Nucleic acid aptamer tet-heR of tetracycline and application thereof

Abstract

The application discloses a nucleic acid aptamer TET-HeR of tetracycline and an application thereof, and belongs to the technical field of nucleic acid aptamer. The nucleic acid aptamer TET-HeR of tetracycline is a combination of an aptamer TET-A1 and an aptamer TET-C1, the nucleotide sequence of the aptamer TET-A1 is shown as SEQ ID NO. 5, and the nucleotide sequence of the aptamer TET-C1 is shown as SEQ ID NO. 7. The nucleic acid aptamer TET-HeR of tetracycline is applied to the detection of tetracycline. The nucleic acid aptamer TET-HeR of tetracycline has an affinity dissociation constant of 62.7 nM with tetracycline, has high affinity, has no obvious affinity with aureomycin and doxycycline, has weak affinity with terramycin, has good specificity, and can be used for the specific detection of tetracycline.

Description

TECHNICAL FIELD

[0001] The present application relates to a nucleic acid aptamer TET-HeR of tetracycline and application thereof, and belongs to the technical field of nucleic acid aptamer. BACKGROUND

[0002] Tetracycline (TET) is an antibiotic that inhibits bacterial protein synthesis by preventing aminoacyl-tRNA from attaching to the ribosome receptor site. It plays a key role in clinical medicine and animal husbandry due to its broad-spectrum antibacterial properties. However, its irrational use and abuse have led to problems such as antibiotic resistance, liver and kidney toxicity in mammals, and other issues, posing a significant threat to the ecological environment and human health. Therefore, it is of great significance to detect tetracycline residues in food.

[0003] Using nucleic acid aptamers for specific detection of targets is one of the current research hotspots. Various nucleic acid aptamers for tetracycline have been disclosed in the prior art, but most of them have the problem of insufficient affinity (small molecule volume, limited epitope). Therefore, developing nucleic acid aptamers with high affinity is of great significance for the detection of tetracycline. SUMMARY

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

[0005] The present application is achieved by the following technical solutions:

[0006] The nucleic acid aptamer TET-HeR of tetracycline is a combination of aptamer TET-A1 and aptamer 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.

[0007] The nucleic acid aptamer TET-HeR of tetracycline is used in the detection of tetracycline.

[0008] The nucleic acid aptamer TET-HeR of tetracycline of the present application has an affinity dissociation constant of 62.7 nM for tetracycline, has high affinity, has no obvious affinity for tetracycline analogs such as aureomycin and doxycycline, has weak affinity for terramycin (affinity dissociation constant is 127 μM), has good specificity, and can be used for specific detection of tetracycline.

[0009] The nucleic acid aptamer TET-HeR of the tetracycline of the application is obtained by modification of the original aptamer, and the modification method is: selecting multiple different tetracycline aptamers, extracting the key recognition sites after splitting, and then performing heterologous recombination. The modification method of the application, on the one hand, extracts the respective advantage key sites from multiple aptamers, avoids the inherent performance bottleneck of a single aptamer, and at the same time eliminates redundant sequences to make the split chain shorter and reduce the synthesis cost; on the other hand, without carrying out time-consuming SELEX screening again, but carrying out "secondary development" on the existing aptamer resources. The modification method of the application breaks the limitation of traditional aptamer "single source design" or "same sequence splitting", and reconstructs the recognition element through "precise integration of multiple source functional sites", which provides a new path for the modification of nucleic acid aptamers of all small molecule targets (such as antibiotics, pesticide residues, biological toxins, etc.).

[0010] Various terms and phrases used in the present application have the general meaning known to those skilled in the art. BRIEF DESCRIPTION OF DRAWINGS

[0011] Figure 1 : Affinity determination results of the aptamer TET-A, wherein the upper part represents the heat change when the aptamer is combined with tetracycline, and the lower part represents the enthalpy change when the aptamer is combined with tetracycline, and the same below.

[0012] Figure 2 : Affinity determination results of the aptamer TET-B.

[0013] Figure 3 : Affinity determination results of the aptamer TET-C.

[0014] Figure 4 : Affinity determination results of the aptamer TET-D.

[0015] Figure 5 : Secondary structure and key sites of the aptamer TET-A.

[0016] Figure 6 : Secondary structure and key sites of the aptamer TET-B.

[0017] Figure 7 : Secondary structure and key sites of the aptamer TET-C.

[0018] Figure 8 : Secondary structure and key sites of the aptamer TET-D.

[0019] Figure 9 : Affinity determination results of the split aptamer TET-A-SP.

[0020] Figure 10 : Affinity determination results of the split aptamer TET-B-SP.

[0021] Figure 11 Affinity assay results of split aptamer TET-C-SP.

[0022] Figure 12 Affinity assay results of split aptamer TET-D-SP.

[0023] Figure 13 Affinity assay results of heterorecombinant aptamer TET-A1+TET-B1.

[0024] Figure 14 Affinity assay results of heterorecombinant aptamer TET-A1+TET-C1.

[0025] Figure 15 Affinity assay results of heterorecombinant aptamer TET-A1+TET-D1.

[0026] Figure 16 Affinity assay results of heterorecombinant aptamer TET-B1+TET-C1.

[0027] Figure 17 Affinity assay results of heterorecombinant aptamer TET-B1+TET-D1.

[0028] Figure 18 Affinity assay results of heterorecombinant aptamer TET-C1+TET-D1.

[0029] Figure 19 Affinity assay results of TET-A1+TET-N1.

[0030] Figure 20 Affinity assay results of TET-C1+TET-N2.

[0031] Figure 21 Affinity assay results of aptamer TET-HeR to terramycin.

[0032] Figure 22 Affinity assay results of aptamer TET-HeR to aureomycin.

[0033] Figure 23 Affinity assay results of aptamer TET-HeR to doxycycline. DETAILED DESCRIPTION

[0034] The application will be further described below with reference to the following examples. However, the scope of the application is not limited to the following examples. Those skilled in the art will understand that various changes and modifications can be made to the application without departing from the spirit and scope thereof.

[0035] The instruments, reagents, materials involved in the following examples are conventional instruments, reagents, materials already existing in the prior art, and can be obtained through regular commercial channels, unless otherwise specified. The experimental methods, detection methods, etc. involved in the following examples are conventional experimental methods, detection methods already existing in the prior art, unless otherwise specified.

[0036] The present application determines the affinity of aptamer to target by using isothermal titration microcalorimeter. Isothermal titration microcalorimeter is a gold standard technology for studying molecular interactions in solution, which directly measures the heat released or absorbed (enthalpy change, ΔH) during the binding reaction of aptamer and target, and then calculates the affinity dissociation constant (Kd) and free energy change (ΔG). K d The test conditions are: tetracycline test concentration is 0.1 mM, aptamer test concentration is 0.002 mM; tetracycline titration initial volume is 50 μL (suction syringe); aptamer titration initial volume is 300 μL (added to sample cell); a total of 25 drops, each drop is 2 μL. Titration interval is 180 s, temperature is 25℃, stirring speed is 350 r / min; experimental group is: tetracycline titration aptamer; control group is: tetracycline titration blank solvent.

[0037] Example 1 Original aptamer affinity determination and key site prediction

[0038] The present application selects four tetracycline aptamers as original aptamers, which are: aptamer TET-A, aptamer TET-B, aptamer TET-C, and aptamer TET-D.

[0039] The nucleotide sequence of aptamer TET-A is shown in SEQ ID NO. 1, as follows (direction 5'-3'):

[0040] GTTTGTGTATTACAGTTATGTTACCCTCATTTTTCTGAAC.

[0041] The nucleotide sequence of aptamer TET-B is shown in SEQ ID NO. 2, as follows (direction 5'-3'):

[0042] CCCCCGGCAGGCCACGGCTTGGGTTGGTCCCACTGCGCGT.

[0043] The nucleotide sequence of aptamer TET-C is shown in SEQ ID NO. 3, as follows (direction 5'-3'):

[0044] GGGGGCACACATGTAGGTGCTGTCCAGGTGTGGTTGTGGT.

[0045] The nucleotide sequence of the aptamer TET-D is shown as SEQ ID NO. 4, as follows (direction 5'-3'):

[0046] GCCGTTCAGAAAAATGAGGGTAACATAACTGTAATACACAAACGGC.

[0047] The affinities of the original aptamers were determined. The results of the affinity determination of the aptamer TET-A are shown in Table 1, the results of the affinity determination of the aptamer TET-B are shown in Table 2, the results of the affinity determination of the aptamer TET-C are shown in Table 3, and the results of the affinity determination of the aptamer TET-D are shown in Table 4. The results show that the four original aptamers all have good affinities for tetracycline, and are suitable for subsequent experiments. Figure 1 Figure 2 Figure 3 Figure 4

[0048] The secondary structures of the four original aptamers were predicted by the online tool "The UNAFold web server", and the tertiary structures of the original aptamers were predicted by RNAcomposer. The tertiary structures of the original aptamers and the target were imported into Autodock for multiple molecular docking to obtain the high-probability distribution positions of the key sites of the original aptamers. The results: the secondary structure and key sites of the aptamer TET-A are shown in Table 5, the secondary structure and key sites of the aptamer TET-B are shown in Table 6, the secondary structure and key sites of the aptamer TET-C are shown in Table 7, and the secondary structure and key sites of the aptamer TET-D are shown in Table 8. The key stem loops are circled by red boxes in the figures. Figure 5 Figure 6 Figure 7 Figure 8

[0049] Example 2 Affinity determination of split aptamers

[0050] In order to obtain nucleic acid aptamers with higher affinities, the original aptamers were modified by using the conventional split method. The split was performed according to the principle of retaining the key sites. Specifically, the aptamer TET-A was split at A23 and C24, the aptamer TET-B was split at C12 and C13, the aptamer TET-C was split at G33 and T34, and the aptamer TET-D was split at A8 and G9 and at A27 and A28; the split positions are marked in Tables 9, 10, 11 and 12 (shown by scissors). The four pairs of split aptamers obtained after splitting the four original aptamers are TET-A-SP, TET-B-SP, TET-C-SP and TET-D-SP, respectively. Figure 5 Figure 6 Figure 7 Figure 8 ​​​​​​​​​​​​

[0051] The affinity of the split aptamer was determined. The affinity determination results of the split aptamer TET-A-SP are shown in Table 1, the affinity determination results of the split aptamer TET-B-SP are shown in Table 2, the affinity determination results of the split aptamer TET-C-SP are shown in Table 3, and the affinity determination results of the split aptamer TET-D-SP are shown in Table 4. It can be seen that the affinity of the split nucleic acid aptamer is reduced, but still retains a high affinity, indicating that it is feasible to split the original aptamer, and the split nucleic acid aptamer will still fold into a conformation that binds to the target, which provides support for the subsequent heterologous recombination of the split aptamer. Figure 9 Figure 10 Figure 11 Figure 12

[0052] Example 3: Heterologous recombination of aptamer and affinity determination

[0053] The results of Example 2 show that the nucleic acid aptamer with higher affinity is not obtained by splitting. In order to obtain a nucleic acid aptamer with higher affinity, the present application proposes a new modification method. After splitting the original aptamer, the fragments containing the key sites in each original aptamer are selected, and these fragments are subjected to heterologous recombination, respectively.

[0054] The fragments containing the key sites were extracted from the original aptamer, which were TET-A1, TET-B1, TET-C1, and TET-D1, respectively.

[0055] The nucleotide sequence of TET-A1 is shown in SEQ ID NO. 5, as shown below (direction 5'-3'):

[0056] GTTTGTGTATTACAGTTATGTTAC.

[0057] The nucleotide sequence of TET-B1 is shown in SEQ ID NO. 6, as shown below (direction 5'-3'):

[0058] CACGGCTTGGGTTGGTCCCACTGCGCGT.

[0059] The nucleotide sequence of TET-C1 is shown in SEQ ID NO. 7, as shown below (direction 5'-3'):

[0060] GGGGGCACACATGTAGGTGCTGTCCAGGTGTGG.

[0061] The nucleotide sequence of TET-D1 is shown in SEQ ID NO. 8, as shown below (direction 5'-3'):

[0062] ​​​​ACTTGCCGCGGCAAACACATAATGTCA.

[0063] The above-mentioned fragments containing key sites were subjected to heterologous random recombination (mixed at a molar ratio of 1:1) to obtain 6 pairs of heterologous recombination aptamers, which are as follows: TET-A1+TET-B1; TET-A1+TET-C1; TET-A1+TET-D1; TET-B1+TET-C1; TET-B1+TET-D1; and TET-C1+TET-D1.

[0064] The affinities of the above-mentioned 6 pairs of heterologous recombination aptamers were determined. The affinity determination results of the heterologous recombination aptamer TET-A1+TET-B1 are shown in Table 2. Figure 13 The affinity determination results of the heterologous recombination aptamer TET-A1+TET-C1 are shown in Table 3. Figure 14 The affinity determination results of the heterologous recombination aptamer TET-A1+TET-D1 are shown in Table 4. Figure 15 The affinity determination results of the heterologous recombination aptamer TET-B1+TET-C1 are shown in Table 5. Figure 16 The affinity determination results of the heterologous recombination aptamer TET-B1+TET-D1 are shown in Table 6. Figure 17 The affinity determination results of the heterologous recombination aptamer TET-C1+TET-D1 are shown in Table 7. Figure 18 It can be seen that the heterologous recombination aptamers still have high affinity to the target.

[0065] The affinity determination results of the split aptamer and the heterologous recombination aptamer are shown in Table 1.

[0066]

[0067] As shown in Table 1, the affinities of the heterologous recombination aptamers TET-A1+TET-C1, TET-A1+TET-D1 and TET-C1+TET-D1 are higher, and the affinities are increased by more than 3.5 times, and the highest can be increased by 22.7 times, compared with the split aptamer (TET-A-SP, TET-C-SP and TET-D-SP). Among them, the affinity of the heterologous recombination aptamer TET-A1+TET-C1 is the highest, and the affinity dissociation constant with tetracycline is 62.7 nM, which is formally named as aptamer TET-HeR.

[0068] 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.

[0069] Example 4: Specificity determination of aptamer TET-HeR

[0070] 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.

[0071] 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 nucleic acid aptamer of tetracycline TET-HeR, characterized in that: For the combination of aptamer TET-A1 and aptamer TET-C1, the nucleotide sequence of aptamer TET-A1 is shown as SEQ ID NO. 5, and the nucleotide sequence of aptamer TET-C1 is shown as SEQ ID NO.

7.

2. Use of the nucleic acid aptamer TET-HeR of tetracycline of claim 1 in detecting tetracycline.

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