifically recognizes tobramycin and its application, and belongs to the field of biotechnology.
  • Tobramycin is an aminoglycoside antibiotic. It is a broad-spectrum antibacterial drug with good antibacterial effect. It is suitable for treating various infection symptoms of the respiratory, digestive, urinary, and reproductive systems. It is widely used in animals and aquaculture. Because tobramycin is a medicine shared by humans and animals, in addition to its direct side effects on humans, tobramycin remaining in food and the environment will eventually affect human health and safety.
  • Traditional antibiotic residue detection methods include microbial detection and instrumental analysis, including gas chromatography (GC), high performance liquid chromatography (HPLC), liquid-mass spectrometry (HPLC-MS), and liquid chromatography-string Polar mass spectrometry (HPLC-MS / MS), etc. Microbial detection method is widely used.
  • the instrumental analysis method has high sensitivity and good accuracy, but often the equipment is expensive, and it also has high requirements for the laboratory personnel. It requires high sample pretreatment, and the analysis is time-consuming and difficult to popularize. It is difficult to meet the needs of field testing.
  • Immunoassay method uses antigen-antibody specific binding reaction to detect various substances, and has been widely used in the field of food antibiotic residue detection.
  • Enzyme-linked immunosorbent assay combines the immune response of the antigen and antibody with the highly efficient catalytic reaction of the enzyme to effectively ensure high selectivity and sensitivity in the analysis.
  • the most reported method is the immunoassay method.
  • ELISA kit products for the detection of antibiotic residues on the market, but they are expensive and the types of enzyme-labeled antibodies are limited. Only a few types of common antibiotic residues can be detected. Because antibiotics are small molecule compounds, they do not have immunogenicity, and there are many restrictions on production and application.
  • antibiotic haptens with weak immunogenicity in antigen preparation are difficult to produce high titer antibodies; they are produced in animals such as mice and rabbits
  • heterogeneous antibodies may produce non-specific reactions (false positives); the preparation of antibodies is expensive, time-consuming and laborious, and clones are not easy to save; the quality varies from batch to batch; the activity of antibodies is difficult to maintain for a long time and is sensitive to temperature. Prone to irreversibility.
  • SELEX technology is a combinatorial chemistry technology that uses random oligonucleotide libraries to screen in vitro oligonucleotide fragments that can specifically bind to various ligands.
  • This technology has no special requirements for target molecules and can be proteins or nucleic acids . Oligopeptides, small molecule organics and even metal ions, the selected oligonucleotides are called aptamers. Due to the advantages of aptamers in many aspects, it has been widely used as a target-specific recognition element in biosensor analysis.
  • the aptamer sequence obtained by SELEX screening is generally long, which increases the uncertainty of the conformation of the aptamer, which is not conducive to its stable binding to the target molecule in different solution environments, and also increases the cost of synthesis.
  • the rational design based on structural analysis can be used to truncate and optimize the aptamer sequence, retain its binding site to the target molecule, and delete a large number of redundant sequences, which can greatly reduce the cost of synthesis.
  • the secondary structure of the original sequence is often destroyed, and the affinity and sensitivity will be reduced.
  • 79-nucleotide tobramycin aptamer ap32 was obtained using magnetic beads SELEX screening. Starting from aptamer ap32, the ap32 sequence was truncated through secondary structure analysis and rational design. By optimization, a piece of aptamer ap32-34nt with a nucleotide length of 34 was obtained, but the affinity of aptamer ap32-34nt was reduced.
  • the present invention on the basis of ap32-34nt, removes part of the terminal base pairs and unpaired bases and shortens the length of the secondary structure stem region to obtain a sequence with greatly shortened sequence length and improved affinity.
  • the aptamer ap32-15nt.
  • the first object of the present invention is to provide a single-stranded DNA aptamer that specifically recognizes tobramycin, the nucleotide sequence of which is shown in SEQ ID NO.1.
  • the 3 'end or 5' end of the aptamer modifies a functional group or molecule.
  • the modified aptamer has the same function as the aptamer described above.
  • the functional group or molecule is used to improve the stability of the aptamer, provide a detection signal, or to connect the aptamer with other substances to form a composition.
  • the functional group or molecule is a fluorescent group, digoxin, isotope, electrochemical label, enzyme label, biotin, amino group, affinity ligand or thiol group.
  • the aptamer further includes the nucleotide sequence shown in SEQ ID No. 1 as a core sequence, and the sequence is extended on both sides or unilaterally, and is adapted to the adaption. Aptamers with the same function.
  • the second object of the present invention is to provide the application of the single-stranded DNA aptamer that specifically recognizes tobramycin for separation and enrichment or analysis and detection of tobramycin.
  • a third object of the present invention is to provide a composition for detecting tobramycin, comprising the aptamer.
  • a fourth object of the present invention is to provide a test paper for detecting tobramycin, including the aptamer.
  • a fifth object of the present invention is to provide a kit for detecting tobramycin, including the aptamer.
  • a sixth object of the present invention is to provide a chip for detecting tobramycin, including the aptamer.
  • the present invention removes part of the terminal base pairs and unpaired bases and shortens the length of the secondary structure stem region to obtain a sequence with a greatly shortened sequence length and improved affinity.
  • Aptamycin aptamer ap32-15nt, and the use of aptamer ap32-15nt for a variety of tobramycin detection methods and detection reagents, aptamers have the advantages of high affinity, high specificity, stable structure, etc.
  • the established detection method has the advantages of short detection period, high sensitivity, low cost, and strong specificity.
  • Figure 2 Simulation of the secondary structure of the truncated aptamer ap32-15nt and its docking with tobramycin molecules;
  • Figure 3 The standard curve of the relationship between the peak current value and the concentration of tobramycin in the DPV curve for electrochemical detection of tobramycin;
  • Figure 4 Standard curve of the relationship between the absorbance of AuNPs solution of tobramycin at 520 nM and the concentration of tobramycin by gold colloid colorimetry;
  • Figure 5 Schematic diagram of test strip detection of tobramycin.
  • the secondary structure of the aptamer ap32-34nt (Kd 58.92nmol ⁇ L -1) obtained from previous research in the laboratory, and its nucleotide sequence is shown in SEQ ID NO. 4 as 5'-CGTCGACGGATCCATGGCACGTTATAGGTCGACG-3 ' Based on the aptamer (ap32-34nt aptamer, see the large paper "Screening of Tobramycin-Specific Single-Stranded DNA aptamers and Sequence Optimization and Application Research", Zhang Yuhong, Jiangnan University). Aptamer sequences, as shown in Table 1:
  • the stem-loop structure is analyzed, and the dissociation constant (K d ) is determined by the fluorescence method.
  • the specific steps are as follows (1)-(11):
  • Epoxy-based magnetic microspheres a particle diameter of 1-2 ⁇ m and a concentration of 10 mg ⁇ mL -1 ;
  • Tobramycin solution configured to a concentration of 10 mmol ⁇ L -1 ;
  • step (1) The epoxy-based magnetic microspheres in step (1) and the tobramycin in step (3) are washed and combined under suitable conditions; the appropriate conditions refer to making the epoxy-based magnetic microspheres compatible with Conditions of specific binding of ofloxacin, including temperature of 37 ° C, action time of 12h, and binding buffer composition;
  • step (5) Blocking the ethanolamine solution in step (5) and step (4) under suitable conditions;
  • suitable conditions include a temperature of 37 ° C and a blocking time of 6 hours;
  • step (2) The steps (6) and the truncated aptamer in step (2) are combined under appropriate conditions; suitable conditions include a temperature of 25 ° C. and a binding time of 1 h;
  • step (8) The aptamer obtained in step (8) is eluted under appropriate conditions; suitable conditions include a temperature of 80 ° C and a binding time of 15 minutes;
  • step (10) collecting the aptamers eluted in step (9) and measuring the fluorescence intensity
  • the fitting curve and Kd value are shown in Figure 1.
  • the secondary structure of the aptamer ap32-15nt is shown in Figure 2.
  • Autodock4.0 software to perform molecular docking simulation with tobramycin. After a series of calculations and simulations, it was determined that the aptamer was mainly composed of tobramycin. When tobramycin interacts with the aptamer, it mainly interacts with bases 2-5, 7, and 9 on the aptamer ( Figure 2).
  • Example 2 aptamer ap32-15nt electrochemical detection of tobramycin
  • the designed hairpin structure sequence is (SEQ ID No. 5): 5'-AAAAAAGACTAGGCACTAGTCAAAAAACCCCGATCCTAGTCTTTCCC-3 '; where the italic portion is the truncated aptamer sequence.
  • the designed signal transduction probe sequence is (SEQ ID NO. 6): 5'-GCGAAAAAAGCG- (CH 2 ) 6 -HS-3 ', and the 3' end of the probe is modified with a thiol group to self-assemble to the surface of a gold electrode.
  • the designed primer sequence is (SQE ID NO.7): 5’-AAAGACTAGGA-3 ’
  • a hairpin structure of Hp
  • signal transduction probe the primers were designed; and formulated concentrations of 10 ⁇ mol ⁇ L -1, 1 ⁇ mol ⁇ L -1, 10 ⁇ mol ⁇ L -1 .
  • step (1) The hairpin structure (Hp) in step (1) is heated at 95 ° C for 5 minutes and placed in a hot water bath at 41 ° C for 2h to form a hairpin structure.
  • Pretreatment of gold electrode Carefully polish the gold electrode (diameter 3mm) with alumina powder (particle diameters 0.5 and 0.05 ⁇ m), and then immerse them in ethanol and ultrapure water for 5min, respectively. Scanning cycle in 0.5M H 2 SO 4 with scanning range: 0.35V to -1.5V and scanning speed of 100mV / s. Electrochemically activated polished electrodes were obtained until a stable CV map was obtained. Rinse with pure water and blow dry with nitrogen for later use.
  • step (4) Formation of triple-stranded structure: The single-stranded DNA obtained in step (4) is further modified on the electrode of step (6) to form a triple-stranded structure on the electrode.
  • step (7) The electrode obtained in step (7) is incubated with the [Ru (NH 3 ) 6 ] 3+ solution in step (2) for 1 h at room temperature for electrochemical detection.
  • the gold electrode obtained in step (8) is a working electrode, and is detected by differential pulse voltammetry (DPV).
  • DPV differential pulse voltammetry
  • Tobramycin of different concentrations is taken.
  • the measurement is performed.
  • DPV curves after reaction under different tobramycin conditions.
  • the relationship between the peak current value and tobramycin in the DPV curve was analyzed, and a linear fitting curve was drawn ( Figure 3).
  • Figure 3 With the increase of tobramycin concentration, the oxidation peak current signal also increases. In the range of tobramycin concentration from 10nmol ⁇ L -1 to 200nmol ⁇ L -1 , the response current is linear with the concentration of tobramycin.
  • the application of the method for detecting tobramycin based on the finally determined truncated aptamer detects the tobramycin in a milk sample.
  • the specific steps are as follows: Trichloroacetic acid is added dropwise to the milk sample. (20%) The pH was adjusted to 4.6, and then a 45 ° C water bath was used for 10 minutes to precipitate the protein, and the coagulated protein and fat were removed by centrifugation at 10,000 r ⁇ min -1 for 25 minutes to obtain a pretreated milk sample. The samples processed in this step are tested according to steps (1)-(8) in the above steps.
  • Solution A AuNPs were prepared using trisodium citrate reduction method and concentrated 5 times;
  • Solution B tobramycin aptamer ap32-15nt, formulated at a concentration of 150nmol ⁇ L -1 ;
  • Liquid C NaCl solution, formulated at a concentration of 120mmol ⁇ L -1 ;
  • step (4) sequentially add tobramycin of different concentrations, and protect from light for 50 minutes;
  • step (6) Add 50 ⁇ L of the C solution of step (3) to the solution obtained in step (5), observe the color change of each centrifuge tube, and perform spectral characterization with a spectrophotometer, and draw the relationship between the absorbance of the AuNPs solution and the concentration of tobramycin.
  • Example 4 Detection of tobramycin by a tobramycin test strip
  • DNA1 (SEQ ID NO.8): 5'-HS- (CH 2 ) 6 -TCAGGACTAGTGCCTGTCCAACGTCAGATCC-3 '
  • DNA2 (SEQ ID NO.9): 5’-Biotin-CCGATGGATCTGACGT-3 ’
  • the gold label pad is a special inert medium. After the synthesized gold label conjugate is dropped on the gold label pad, it will be adsorbed in the special inert medium to make the product. In order to make the gold label couple added on the gold label pad dropwise, The complex can be completely released after rehydration, and the gold label pad needs to be pretreated. First, cut the gold label pad and sample pad to the appropriate size, soak them in the sample pad and gold label pad pretreatment buffer for 30 minutes, and then place them in a 45 ° C constant temperature drying oven to dry. Drop on the treated gold label pad Add AuNPs-ap32-15nt and AgNPs-DNA1, dry in a 37 ° C incubator, and store at 4 ° C in the dark under dry conditions for future use;
  • NC membrane nitrocellulose membrane
  • biotinylated DNA2 was combined with streptavidin (SA). Mix 10 ⁇ L, 1mg ⁇ mL -1 streptavidin and 20 ⁇ L, 10 ⁇ mol ⁇ L -1 biotinylated DNA2 and mix for 2h at 4 °C. Due to the strong binding force between SA and biotin The SA and biotinylated DNA2 were fully coupled. Continue adding 10 ⁇ L of 5 mmol ⁇ L -1 biotin to the above solution to supplement unbound sites on SA.
  • the PVC base plate, sample pad, gold label pad, nitrocellulose membrane, and water absorption pad are the five components of the colloidal gold test strip.
  • the PVC base plate is a sticky base plate, and other film materials can be directly pasted and assembled on the base plate.
  • the treated sample pad and gold label pad are respectively adhered to the PVC bottom plate, of which the sample pad and gold label pad ,
  • the gold label pad and the NC film overlap each other by 2mm
  • the water absorption pad and the NC film are overlapped by 2mm and pasted on the PVC base plate, and the excess part is cut off.
  • Each part is firmly adhered, cut into test strips with a size of 60mm ⁇ 4mm, and placed in an aluminum foil bag with a desiccant, and stored at 4 ° C in a refrigerator for future use (Figure 5A);
  • the test strip First put the prepared test strip in the test strip cartridge. After the solution to be tested is added dropwise to the sample loading hole, the liquid moves to the water absorption pad with the siphon action of the capillary, and the test solution passes the test line on the NC membrane and The quality control line, react with it, let it stand for 10 minutes. After the test strip is completely colored, when the tobramycin is not present, the detection line can capture the AgNPs-DNA1-ap32-15nt-AuNPs complex, which makes AuNPs accumulate and display. color. The SA modified on the C line can directly capture AuNPs-ap32-15nt under the action of biotin-avidin, and make the C line develop color. At this time, both T and C lines showed negative results ( Figure 5B).

Abstract

L'invention concerne un aptamère d'ADN simple brin reconnaissant spécifiquement la tobramycine et une application associée, qui se rapportent au domaine de la biotechnologie. Sur la base de l'ap32-34 nt, un aptamère de tobramycine ap32-15nt ayant une longueur de séquence considérablement raccourcie et une affinité améliorée est obtenu au moyen d'une manière d'éliminer une partie des paires de bases terminales et des bases non appariées et de raccourcir la longueur d'une région de tige de structure secondaire, et l'aptamère ap32-15nt est utilisé dans une pluralité de procédés de détection de tobramycine ainsi que la construction de réactifs de détection. L'aptamère présente les avantages d'une affinité élevée, d'une spécificité élevée, d'une structure stable, etc, et les procédés de détection établis présentent les avantages d'une courte période de détection, d'une sensibilité élevée, de faibles coûts, d'une forte spécificité, etc.
PCT/CN2018/096048 2018-07-16 2018-07-18 Aptamère d'adn simple brin reconnaissant spécifiquement la tobramycine et application associée WO2020014883A1 (fr)

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CN201810778296.8A CN108841828B (zh) 2018-07-16 2018-07-16 一种特异性识别妥布霉素的单链dna适配体及其应用
CN201810778296.8 2018-07-16

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CN113278684B (zh) * 2021-04-01 2022-11-25 江南大学 基于适配体和铂修饰金纳米粒子的妥布霉素检测试纸
CN113341128B (zh) * 2021-06-02 2023-05-16 江苏第二师范学院 一种检测妥布霉素的生物传感器及检测方法
CN113640268B (zh) * 2021-08-30 2023-03-28 南京林业大学 一种基于CRISPR-Cas12a的妥布霉素检测系统及检测方法

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