Screening method of tilmicosin specific aptamer

Abstract

The invention relates to a nucleic acid aptamer for specifically recognizing tilmicosin as well as screening and application of the nucleic acid aptamer, and belongs to the technical field of food safety detection and biosensing. Aiming at the defects of the existing method for detecting tilmicosin residues in food, the invention provides a high-affinity and high-specificity nucleic acid aptamer obtained by screening based on a graphene oxide index enrichment ligand systematic evolution technology. The aptamer is obtained by multiple rounds of screening from a random single-stranded DNA library through a GO-SELEX technology, and a core sequence Apt-2-1 with the length of 33 nt is finally obtained through sequence analysis and structure optimization. The aptamer shows excellent binding capacity to tilmicosin, the dissociation constant reaches 9.82 nM, and the aptamer has good specificity. The nucleic acid aptamer provided by the invention can be used for constructing a rapid and sensitive tilmicosin residue detection method, and has important application value in monitoring of livestock and poultry products and food safety.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and food safety testing, specifically relating to a method for screening high-affinity and high-specificity aptamers for the antibiotic tilmicosin, and its application in residue detection. Background Technology

[0004] Existing detection methods (such as HPLC and LC-MS / MS) suffer from problems such as expensive equipment and cumbersome sample preparation. Aptamers, as novel molecular recognition elements, have advantages such as ease of modification and good stability; however, screening studies of tilmicosin aptamers are scarce, mainly facing the following technical bottlenecks:

[0005] 1. Difficulty in immobilizing small molecules: Traditional SELEX requires immobilization of the target, which may alter the structure of tilmicosin;

[0006] 2. Lack of specificity: Macrolide antibiotics have similar structures and are prone to cross-reactions;

[0007] 3. Lack of affinity optimization: The existing aptamer length is redundant and has not been truncated to improve performance.

[0008] Aptamers are a class of single-stranded DNA or RNA molecules typically obtained through phylogenetic ligand enrichment (SELEX) screening. They are short, single-stranded oligonucleotide sequences that can form partially complementary secondary structures. Common aptamer secondary structures include stem-loop structures, hairpin structures, and G-quadruplex structures. In addition, the tertiary structures of aptamers can form specific binding pockets or surfaces, allowing them to bind tightly to target molecules (such as proteins, small molecules, and ions) with high affinity and specificity. Therefore, aptamers have been widely used to build various types of biosensors, enabling real-time, rapid, and sensitive detection of target substances. Summary of the Invention

[0009] The purpose of this invention is to provide a GO-SELEX-based tilmicosin aptamer screening method to solve the problem of small molecule immobilization, and to obtain high affinity (K) through reverse screening and truncation optimization. d ≤10 nM), highly specific aptamers.

[0010] To achieve the above-mentioned technical objectives, the technical solution adopted by the present invention includes the following steps:

[0011] 1. GO-SELEX screening: Using graphene oxide (GO) as the separation medium, it utilizes π-π stacking to non-specifically adsorb unbound ssDNA; alternating forward screening (target: tilmicosin) and reverse screening (reverse screening target: erythromycin and 4 other structural analogs); the DNA recovery rate tends to stabilize through 11 rounds of screening.

[0012] 2. High-throughput sequencing and candidate screening: The enriched library was sequenced to obtain 23 high-frequency sequences; they were divided into 4 families according to homology, and 4 candidate aptamers (Apt-2, 13, 16, 21) were selected based on secondary structure stability (ΔG value).

[0013] 3. Affinity and specificity verification: K was determined by GO fluorescence competitive assay. d Value, Apt-2 has the best affinity (K d =21.86 nM); specificity tests showed that Apt-2 binds only to tilmicosin.

[0014] 4. Molecular docking and truncation optimization: Simulating the binding mode of tilmicosin to Apt-2, key binding sites were identified (hydrogen bonds: DC52; hydrophobic interactions: DG53 and 9 other nucleotides); non-essential sequences were truncated to obtain a 33 nt truncated version, Apt-2-1, K. d The specificity was reduced to 9.82 nM, which significantly improved the specificity.

[0015] This invention offers the following advantages: It is the first to introduce reverse screening into GO-SELEX, eliminating cross-reactions; based on precise truncation via molecular docking, it increases aptamer affinity by more than two times. The aptamer length is shortened by 58% (79 nt → 33 nt), reducing synthesis costs; K d Its value reaches nanomolar levels (9.82 nM), which is superior to similar antibiotic aptamers; it provides a core identification element for rapid on-site detection. Attached Figure Description

[0016] Figure 1 Schematic diagram of aptamer screening principle, where GO represents graphene oxide.

[0017] Figure 2 Recovery rate during aptamer screening process.

[0018] Figure 3 Candidate aptamer sequence diagram.

[0019] Figure 4 Affinity analysis of truncated aptamers.

[0020] Figure 5 Specificity analysis of truncated aptamers, where TET is tetracycline, PEN is penicillin, CHL is chloramphenicol, ERY is erythromycin, and SUL is sulfadiazine. Detailed Implementation

[0021] Example 1: GO-SELEX screening of tilmicosin aptamers ( Figure 1 )

[0022] (1) Library design and pretreatment: Initial ssDNA library: 79 nt (5'-ACCGACTCGTATGCCGT-N35-CGGTAGCGTACTCGCA-3'); 1 μL of the initial 1000 μM DNA library was added to 199 μL of binding buffer and incubated at 95 °C for 10 min. The mixture was then rapidly cooled on ice for 10 min and equilibrated at 25 °C for 10 min to complete denaturation.

[0023] (2) Forward screening (rounds 1-6, 9-11): The denatured library was mixed with 1 μL of 1000 μM tilmicosin and incubated at room temperature for 1 h; 500 μL of GO (2 mg / mL) was added and stirred for 2 h to adsorb unbound ssDNA; the supernatant (containing the target-aptamer complex) was collected by centrifugation (12,000 rpm, 15 min); PCR amplification: 1 µL of 20 µM upstream primer, 1 µL of 20 µM biotinylated downstream primer, 1 µL of purified ssDNA template, and 12.5 µL of PCR premix were added to 9.5 µL of ultrapure water to prepare a 25 µL PCR mixture. The mixture was then amplified in a PCR amplification instrument through a series of reactions. Procedure: Pre-denaturation (95℃, 5 min), denaturation (95℃, 30 s), annealing (55℃, 30 s), extension (72℃, 15 s), final extension (72℃, 10 min), then cooled at 4℃ for later use. ssDNA was isolated using streptavidin magnetic beads for the next round of screening.

[0024] (3) Reverse screening (rounds 7-8): Incubate the ssDNA library with erythromycin, gamimycin, tylosin, and terbinafine (10 μL each, 1000 μM); add GO to adsorb and bind the ssDNA of the reverse screening target, and discard the supernatant; after resuspending the precipitate, add tilmicosin to dissociate the high-affinity ssDNA.

[0025] (4) Termination condition: Stop screening when the DNA recovery rate stabilizes (reaching 22.3% in round 11). Figure 2 ).

[0026] Example 2: Candidate Aptamer Screening and Validation

[0027] (1) High-throughput sequencing and analysis: Sequencing of the 11th round of products yielded 23 high-frequency sequences; DNAMAN software clustered them into 4 families; based on the ΔG values ​​predicted by M-fold sequencing, Apt-2, 13, 16, and 21 were selected. Figure 3 )

[0028] (2) Affinity assay (GO fluorescence competition method): FAM-labeled aptamers (100–400 nM) were incubated with tilmicosin (1000 nM) for 2 h; GO (50 μL, 2 mg / mL) was added to quench the fluorescence of unbound aptamers; after centrifugation, the fluorescence intensity (λ) of the supernatant was measured. ex / λ em =490 / 520 nm); nonlinear fitting calculation of Kd: Y=B max ×X / (K d +X). Specificity test: Apt-2 (100 nM) was incubated with each antibiotic (1000 nM); the fluorescence intensity of the tilmicosin group was significantly higher than that of the other groups.

[0029] Example 3: Molecular docking and truncation optimization

[0030] (1) Molecular docking simulation: The tilmicosin structure was obtained from PubChem, and the energy was optimized and saved in mol2 format; the Apt-2 three-dimensional model was constructed using RNAComposer; AutoDock 4.2 docking was performed, and the binding energy ΔG = -8.4 kcal / mol was determined; key binding sites were identified: hydrogen bond (DC52) and hydrophobic interaction (9 nucleotides including DG53).

[0031] (2) Truncated aptamer design: The stem-loop region containing the binding site (33 nt) was retained; sequence: 5'-CGGTAGCGTACTCGCACTGGCGACGGTTGGGCGA-3' (Apt-2-1); verified K d =9.82±0.97 nM ( Figure 4 ), with better specificity than full-length ( Figure 5 ).

Claims

1. A method of screening for tilmicosin specific aptamer, characterized in that, The graphene oxide systematic evolution of ligands by exponential enrichment (GO-SELEX) includes the following steps: (a) Forward screening: denatured single-stranded DNA (ssDNA) initial library is incubated with tilmicosin solution, and graphene oxide (GO) is added to adsorb unbound ssDNA. The supernatant containing "tilmicosin-ssDNA complex" is collected by centrifugation; (b) Reverse screening: the ssDNA library is incubated with a reverse screening target solution containing erythromycin, gamithromycin, kitasamycin, and telithromycin. After adsorption, the supernatant is discarded, the precipitate is resuspended and incubated with tilmicosin for the second time; (c) Iterative enrichment: steps (a)-(b) are repeated for multiple rounds of screening, and the DNA recovery rate is monitored in real time. When the recovery rate is stable, the screening is terminated; (d) High-throughput sequencing: the final enriched ssDNA is subjected to PCR amplification and high-throughput sequencing; (e) Sequence analysis: high stability candidate aptamers are screened by homology comparison, secondary structure prediction, and Gibbs free energy (ΔG) evaluation.

2. The method of claim 1, wherein, The ssDNA initial library in step (a) consists of 79 nucleotides, including 35 random sequences, and the denaturation conditions are: incubation at 95°C for 10 minutes, ice bath for 10 minutes, and equilibration at 25°C for 10 minutes.

3. The method of claim 1, wherein, The reverse screening in step (b) is performed in the seventh and eighth rounds, and the reverse screening target concentration is 1000 μM, which is 10 times the concentration of tilmicosin.

4. The method of claim 1, wherein, The calculation formula of the DNA recovery rate in step (c) is: Recovery rate (%) = (bound ssDNA amount / initial added ssDNA amount) × 100%.

5. The method of claim 1, wherein, In step (e), the M-fold program is used to predict the secondary structure, and the screening criteria include: ΔG < -20 kcal / mol, formation of stem loop / hairpin / G-quadruplex structure, and distribution in different homologous families.

6. A high affinity tilmicosin aptamer selected by the method of any one of claims 1 to 5, characterized in that, The nucleotide sequence is shown as SEQ ID NO: 1: 5'-GGCTGTTGGTGGTGTTGGCTTGGTTTGGTATTGTT-3' Note: The aptamer is a 33 nt truncated version, and the dissociation constant Kd = 9.82 ± 0.97 nM.

7. A method for truncation optimization of a telithromycin aptamer, characterized by, It includes: (a) Molecular docking simulation: molecular docking of tilmicosin and the original aptamer to determine the binding site and interaction mode; (b) Key region identification: based on the docking results, the hydrogen bond binding site and hydrophobic interaction region are located; (c) Sequence truncation: unnecessary nucleotides are removed, and the core region containing DC52, DG53, DG54, DT55, DG56, DC69, DF67, DF70, DC71, and DA72 binding sites is retained; (d) Function verification: the Kd value and specificity of the truncated aptamer are determined by GO fluorescence competition method.

8. The method of claim 7, wherein, In step (a), molecular docking uses AutoDock 4.2 software, with a binding free energy ΔG = -8.4 kcal / mol, in which tilmicosin forms two hydrogen bonds with DC52 (bond length 2.87 Å and 2.90 Å).

9. A test reagent for detecting tilmicosin for non-diagnostic purposes, characterized in that It comprises the aptamer of claim 6.

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