Nucleic acid aptamer for specifically recognizing staphylococcus aureus enterotoxin A as well as design method and application of nucleic acid aptamer

Abstract

The invention discloses a nucleic acid aptamer for specifically recognizing staphylococcus aureus enterotoxin A as well as a design method and application of the nucleic acid aptamer. A binding mechanism of the aptamer and SEA is analyzed through bioinformatics. Meanwhile, a sequence fusion design experiment is carried out under the assistance of molecular docking, so that an aptamer core sequence related to target binding can be clearly obtained, the working efficiency is improved, the cost is saved, and a high-affinity aptamer of which the affinity is about 1.79 times that of an initial aptamer is finally obtained and is successfully applied to SEA detection. According to the method, the optimal aptamer for SEA is obtained, a foundation is laid for practical application of the aptamer, and a good reference is provided for optimization of the aptamer. Meanwhile, the fusion design method used in the invention may provide an effective and universal strategy for stable, low-cost and high-affinity aptamers of different targets.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and more particularly to a nucleic acid aptamer specifically recognizing Staphylococcus aureus enterotoxin A and a design method and application thereof. Background Art

[0002] Staphylococcus aureus enterotoxins (SEs) are bacterial exotoxins secreted by Staphylococcus aureus. They can cause major foodborne diseases including food poisoning and toxic shock syndrome, and then cause gastroenteritis. They have a short incubation period (about 6-8 hours), and symptoms include abdominal pain, vomiting, nausea, and diarrhea. In addition, SEs also act as superantigens in the immune system response by activating the ability of T cells to proliferate. Among them, Staphylococcus aureus enterotoxin A (SEA) is one of the most widely studied SEs, mainly because the proportion of food poisoning incidents caused by it accounts for 80% of the reported cases of staphylococcal poisoning. SEA is resistant to most treatments that eliminate bacterial cells, such as heat treatment and low pH, which will not cause its inactivation and denaturation, so it may exist in food even after processing. At the same time, SEA's resistance to proteolytic enzymes also allows them to pass through the digestive tract unaffected, thereby increasing the risk of illness. In addition, for adults (weight 70kg), ingestion of 50ng of SEA can cause food poisoning and even life-threatening toxic shock. Therefore, the detection of SEA is of great significance to food safety.

[0003] At present, the detection technologies of SEA can be divided into animal experiments, molecular biology techniques and immunological analysis. However, due to the lack of availability of experimental animals and ethical issues, animal experiments have been gradually eliminated. Molecular biology methods can solve these problems, but their application is also limited due to the need for high-quality samples, expensive equipment and professional technicians. With the development of emerging technologies, biosensors play an increasingly important role in the ultrasensitive detection of SEA. However, the preparation cost of antibodies used in biosensor technology is high and the cycle is long. In order to improve this defect, aptamers have received increasing attention due to their excellent sensitivity and cost-effectiveness.

[0004] The main method for screening nucleic acid aptamers is the systematic evolution of ligands by exponential enrichment (SELEX). 15 In order to screen out high-affinity aptamers from the initial random sequence library of nucleotide molecules, multiple aptamer sequences need to be synthesized and identified during the screening process, which consumes a lot of time and resources. In order to solve this problem, people have developed a computer screening method to optimize and design aptamers, which helps to design high-affinity nucleic acid aptamers with targeted binding sites.

[0005] Based on the above problems, the present invention analyzes the binding mechanism between aptamers and Staphylococcus aureus enterotoxin A (SEA) with the assistance of bioinformatics, and obtains high-affinity aptamers by fusion design of sequences with the assistance of molecular docking. Our data proves the successful design and application of SEA aptamers, and proposes a method for aptamer design applicable to other target molecules. Summary of the invention

[0006] In view of this, the present invention provides a nucleic acid aptamer that specifically recognizes Staphylococcus aureus enterotoxin A, and a design method and application thereof.

[0007] In order to achieve the above purpose, the present invention adopts the following technical solutions:

[0008] The present invention provides an aptamer that specifically recognizes SEA, and the nucleotide sequence of the aptamer is shown in SEQ ID NO.1.

[0009] 5'-CCTAACCGATATCAGATGTCTTACTACGTTGGTACTTTTATGAT CTCTATTCGT-3', SEQ ID NO. 1, is an artificial sequence.

[0010] The present invention provides a method for designing an aptamer that specifically recognizes SEA:

[0011] (1) Construction of the initial aptamer secondary structure, tertiary structure and 3D structure of SEA;

[0012] (2) Molecular docking and conformational analysis of the initial aptamer tertiary structure:

[0013] The tertiary structure of the initial aptamer was used as the receptor, and the 3D structure of SEA in step (1) was used as the ligand. The receptor and the ligand were molecularly docked to obtain the SEA-initial aptamer, and the SEA-initial aptamer with high docking score and low root mean square deviation (RMSD) was subjected to conformational analysis. The molecular docking results and the analysis graph of LigPlot+v2.2 were helpful to determine the nucleotides involved in the SEA interaction.

[0014] (3) Construction of SEA-aptamer after fusion design:

[0015] According to the obtained important binding sites, the nucleotide sequences of the initial aptamers in step (1) are fused. These important binding nucleotides are selected, and the nucleotides that do not participate in the action are cut off. Then, the nucleotides involved in the action of multiple aptamers are fused, and finally 10 sequences are generated. These aptamers and the 3D structure of SEA are molecularly docked to form SEA-aptamers;

[0016] (4) Affinity determination:

[0017] The first four aptamers with a larger number of involved nucleotides, a lower RMSD value and a higher docking score in step (3) were selected for microthermophoresis experiments to verify their affinity and obtain high-affinity aptamers.

[0018] Furthermore, the nucleotide sequence of the initial aptamer in step (1) is shown in SEQ ID NO.2:

[0019] A29: 5'-AGCAGCAGAGGTCAGATGTATTATCTTACTACGTTGCATC CTACTTTTATAGTCCCCTCCTATGCGTGCTACCGTGAA-3', SEQ ID NO. 2, is an artificial sequence;

[0020] C7: 5'-CCTAACCGATATCACACTCACAGTATACCGCTCCACCAGTG TGATATCGGGATCTGCTGACGTTGGTCGTCATTGGAGTATC-3', SEQ ID NO.3, is an artificial sequence;

[0021] Furthermore, the initial aptamer in step (2) is used as a template, and according to the molecular docking results, the DS (Discovery Studio) and LigPlot+v2.2 tools are used for visualization to determine the docking site between the initial aptamer and SEA. Thus, multiple aptamers are fused to design a new aptamer sequence;

[0022] Furthermore, the affinity determination in step (4) requires modification of the 3' end of the aptamer with a 6-FAM group;

[0023] The present invention provides an application of the above-mentioned nucleic acid aptamer in detecting SEA.

[0024] It can be seen from the above technical solution that compared with the prior art, the beneficial effects achieved by the present invention are as follows: the present invention analyzes the binding mechanism of the aptamer and SEA through bioinformatics. At the same time, by conducting sequence fusion design experiments with the assistance of molecular docking, the core sequence of the aptamer related to target binding can be clearly obtained, which improves work efficiency and saves costs. Finally, a high-affinity aptamer with an affinity of about 1.79 times that of the initial aptamer was obtained, and it was successfully applied to the detection of SEA. The present invention not only obtains the best aptamer for SEA, laying the foundation for its practical application, but also provides a good reference for the optimization of aptamers. At the same time, the fusion design method used in the present invention may provide an effective and universal strategy for stable, low-cost and high-affinity aptamers for different targets. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0026] Figure 1 The attached figure is a schematic diagram of the aptamer fusion design;

[0027] Figure 2 The attached drawings show the establishment of secondary and tertiary structures of the initial aptamer and the new aptamer designed by fusion, wherein Figures (a) and (b) are the secondary and tertiary structures of the initial aptamer, and Figures (c) and (d) are the secondary and tertiary structures of the new aptamer designed by fusion;

[0028] Figure 3 The attached figure shows the molecular docking results of the initial aptamer and SEA and the corresponding docking sites;

[0029] Figure 4 The attached figure shows the molecular docking results of the fusion-designed new aptamer and SEA and the corresponding docking sites;

[0030] Figure 5 Figure (a) shows the interaction between the new aptamer and SEA measured by microscale thermophoresis (MST). Figure 5 (b)(c)(d) are the applications of the new aptamer in detecting SEA. Figure 5 (b) shows the color change and standard curve of SEA at different concentrations. Figure 5 (c) is the specific detection of SEA by the aptamer colorimetric sensor. Figure 5 (d) Specificity and stability of aptamer colorimetric sensors;

[0031] Figure 6 The attached figure is a summary figure. DETAILED DESCRIPTION

[0032] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0033] The drugs required for the present invention are conventional experimental drugs, which are purchased from commercial channels; the experimental methods not mentioned are conventional experimental methods and will not be described in detail here.

[0034] Example 1

[0035] (1) First, the structure of the aptamer was modeled. The UNAFold (http: / / www.unafold.org / ) website was used to predict the secondary structure of the aptamer. The folding temperature was set to 25°C and the ionic condition was 50 mM Na+ solution. The secondary structure of the aptamer was converted into a dot-bracket format and input into the Xiao Lab website (http: / / biophy.hust.edu.cn / new / 3dRNA / DNA / ) to obtain the tertiary structure of the aptamer with the minimum energy.

[0036] All nucleic acid aptamers of the present invention were synthesized by Sangon Biotechnology (Shanghai) Co., Ltd., and the sequences of the nucleic acid aptamers are listed in Table 1.

[0037] Table 1. Aptamer sequences used in the experiment

[0038]

[0039]

[0040] like Figure 1 , 2 , Figure 1 Schematic diagram for aptamer fusion design, Figure 2 The secondary and tertiary structures of the initial aptamer and the new aptamer designed by fusion are established, where Figures (a) and (b) are the secondary and tertiary structures of the initial aptamer, and Figures (c) and (d) are the secondary and tertiary structures of the new aptamer designed by fusion. These structures prepare for the subsequent molecular docking experiment.

[0041] (2) Molecular docking

[0042] In order to more intuitively determine the binding mechanism between the optimized aptamer and SEA, molecular docking analysis was performed to study the interaction between the initial aptamer and SEA. With SEA as the ligand (the initial structure comes from the PDB database ID: 1ESF) and the initial aptamer as the receptor, molecular docking was performed using the HDOCK Web server. After the docking was completed, the complexes with high scores and low root mean square deviation (RMSD) were selected for the next step of verification. The aptamer-protein complex (target-ligand complex) was further visualized by the LigPlot+ v2.2 tool to search for nucleotides directly involved in the interaction with the target in the complex.

[0043] Table 2 shows the number of nucleotides involved in the docking of the 20 initial template aptamers with the SEA molecule, the RMSD value and the docking score of the docking complex. The results show that 8 of the aptamers have more nucleotides involved after docking with SEA, and have lower RMSD and higher docking scores. The number of nucleotides involved may determine the strength of the interaction between the aptamer and the target. The more nucleotides there are, the higher the affinity with the target may be. A high binding score indicates the maximum interaction between the ligand and the target, with better shape complementarity, while a low RMSD indicates that the modeled complex has a smaller deviation from the reference. Therefore, a high docking score and a low RMSD value indicate that a stable complex has been formed. Finally, 8 aptamers such as C7 were selected as templates for the fusion design study of aptamers. Among the selected aptamers, C7 has a higher affinity with SEA, and K d The value was 7.44±0.6nM, so C7 was used as the main template to fuse with other aptamers. The docking results of these 8 aptamers and SEA were visualized by DS and further analyzed by LigPlot+v2.2 tool. Figure 3 It is the action site of the docking complex of the template aptamer and SEA. It is observed that the aptamer binds to SEA through hydrogen bonding and hydrophobic interaction. Table 3 lists the types of bonding formed between the template aptamer nucleotide and SEA.

[0044] Table 2. Initial docking results of aptamers and SEA molecules

[0045]

[0046]

[0047]

[0048]

[0049] Table 3. Types of bonds formed between template aptamer nucleotides and SEA

[0050]

[0051]

[0052]

[0053] Example 2

[0054] (1) Fusion design of new aptamers

[0055] Ten aptamer sequences were designed based on the molecular docking results of the template aptamer and the target SEA, and the 10 aptamer sequences designed by fusion are listed in Table 4. All sequences were characterized similarly to the previous ones, and stable structure modeling of the aptamer sequences was performed using UNAFold and Xiao Lab / 3D RNA / DNA.

[0056] (2) Molecular docking

[0057] After modeling, the designed aptamers were molecularly docked with the target SEA using HDOCK, and the number of nucleotides involved, RMSD values, and docking scores of the docked complexes after the 10 aptamers were docked with the SEA molecule were compared (Table 4). Among them, 4 aptamers had a larger number of nucleotides involved, a lower RMSD value, and a higher docking score. The docking results of these 4 aptamers with SEA were visualized by DS and further analyzed by the LigPlot+v2.2 tool. Figure 4 The action site of the docking complex of the new aptamer designed for fusion and SEA.

[0058] The results showed that the aptamers bind to SEA through hydrogen bonding and hydrophobic interactions. Table 5 lists the types of bonds formed between the fused designed new aptamer nucleotides and SEA. The results showed that among these aptamers, there were many more nucleotides that formed hydrophobic interactions with the target SEA than those that formed hydrogen bonds. The reason for this result may be that the hydrophobic regions of the aptamers and the target SEA are more matched, and hydrophobic interactions are usually closely related to the shape and surface properties of the molecules, so they are more likely to form hydrophobic interactions in structure. Hydrophobic interactions are usually stronger than hydrogen bonds, so when forming molecular complexes, hydrophobic interactions can provide more stable binding forces.

[0059] Table 4. Docking results of the new aptamers designed by fusion and SEA molecules

[0060]

[0061]

[0062] Table 5. Types of interactions between the fusion designed new aptamer nucleotides and SEA

[0063]

[0064] (3) Affinity experiment

[0065] According to the molecular docking results of SEA-aptamers in step (2), the top four BX1, BX7, BX8, and BXC were selected for affinity determination. Microthermophoresis experiments of SEA and nucleic acid aptamers were performed in the Monolith NT.15 system (Nano Temper Technologies, Germany) to detect the binding affinity between the two. The aptamer labeled with 6-FAM at the 3 ends was diluted to 200nM with Binding-Buffer (50mM Tris, 15mM NaCl, 2mM MgCl2, pH 7.4). Then heated at 95°C for 10min and slowly cooled to room temperature. SEA was continuously diluted to 16 concentration gradients with MST-Buffer (Binding-Buffer containing 0.05% Tween-20), each gradient was 10uL, and an equal volume of nucleic acid aptamers was added. After incubation at room temperature for 2h, each concentration gradient sample was transferred to a MO-K022 capillary. Each experiment was repeated at least three times, and the final data was the average of multiple measurements, and the relative standard deviation (RSD) was calculated. The data were imported into MO.Affinity analysis software to calculate the Kd value of the aptamer binding to SEA.

[0066] The results are as follows Figure 5 (a) The dissociation equilibrium constants K of aptamers BX1, BX7, BX8 and BXC d The K values ​​of aptamer C7 with the strongest affinity reported were 4.16±0.22nM, 12.61±0.66nM, 58.42±0.81nM and 168.99±1.74nM, respectively. d The K value of the designed aptamer BX1 was 7.44±0.6 nM. d The value has increased significantly.

[0067] Application of the high-affinity SEA nucleic acid aptamer designed in Example 3

[0068] (1) Detection of SEA based on colloidal gold aptamer colorimetry

[0069] Colloidal gold was prepared by the classic sodium citrate reduction method of chloroauric acid. 2 mL of colloidal gold (AuNPs) solution was mixed with 50 μL of 600 mM aptamer solution and incubated in a 37°C shaking incubator for 10 min. Then different concentrations of SEA solution were added and incubated in a 37°C shaking incubator for 30 min. Finally, 200 μL of 350 mmol / L NaCl solution was added and mixed thoroughly. After standing at room temperature for 12 min, the color change of the solution was observed and the ratio of its absorbance at 620 nm to that at 520 nm was recorded (denoted as: A 620 / A 520 ). Each experiment was performed in triplicate.

[0070] The results are as follows Figure 5 As shown in (b), with the increase of SEA concentration, more and more aptamers on the colloidal gold particles are competed off, resulting in the A 620nm / A 520nm As the ratio continues to rise, the color of the system changes from wine red to blue-gray ( Figure 5 ). 620nm / A 520nm With the concentration of SEA solution as the ordinate and the concentration of SEA solution as the abscissa, a correlation curve is drawn between the two, as shown in Figure 5 As shown, SEA is in the concentration range of 0.5-250ng / mL, A 620nm / A 520nm The ratio and its concentration show a good linear relationship, the linear equation is y = 0.00116x + 0.2142, and the correlation coefficient is R 2 Under the optimized conditions, the blank control was measured 10 times in parallel, and the detection limit of the method was determined to be 3.41 ng / mL (S / N=3).

[0071] (2) Specificity and stability analysis

[0072] In order to verify whether the designed aptamer can specifically recognize SEA, enterotoxin B (SEB) and enterotoxin C (SEC) homologous to SEA were selected to study the specificity of the aptamer. The determination was carried out according to the method (1) above, and each experiment was carried out in triplicate, and a blank control was also performed. In order to evaluate the stability of the designed sensor, the sensor containing SEA was stored at 4°C for 7 days, and the SEA content was monitored every day under the same conditions.

[0073] The results are as follows Figure 5 As shown in (c), under the optimal conditions, the aptamer binds to SEA and the absorbance ratio is significantly higher than that of the other two toxins. This is because the designed SEA aptamer has strong specificity. The partial binding of the aptamer to other SEs (SEB and SEC) may be attributed to the 40-60% sequence homology between SEA, SEB and SEC. At the same time, the absorbance ratio of the blank group is lower, because the surface charge of the gold nanoparticles in the blank group solution will not be affected, and the dispersed state of the gold nanoparticles can still be maintained. Combined with the results of these confirmatory analyses, it is proved that the nucleic acid aptamer selected in this experiment has good selectivity for SEA, and other substances have little effect on this experiment, so the specificity is good.

[0074] like Figure 5 As shown in (d), the relative standard deviation of the measurement results is less than 5%, which indicates that the sensor can be stored for at least 1 week, indicating strong stability.

[0075] (3) Milk sample testing

[0076] Take 2 mL of milk and add a certain amount of SEA to make the final concentration of SEA in the sample 50 ng / mL, 100 ng / mL, and 200 ng / mL. Then centrifuge at 15°C, 3500g for 10 min, discard the fat, and take the supernatant. Dilute it with distilled water (1:20), take 100 μL of the diluted sample, and measure it according to method (1). Perform three parallel experiments for each concentration, and do a blank control at the same time.

[0077] The results are shown in Table 6. The accuracy of the method for quantifying SEA in milk was evaluated by recovery analysis. The recovery rates ranged from 78.42% to 92.38% (see Table 6), which indicated that the designed aptamer was successfully applied to the detection of actual samples.

[0078] Table 6. Detection of SEA in milk samples

[0079]

[0080]

Claims

1. A nucleic acid aptamer that specifically recognizes Staphylococcus aureus enterotoxin A, characterized in that: The oligonucleotide sequence is shown in SEQ ID NO.

1.

2. A method for designing a nucleic acid aptamer that specifically recognizes Staphylococcus aureus enterotoxin A as claimed in claim 1, characterized in that: The following steps are involved: (1) constructing the secondary and tertiary structures of the initial aptamer and the 3D structure of Staphylococcus aureus enterotoxin A; (2) molecular docking of the 3D structure of S. aureus enterotoxin A with the tertiary structure of the initial aptamer was performed to obtain the complex of S. aureus enterotoxin A and the initial aptamer, and conformational analysis was performed to identify the nucleotides involved in the interaction of S. aureus enterotoxin A as important binding sites; (3) fusing the nucleotide sequence of the initial aptamer according to the important binding sites obtained in step (2) to generate a fusion sequence, and performing molecular docking on the fusion sequence and the 3D structure of Staphylococcus aureus enterotoxin A to form a new aptamer; (4) First, the new aptamer obtained in step (3) is modified, and then a micro-thermophoresis experiment is performed to select the aptamer with the strongest affinity, which is the nucleic acid aptamer that specifically recognizes Staphylococcus aureus enterotoxin A.

3. The design method according to claim 2, characterized in that: In step (1), the nucleotide sequences of the initial aptamers are shown in SEQ ID NO.2 and SEQ ID NO.

3.

4. The design method according to claim 2, characterized in that: In step (2), conformational analysis was performed using DiscoveryStudio and LigPlot+ v2.2 tools.

5. The design method according to claim 2, characterized in that: In step (4), the modification refers to modifying the 3' end of the new aptamer obtained in step (3) with a 6-carboxyfluorescein group.

6. Use of the nucleic acid aptamer that specifically recognizes Staphylococcus aureus enterotoxin A as claimed in claim 1 in detecting Staphylococcus aureus enterotoxin A.

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