WO2020014883A1 - Single-stranded dna aptamer specifically recognizing tobramycin and application thereof - Google Patents

Abstract

Provided are a single-stranded DNA aptamer specifically recognizing tobramycin and an application thereof, which relate to the field of biotechnology. On the basis of ap32-34nt, a tobramycin aptamer ap32-15 nt having a greatly shortened sequence length and improved affinity is obtained by means of a manner of removing some of the terminal base pairs and unpaired bases and shortening the length of a secondary structure stem region, and the aptamer ap32-15nt is used in a plurality of tobramycin detection methods as well as the construction of detection reagents. The aptamer has the advantages of high affinity, high specificity, a stable structure, and so on, and the established detection methods have the advantages of a short detection period, high sensitivity, low costs, strong specificity, and so on.

Description

Single-stranded DNA aptamer specifically recognizing tobramycin and application thereof Technical field

The invention relates to a single-stranded DNA aptamer that specifically recognizes tobramycin and its application, and belongs to the field of biotechnology.

Background technique

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. Its advantages are low cost and can be operated in general laboratories, but the measurement time is long, the results are large, and the operation is complicated. Therefore, it cannot meet the needs of high-throughput and sensitive quantitative detection. 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 (ELISA) 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. There are currently some 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. These include: 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 The use of 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. These have largely limited the application of immunoassay methods in the detection of antibiotics.

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. However, 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. However, in the truncation process, the secondary structure of the original sequence is often destroyed, and the affinity and sensitivity will be reduced.

In our previous research, 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.

Summary of the invention

In order to solve the above problems, 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.

In one embodiment of the present invention, the 3 'end or 5' end of the aptamer modifies a functional group or molecule.

In one embodiment of the present invention, the modified aptamer has the same function as the aptamer described above.

In one embodiment of the present invention, 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.

In one embodiment of the present invention, the functional group or molecule is a fluorescent group, digoxin, isotope, electrochemical label, enzyme label, biotin, amino group, affinity ligand or thiol group.

In one embodiment of the present invention, 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.

Beneficial effects of the present invention: On the basis of ap32-34nt, 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.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1: K _d fitting curve of aptamer ap32-15nt;

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.

detailed description

In order to better understand the essence of the invention, the technical content of the invention will be described in detail through the following embodiments.

Example 1: Tobramycin ssDNA aptamer truncation

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:

Table 1 Aptamers and corresponding nucleotide sequences

Aptamer	Nucleotide sequence (SEQ ID No. 1 to 3)
ap32-15nt	5’-GACTAGGCACTAGTC-3 ’
ap32-13nt	5’-GACGGGCACCGTC-3 ’
ap32-15nt-2	5’-GACGTGGCACACGTC-3 ’

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):

(1) Epoxy-based magnetic microspheres: a particle diameter of 1-2 μm and a concentration of 10 mg · mL ^-1 ;

(2) truncated aptamer: the three truncated aptamers are modified with a fluorescent group (FAM);

(3) Tobramycin solution: configured to a concentration of 10 ^{mmol·L -1} ;

(4) ethanolamine solution: configured to a concentration of 0.5mol·L ^-1 ;

(5) 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;

(6) 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;

(7) 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;

(8) collecting the aptamer obtained through the processing in step (7);

(9) 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;

(10) collecting the aptamers eluted in step (9) and measuring the fluorescence intensity;

(11) Using the aptamer concentration as the abscissa and the fluorescence intensity as the ordinate, Grapad Prism 5.0 was used for non-linear fitting to analyze the dissociation constant K _d of the aptamer.

In the end, the affinity of the aptamer ap32-15nt increased, while the affinity of the other two aptamers decreased significantly. The fitting curve and Kd value are shown in Figure 1. The Kd value of the aptamer ap32-15nt is 42.12nmol. · L ^-1 , compared with ap32-34nt (Kd = 58.92nmol·L ^-1 ), the affinity is greatly improved, and compared with the original aptamer ap32 (Kd = 52.32nmol·L ^-1 ), Affinity has also been improved.

The secondary structure of the aptamer ap32-15nt is shown in Figure 2. Using 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 ’

(1) a hairpin structure (of Hp), signal transduction probe, the primers were designed; and formulated concentrations of ^{10μmol·L -1, 1μmol·L -1, 10μmol·L -1} .

(2) Trichlorohexamine ruthenium ([Ru (NH ₃ ) ₆ ] ³⁺ ) solution: 10 ^{mmol·L -1} Tris-HCl was used to prepare a concentration of 10 μmol·L ^-1 .

(3) 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.

(4) Replacement of single-stranded DNA: in 15.6 μL of 1 × CutSmart buffer containing ¹ μmol·L ^-1 primer, 5U phi29 DNA polymerase, ^{1 mmol·L -1} dNTP and 5U Nt.AlwI nicking endonuclease Add 2.4 μL of the hairpin structure (Hp) in step (3). Then a tobramycin-containing sample solution (2 μL) was added to the buffer, and incubated at 37 ° C. for 120 min to perform an enzymatic reaction. The resulting mixture was then immersed in a hot water bath at 65 ° C for 10 min to inactivate the enzyme. Finally, a large number of single-stranded DNA target sequences were obtained.

(5) Pretreatment of gold electrode: Carefully polish the gold electrode (diameter 3mm) with alumina powder (particle diameters 0.5 and 0.05 μm), 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.

(6) Fixation of the signal transduction probe: 10 ^{mmol·L -1} Tris-HCl (10 ^{mmol·L -1} tris (2-carboxyethyl) phosphine TCEP, 100 mmol · L ^-1 NaCl, pH 7.4). After incubation for 1 h, immerse the gold electrode obtained in step (5) in 100 μL of the incubation auxiliary probe, and incubate at 37 ° C for 13 h, so that the capture probe will form on the surface of the gold electrode. Assemble the monolayer; block the electrode with ^{1 mmol·L -1} mercaptohexanol for 1 h to obtain a gold electrode with modified capture probe.

(7) 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.

(8) 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). Tobramycin of different concentrations is taken. After the same operations as in steps (1) to (8) above and the reagents are reacted, 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). 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. Correlation, the fitting curve equation y = 0.0005x + 0.0788 (x is the concentration of tobramycin / nmol·L ^-1 , y is the peak current value / μA), and the linear correlation coefficient R ² = 0.994.

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.

Example 3: Detection of Tobramycin by Gold Gel Colorimetric Kit

(1) Solution A: AuNPs were prepared using trisodium citrate reduction method and concentrated 5 times;

(2) Solution B: tobramycin aptamer ap32-15nt, formulated at a concentration of 150nmol·L ^-1 ;

(3) Liquid C: NaCl solution, formulated at a concentration of 120mmol·L ^-1 ;

(4) Take 50 μL of the liquid A in step (1) and 100 μL of the liquid B in step (2) at room temperature in the dark for 1 h;

(5) To the step (4), sequentially add tobramycin of different concentrations, and protect from light for 50 minutes;

(6) Add 50 μL 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.

In the presence of different concentrations of tobramycin, as the concentration of tobramycin increases, the color of the solution can be observed by the naked eye from the original wine red to blue (Figure 4); the absorption of AuNPs solution at 520nm The relationship between the value and the concentration of tobramycin. When the concentration of tobramycin is in the range of 100-1400 nmol·L ^-1 , the absorbance at 520 nm of the AuNPs solution gradually decreases as the concentration of tobramycin increases. Small, showing a good linear relationship (Figure 4). The linear relationship is: y = -2.599e-4x + 1.02186 (x is the concentration of tobramycin / nmol·L ^-1 , and y is the absorbance value), and the linear correlation coefficient R ² = 0.99047.

Example 4: Detection of tobramycin by a tobramycin test strip

(1) Modified tobramycin aptamer (ap32-15nt): 5'-HS- (CH ₂ ) ₆ -GACTAGGCACTAGTC-Biotin-3 '

DNA1 (SEQ ID NO.8): 5'-HS- (CH ₂ ) ₆ -TCAGGACTAGTGCCTGTCCAACGTCAGATCC-3 '

DNA2 (SEQ ID NO.9): 5’-Biotin-CCGATGGATCTGACGT-3 ’

(2) Pretreatment of sample pad and gold label pad

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;

(3) Pretreatment of nitrocellulose membrane (NC membrane)

First, biotinylated DNA2 was combined with streptavidin (SA). Mix 10μL, 1mg · mL ^-1 streptavidin and 20μL, 10μmol·L ^-1 biotinylated DNA2 and mix for 2h at 4 ℃. Due to the strong binding force between SA and biotin The SA and biotinylated DNA2 were fully coupled. Continue adding 10 μL of 5 mmol·L ^-1 biotin to the above solution to supplement unbound sites on SA. After incubation for 1 h, remove unbound biotin and DNA with ultrafiltration centrifuge tubes (30 kDa), and take 1 μL of bound SA -Biotin-DNA2 Prepare a detection line (T line) on the NC membrane 6mm near the end of the gold label pad. Take 1μLSA and draw a quality control line (line C) 6mm near the end of the absorbent pad. Dry the prepared nitrocellulose membranes at T and C lines at 37 ° C to fully fix them on the NC membrane, and store them at 4 ° C under dry conditions for future use;

(4) Assembly of test strips

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. First, paste the NC film to the middle position of the PVC bottom plate, and press gently to make the NC film and the bottom plate closely adhere to each other. Then, 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, and finally 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);

(5) Test strip test

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). When tobramycin was present, ap32-15nt had much higher affinity with tobramycin than its complementary sequence, so ap32- 15nt binds tobramycin and falls off on the test line. Therefore, the color of the T line becomes light or completely faded, and the C line can still capture the normal coloration of the biotinylated ap32-15nt, which shows a positive result at this time (Figure 5C ).

The embodiments described above are merely preferred embodiments for fully explaining the present invention, and the protection scope of the present invention is not limited thereto. Equivalent substitutions or changes made by those skilled in the art on the basis of the present invention are all within the protection scope of the present invention. The protection scope of the present invention is subject to the claims.

Claims (9)

1. A single-stranded DNA aptamer that specifically recognizes tobramycin is characterized in that its nucleotide sequence is shown in SEQ ID NO.1.
2. The single-stranded DNA aptamer that specifically recognizes tobramycin according to claim 1, wherein the 3 'end or the 5' end of the aptamer modifies a functional group or molecule.
3. The single-stranded DNA aptamer that specifically recognizes tobramycin according to claim 2, wherein the functional group or molecule is a fluorescent group, digoxin, an isotope, an electrochemical label, an enzyme Label, biotin, amino, affinity ligand, or thiol.
4. The single-stranded DNA aptamer that specifically recognizes tobramycin according to claim 1, characterized in that the aptamer further comprises a nucleotide sequence as shown in SEQ ID NO.1 as a core sequence An aptamer that extends the sequence on both sides or on one side and has the same function as the aptamer according to claim 1.
5. The use of a single-stranded DNA aptamer that specifically recognizes tobramycin according to any one of claims 1 to 4 in the isolation, enrichment, or analysis of tobramycin.
6. A composition for detecting tobramycin, comprising the aptamer according to any one of claims 1 to 4.
7. A test paper for detecting tobramycin, comprising the aptamer according to any one of claims 1 to 4.
8. A kit for detecting tobramycin, comprising the aptamer according to any one of claims 1 to 4.
9. A chip for detecting tobramycin, comprising the aptamer according to any one of claims 1 to 4.

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