Aptamer specifically binding to GYM-B and its application

Abstract

The present invention discloses an aptamer specifically binding to GYM‑B and its application, belonging to the technical field of detection of naked dinoflagellate imine toxins, two aptamers specifically binding to GYM‑B are G15 and G15a, and the sequences are shown in SEQ ID NO.1 and SEQ ID NO.2, respectively. The present invention fills the gap that there is no aptamer specifically binding to GYM‑B, and the provided aptamer can specifically bind to GYM‑B, has high detection sensitivity, strong affinity and good stability, wherein G15a has the highest affinity to GYM‑B, reaching 64nM, and can be used for the detection of GYMs and GYM‑B, thereby effectively preventing poisoning caused by contact with and accidental ingestion of water and aquatic products contaminated by GYMs; the provided aptamer has low preparation cost, low immunogenicity, stable properties and convenient modification, which can greatly reduce the detection cost of GYMs, simplify the detection process, and improve the detection accuracy and efficiency.

Description

Technical Field

[0001] The invention relates to the technical field of detection of gymnodinium imide toxins, and in particular to an aptamer specifically binding to GYM-B and an application thereof. Background Art

[0002] Affected by global climate change and marine pollution, harmful algal blooms have become a common ecological disaster in coastal waters around the world. Shellfish accumulate high doses of toxins in their bodies by filtering toxic algae, which potentially threatens the safety of aquatic products and human health. According to the chemical structure, marine biotoxins can be divided into polyether toxins, alkaloid toxins, polypeptide toxins and cyclic imine toxins (Cyclicimines). Cyclicimine toxins are a new type of "fast-acting" fat-soluble toxins discovered in recent years. They have been found in the waters and seafood of many countries. Members of this family include gymnodimines (GYMs), spirolides (SPXs), pinatoxins (PnTXs), etc.

[0003] GYMs is the earliest discovered and smallest molecular weight of the cyclic imine toxin family. It is a type of "rapid-acting toxin" that can produce neurotoxicity by blocking acetylcholine receptors. It is very toxic. Mice will die within 15 minutes after intraperitoneal injection of GYM. 50 The highest is 80μg / kgb.w. Other studies have shown that GYMs can enter organisms through the food chain, aerosols, and skin contact, causing great harm to human health and aquaculture. The EU stipulates that the maximum content of GYM in aquatic products is 200μg / kg. Therefore, it is urgent to establish an accurate and sensitive GYM detection method to better monitor the level of GYM in water and aquatic products, thereby ensuring the safety of consumers and aquatic organisms.

[0004] At present, the commonly used methods for detecting marine biotoxins mainly include bioanalysis, chemical analysis and immunoassay. The mouse biological method (MBA) is the most traditional bioanalysis method for detecting GYM. However, this method is not only costly, has low specificity and poor reproducibility, but also has ethical issues. Chemical analysis methods, such as high performance liquid chromatography (HPLC) and liquid chromatography-mass spectrometry (LC-MS), have also been gradually improved, among which liquid chromatography-mass spectrometry has been identified by the European Union as the primary detection method. However, this method requires a complex sample pretreatment process, the related instruments and equipment are expensive, and the operator has high technical requirements. Therefore, it is urgent to establish a new rapid and sensitive detection method.

[0005] In recent years, biosensor detection has attracted much attention from researchers because it can overcome many shortcomings of traditional detection methods. The core component of biosensors is molecular recognition elements. Antibodies are a well-developed and highly regarded molecular recognition element. However, antibodies are not only expensive, but also easy to denature and have batch-to-batch differences. Therefore, it is necessary to find a new molecular recognition element.

[0006] Aptamers are a new type of molecular recognition element, known as "chemical antibodies". They are single-stranded oligonucleotides that can specifically bind to a target and are screened through the systematic evolution of ligands by exponential enrichment (SELEX) technology. They have the advantages of good specificity, high stability, easy synthesis, easy modification, and low immunogenicity. As a new type of molecular recognition element, aptamers are similar to antibodies, but superior to antibodies. For example, aptamers can recognize a variety of targets, such as proteins, amino acids, metal ions, and even cells and viruses; aptamers can be connected to a variety of groups and are easy to modify; aptamers have stable chemical properties and are not easy to denature; aptamers can be directly chemically synthesized at a low cost.

[0007] At present, the structures of GYMs that have been elucidated include GYM-A, 12-Me GYM-A, GYM-B, 12-Me GYM-B, GYM-C, GYM-D, 16-desMe GYM-D and GYM-E. They have spirocyclic and imine structures, among which the imine structure has biological activity. Among the GYMs with elucidated structures, GYM-A and GYM-B are the most toxic, but there are no reports of GYM-B aptamers. Conventional aptamer screening adopts the strategy of fixed target, but GYM-B is a fat-soluble molecule with no charge, small molecular weight, limited number of chemical groups, lack of the groups required for fixation, and is difficult to fix. Screening its aptamers is challenging. Summary of the invention

[0008] The purpose of the present invention is to provide an aptamer that specifically binds to GYM-B and its application, so as to provide an aptamer for GYM detection with low cost and good stability, so as to make it possible to detect GYM using a biosensor detection method, greatly improve the sensitivity and accuracy of GYM detection, reduce the detection cost, and contribute to better protecting the life safety of consumers.

[0009] To achieve the above object, the present invention provides two aptamers that specifically bind to GYM-B, the aptamers are G15 and G15a, and their sequences are shown in SEQ ID NO.1 and SEQ ID NO.2, respectively.

[0010] The two aptamers described above are used in the preparation of GYM-B detection reagents, kits and sensors. The aptamers may be unmodified aptamers or modified aptamers.

[0011] Preferably, the modification includes modification of the 3′ end, 5′ end or middle sequence of the aptamer with biotin, FITC, thiol or fluorescent group.

[0012] The two aptamers described above are used in the preparation of GYMs detection reagents, separation and enrichment reagents, detection / separation and enrichment kits and sensors. The aptamers may be unmodified aptamers or modified aptamers.

[0013] The two aptamers mentioned above are used in the detection, separation and enrichment of GYMs and GYM-B in water bodies and aquatic products.

[0014] Therefore, the aptamer specifically binding to GYM-B and its application provided by the present invention have the following characteristics:

[0015] Beneficial effects:

[0016] (1) The present invention fills the gap of the lack of aptamers that specifically bind to GYM-B. The provided aptamers can specifically bind to GYM-B, have high detection sensitivity, strong affinity, and good stability. Among them, G15a has the highest affinity for GYM-B, reaching 64nM, and can be used for the detection of GYMs and GYM-B, thereby effectively preventing poisoning caused by contact with or accidental ingestion of water and aquatic products contaminated by GYMs;

[0017] (2) The aptamers provided by the present invention have low preparation cost, low immunogenicity, stable properties, and are easy to modify, which can significantly reduce the detection cost of GYMs, simplify the detection process, and improve detection accuracy and efficiency.

[0018] The technical solution of the present invention is further described in detail below through the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings required for use in the description of the embodiments of the present invention will be briefly introduced below. Obviously, the accompanying drawings in the following description are only some embodiments of the present invention. For ordinary technicians in this field, other accompanying drawings can be obtained based on these accompanying drawings without paying creative labor.

[0020] Figure 1 is a flow chart of screening aptamers for GYM-B in Example 2 of the present invention;

[0021] Figure 2is the secondary structure prediction diagram of aptamers G15 (part A) and G15a (part B) in Example 2 of the present invention;

[0022] Figure 3 This is the test result of the interaction between the aptamer G15a and different toxins in Example 4 of the present invention. DETAILED DESCRIPTION

[0023] The technical solution of the present invention is further described below through the accompanying drawings and embodiments.

[0024] In order to make the purpose, technical scheme and advantages of the present application clearer, more thorough and more complete, the technical scheme of the present invention is clearly and completely described below through the accompanying drawings and examples. The following detailed descriptions are all descriptions of the embodiments, and are intended to provide further detailed descriptions of the present invention. Unless otherwise specified, all technical terms used in the present invention have the same meaning as those generally understood by those skilled in the art to which the present application belongs.

[0025] The experimental methods in the following examples, for which specific conditions are not specified, are generally carried out under conventional conditions such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer. Unless otherwise specified, percentages and parts are calculated by weight. Unless otherwise defined, all professional and scientific terms used herein have the same meanings as those familiar to those skilled in the art. In addition, any methods and materials similar or equivalent to those described herein can be applied to the present invention. The preferred embodiments and materials described herein are for exemplary purposes only.

[0026] The instruments, equipment, reagents and materials used in the examples were all obtained through commercial channels.

[0027] Embodiment 1

[0028] Construction of ssDNA library:

[0029] The library consists of 69 bases, with fixed regions containing 20 and 22 bases at both ends, and a random region containing 30 bases in the middle, as shown in the sequence SEQ ID NO.3, where N is any one of the four deoxyribonucleotide bases A, T, G, and C, and 30 represents the number of random bases.

[0030] Embodiment 2

[0031] The aptamers of GYM-B were screened by magnetic bead method. The specific steps are as follows:

[0032] S2.1, library hybridization incubation: In each round of screening, the libraries at each level and the capture sequence (sequence as shown in SEQ ID NO.4) were mixed at a ratio of 1:3. The mixed mixture was then subjected to denaturation treatment, and the treatment conditions were: heating at 95°C for 10 minutes, slowly cooling to 60°C at a rate of 0.1°C per second, maintaining for 1 minute, and then slowly cooling to 25°C at a rate of 0.1°C per second, and maintaining at 25°C for 20 minutes. The entire denaturation process was carried out on the PCR instrument by setting a cooling program, and finally a hybrid formed by the library and the capture sequence was obtained.

[0033] In the first round, 3 nmol of ssDNA library was used, 200 pmol of library was used from the second to the fifth round, and 100 pmol of library was used from the sixth to the tenth round.

[0034] S2.2, SA magnetic beads and library coupling: add the denatured library to the SA magnetic beads washed with anhydrous ethanol, and incubate overnight at room temperature on a four-dimensional rotating mixer. After magnetic separation, wash the magnetic beads once with 1000 μL screening buffer and magnetically separate for 2 minutes.

[0035] The coupled magnetic beads were mixed with 200 pmol of GYM-AB in a screening buffer with a volume of 1000 μL, and incubated on a four-dimensional rotator at room temperature for 2 hours, which was recorded as the experimental group. At the same time, a control group was set up, in which GYM-A was not added, and other conditions were the same as those of the experimental group. After incubation, magnetic separation was performed, and the eluted ssDNA in the experimental group and the control group was quantitatively analyzed using a nucleic acid quantitative fluorometer Qubit, and the recovery rate was calculated to monitor the progress of the screening. The input amount of GYM-A in each round was 200 pmol.

[0036] S2.3, amplification: PCR amplification was performed using the ssDNA eluted in the experimental group of step S2.2 as a template. The PCR reaction system (50 μL) was: template 2.5 μL; PCRmix (2×) 25 μL; upstream primer (10 μM) 2.5 μL; downstream primer A (10 μM) 2.5 μL; enzyme-free water 17.5 μL. The PCR amplification program was: 98°C, 5 min; 95°C, 30 s, 57°C, 40 s, 72°C, 30 s, a total of 26 cycles; 72°C, 5 min.

[0037] The upstream primer sequence is shown in SEQ ID NO.5, and the downstream primer A sequence is shown in SEQ ID NO.6.

[0038] In order to improve the efficiency of screening, reverse screening was carried out from the sixth round. The difference from the forward screening is that before incubating the ssDNA library with GYM-B magnetic beads, the ssDNA library and the negative magnetic beads were rotated and incubated at room temperature. After the incubation, the ssDNA in the solution and the ssDNA bound to the blank magnetic beads were separated by a magnetic stand. The ssDNA in the solution recovered was the ssDNA library for the forward screening. The screening scheme is shown in Table 1.

[0039] Table 1

[0040]

[0041] S2.4, electrophoresis gel recovery: The DNA amplified by PCR in step S2.3 is dsDNA, while the aptamer is ssDNA. In order to obtain the ssDNA secondary library, a downstream primer A containing 20 guanines (sequence as shown in SEQ ID NO.6) was specially designed so that the two chains after PCR differed by 20 bases, and then the two chains were separated by urea denatured polyacrylamide gel electrophoresis, and the target chain was cut and recovered.

[0042] After the recovered gel was centrifuged and crushed, 1 mL of buffer was added and incubated in a 50°C water bath for 20 min (repeat twice). After the incubation, the sample was centrifuged (12000 rpm, 5 min) and the supernatant was collected. The recovered ssDNA was purified using a gel extraction kit. After purification, the nucleic acid quantitative fluorometer Qubit was used for quantitative analysis. At this point, the secondary library preparation was completed.

[0043] S2.5, cloning sequencing and analysis: When the screening reached the 12th round, the recovery rate of ssDNA no longer increased, indicating that the screening endpoint was reached. Therefore, the ssDNA eluted from the 12th round was amplified by PCR using the upstream primer (sequence shown in SEQ ID NO.5) and the downstream primer B (sequence shown in SEQ ID NO.7), and the dsDNA recovered by polyacrylamide gel electrophoresis was purified using a nucleic acid recovery kit, and then the cloning sequencing was commissioned to Shanghai Sangon Biotechnology Co., Ltd., and a total of 50 sequences were measured.

[0044] After comparing and analyzing all the sequences using Clustal X 2.1 software, 8 representative sequences with the highest sequencing frequency were selected for affinity determination (biomembrane interferometry), among which G15 had the highest affinity and was used for subsequent optimization. The sequence of G15 is shown in SEQ ID NO.1, wherein the underlined part is the non-primer region, and the ununderlined part is the primer region.

[0045] Embodiment 3

[0046] The optimization steps of aptamer G15 are as follows:

[0047] The affinity constant (KD) between aptamer G15 and GYM-B is 0.43 μM. In order to obtain the core structure of aptamer G15 and optimize the aptamer, the aptamer was truncated according to the prediction of the secondary structure of G15 by the online tool the mfold web server. Figure 2 This is the secondary structure prediction diagram of the truncated product of aptamer G15 by the online tool the mfold web server.

[0048] After the primer regions at both ends of the aptamer G15 were truncated (named G15a, the sequence is shown in SEQ ID NO.2, where the underlined part is the non-primer region), the free energy of the secondary structure increased to a certain extent, and the structural stability decreased, but the results of biomembrane interference analysis showed that the aptamer (G15a) can still bind to GYM-B, and the KD value did not change much, which was 0.46 μM. Therefore, it can be inferred that the primer region of the aptamer G15 does not participate in the binding between GYM-B, and has a supporting effect on the structural stability of the aptamer. The stem-loop structure in the aptamer G15a is of great significance during binding and is very likely to be its core structure.

[0049] Embodiment 4

[0050] The biomembrane interferometry technique is used to analyze the interaction between molecules. The steps are as follows:

[0051] Biomembrane interferometry is a label-free technology that can provide real-time, high-throughput information on biomolecular interactions. The basic principle of biomembrane interferometry is that the instrument emits white light to the sensor surface and collects the reflected light from the two surfaces of the optical film layer at the same time. Because the reflected light of different frequencies is affected differently by the thickness and mass density of the optical film layer, some frequencies of reflected light form constructive interference (blue) and others form destructive interference (red). After the spectrometer detects these interference lights, an interference spectrum is formed and displayed as the phase shift intensity (Δλ) of the interference spectrum.

[0052] Therefore, when the number of molecules bound to the sensor surface changes, the shift of the interference spectrum will be detected by the spectrometer. This shift can directly reflect the change in the thickness and mass density of the biofilm on the sensor surface, thereby accurately and quantitatively measuring the interaction process between the molecules to be tested. As a new type of label-free technology, biomembrane interferometry has occupied an important position in the study of interactions between biomolecules.

[0053] The 5′ end of the nucleic acid aptamer is modified with biotin, and the aptamer is fixed on the surface of the super streptavidin (SSA) sensor through the interaction between biotin and streptavidin. Before fixation, the aptamer must first be renatured (95°C water bath for 10 minutes, ice bath for 5 minutes, and room temperature for 10 minutes) to help the aptamer refold into a stable spatial structure.

[0054] Before detection using the biofilm interferometer OctetRED 96, buffer, biotin-labeled aptamer solution, buffer, and GYM-B solution were added to different columns of a 96-well plate in sequence, with a sample volume of 200 μL. The program of the Octet RED 96 system was set as follows: sensor equilibration; aptamer coupling; sensor reequilibration; GYM-B binding; GYM-B dissociation. All steps were completed at room temperature.

[0055] After the test is completed, the Octet data analysis software CFR Part 11 Version 6.x is used to deduct the response value of the control group sensor from the response value of the experimental group sensor to obtain the corrected actual response value. By fitting the response data, the affinity constant KD value can be calculated.

[0056] Using the above method, the specificity of the interaction between aptamer G15a and GYM-A, GYM-B, PnTX, OA, STX, DTX, PTX, and between random sequence and GYM-B was tested. The results are as follows: Figure 3 G15a can specifically bind to GYM-B (response value is 0.35±0.02nm), which is significantly higher than the response value produced by other toxins. Therefore, G15 and G15a have great practical application value in GYM-B detection.

[0057] Therefore, the present invention fills the gap of the lack of aptamers that specifically bind to GYM-B. The provided aptamer can specifically bind to GYM-B, has high detection sensitivity, strong affinity and good stability. Among them, G15a has the highest affinity for GYM-B, reaching 64nM, and can be used for the detection of GYMs and GYM-B, thereby effectively preventing poisoning caused by contact with and accidental ingestion of water and aquatic products contaminated by GYMs; the provided aptamer has low preparation cost, low immunogenicity, stable properties and convenient modification, which can greatly reduce the detection cost of GYMs, simplify the detection process, and improve the detection accuracy and efficiency.

[0058] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that they can still modify or replace the technical solution of the present invention with equivalents, and these modifications or equivalent replacements cannot cause the modified technical solution to deviate from the spirit and scope of the technical solution of the present invention.

Claims

1. Two aptamers that specifically bind to GYM-B, characterized in that: The aptamers are G15 and G15a, and their sequences are shown in SEQ ID NO.1 and SEQ ID NO.2 respectively.

2. The use of the two aptamers according to claim 1 in the preparation of a GYM-B detection reagent, a kit and a sensor, characterized in that: The aptamer may be an unmodified aptamer or a modified aptamer; the modification includes modification of the 3′ end, 5′ end or middle sequence of the aptamer with biotin, FITC, thiol or fluorescent group.

3. Use of the two aptamers according to claim 1 in the preparation of GYM-B detection reagents, separation and enrichment reagents, detection / separation and enrichment kits and sensors, characterized in that: The aptamer may be an unmodified aptamer or a modified aptamer.

4. Use of the two aptamers as described in claim 1 in the detection, separation and enrichment of GYM-B in water or aquatic products.

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