CN112899281B - Optimized nucleic acid aptamer capable of specifically recognizing T-2 toxin, optimization method and application of aptamer - Google Patents
Optimized nucleic acid aptamer capable of specifically recognizing T-2 toxin, optimization method and application of aptamer Download PDFInfo
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Abstract
The invention discloses an aptamer capable of specifically recognizing T-2 toxin and application thereof, belonging to the technical field of food safety biology. The T-2 toxin is taken as a target, and on the basis of an original sequence obtained by SELEX screening, the aptamer sequence is guided to be cut by using a molecular docking technology, so that the aptamer with good affinity and specificity for the T-2 toxin is obtained. And SYBR Green I is used for designing a novel fluorescence method for detecting T-2 toxin, and MnO is added to the FAM-labeled aptamer 2 Adsorption, simultaneous presence of SG in solution for amplification of fluorescent signal, and in the presence of T-2 toxin, aptamer from MnO 2 The surface dissociation fluorescence is recovered, and simultaneously, the fluorescence is non-specifically combined with free SG in the solution, the fluorescence signal is further enhanced, the aim of quantitatively/qualitatively detecting the T-2 toxin is achieved, feasibility verification is provided for applying the optimized aptamer to other detection methods, and the method has wide application prospect.
Description
Technical Field
The invention belongs to the technical field of food safety biology, and particularly relates to an optimized aptamer capable of specifically recognizing T-2 toxin, an optimization method and application of the aptamer.
Background
The T-2 toxin is mainly a toxic secondary metabolite produced by fusarium trilobate, and the molecular formula is C 24 H 34 O 9 . It is widely distributed in nature and is a common major toxin contaminating field crops and stored grains. Can cause serious inflammatory reaction in animal body, has teratogenic effect, and has great harm to human and livestock. Thus, accurate and rapid T-2 toxin in food is realizedAnd sensitive detection, which is of great significance for guaranteeing food safety and protecting human health. To date, the determination of trace amounts of T-2 toxin in food or feed has been a difficult task. The instrumental analysis method is a main means for detecting T-2 toxin, and comprises LC-MS, UPLC-MS, GC-MS and the like. These instrument-based analytical methods rely on expensive precision instrumentation and trained staff and are therefore not widely available on a large scale. Another type of T-toxin detection technique is immunoassay, which has the advantages of high sensitivity and wide applicable range, but immunoassay methods cannot leave high quality antibodies, and instability of the antibodies may lead to the occurrence of false negative or false positive detection results.
The aptamer is also called a chemical antibody, and compared with the antibody, the aptamer has the advantages of good stability, easiness in chemical labeling, small batch-to-batch difference and wide application in the field of analysis and detection. In general, aptamers obtained by SELEX screening have the number of 70-130 bases, and comprise two primer regions and a middle random sequence region. However, not all bases participate in the recognition process of the aptamer and the target, and the sequence of the aptamer is truncated and optimized, so that the synthesis and use cost of the aptamer can be reduced, and the application range of the aptamer can be expanded. At present, the truncation optimization method of the aptamer sequence mainly removes primer regions at two ends according to the secondary structure of the aptamer, and is a time-consuming trial-and-error method. Therefore, a method for precisely guiding the optimization of the aptamer sequence is urgently needed.
Molecular docking is a theoretical simulation method to study intermolecular interactions such as ligand and receptor-interactions and predict binding patterns and affinities. Molecular docking places small molecule ligands at the active sites of the receptor and finds their reasonable orientation and conformation, optimizing the match of the shape and interaction of the ligand with the receptor. In the field of aptamer research, the molecular docking technology is mostly used for characterizing the interaction mode of an aptamer and a target at present, and no report for guiding the sequence of the truncated and optimized aptamer by utilizing the molecular docking technology exists. Manganese dioxide nanosheet (MnO) 2 ) Is an ultrathin two-dimensional plane nano material and has excellent fluorescence quenching capability. SY (simple electronic system)BR Green I (SG) is a dye with a Green excitation wavelength that binds to all dsDNA duplex minor groove regions. In the free state, SG emits weak fluorescence, but once bound to double-stranded DNA, the fluorescence increases by about 1000 times, and since single-stranded aptamers have a secondary structure, the fluorescence signal also increases after SG binds to aptamers non-specifically.
The invention takes T-2 toxin as a target, obtains an optimized aptamer sequence which can recognize and bind the T-2 toxin with high affinity and specificity based on the guidance cutting of a molecular docking technology on the basis of an original sequence obtained by SELEX screening, and uses the optimal aptamer for the detection of the T-2 toxin. The invention provides a high-specificity detection recognition element which has high affinity, good specificity, easy preparation and easy labeling and an application example for the detection of T-2 toxin.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a method for directionally optimizing an aptamer sequence and quickly and sensitively detecting T-2 toxin by utilizing a molecular docking technology and an aptamer technology. The invention takes T-2 toxin as a target, and utilizes the molecular docking technology to guide cutting on the basis of an original aptamer sequence T80 obtained by SELEX screening to obtain 1 aptamer T40 which can be combined with the T-2 toxin with high affinity and high specificity, wherein the nucleotide sequence is shown in SEQ ID NO.2. Based on the detection, a novel method for detecting T-2 toxin based on SG amplified fluorescence signals is designed. When T-2 toxin is present, T40 is bound to T-2 toxin by FAM-labeled aptamer T40, so that T40 is separated from MnO 2 Surface dissociation and fluorescence recovery of FAM, and at the same time, free SG in the solution is non-specifically bound to the aptamer, and the fluorescence signal in the solution is further enhanced. Wherein the nucleotide sequence of T80 is shown in SEQ ID NO.3, and the sequence is disclosed in patent No. 2014103576499.
The technical scheme of the invention is as follows:
an optimized aptamer capable of specifically recognizing T-2 toxin, wherein the nucleotide sequence of the aptamer is shown as SEQ ID No.1 and SEQ ID No. 2;
further, the nucleotide sequence of the aptamer is shown as SEQ ID NO.2.
Further, the nucleotide sequence of the aptamer is modified by a modifier, wherein the modifier comprises at least one of a fluorescent group, an isotope, an electrochemical label, an enzyme label, an affinity ligand or a sulfhydryl group.
Use of an optimized aptamer for the detection of a T-2 toxin.
Further, the nucleic acid aptamers can be used for detecting T-2 toxin by using modification or non-modification singly or in combination.
An application of an optimized nucleic acid aptamer in preparing a kit for detecting T-2 toxin for non-diagnostic purposes.
A kit comprising the aptamer.
Further, the kit also comprises MnO 2 Nanosheets.
Further, the kit also comprises a FAM marker SYBR Green I.
An optimizing method for the nucleic acid aptamer capable of specifically recognizing T-2 toxin includes such steps as obtaining the original nucleic acid aptamer sequence by SELEX screening, predicting the aptamer by molecular butt joint technique, and directionally removing the basic groups irrelevant to binding domain to obtain the truncated optimized nucleic acid aptamer sequence.
The beneficial technical effects of the invention are as follows:
(1) Compared with an antibody, the aptamer has the advantages of capability of in vitro screening, short screening period, convenience in synthesis, easiness in marking various functional groups and reporter molecules, stable property, long-term storage and use and the like;
(2) The sequence is based on an original sequence, and is cut by the guidance of a molecular docking technology, so that non-directional trial and error in the truncation process of an aptamer sequence are reduced, the obtained aptamer sequence with high affinity and specificity is obtained, and the T-2 toxin can be specifically identified;
(3) Compared with the original T-2 toxin aptamer obtained by screening, the sequence has ideal affinity, good specificity and lower synthesis cost, and the constructed detection method can detect T-2 toxin in environment and food more sensitively.
Drawings
FIG. 1 is a flow chart of the optimization of aptamer sequence truncation based on molecular docking guidance.
FIG. 2 is the secondary structure (a) of the Mfold-fitted aptamer T40 of the invention; t40 and T-2 toxin binding tertiary space structure (b).
FIG. 3 is a saturation binding curve (a) for an aptamer T40 of the invention; specificity of aptamer T40 (b).
FIG. 4 is a schematic diagram of the detection of T-2 toxin by fluorescence method based on SG amplification signals.
FIG. 5 is a graph showing the standard test for T-2 toxin according to the present invention.
FIG. 6 is a schematic diagram of the application of the aptamer of the present invention to the detection of T-2 toxin by fluorescence.
Detailed Description
The present invention will be described in detail with reference to the accompanying drawings and examples. These examples are given solely for the purpose of illustration and are not to be construed as limitations of the present invention, as any equivalent or partial modifications thereof within the spirit and principles of the invention are deemed to fall within the scope of the invention.
The aptamer of the invention is an aptamer labeled by a 5' end FAM group synthesized by Shanghai Biotechnology engineering service company Limited.
Example 1 directed tailoring of aptamer sequences based on molecular docking techniques
(1) Molecular docking guided aptamer sequence truncation optimization process
After the aptamer sequence predicts the secondary structure on line by using the MFold, the tertiary space structure of the aptamer is constructed based on the secondary structure of the aptamer. Molecular docking was performed using UCSF DOCK 6.9 software, and docking results were displayed using Discovery Studio Visualizer software. Based on the binding domains of the aptamer and the target predicted by molecular docking, bases unrelated to the binding domains or farther away from the binding domains are directionally removed, and the truncated and optimized aptamer is obtained.
The directional cropping process is shown in fig. 1. T80 (FIG. 1) is a T-2 toxin-specific aptamer obtained by SELEX screening, and measured by fluorescence polarizationK binding to the target d The value is 213.2 +/-15.9 nmol/L, the specificity is good, the sequence contains 80 bases, the secondary structure is relatively complex, the sequence is optimized by considering the subsequent practical application, and the sequence comprises 40 bases in primer regions at two ends and 40 bases in a middle random region.
(2) Aptamer affinity assay
The affinity measurement uses fluorescence polarization. The synthesized aptamers were diluted with TE buffer to prepare 10. Mu.M solution and stored at-20 ℃ for further use. Heating and denaturing the FAM-labeled aptamer at 95 ℃ for 5min, and standing at room temperature for 30min to form a stable spatial structure. Then, aptamers (25, 50, 100, 150, 200, 300 nmol/L) at different final concentrations and T-2 toxin at a constant final concentration (1. Mu.M) were incubated in aptamer binding buffer for 40min at room temperature in the absence of light to allow sufficient binding of the aptamers to the T-2 toxin. Then adding manganese dioxide nano-sheets with corresponding concentration, adsorbing the aptamer which is not combined with the T-2 toxin, and taking out 100 mu l of solution after 20 minutes and placing the solution in an ELISA plate to measure the fluorescence polarization value. The group without T-2 toxin was used as a control group. All experiments were repeated 3 times. The fluorescence polarization difference of the sample represents the affinity, and the dissociation constant K of the aptamer is calculated by utilizing GraphPad Prism 5 software d Values and saturation binding curves were plotted.
(2) Aptamer specificity assay
Respectively incubating a solution of aptamer (T40) with a final concentration of 50nmol/L with fumonisins B1 (FB 1), zearalenone (ZEN), ochratoxins A (OTA) and aflatoxins B1 (AFB 1) in an aptamer combination buffer solution at room temperature for 40min, then adding manganese dioxide nanosheets with corresponding concentrations, incubating at room temperature for 20min, adsorbing free aptamers, and finally taking 100 mu L of the solution out and placing in an ELISA plate to measure a fluorescence polarization value.
Usually, the sequence optimization of the aptamer can be carried out by trial and error cutting according to the secondary structure of the aptamer, and a large amount of manpower and material cost is consumed, in the invention, the sequence truncation optimization method is shown in figure 1, namely, according to the binding domain of T80 and T-2 toxin predicted by a molecular docking technology, 22 bases irrelevant to the binding domain are firstly removed in a targeted mode, and the aptamer T58 containing 58 bases is obtained, wherein the nucleotide sequence is shown in SEQ ID NO.1. The affinity results are shown in table 1, after 22 bases unrelated to the binding domain are removed, the sequence affinity is enhanced to a certain extent, which indicates that the removed bases do not participate in the identification and binding process of the target at all, and the molecular docking prediction of the binding domain is more accurate. And (3) performing molecular docking on the T58 and the T-2 toxin again, and directionally removing 18 bases irrelevant to the binding domain again according to the predicted binding domain to obtain the aptamer T40 containing 40 bases. The affinity of T40 is enhanced by several times compared with that of T80, and the sequence saturation binding curve of T40 is shown in FIG. 3 (a), which indicates that the removed 18 bases play a role in hindering the binding process of the target and the aptamer. The T40 and T-2 toxin were again subjected to molecular docking, and the predicted binding domains indicated that the binding domains of the aptamer and the target were at the 5 'and 3' ends of the T40 secondary structure (shown in fig. 2), so the 19 base-containing stem-loop structure at the top of the T40 secondary structure was removed, resulting in the sequence T21, and the reduced affinity of T21, indicating that the stem-loop region at the top supported or assisted recognition of the aptamer and the target, which is consistent with the binding domains of the sequence below the T40 secondary structure predicted by molecular docking. The binding domains predicted by the three molecular docking are consistent, which illustrates the accuracy of the molecular docking technique, since the binding domain of the aptamer bound to the target is not changed during the optimization process of aptamer sequence truncation.
As shown in FIG. 3 (b), the specificity determination result shows that the aptamer T40 after the truncation optimization through molecular docking guidance has weak or no recognition capability on other mycotoxins, which indicates that the T40 has good specificity, so that the T40 is selected as an aptamer sequence capable of binding the T-2 toxin with high specificity for subsequent experiments.
TABLE 1
Example 2 detection of T-2 toxin (detection of T-2 toxin by fluorescence method based on SG amplification Signal)
(1) Synthesis of manganese dioxide nanosheet
1mL of Bovine Serum Albumin (BSA) with the mass concentration of 1mg/mL and 1mL of manganese acetate with the mass concentration of 0.05g/mL are taken and placed in a 250mL beaker, and 98mL of ultrapure water is added into the beaker until the total volume of the solution in the beaker is 100mL; placing the beaker on a magnetic stirrer, and uniformly stirring at normal temperature; after 50min, adding a sodium hydroxide solution with the concentration of 1mol/L into the solution, and adjusting the pH value of the solution to 8.0; placing the solution on a magnetic stirrer, continuously stirring for 6h to obtain a light yellow solution, carrying out centrifugal precipitation on the solution, washing the obtained material precipitate twice with ultrapure water, and storing the material precipitate in a refrigerator at 4 ℃ for later use.
(2) Fluorescence method for detecting T-2 toxin based on SG amplification signal
In a 200. Mu.L assay system, the aptamer (T40) of FAM-labeled T-2 toxin at a concentration of 100nmol/L and MnO at 0.1mg/mL 2 The nanoplatelets were incubated at room temperature for 20min so that there were no free aptamers in the solution. SG (1X) and different concentrations of T-2 toxin were then added, incubated in aptamer binding buffer for 40min, and finally 100. Mu.L of the solution was taken out and measured for fluorescence intensity on a microplate reader.
The detection scheme is shown in FIG. 4, and the detection system comprises MnO 2 Nanoplatelets, FAM-labeled T40 and SG. T40 from MnO due to T40 binding to T-2 toxin when T-2 toxin is present 2 And (4) dissociation of the surface of the nanosheet and recovery of fluorescence of FAM. Meanwhile, free SG in the solution is non-specifically combined with the aptamer dissociated from the surface of the nanosheet, and the fluorescence intensity of the SG after being combined with the aptamer is enhanced by 1000 times, so that the fluorescence signal in the solution is further enhanced. The result of a standard curve for T-2 toxin is drawn according to the fluorescence intensity and the concentration of the T-2 toxin and is shown in figure 5, so that the method for detecting the T-2 toxin by the aptamer has good linearity and can meet the requirement of quantitative determination.
EXAMPLE 3 detection of T-2 toxin in actual samples
Placing the beer sample in an ultrasonic machine, performing ultrasonic treatment for 30min until degassing is completed, placing 20g of the degassed beer sample in a 50mL volumetric flask, fixing the volume to the scale with 70% methanol, shaking up, filtering with quantitative filter paper to obtain filtrate, diluting the filtrate by 4 times with ultrapure water, and filtering with a 0.22 μm filter membrane until the filtrate is clear, wherein the filtrate is ready for use.
After the actual samples were pretreated, T-2 toxins were added to the filtrates at final concentrations of 1, 5, and 10ng/mL, respectively, and the recovery rates of T-2 toxin addition in the actual samples were measured by a fluorescence method based on SG amplification signals, and the results are shown in Table 2.
TABLE 2 detection and recovery of T-2 toxin in beer samples
As can be seen from the data in the table, the T40 has high recovery rate within a certain concentration range in the process of detecting the real object, and the method is proved to have good accuracy.
The present invention has been described in detail in order to enable those skilled in the art to understand the invention and to practice it, and it is not intended to limit the scope of the invention, and all equivalent changes and modifications made according to the spirit of the present invention should be covered by the present invention.
SEQUENCE LISTING
<110> university of south of the Yangtze river
<120> optimized aptamer capable of specifically recognizing T-2 toxin, optimization method and application of aptamer
<130> 3
<160> 3
<170> PatentIn version 3.3
<210> 1
<211> 58
<212> DNA
<213> Artificial Synthesis
<400> 1
cagctcagaa gcttgatcct gtatatcaag catcgcgtgt ttacacatgc gagaggtg 58
<210> 2
<211> 40
<212> DNA
<213> Artificial Synthesis
<400> 2
cagctcagaa gcttgatcct gtatatcaag catagaggtg 40
<210> 3
<211> 80
<212> DNA
<213> Artificial Synthesis
<400> 3
cagctcagaa gcttgatcct gtatatcaag catcgcgtgt ttacacatgc gagaggtgaa 60
Claims (7)
1. An optimized aptamer capable of specifically recognizing T-2 toxin, wherein the nucleotide sequence of the aptamer is shown as SEQ ID NO.2.
2. The optimized aptamer according to claim 1, wherein the nucleotide sequence of the aptamer is labeled with FAM.
3. Use of an optimized aptamer according to any of claims 1-2 for the detection of a T-2 toxin.
4. The use according to claim 3, wherein the aptamer, alone or modified, is useful for the detection of T-2 toxin.
5. Use of the optimized aptamer of any one of claims 1-2 in the preparation of a kit for detecting a T-2 toxin.
6. A kit comprising an aptamer according to any one of claims 1 to 2.
7. The kit of claim 6, wherein the kit further comprises MnO 2 Nanosheets and SYBR Green I.
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| MnO2 nanosheets based fluorescent sensing platform with organic dyes as a probe with excellent analytical properties;Chunxia Wang等;《Analyst》;20151231;摘要和实验部分 * |
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