Aptamer TET-HeR of tetracycline and application thereof

Abstract

The invention discloses a nucleic acid aptamer TET-HeR of tetracycline and application of the nucleic acid aptamer TET-HeR, and belongs to the technical field of nucleic acid aptamers. The nucleic acid aptamer TET-HeR of the tetracycline is a combination of an aptamer TET-A1 and an aptamer TET-C1, the nucleotide sequence of the aptamer TET-A1 is as shown in SEQ ID NO.5, and the nucleotide sequence of the aptamer TET-C1 is as shown in SEQ ID NO.7. The nucleic acid aptamer TET-HeR of the tetracycline is a combination of the aptamer TET-A1 and the aptamer TET-C1. The invention further discloses the application of the nucleic acid aptamer TET-HeR of the tetracycline in detection of the tetracycline. The affinity and dissociation constant of the nucleic acid aptamer TET-HeR of the tetracycline and the tetracycline is 62.7 nM, and the nucleic acid aptamer TET-HeR of the tetracycline has high affinity; the compound has no obvious affinity to tetracycline analogues such as aureomycin and doxycycline, has weak affinity to oxytetracycline, and has good specificity; the method can be used for specific detection of tetracycline.

Description

Technical Field

[0001] This invention relates to a tetracycline nucleic acid aptamer, TET-HeR, and its applications, belonging to the field of nucleic acid aptamer technology. Background Technology

[0002] Tetracycline (TET) is an antibiotic that inhibits bacterial protein synthesis by preventing aminoacyl-tRNA from attaching to ribosome receptor sites. Due to its broad-spectrum antibacterial activity, it plays a crucial role in clinical medicine and animal husbandry. Currently, its irrational use and abuse have led to problems such as antibiotic resistance, hepatotoxicity in mammals, and nephrotoxicity, posing a significant threat to the ecological environment and human health. Therefore, the detection of tetracycline residues in food is of great importance.

[0003] The specific detection of targets using nucleic acid aptamers is currently a hot research topic. Various nucleic acid aptamers for tetracyclines have been disclosed in existing technologies, but most suffer from insufficient affinity (small molecule size, limited epitopes). Therefore, developing nucleic acid aptamers with high affinity is of great significance for the detection of tetracyclines. Summary of the Invention

[0004] In view of the above-mentioned prior art, the present invention provides a tetracycline nucleic acid aptamer TET-HeR and its application, which belongs to the field of nucleic acid aptamer technology.

[0005] This invention is achieved through the following technical solution: A tetracycline aptamer, TET-HeR, is a combination of aptamers TET-A1 and TET-C1. The nucleotide sequence of aptamer TET-A1 is shown in SEQ ID NO.5, and the nucleotide sequence of aptamer TET-C1 is shown in SEQ ID NO.7.

[0006] The application of the tetracycline aptamer TET-HeR in the detection of tetracycline.

[0007] The tetracycline aptamer TET-HeR of the present invention has a high affinity for tetracycline with an affinity dissociation constant of 62.7 nM; it has no significant affinity for tetracycline analogs chlortetracycline and doxycycline, and a weak affinity for oxytetracycline (affinity dissociation constant of 127 μM), exhibiting good specificity; it can be used for the specific detection of tetracycline.

[0008] The tetracycline aptamer TET-HeR of this invention is obtained by modifying the original aptamer. The modification method involves selecting multiple different tetracycline aptamers, splitting them, extracting key recognition sites, and then performing heterologous recombination. This modification method avoids the inherent performance bottlenecks of a single aptamer by extracting advantageous key sites from multiple aptamers, while eliminating redundant sequences to shorten the split chain and reduce synthesis costs. Furthermore, it eliminates the need for time-consuming SELEX screening, instead allowing for the "secondary development" of existing aptamer resources. This modification method breaks through the limitations of traditional aptamer "single-source design" or "iso-sequence splitting," reconstructing recognition elements through "precise integration of multiple functional sites," providing a new pathway for the modification of nucleic acid aptamers for all small molecule targets (such as antibiotics, pesticide residues, and biotoxins).

[0009] The various terms and phrases used in this invention have their general meanings known to those skilled in the art. Attached Figure Description

[0010] Figure 1 The results of affinity determination of aptamer TET-A are shown below. The upper part represents the heat change when the aptamer binds to tetracycline, and the lower part represents the enthalpy change when the aptamer binds to tetracycline.

[0011] Figure 2 Affinity test results for aptamer TET-B.

[0012] Figure 3 Affinity test results for aptamer TET-C.

[0013] Figure 4 Affinity test results for aptamer TET-D.

[0014] Figure 5 Secondary structure and key sites of aptamer TET-A.

[0015] Figure 6 Secondary structure and key sites of aptamer TET-B.

[0016] Figure 7 Secondary structure and key sites of aptamer TET-C.

[0017] Figure 8 Secondary structure and key sites of aptamer TET-D.

[0018] Figure 9 Affinity determination results of the split aptamer TET-A-SP.

[0019] Figure 10 Affinity determination results of the split aptamer TET-B-SP.

[0020] Figure 11 Affinity determination results of the split aptamer TET-C-SP.

[0021] Figure 12 Affinity determination results of the split aptamer TET-D-SP.

[0022] Figure 13 Affinity test results of the heterologous recombinant aptamer TET-A1+TET-B1.

[0023] Figure 14 Affinity determination results of the heterologous recombinant aptamer TET-A1+TET-C1.

[0024] Figure 15 Affinity test results of the heterologous recombinant aptamer TET-A1+TET-D1.

[0025] Figure 16 Affinity determination results of the heterologous recombinant aptamer TET-B1+TET-C1.

[0026] Figure 17 Affinity test results of the heterologous recombinant aptamer TET-B1+TET-D1.

[0027] Figure 18 Affinity determination results of heterologous recombinant aptamers TET-C1+TET-D1.

[0028] Figure 19 Affinity test results for TET-A1+TET-N1.

[0029] Figure 20 Affinity test results for TET-C1+TET-N2.

[0030] Figure 21 Results of affinity determination of aptamer TET-HeR for oxytetracycline.

[0031] Figure 22 Results of affinity assay for chlortetracycline by aptamer TET-HeR.

[0032] Figure 23 Results of affinity determination of doxycycline by aptamer TET-HeR. Detailed Implementation

[0033] The present invention will be further described below with reference to embodiments. However, the scope of the present invention is not limited to the following embodiments. Those skilled in the art will understand that various changes and modifications can be made to the present invention without departing from the spirit and scope thereof.

[0034] Unless otherwise specified, the instruments, reagents, and materials used in the following embodiments are all conventional instruments, reagents, and materials already available in the prior art and can be obtained through legitimate commercial channels. Unless otherwise specified, the experimental methods and detection methods used in the following embodiments are all conventional experimental methods and detection methods already available in the prior art.

[0035] This invention employs isothermal titration microcalorimetry to determine the affinity of nucleic acid aptamers for their targets. Isothermal titration microcalorimetry is a gold standard technique for studying molecular interactions in solution. It directly measures the heat released or absorbed (enthalpy change, ΔH) during the binding reaction between the nucleic acid aptamer and its target, thereby calculating the affinity dissociation constant. K d The test conditions were as follows: tetracycline concentration 0.1 mM, aptamer concentration 0.002 mM; initial titration volume of tetracycline 50 μL (using a syringe); initial titration volume of aptamer 300 μL (added to the sample cell); a total of 25 drops, 2 μL per drop. The titration interval was 180 s, the temperature was 25℃, and the stirring speed was 350 r / min. The experimental group consisted of tetracycline titration of the aptamer; the control group consisted of tetracycline titration of the blank solvent.

[0036] Example 1: Determination of primitive aptamer affinity and prediction of key sites In this invention, four tetracycline nucleic acid aptamers were selected as the original aptamers: aptamer TET-A, aptamer TET-B, aptamer TET-C, and aptamer TET-D.

[0037] The nucleotide sequence of aptamer TET-A is shown in SEQ ID NO.1, as shown below (direction 5'-3'): GTTTGTGTATTACAGTTATGTTACCCTCATTTTTCTGAAC.

[0038] The nucleotide sequence of aptamer TET-B is shown in SEQ ID NO.2, as shown below (direction 5'-3'): CCCCCGGCAGGCCACGGCTTGGGTTGGTCCCACTGCGCGT.

[0039] The nucleotide sequence of aptamer TET-C is shown in SEQ ID NO.3, as shown below (direction 5'-3'): GGGGGCACACATGTAGGTGCTGTCCAGGTGTGGTTGTGGT.

[0040] The nucleotide sequence of aptamer TET-D is shown in SEQ ID NO.4, as shown below (direction 5'-3'): GCCGTTCAGAAAAATGAGGGTAACATAACTGTAATACACAAACGGC.

[0041] The affinity of the original aptamer was determined. The affinity determination results for aptamer TET-A are as follows: Figure 1 As shown, the affinity determination results for aptamer TET-B are as follows: Figure 2 As shown, the affinity determination results for aptamer TET-C are as follows: Figure 3 As shown, the affinity determination results for aptamer TET-D are as follows: Figure 4 As shown in the figure. The results indicate that all four primitive aptamers have good affinity for tetracycline and are suitable for subsequent experiments.

[0042] The secondary structures of four primitive aptamers were predicted using the online tool "The UNAFold web server," and the tertiary structures were predicted using RNAcomposer. The tertiary structures of the primitive aptamers and their targets were then imported into Autodock for multiple molecular docking experiments to obtain the high-probability distribution locations of key sites on the primitive aptamers. Results: The secondary structure and key sites of aptamer TET-A are shown below. Figure 5 As shown, the secondary structure and key sites of aptamer TET-B are as follows: Figure 6 As shown, the secondary structure and key sites of aptamer TET-C are as follows: Figure 7 As shown, the secondary structure and key sites of aptamer TET-D are as follows: Figure 8 As shown in the figure, the key stem ring is circled in red.

[0043] Example 2 Determination of apposition affinity of aptamers To obtain nucleic acid aptamers with higher affinity, the original aptamers were modified using a conventional splitting method. Splitting was performed with the principle of preserving key sites. Specifically, aptamer TET-A split at A23 and C24, aptamer TET-B split at C12 and C13, aptamer TET-C split at G33 and T34, and aptamer TET-D split at A8 and G9, and A27 and A28; the splitting locations are... Figure 5 , Figure 6 , Figure 7 , Figure 8 The following are marked (shown by scissors). The four pairs of split aptamers obtained after the four original aptamers split are: TET-A-SP, TET-B-SP, TET-C-SP, and TET-D-SP.

[0044] The affinity of the split aptamer was determined. The affinity determination results for the split aptamer TET-A-SP are as follows: Figure 9 As shown, the affinity determination results for the split aptamer TET-B-SP are as follows: Figure 10 As shown, the affinity determination results for the split aptamer TET-C-SP are as follows: Figure 11 As shown, the affinity determination results for the split aptamer TET-D-SP are as follows: Figure 12 As shown, the affinity of the split nucleic acid aptamer is reduced, but it still retains a high affinity, indicating that splitting the original aptamer is feasible. The split nucleic acid aptamer will still fold into a conformation that binds to the target, which provides support for subsequent heterologous recombination of the split aptamer.

[0045] Example 3: Determination of Heterologous Recombinant Aptamers and Affinity The results of Example 2 show that the splitting method did not yield nucleic acid aptamers with higher affinity. To obtain nucleic acid aptamers with higher affinity, this invention proposes a new modification method: after splitting the original aptamer, fragments containing key sites are selected from each original aptamer, and these fragments are then heterologously recombinated.

[0046] Fragments containing key sites were extracted from the original aptamers, namely: TET-A1, TET-B1, TET-C1, and TET-D1.

[0047] The nucleotide sequence of TET-A1 is shown in SEQ ID NO.5, as shown below (direction 5'-3'): GTTTGTGTATTACAGTTATGTTAC.

[0048] The nucleotide sequence of TET-B1 is shown in SEQ ID NO.6, as shown below (direction 5'-3'): CACGGCTTGGGTTGGTCCCACTGCGCGT.

[0049] The nucleotide sequence of TET-C1 is shown in SEQ ID NO.7, as shown below (direction 5'-3'): GGGGGCACACATGTAGGTGCTGTCCAGGTGTGG.

[0050] The nucleotide sequence of TET-D1 is shown in SEQ ID NO.8, as shown below (direction 5'-3'): ACTTGCCGCGGCAAACACATAATGTCA.

[0051] The fragments containing the key sites were subjected to heterologous random recombination (mixed at a molar ratio of 1:1) to obtain 6 pairs of heterologous recombination aptamers: TET-A1+TET-B1; TET-A1+TET-C1; TET-A1+TET-D1; TET-B1+TET-C1; TET-B1+TET-D1; TET-C1+TET-D1.

[0052] The affinity of the above six pairs of heterorecombinant aptamers was determined. The affinity determination results of the heterorecombinant aptamer TET-A1+TET-B1 are as follows: Figure 13 As shown. The affinity determination results of the heterologous recombinant aptamer TET-A1+TET-C1 are as follows. Figure 14 As shown. The affinity determination results of the heterologous recombinant aptamer TET-A1+TET-D1 are as follows. Figure 15 As shown. The affinity determination results of the heterologous recombinant aptamer TET-B1+TET-C1 are as follows. Figure 16 As shown. The affinity determination results of the heterologous recombinant aptamer TET-B1+TET-D1 are as follows. Figure 17 As shown. The affinity determination results of the heterologous recombinant aptamer TET-C1+TET-D1 are as follows. Figure 18 As shown, the heterologous recombinant aptamer still exhibits high affinity for the target.

[0053] The affinity determination results of the split aptamer and the heterologous recombination aptamer are shown in Table 1.

[0054]

[0055] As shown in Table 1, the heterorecombinant aptamers TET-A1+TET-C1, TET-A1+TET-D1, and TET-C1+TET-D1 exhibit high affinity, showing an increase of more than 3.5 times compared to the split aptamers (TET-A-SP, TET-C-SP, and TET-D-SP), with a maximum increase of 22.7 times. Among them, the heterorecombinant aptamer TET-A1+TET-C1 shows the highest affinity, with an affinity dissociation constant of 62.7 nM with tetracycline. This invention formally names it aptamer TET-HeR.

[0056] The binding of the aptamer TET-HeR to tetracycline was verified by testing random sequences (i.e., TET-A1+TET-N1, TET-C1+TET-N2). TET-N1 is a random fragment of 33 arbitrary bases (A, C, G, or T) (the same number of bases as TET-C1), and TET-N2 is a random fragment of 24 arbitrary bases (A, C, G, or T) (the same number of bases as TET-A1). The affinities of TET-A1+TET-N1 and TET-C1+TET-N2 were determined. The affinity results for TET-A1+TET-N1 are as follows: Figure 19 As shown, the affinity test results for TET-C1+TET-N2 are as follows: Figure 20 As shown, neither of the two random sequence combinations binds to tetracycline, indicating that the high affinity of the heterologous recombination aptamer of this invention depends on the "synergistic effect of functional fragments" rather than random combination. The heterologous recombination aptamer can achieve synergistic complementarity of multiple binding sites, which can enhance target binding strength and eliminate redundant sequences, thus optimizing the folding efficiency of the three-dimensional conformation.

[0057] Example 4: Specificity determination of aptamer TET-HeR To avoid non-specific interactions between fragments after heterologous recombination, specificity assays were performed. The affinity of the aptamer TET-HeR for tetracycline analogs (oxytetracycline, chlortetracycline, doxycycline) was determined. The results of the affinity assay for oxytetracycline by the aptamer TET-HeR are as follows: Figure 21 As shown, the affinity assay results for aptamer TET-HeR to chlortetracycline are as follows: Figure 22 As shown, the affinity determination results of aptamer TET-HeR for doxycycline are as follows: Figure 23 As shown, the aptamer TET-HeR does not bind to chlortetracycline or doxycycline, but because oxytetracycline is structurally most similar to tetracycline, the aptamer TET-HeR shows a weak binding to oxytetracycline. K d =1.27×10 -4 This indicates that the aptamer TET-HeR has high specificity for tetracycline and no cross-reactivity.

[0058] The above embodiments are provided to those skilled in the art to fully disclose and describe how the claimed implementations can be carried out and used, and are not intended to limit the scope of the disclosure herein. Modifications that will be obvious to those skilled in the art will be within the scope of the appended claims.

Claims

1. A tetracycline aptamer, TET-HeR, characterized in that: The combination of aptamers TET-A1 and TET-C1 is shown in SEQ ID NO.5, and the nucleotide sequence of aptamer TET-C1 is shown in SEQ ID NO.

7.

2. The application of the tetracycline aptamer TET-HeR as described in claim 1 in the detection of tetracycline.

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