Broad-spectrum aptamers for 22 synthetic cannabinoids and uses thereof

Abstract

The present application belongs to the technical field of drug detection, and provides a broad-spectrum aptamer for recognizing 22 synthetic cannabinoids and an application thereof to solve the problem of detecting synthetic cannabinoids. The broad-spectrum aptamer is a single-stranded DNA molecule sequence as shown in SEQ ID NO: 5. The present application takes two existing nucleic acid aptamers as original aptamers, preliminarily predicts the binding mechanism of the nucleic acid aptamers and synthetic cannabinoids through molecular docking, further analyzes the interaction between the nucleic acid aptamers and the target in the base level through molecular dynamics simulation, optimizes the nucleic acid aptamers by truncation, and screens out aptamers capable of highly specifically recognizing 22 synthetic cannabinoids, which are few in base number, good in stability, easy to synthesize, and easy to modify or label functional groups. The aptamers can distinguish synthetic cannabinoids from other common drugs, verify the reliability thereof, can stably bind to 22 synthetic cannabinoids, and exhibit broad-spectrum recognition ability for synthetic cannabinoids.

Description

Technical Field

[0001] This invention belongs to the field of drug detection technology, specifically relating to a broad-spectrum nucleic acid aptamer that can identify 22 synthetic cannabinoids and its application. Background Technology

[0002] Synthetic cannabinoids (SCs) are a new type of psychoactive substance, similar to the active ingredient in cannabis, Δ9-tetrahydrocannabinol (Δ9-THC). SCs can activate human cannabinoid receptors CB1 and CB2, exerting a stronger new psychoactive effect. The diverse structural variations of SCs have long posed significant challenges to regulation. Recently, it has been discovered that criminals are using SCs to evade regulation by mixing them into e-cigarette liquids or applying them to dried plant stems, disguised as fragrances and sleep aids. Current detection methods primarily use gas chromatography-mass spectrometry (GC-MS), liquid chromatography combined with quadrupole time-of-flight mass spectrometry (LC-QTOF-MS), and ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS) to detect SCs or their metabolites in samples. While these methods are accurate and reliable, they require highly skilled operators and equipment and are difficult to implement rapidly on-site. Therefore, there is an urgent need to develop a simple, portable, and rapid detection method.

[0003] Aptamers are a class of biomolecules composed of single-stranded DNA or RNA oligonucleotide sequences that can specifically bind to specific target molecules (such as proteins, small molecules, toxins, viruses, and cells) through their unique three-dimensional structure. They are typically generated from randomized nucleic acid libraries using phylogenetic ligand index enrichment (SELEX) technology, exhibiting high affinity and specificity, and are considered "chemical antibodies." Because aptamers usually have small molecular weights, are easy to synthesize and modify, and possess better stability under various environmental conditions, they can be mass-produced in a simple, rapid, and low-cost manner. Biosensors constructed using nucleic acid aptamers as recognition elements have wide applications in biomedicine and detection fields. A biosensor is an analytical device that combines a biorecognition element with a physicochemical signal converter. Its core function is to convert the specific binding or reaction of the biorecognition element with the target into a measurable signal to achieve qualitative or quantitative measurement of the target.

[0004] The traditional SELEX process involves designing a random oligonucleotide library, incubating the target with the library, removing unbound nucleotides, and performing PCR amplification and purification of the remaining nucleotides. This process typically takes weeks or even months, involving multiple rounds of screening to gradually obtain aptamers with higher affinity and specificity. Finally, the library is sequenced to obtain the aptamer sequences, and parameters such as affinity and specificity are experimentally tested.

[0005] SCs (synthetic cannabinoids) are generally poorly soluble in water, have small molecular weights, and few functional groups that can be recognized by aptamers, making aptamer screening difficult. Screening for broad-spectrum nucleic acid aptamers that can simultaneously recognize multiple synthetic cannabinoids is even more challenging. Therefore, truncating and optimizing reported synthetic cannabinoid nucleic acid aptamers is a feasible approach. A common optimization method for nucleic acid aptamers is to retain the neck-loop structure and perform affinity tests with the target molecule, removing redundant bases. Molecular docking and molecular simulation are computational methods widely used in structural biology and drug design. They can be used to predict the binding mode and binding site between two molecules and analyze the binding process. In recent years, with the development of computer and bioinformatics technologies, molecular docking and molecular simulation techniques have been used in the field of nucleic acid aptamers to study the interaction mechanism between aptamers and target molecules and to guide nucleic acid aptamer optimization, improving the affinity and specificity of nucleic acid aptamers. Summary of the Invention

[0006] To address the current problem of detecting synthetic cannabinoids, this invention provides a broad-spectrum nucleic acid aptamer for identifying 22 synthetic cannabinoids and its applications.

[0007] This invention is achieved by the following technical solution: a broad-spectrum nucleic acid aptamer that recognizes 22 synthetic cannabinoids, wherein the 22 synthetic cannabinoids are: MDMB-4en-PINACA, 5F-MDMB-PICA, JWH-307, AMB-FUCBICA, ADB-PINACA, 5F-EMB-PINACA, ADB-BUTINACA, AB-FUCBICA, AB-005, HEXINACA, ADB-4en-PINACA, ADB-CHMINACA, EDMB-PINACA, EMB-FUCBINACA, BIM-018, FUBIMINA, JWH-030, JWH-370, JWH-019, UR-144, JWH-200, and FUB-144; the broad-spectrum nucleic acid aptamer is a single-stranded DNA molecule sequence as shown in SEQ ID NO: 5.

[0008] Furthermore, the single-stranded DNA molecule sequence is modified or labeled with a modifier, which is at least one of the fluorescent groups FAM, Cy3, Cy5, TAMRA, or Texas Red.

[0009] The present invention also provides the application of the broad-spectrum nucleic acid aptamer for identifying 22 synthetic cannabinoids, and the application of the nucleic acid aptamer in the preparation of products for detecting synthetic cannabinoids.

[0010] Furthermore, the application of the nucleic acid aptamer in the preparation of drugs or reagents for the qualitative and / or quantitative detection of synthetic cannabinoids.

[0011] Furthermore, the application of the nucleic acid aptamer in the preparation of kits for the qualitative and / or quantitative detection of synthetic cannabinoids.

[0012] The product used for detecting synthetic cannabinoids is a reagent or tool for in vitro detection of synthetic cannabinoids. The tool for in vitro detection of synthetic cannabinoids is a berberine-molybdenum disulfide nanosheet fluorescent sensor.

[0013] This invention uses two existing nucleic acid aptamers as the original aptamers and employs various computer technologies, especially AutoDock 4.2.2 software, for molecular docking. It preliminarily predicts the binding mechanism between the nucleic acid aptamers and synthetic cannabinoids. It uses GROMACS software to conduct molecular dynamics studies to predict the binding pocket of the original nucleic acid aptamers for synthetic cannabinoids. Then, it retains the binding bases and truncates and mutates the non-binding bases by removing redundant bases and explores the binding mechanism.

[0014] The results showed that the key binding base of the original aptamer XA to MDMB-4en-PINACA was located at the front end of the second stem-loop at the 5' end. The affinity of the two stem-loop structures for MDMB-4en-PINACA was measured, and the second stem-loop with stronger affinity was truncated and mutated to make the binding more stable. Further systematic truncation and mutation of the stem region bases yielded a new aptamer 7C capable of recognizing synthetic cannabinoids. Molecular dynamics (MD) was used to study the binding driving force and key bases of 7C to MDMB-4en-PINACA, circular dichroism (CD) chromatography was used to study the conformational changes of 7C during the recognition of MDMB-4en-PINACA, gel electrophoresis was used to study the binding of the nucleic acid aptamer to the target, and Raman spectroscopy and fluorescence experiments were used to verify the affinity and specificity of the nucleic acid aptamer to the synthetic cannabinoid MDMB-4e-PINACA. Finally, molecular docking technology was used to verify the binding of the optimal nucleic acid aptamer 7C to 22 synthetic cannabinoids, ultimately yielding a broad-spectrum aptamer sequence that can specifically bind to synthetic cannabinoids.

[0015] This invention utilizes fluorescence to screen for the optimal aptamer, SEQ ID NO: 5, which exhibits high specificity and high affinity for the target substance, using MDMB-4en-PINACA as a representative of synthetic cannabinoids.

[0016] The aptamer of this invention, compared with other truncated nucleic acid aptamers that can recognize MDMB-4en-PINACA, has fewer bases in its sequence SEQ ID NO: 5, better stability, and is easier to synthesize and modify or label with functional groups.

[0017] This invention verifies the affinity and specificity of nucleic acid aptamers for synthetic cannabinoids using fluorescence, Raman spectroscopy, and circular dichroism spectroscopy. It further verifies that the aptamers can distinguish synthetic cannabinoids from other common drugs, and further verifies the reliability of the aptamers. The aptamers can form complexes with 22 synthetic cannabinoids, and the binding pockets are approximately located at 4-9, 16-20 bases, demonstrating a broad-spectrum recognition ability of 7C for synthetic cannabinoids. Attached Figure Description

[0018] Figure 1 The 2D and 3D chemical structures of the MDMB-4en-PINACA molecule are shown. Figure 2 The diagram shows the molecular docking of XA, XB and MDMB-4en-PINACA. In the diagram: a is the molecular docking diagram of XA and MDMB-4en-PINACA; b is the molecular docking diagram of XB and MDMB-4en-PINACA; the blue bases are binding pockets. In the three-dimensional binding pocket diagram, the blue bases are hydrogen-bonded bases, and the green bases are hydrophobic binding pockets. Figure 3 This is a diagram illustrating the truncation of XA; Figure 4 This is a schematic diagram of the secondary structure of 7C and the 7C / MDMB-4en-PINACA binding base of the present invention. In the diagram: blue represents hydrogen-bonded bases, and green represents hydrophobic binding pockets. Figure 5 This is a schematic diagram of the XB truncation of the present invention; Figure 6 For fluorescence affinity testing (all nucleic acid aptamer concentrations were 1 µM, and MDMB-4en-PINACA concentration was 1 µM) (error bars represent the standard deviation of three parallel samples). Figure 7 For fluorescence specificity testing (all nucleic acid aptamer concentrations were 1µM, MDMB-4en-PINACA concentration was 1µM, and the other six drugs concentrations were 10µM) (error bars represent the standard deviation of three parallel samples). Figure 8 The dissociation constant k was determined for the fluorescence assays of this invention (a: berberine system, nucleic acid aptamer concentration of 1 µM) (b: berberine-molybdenum disulfide nanosheet system, MDMB-4en-PINACA concentration of 1 µM). d (The error bars represent the standard deviation of three parallel samples.) Figure 9In the diagram: (A) shows the root mean square deviation (RMSD) curves of the XA / MDMB-4en-PINACA, XA7 / MDMB-4en-PINACA, XA8 / MDMB-4en-PINACA, and 7C / MDMB-4en-PINACA complexes in the MD process of this invention; (B) shows the root mean square fluctuation (RMSF) curves of 7C and 7C / MDMB in the MD process; (C) shows the energy decomposition of the target and bound bases in the 7C / MDMB-4en-PINACA complex using MM / PBSA; (D) shows the radius of gyration curves of 7C and 7C / MDMB-4en-PINACA in the MD process; (E) shows the structural changes of 7C / MDMB-4en-PINACA during the MD process. Figure 10 In the image: (a) shows the effect of the reaction time of the sensing system on the relative Raman spectral intensity; (b) shows the reaction time for testing protamine; (c) shows the fitting plot of the dissociation constants for nucleic acid aptamers 7C, XB2, XB3, XA7, and XA8 using Raman spectroscopy; (d) shows the Raman spectra of 7C with different concentrations of MDMB-4en-PINACA; (e) shows the affinity test plot; (f) shows the Raman spectra of 7C, XB2, XB3, XA7, XA8, and 7C / MDMB-4en-PINACA, XB2 / MDMB-4en-PINACA, XB3 / MDMB-4en-PINACA, XA7 / MDMB-4en-PINACA, and XA8 / MDMB-4en-PINACA. (Error bars represent the standard deviation of three parallel samples). Figure 11 The circular dichroism spectrum of the 7C (1μM) of the present invention is shown in the absence of MDMB-4en-PINACA (black line) and in the presence of MDMB-4en-PINACA (100μM, red line). Figure 12 This is an agarose gel electrophoresis image; in the image: lane 1, DNA marker; lane 2, 7C / MDMB-4en-PINACA; lane 3, 7C; lane 4, XA7; lane 5, XA7 / MDMB-4en-PINACA; lane 6, XA8 / MDMB-4en-PINACA; lane 7, XA8; lane 8, XA / MDMB-4en-PINACA; lane 9, XA; Figure 13 In the middle: (a) shows the secondary structure and molecular docking prediction results of 22 synthetic cannabinoid molecules. The red box represents the hydrogen bonded atoms between the target and 7C, and the green box represents the atoms that form π-π conjugation between the target and 7C; (b) shows the molecular docking results between 7C and 22 synthetic cannabinoids. Figure 14This is a visualization of the docking results of 7C with 22 synthetic cannabinoid molecules in this invention. Detailed Implementation

[0019] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are some embodiments of the present invention, but not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0020] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains, and all materials publicly cited herein and cited by them are incorporated herein by reference.

[0021] Equivalent technologies of the specific embodiments described herein that are readily apparent to those skilled in the art through routine experimentation are included in this application.

[0022] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the instruments and equipment used in the following examples are all standard laboratory instruments and equipment; unless otherwise specified, the experimental materials used in the following examples were all purchased from regular biochemical reagent stores.

[0023] The original and truncated optimized sequences of the synthetic aptamers used in this invention were completed by Shanghai Sangon Biotech Co., Ltd.

[0024] I. Screening of Broad-Spectrum Nucleic Acid Aptamers for Synthetic Cannabinoids Based on Simulation Calculation 1. Experimental Methods (1) Nucleotide sequence: The original sequence of the aptamer is: XA (SEQ ID NO.1): 5'-CTTACGACTGTGGTCGGGTGGTGGGCCTCTAGAGGGGTGTCGTAAG-3'; XB (SEQ ID NO.6): 5'-CTTACGACTGCGGGCATTTGTGGGGGGCGTCGGTGGGCGTCGTAAG-3'.

[0025] XA and XB, both 46 bases in length, are two known aptamers identified through SELEX screening for synthetic cannabinoids UR-144 and XLR-11. Given the structural similarities of synthetic cannabinoids, we hypothesized that aptamers XA and XB might have the potential to bind to other synthetic cannabinoids. Based on this, we pruned redundant bases from the aptamers (excluding those involved in binding) and performed base mutations to obtain a series of aptamers, the specific sequences of which are shown in Table 1.

[0026] Table 1: Oligonucleotide sequences of nucleic acid aptamers (2) Software tools: Source of molecular structures of synthetic cannabinoids: PubChem database (http: / / pubchem.ncbi.nlm.nih.gov / ); Nucleic acid aptamer secondary structure prediction software: Mfold Web Server (http: / / www.mfold.org / mfold / applications / dna-folding-form.php); Software for predicting and modifying the tertiary structure of nucleic acid aptamers: Xiao Lab (http: / / biophy.hust.edu.cn / new / ); Format conversion software: OpenBabel 2.4.1 (https: / / openbabel.org / ); Molecular docking software: AutoDock 4.2.6 (https: / / Autodock.scripps.edu / ); Molecular simulation software: GROMACS 2023.6 (https: / / www.gromacs.org / ); Visualization and plotting software: Pymol 2.3.3 (https: / / pymol.org / 2 / ), LigPlot+ (https: / / www.eb i.ac.uk / thornton-srv / software / LigPlus / ).

[0027] (3) Truncating and Mutation of Nucleic Acid Aptamers: The secondary structure of nucleic acid aptamers was predicted using the online software Mfold. Following the 5'→3' input sequence, the conformation with the lowest free energy in the output results was retained as the dominant secondary structure of that sequence. The secondary structure was then input into the Xiao Lab website to construct a three-dimensional structural model. Five three-dimensional models were built for each nucleic acid aptamer, and the model with the lowest score and best prediction effect was selected as the output PDB file for later use. Taking MDMB-4en-PINACA as an example, PubChem CID:131762465 was queried on the PubChem website, and the three-dimensional structure SDF format file was saved. This file was then imported into OpenBabel software and converted to mol2 format. The 2D and 3D chemical structures of the MDMB-4en-PINACA molecule are shown below. Figure 1 As shown.

[0028] Upload the 3D structure files of the MDMB-4en-PINACA molecule and nucleic acid aptamer to AutoDock 4.2.2 software. Remove water molecules from the nucleic acid aptamer, add polar hydrogen atoms and Kollman charges to confirm the torque center of the MDMB-4en-PINACA molecule, and save the structure as a PDBQT file. Open the PDBQT file of the nucleic acid aptamer and target to construct a docking box, adjust the box size to completely cover the aptamer and target molecules, and save the mesh file as a GPF file. Run Auto Grid to generate a mesh diagram. Select the Lamarck genetic algorithm (LGA) as the search method, perform 100 dockings on the nucleic acid aptamer and target, sort the docking results from low to high binding energy, retain the result with the lowest binding energy as the optimal conformation, and save it as a PDB file. Use LigPlot+ and Pymol 2.3.3 software to analyze key parameters such as hydrogen bonding, hydrophobic interactions, and π-π stacking of the aptamer and target molecules, and visualize the results.

[0029] (4) Fluorescence assay for aptamer affinity and specificity: This invention uses a molybdenum disulfide nanosheet-berberine fluorescence method to test the affinity and specificity of nucleic acid aptamers with synthetic cannabinoids. Berberine is used as a fluorescent probe. When the nucleic acid aptamer binds to the target, it provides a rigid structure to berberine, thereby enhancing the fluorescence of berberine. In the system, molybdenum disulfide nanosheets are used to adsorb free nucleic acid aptamers and berberine through π-π stacking, reducing background interference and improving the sensitivity of the sensor.

[0030] In the specificity detection, the effects on other drugs and the synthetic cannabinoid MDMB-4en-PINACA were investigated. Morphine, ketamine, methcathinone, methamphetamine, cocaine, and fentanyl were selected for detection, with standards provided by the Third Research Institute of the Ministry of Public Security. Tris, sodium chloride (NaCl), magnesium chloride hexahydrate (MgCl2·6H2O), potassium chloride (KCl), sodium hydroxide (NaOH), and hydrochloric acid (HCl) were all analytical grade and purchased from Tianjin Kemei Chemical Reagent Co., Ltd. Methanol (chromatographic grade) was purchased from Sigma-Aldich, USA, and copper sulfate, sodium molybdate, and thioacetyl were purchased from Aladdin Biochemical Technology Co., Ltd. Oligonucleotides were provided by Sangon Biotech (Shanghai) Co., Ltd. Ultrapure water used in the experiments was obtained from the Millipore ultrapure water system (18.2 MΩ·cm, USA). Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) images were acquired using a JEM-7900F scanning electron microscope (JEOL, Japan). Molybdenum disulfide nanosheets were dried using a Scientz-12N vacuum freeze dryer (Ningbo Xinzhi Biotechnology Co., Ltd., China). Fluorescence intensity of the samples was obtained using a TECAN Infinite M200 PRO microplate reader (TECAN, Austria).

[0031] Preparation of buffer solution: Tris buffer (5mM, 20mM NaCl, 0.5mM MgCl2), adjust pH to 7.4 with HCl, store at 4℃ for later use.

[0032] Preparation of aptamer stock solution: Dissolve nucleic acid aptamer powder (XA, XB, XA7, XA8, 7C, XB2, XB3) in freshly boiled and cooled distilled water to prepare a 10 μM nucleic acid aptamer solution. Heat at 95°C for 15 min and immediately place in an ice box to cool to room temperature. Store at 4°C for later use.

[0033] Preparation of molybdenum disulfide nanosheets (MoS2NPs): 60 mg sodium molybdate and 120 mg thioacetamide were dissolved in 50 mL ultrapure water and magnetically stirred for 60 min. The mixture was then transferred to a 100 mL high-pressure reactor and reacted at 200°C for 24 h. The resulting product was washed three times each with ultrapure water and ethanol, freeze-dried, and stored for later use.

[0034] Nucleic acid aptamers and berberine were mixed in Tris buffer (pH 7.4), and synthetic cannabinoid solutions of different concentrations were added and mixed well. Finally, 125 nM molybdenum disulfide nanosheets (MoS2NPs) were added, and 100 μL of the supernatant was taken and the fluorescence intensity was measured on an ELISA reader.

[0035] (5) Molecular dynamics: Molecular dynamics (MD) simulations were used to further verify the accuracy of the molecular docking results and to explore the interaction between aptamers XA, XA7, XA8, 7C and MDMB-4en-PINACA.

[0036] PyMOL 2.3.3 software converts molecular docking results into PDB format. By selecting appropriate force fields and water molecule modes, energy optimization of the ligand structure is performed to eliminate steric hindrance. After energy minimization, heating, and equilibration of the nucleic acid aptamer / MDMB-4en-PINACA complex, the trajectory within 20 ns is analyzed, and the resulting trajectory is further analyzed using root mean square fluctuation and radius of gyration.

[0037] (6) Raman spectroscopy analysis: Raman spectra were measured on a Thermo Fisher portable Raman spectrometer with and without the target molecule at 7C (0.1 μM), with a scanning range of 980-1800 cm⁻¹. -1 The excitation wavelength was 785 nm. Before the test, 10 μL (10 μM) of nucleic acid aptamer solution and 10 μL of MDMB-4en-PINACA solution with concentration gradient were added to 20 μL of PBS buffer (10 mM, 50 mM NaCl, 2.5 mM MgCl2, pH=7.4) and mixed well. 10 μL (0.1 mM) of crystal violet solution was added as a Raman spectroscopy probe, and the mixture was allowed to stand for 3 min. Then, 200 μL (5 mM) of AuNPs solution was added, mixed by pipetting, and incubated at room temperature for 20 min. 25 μL (1.5 μg / mL) of protamine was added, and the mixture was incubated at room temperature for 8 min. The solution was then diluted to 1 mL with deionized water, and the crystal violet was measured at 1172 and 1635 cm⁻¹. -1 The intensity variation of the Raman spectral peak at that location.

[0038] (7) Circular dichroism spectroscopy: Circular dichroism spectroscopy is commonly used to study conformational changes when nucleic acid aptamers bind to targets. The morphological changes of DNA upon aptamer binding to the target were measured. Circular dichroism spectroscopy of 7C (1 μM) in the presence or absence of MDMB-4en-PINACA (10 μM) was measured on a BioLogic MOS-500 spectrometer (Bio-Logic, France). The scanning range was 220-340 nm, and the scanning rate was 1 second / point at room temperature. The spectral bandwidth was set to 2 nm, and a 1 cm quartz cuvette was used as the liquid cell for detection. Before the test, the aptamer and MDMB-4en-PINACA solution were added to Tris (pH 7.4) buffer and incubated at room temperature for 10 min.

[0039] (8) Gel electrophoresis analysis: The binding ability of nucleic acid aptamers of different lengths to small molecule targets was investigated by agarose gel electrophoresis. 5 μL of MSHipure Next III Gelred nucleic acid dye was added to a 1% agarose gel after cooling to 60°C at room temperature. After mixing, the mixture was poured into a mold and allowed to solidify at room temperature. The agarose gel was placed in a horizontal electrophoresis tank, and 1×TBE electrophoresis buffer was added to submerge the agarose gel. 10 μL of sample was loaded into each well, and the gel was run at 50V for 90 min at room temperature. Images were taken using a gel imaging system (Tanon 2500 R, Tianneng Co., Ltd., Shanghai, China).

[0040] 2. Experimental Results (1) Truncating and Mutating Nucleic Acid Aptamers: The molecular docking results of the original aptamers and their truncated aptamers are shown in Table 2, such as... Figure 2 As shown in figure a, the base 22T of the original aptamer XA forms a 3.4 Å long hydrogen bond with MDMB-4en-PINACA, and 22T together with the three surrounding bases forms a binding pocket. The base 23G in the pocket forms a dislocated parallel structure with the indole ring in the MDMB-4en-PINACA structure, with a ring spacing of 4.6 Å, satisfying π-π conjugation, which is conducive to the binding between XA and MDMB-4en-PINACA. However, in terms of Gibbs free energy, XA has a ΔG of -5.49 kcal / mol, which increases to -2.17 kcal / mol after binding with MDMB-4en-PINACA, indicating that the binding is not stable.

[0041] Table 2: Molecular docking results of original aptamers and truncated aptamers Since the binding pocket of XA is located near the second stem-ring, the second stem-ring structure was preserved during truncation optimization. Multiple molecular docking experiments were performed, retaining both the first and second stem-ring structures. The results showed that XA7 and XA8, obtained by retaining the second stem-ring structure and removing the 3' end base, yielded the best results.

[0042] When both aptamers bound to MDMB-4en-PINACA, the ΔG decreased. XA7 and XA8 each had one base forming π-π conjugation with the target. XA7 formed three hydrogen bonds with the target, with the shortest hydrogen bond length being 2.0 Å, and the binding pocket expanded to five bases. XA8 formed four hydrogen bonds with the target, with the shortest hydrogen bond length being 2.6 Å, and the binding pocket included eight bases. Compared to XA, the truncated aptamers XA7 and XA8 showed a lower ΔG when binding to MDMB-4en-PINACA, forming more and shorter hydrogen bonds, and involving more bases in the formation of the hydrophobic binding pocket, indicating a more readily occurring and stable binding. However, during secondary structure prediction, we noted a higher ΔG for XA7 and XA8. To ensure the stability of the nucleic acid aptamers, we appropriately increased the number of bases in the aptamers and increased the number of complementary bases in the stem-loop structure through base mutation, ultimately selecting a 22-base nucleic acid aptamer, 7C.

[0043] Based on the lower number of bases, lower ΔG, and further reduction of ΔG when binding to MDMB-4en-PINACA, 7C binds to the target and forms 7 potential hydrogen bonds, with the shortest hydrogen bond length being 2.1 Å. The binding pocket contains 8 bases, and the 17G base can form two pairs of π-π conjugations with the indole ring of MDMB-4en-PINACA (e.g., ...). Figure 4 As shown), it exhibits optimal binding properties, and the truncation process is as follows: Figure 3 As shown.

[0044] The molecular docking results of XB are superior to those of XA. MDMB-4en-PINACA forms four hydrogen bonds with the bases 28C, 33G, and 34T of XB, with the shortest hydrogen bond length being 1.9 Å and the binding pocket expanding to eight bases (e.g., Figure 2 As shown in b), the target forms π-π conjugation with the 28C base. Unlike XA, after XB binds to MDMB-4en-PINACA, ΔG decreases from -4.82 kcal / mol to -5.9 kcal / mol, indicating that XB tends to bind to MDMB-4en-PINACA. Based on the location of the binding pocket, MDMB-4en-PINACA binds to the ring structure on XB, and truncation optimization is performed by removing some paired bases. XB2 and XB3, which performed well in the XB cleavage chain, failed to retain the ring structure, resulting in a reduction in the number of hydrogen bonds and the disappearance of π-π conjugation. The truncation process is as follows: Figure 5As shown in the diagram. We hypothesize that the base pairing of XB is beneficial to the stability of the upper ring structure, and that removing non-binding bases disrupts this stability, leading to the disappearance of the XB2 and XB3 ring structures. To preserve the XB ring structure while reducing redundant bases, we optimized the truncation by removing one complementary base from the stem of XB at a time, aiming to obtain the shortest aptamer sequence that maintains the XB ring structure. The results show that to preserve the XB ring structure, a maximum of two complementary base pairs can be removed from the stem. By adding a base A to the XB stem to form more complementary bases, a maximum of five complementary base pairs can be truncated while maintaining the ring structure. Although the molecular docking results of the original aptamer XB are better than those of XA, XA shows better binding pocket stability in the truncation optimization, demonstrating greater optimization potential.

[0045] (2) Fluorescence assay for affinity and specificity: The affinity and specificity of nucleic acid aptamers to synthetic cannabinoids were tested using a molybdenum disulfide nanosheet-berberine fluorescence assay. Berberine was used as a fluorescent probe. When the nucleic acid aptamer binds to the target, it provides a rigid structure to berberine, thereby enhancing the fluorescence of berberine. In the system, molybdenum disulfide nanosheets are used to adsorb free nucleic acid aptamers and berberine through π-π stacking interactions, reducing background interference and improving sensitivity.

[0046] MDMB-4en-PINACA, a representative synthetic cannabinoid, was selected as the target. Fluorescence affinity tests were performed on the obtained candidate aptamers with MDMB-4en-PINACA. The results are as follows: Figure 6 As shown, after the addition of MDMB-4en-PINACA, the fluorescence intensity of XB in the original aptamer increased by the largest amount (0.064), while the fluorescence intensity of XA showed a weaker change. Most truncated aptamers showed a greater increase in fluorescence intensity after binding to MDMB-4en-PINACA, demonstrating that truncating the non-binding bases is beneficial for the binding of nucleic acid aptamers to synthetic cannabinoids. Among them, 7C, XA7, XA8, XB2, and XB3 all showed an increase in fluorescence intensity greater than 0.1 after binding to MDMB-4en-PINACA, with 7C showing the largest increase (0.6240), indicating that 7C has the best affinity for MDMB-4en-PINACA.

[0047] In specificity tests, four aptamers (7C, XA7, XA8, and XB2) with good affinity for synthetic cannabinoids were examined against six common drugs other than MDMB-4en-PINACA: morphine, ketamine, methcathinone, methamphetamine, cocaine, and fentanyl. Results are as follows: Figure 7As shown, the fluorescence response of 7C to MDMB-4en-PINACA was significantly higher than that of the other six drugs. It is noteworthy that the XA8 molecular docking results showed that 8 of its 13 bases participated in forming a binding pocket, but the fluorescence test showed poor specificity. This may be because excessive truncation disrupted the secondary structure for the specific binding of the nucleic acid aptamer to synthetic cannabinoids. Clearly, the nucleic acid aptamer 7C, obtained by appropriately retaining the number of bases and performing base mutations, is the nucleic acid aptamer with the best affinity and specificity.

[0048] dissociation constant k d This is an important parameter for evaluating the affinity between nucleic acid aptamers and targets. We tested the dissociation constants between the five nucleic acid aptamers with the highest response values ​​in the affinity test and MDMB-4en-PINACA, such as... Figure 8 a shows 7C(k) d The affinity (0.030±0.011µM) was significantly lower than the other four nucleic acid aptamers, exhibiting the strongest affinity. Subsequently, we introduced molybdenum disulfide nanosheets into the fluorescence detection system to quench the fluorescence of the free nucleic acid aptamer-berberine complex, reducing background interference and improving the signal-to-noise ratio. Figure 8 As shown in b, 7C(k) d The molybdenum disulfide nanosheets (0.097±0.017µM) still exhibit the smallest dissociation constant, indicating that while amplifying the signal, they have little impact on the specific binding of 7C to MDMB-4en-PINACA. Therefore, they can be used to construct a berberine-molybdenum disulfide nanosheet fluorescent sensor for detecting synthetic cannabinoids.

[0049] (3) Molecular dynamics studies: Molecular dynamics simulations are often used to predict conformational changes when nucleic acid aptamers bind to targets. The root mean square bias (RMSD) of the aptamer is an important parameter for measuring the stability of the system. When the RMSD curve tends to be stable, it indicates that the nucleic acid aptamer binds stably to the target. Figure 9 A shows the RMSD curves of the series of nucleic acid aptamers obtained by XA truncation optimization and their complexes with MDMB-4en-PINACA. It can be seen that the RMSD curve of XA / MDMB-4en-PINACA continuously increases from 0-3 ns during the simulated 20 ns process, then decreases from 3-20 ns, but does not reach stability within 20 ns. This indicates that XA / MDMB-4en-PINACA binds slowly, the complex is not very stable, and the results are consistent with the small binding pocket, fewer hydrogen bonds formed, and higher binding free energy in molecular docking, as well as the low fluorescence response value in the fluorescence affinity test.

[0050] Shortening the XA7 by 26 bases significantly improved its stability. The RMSD curve showed an increase and dramatic fluctuation in the 0-2 ns range, followed by another increase and stabilization at around 0.82 at 4 ns. The curve remained stable from 4-14 ns, fluctuated again from 14-17 ns, and then stabilized until 20 ns. Observation of the MD process revealed that when the RMSD curve changed significantly, the unpaired bases at the 5' and 3' ends and on the loop of XA7 were closer together, which may affect the binding of the nucleic acid aptamer to the target.

[0051] XA8, obtained by removing seven bases from both ends, showed a rapid increase in its RMSD curve from 0 to 1 ns and stabilized at around 0.35. It also exhibited high affinity but poor specificity in fluorescence experiments. 7C, with the addition of unpaired bases at both ends and base mutations to increase the number of complementary base pairs, showed a similar RMSD curve to XA8, with a rapid increase in the 0-1 ns curve and stabilization at around 0.42. XA7 has seven more bases than 7C, but the RMSD difference is only 0.07, demonstrating that 7C / MDMB-4en-PINACA is more stable than XA8 / MDMB-4en-PINACA. Fluctuations were observed in the RMSD curve of 7C at 15 ns. Visualizing the simulation process, we obtained the time points at which significant structural changes occurred in 7C (see [link to simulation]). Figure 9 E. Using the 7C / MDMB-4en-PINACA structure at 0 ns as a baseline, it was found that at 5 ns, the 1T base was attracted by MDMB-4en-PINACA to form a "pocket" structure. At 15 ns, the "pocket" further shrank, and the distance between 1T and the 17G on the other side decreased, which was visualized as a "surface" fusion. However, the two bases did not satisfy the complementarity condition and quickly moved away from each other, consistent with the fluctuations in the RMSD curve. When MDMB-4en-PINACA binds to 7C, it changes the position of the aptamer bases through interactions, forming a more stable complex structure.

[0052] Root mean square fluctuations (RMSF) can describe the total displacement of each base of a nucleic acid aptamer during the MD process, and thus infer the flexibility of the structure. Figure 9 B shows the RMSF of nucleic acid aptamer 7C and complex 7C / MDMB-4en-PINACA in 20 ns. The RMSF curves of 7C / MDMB-4en-PINACA are all lower than those of 7C, proving that the binding of the target MDMB-4en-PINACA causes 7C to undergo a smaller shift in the simulation. The 11-15 bases with the larger difference are the ring region in the secondary structure of 7C. The binding of MDMB-4en-PINACA makes the highly flexible unpaired bases at both ends and the ring region bases of 7C more stable.

[0053] The radius of gyration is used to study the distribution characteristics of atoms in a system along a specified center, and can be used to assess the compactness or stability of nucleic acid aptamers. Figure 9 As can be observed in D, the rotation radius curve of the 7C / MDMB-4en-PINACA complex decreases and stabilizes more quickly compared to 7C. This means that the binding of 7C to MDMB-4en-PINACA is rapid and stable, which is consistent with the conclusion of the RMSD curve.

[0054] To further elucidate the binding mechanism between nucleic acid aptamer 7C and MDMB-4en-PINACA, the binding free energy of the 7C base and MDMB-4en-PINACA was calculated using the molecular mechanics / Poisson-Boltzmann surface area (MM / PBSA) method. Figure 9 As shown in Figure C, the change in binding free energy of 7C binding to MDMB-4en-PINACA mainly comes from the van der Waals interaction energy (vdm). The figure shows four bases with the highest energy contributions (1T, 17G, 18G, 19G), which play important roles in the 7C / MDMB-4en-PINACA binding. The base with the highest energy contribution is 1T (vdm = -7.59 kcal / mol, electrostatic interaction Elec = -0.46 kcal / mol). 17G, 18G, and 19G are bases located at the stem of 7C and bind to MDMB-4en-PINACA via vdm, participating in the formation of the binding pocket.

[0055] (4) Raman spectroscopy analysis: Raman spectroscopy has advantages such as fingerprint recognition characteristics, high sensitivity, and non-destructive detection. We used Raman spectroscopy to detect the binding of XA7, XA8, 7C, XB2, and XB3 with different concentrations of MDMB-4en-PINACA at 1172 and 1635 cm⁻¹. -1 The intensity changes of the SERS peaks at 1172 cm⁻¹ show that the intensities of the two characteristic Raman peaks of crystal violet decrease at different rates with increasing MDMB-4en-PINACA concentration. -1 The intensity change of the SERS peak is relatively small. In this experiment, the ratio of the intensities of the two characteristic peaks, i.e., I, is used. 1635 / I 1172 The relative Raman intensity change was considered. Factors affecting the system were investigated. First, the reaction time was optimized. The nucleic acid aptamer, target, gold nanoparticles, and crystal violet showed the strongest Raman signals after 20 minutes of incubation, indicating the system had reached stability. Figure 10 b. The increased relative Raman intensity change within 0-8 minutes after the addition of protamine indicates that protamine forms "hot spots" with nucleic acid aptamers and gold nanoparticles-crystal violet through electrostatic adsorption, thereby enhancing the Raman signal. (See [link]). Figure 10 a.

[0056] The dissociation constant k of nucleic acid aptamers 7C, XB2, XB3, XA7, and XA8 with MDMB-4en-PINACA was determined respectively.d ,from Figure 10 c shows that 7C's k d Significantly smaller than the k of the other four nucleic acid aptamers d In affinity tests, 7C also exhibited the highest response value, such as... Figure 10 As shown in d, this confirms that 7C has a stronger affinity for the target.

[0057] (5) Circular dichroism (CD) analysis: Circular dichroism (CD) can determine the morphological changes of DNA when nucleic acid aptamers bind to targets. For example... Figure 11 As shown, 7C exhibits positive and negative Cotton effect peaks at 240 nm and 280 nm, respectively, indicating that the DNA has a B-type structure. It is important to note that the negative Cotton effect peak is generated by the double helix structure of DNA, while the positive Cotton effect peak originates from DNA base stacking. When 7C binds to MDMB-4en-PINACA, the intensity of the Cotton effect peak at 240 nm decreases, while the intensity of the Cotton effect peak at 280 nm increases and undergoes a red shift. This indicates that MDMB-4en-PINACA intercalates into the base pairs of 7C, weakening the helicality of 7C and enhancing base stacking, thus inducing 7C to transform into a conformation more closely matching the target.

[0058] (6) Gel electrophoresis analysis: The binding affinity of nucleic acid aptamers of different lengths to small molecule targets was investigated by agarose gel electrophoresis. The electrophoresis results are as follows: Figure 12 As shown, in the absence of the target, each nucleic acid aptamer is a single band, and the migration rate is negatively correlated with molecular weight. Among them, XA8 (13nt) migrates the fastest, and XA (46nt) migrates the slowest. After adding the target MDMB-4en-PINACA, lanes 7C, XA7, and XA8 show tails or new bands above the original bands, indicating the formation of nucleic acid aptamer / target complexes; while XA shows no significant change in band size under both target-free and non-target conditions, indicating that it fails to bind effectively to MDMB-4en-PINACA under these conditions.

[0059] (7) Determination of the affinity of 7C for synthetic cannabinoids using molecular docking technology: Synthetic cannabinoids are a class of numerous and rapidly evolving new psychoactive substances. We performed molecular docking with 7C on 22 synthetic cannabinoids. The molecular structures of the synthetic cannabinoids and the molecular docking results are as follows: Figure 13 and Figure 14As shown, 7C can form complexes with all 22 synthetic cannabinoids, with the binding pockets roughly located at bases 4-9 and 16-20. All synthetic cannabinoids can form hydrogen bonds with 7C. Among them, six synthetic cannabinoids (MDMB-4en-PINACA, 5F-MDMB-PICA, 5F-EMB-PINACA, ADB-HEXINACA, EDMB-PINACA, and AB-005) form a π-π co-bonding with 7C. Five synthetic cannabinoids (AMB-FUBICA, EMB-FUBINACA, JWH-030, FUBIMINA, FUB-144) bind with 7C to form two π-π conjugations, and five synthetic cannabinoids (BIM-018, JWH-019, JWH-200, JWH-370, JWH-307) bind with 7C to form three π-π conjugations, demonstrating 7C's broad-spectrum recognition ability for synthetic cannabinoids.

[0060] This invention employs various computer simulation techniques, particularly using AutoDock 4.2.2 software for molecular docking, and GROMACS software for molecular dynamics prediction of the binding pocket of the synthetic cannabinoid aptamer. Then, binding bases are retained, and redundant bases are truncated and mutated to investigate the binding mechanism. Results show that the key binding base of the aptamer XA to MDMB-4en-PINACA is located at the 5' end of the second stem-loop. The affinity of the two stem-loop structures for MDMB-4en-PINACA was measured, and truncation and mutation of the second stem-loop with stronger affinity resulted in a more stable binding. Systematic truncation and mutation of the stem region bases yielded a new aptamer, 7C, capable of recognizing synthetic cannabinoids. The binding driving force and key bases of 7C to MDMB-4en-PINACA were investigated using molecular spectroscopy (MD). Conformational changes of 7C during MDMB-4en-PINACA recognition were studied using CD (conformal chromosomal) technology. Gel electrophoresis was used to investigate the binding of the nucleic acid aptamer to the target. Raman spectroscopy and fluorescence experiments were used to verify the affinity and specificity of the nucleic acid aptamer to the synthetic cannabinoid MDMB-4e-PINACA. Finally, molecular docking technology was used to verify the binding of the optimal nucleic acid aptamer 7C to 22 synthetic cannabinoids, ultimately yielding a broad-spectrum aptamer sequence capable of specifically binding to synthetic cannabinoids.

[0061] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A broad-spectrum nucleic acid aptamer for recognizing 22 synthetic cannabinoids, wherein the 22 synthetic cannabinoids are: MDMB-4en-PINACA, 5F-MDMB-PICA, JWH-307, AMB-FUBICA, ADB-PINACA, 5F-EMB-PINACA, ADB-BUTINACA, AB-FUBINACA, AB-005, HEXINACA, ADB-4en-PINACA, ADB-CHMINACA, EDMB-PINACA, EMB-FUBINACA, BIM-018, FUBIMINA, JWH-030, JWH-370, JWH-019, UR-144, JWH-200, and FUB-144; characterized in that: The broad-spectrum nucleic acid aptamer is a single-stranded DNA molecule sequence as shown in SEQ ID NO:

5.

2. The broad-spectrum nucleic acid aptamer for recognizing 22 synthetic cannabinoids according to claim 1, characterized in that: The single-stranded DNA molecule sequence is modified or labeled with a modifier, which is at least one of the fluorescent groups FAM, Cy3, Cy5, TAMRA, or TexasRed.

3. The application of the broad-spectrum nucleic acid aptamer for recognizing 22 synthetic cannabinoids as described in claim 1 or 2, characterized in that: The application of the nucleic acid aptamer in the preparation of products for detecting synthetic cannabinoids.

4. The application according to claim 3, characterized in that: The application of the nucleic acid aptamer in the preparation of drugs or reagents for the qualitative and / or quantitative detection of synthetic cannabinoids.

5. The application according to claim 4, characterized in that: Application of the nucleic acid aptamer in the preparation of kits for qualitative and / or quantitative detection of synthetic cannabinoids.

6. The application according to claim 3, characterized in that: The product used to detect synthetic cannabinoids is a reagent or tool for in vitro detection of synthetic cannabinoids.

7. The application according to claim 6, characterized in that: The tool used for in vitro detection of synthetic cannabinoids is a berberine-molybdenum disulfide nanosheet fluorescent sensor.

Images