Nucleic acid aptamer capable of being specifically combined with danmarin and application of nucleic acid aptamer

Abstract

The invention discloses a nucleic acid aptamer capable of being specifically combined with danmarin and application of the nucleic acid aptamer, and belongs to the technical field of food science and engineering. The sequence of the nucleic acid aptamer DHU1 is as shown in SEQ ID NO. 1, and the truncated optimized sequence DHU1-23 of the nucleic acid aptamer DHU1 is as shown in SEQ ID NO. 2. The nucleic acid aptamer DHU1 is obtained through screening by virtue of a Capture-SELEX technology, the dissociation constant (Kd) of the nucleic acid aptamer DHU1 to the daughter glycoside is 66.06 + / -3.92 nmol / L, and the Kd value of the truncated sequence DHU1-23 to the daughter glycoside is 41.2 + / -2.05 nmol / L. The nucleic acid aptamer can be specifically combined with the danioside and can be applied to rapid detection of the danioside in baijiu brewing raw materials such as sorghum, so that generation of a metabolite ethyl carbamate is controlled. The method provided by the invention solves the problems of dependence on expensive equipment, complex operation and the like in the existing daumatin detection technology, and can provide a simple, convenient and low-cost solution for on-site rapid detection of cyanoglycoside hazardous substances.

Description

TECHNICAL FIELD

[0001] The present application relates to a nucleic acid aptamer specifically binding to dhurrin and its application, and belongs to the technical field of food science and engineering. BACKGROUND

[0002] Dhurrin is a cyanogenic glycoside in the main raw material of liquor brewing, i.e. sorghum, and its metabolism is closely related to the generation of ethyl carbamate (EC), a 2A carcinogen in liquor. Specifically, during the processing or fermentation of sorghum, dhurrin is hydrolyzed by β-glucosidase or decomposed under acidic and high-temperature conditions to generate β-D-glucose and the intermediate p-hydroxy-(S)-mandelonitrile; the latter is further decomposed into p-hydroxybenzaldehyde and hydrogen cyanide (HCN) under the action of endogenous α-hydroxyl cleavage enzyme or alkaline conditions. As a key precursor, HCN can react with ethanol through a cyanate intermediate to ultimately form EC. Therefore, effective monitoring of the dhurrin content in sorghum is a key link for controlling EC pollution in liquor and ensuring food safety. At present, the detection of dhurrin mainly relies on techniques such as Raman spectroscopy imaging, high-performance liquid chromatography (HPLC), and ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS / MS). However, these methods require expensive instruments, complex sample pretreatment, and professional operation, making it difficult to meet the needs of on-site rapid detection. Therefore, it is of urgent practical significance to develop a new method for detecting dhurrin that is simple, rapid, and low-cost.

[0003] Aptamer is a single-stranded DNA or RNA molecule that can fold into a specific three-dimensional structure and bind to a target with high affinity and specificity, and is known as a "chemical antibody". Compared with traditional antibodies, aptamers have the advantages of high stability, small batch difference, easy chemical modification, and no immunogenicity, and have shown great potential in the detection of small molecules such as pesticides, heavy metals, and biological toxins. However, there is still no report on the screening of dhurrin nucleic acid aptamers. SUMMARY

[0004] The present application aims to provide a nucleic acid aptamer specifically binding to dhurrin and its application. The nucleic acid aptamer DHU1 and DHU1-23 of the present application can simply, sensitively, and conveniently bind to, recognize, and inhibit dhurrin, which is of great significance for preventing the generation of the 2A carcinogen ethyl carbamate in liquor.

[0005] The technical scheme of the present application is: a nucleic acid aptamer specifically combined with dhurrin, the nucleic acid aptamer comprises a nucleic acid aptamer DHU1, the nucleic acid aptamer sequence of which is shown as SEQ ID NO. 1, and the sequence of SEQ ID NO. 1 is: 5'-ACCGACCGTGCTGGACTCTCGAGCTCATCTCATCGTTGCGAGTTCAATGAGTATGAGCGAGCGTTGCG-3'.

[0006] In the aforementioned nucleic acid aptamer specifically combined with dhurrin, the nucleic acid aptamer further comprises a nucleic acid aptamer DHU1-23, which is obtained by optimizing and truncating the nucleic acid aptamer DHU1, and the sequence of the nucleic acid aptamer DHU1-23 is shown as SEQ ID NO. 2, and the sequence of SEQ ID NO. 2 is: 5'-GCTCATCTCATATGAGTATGAGC-3'.

[0007] The application of the aforementioned nucleic acid aptamer in specific recognition of dhurrin.

[0008] The beneficial effects of the present application are: compared with the prior art, the present application uses the Capture-SELEX technology to first screen the nucleic acid aptamer DHU1 specifically combined with dhurrin, the nucleotide sequence of which is shown as SEQ ID NO. 1, and the DHU1 is optimized and truncated to obtain DHU1-23. It is verified that DHU1-23 can recognize and combine dhurrin with higher affinity and higher specificity than DHU1. The secondary structure of the nucleic acid aptamer DHU1 specifically combined with dhurrin in the present application has a ring and a stem, and the Gibbs free energy is ΔG=-5.02, and the dissociation constant Kd of DHU1 to dhurrin is 66.06±3.92 nmol / L, and the Kd value of the truncated sequence DHU1-23 is 41.2±0.25 nmol / L. The nucleic acid aptamer DHU1 and DHU1-23 described in the present application can simply, sensitively and conveniently combine, recognize and inhibit dhurrin, and have important significance for preventing the 2A-grade carcinogen ethyl carbamate produced in Baijiu (Chinese liquor). BRIEF DESCRIPTION OF DRAWINGS

[0009] Figure 1 The nucleic acid aptamer structure of the present application;

[0010] Figure 2 The elution rate of each round of screening;

[0011] Figure 3 The electrophoresis map of PCR product of each round of screening;

[0012] Figure 4 The sequence of the nucleic acid aptamer screened;

[0013] Figure 5Phytate aptamer development tree analyzed by MEGA software;

[0014] Figure 6 Dissociation constant (Kd) of phytate affinity sequence DHU1;

[0015] Figure 7 Secondary structure prediction of DHU1 affinity sequence by Mfold software;

[0016] Figure 8 Molecular docking diagram of DHU1 aptamer;

[0017] Figure 9 Secondary structure prediction diagram of truncated sequence DHU1-23;

[0018] Figure 10 Molecular docking diagram of truncated sequence DHU1-23

[0019] Figure 11 Biomembrane interference diagram of aptamer DHU1;

[0020] Figure 12 Biomembrane interference diagram of truncated sequence DHU1-23. DETAILED DESCRIPTION

[0021] The present application is further described below in conjunction with the accompanying drawings and examples, but is not limited to the basis of the application.

[0022] Embodiment of the present application: a nucleic acid aptamer specifically binding to phytate, the nucleic acid aptamer comprising nucleic acid aptamer DHU1, the nucleic acid aptamer sequence of which is shown in SEQ ID NO. 1, and the sequence of SEQ ID NO. 1 is: 5'-ACCGACCGTGCTGGACTCTCGAGCTCATCTCATCGTTGCGAGTTCAATGAGTATGAGCGAGCGTTGCG-3'.

[0023] The nucleic acid aptamer further comprises nucleic acid aptamer DHU1-23, which is obtained by optimizing and truncating nucleic acid aptamer DHU1, and the sequence of which is shown in SEQ ID NO. 2, and the sequence of SEQ ID NO. 2 is: 5'-GCTCATCTCATATGAGTATGAGC-3'.

[0024] The present application also protects the use of the above-mentioned nucleic acid aptamer in the specific recognition of phytate.

[0025] The nucleic acid aptamer to be protected by the present application is obtained by the following experimental method (all nucleic acid sequences in the experiment are purchased from Shanghai Sangon).

[0026] 1. In vitro screening steps of aptamer

[0027] (1) Fixation of random ssDNA library. A certain amount of random ssDNA library with the sequence of 5'-ACCGACCGTGCTGGACTCT-N30-AGTATGAGCGAGCGTTGCG-3', wherein N represents any of A, T, C, G, is mixed with P3 sequence in a molar ratio of 1:2, and the P3 sequence is 5'-Biotin-CGCAACGCTCGC-3'. After annealing treatment at 95°C for 5 min and incubation at 30°C for 60 min, the 12-base sequence at the 3' end of the random library is paired with P3 through hydrogen bond complementation. The magnetic beads after pretreatment are added to the centrifuge tube and incubated at 30°C for 60 min. Since the surface of the magnetic beads is modified with streptavidin, the random library is fixed on the magnetic beads through biotin-avidin interaction. Finally, the unbound random library and P3 are removed by washing with Binding Buffer (BB buffer, 100 mmol / L NaCl; 20 mmol / L Tris-HCl; 5 mmol / L KCl; 2 mmol / L MgCl2·6(H2O); 1 mmol / L CaCl2; 0.02% Tween-20, pH=7.4).

[0028] (2) Elution of ssDNA sequence. In the first round of screening, a solution of 100 μg / mL of mairein is added to the random library fixed on the magnetic beads, and the supernatant is collected by magnetic separation after incubation at 30°C (200 rpm) for 1 h. The DNA content is determined by nucleic acid quantification. Then the magnetic beads are separated with a magnetite, and the ssDNA supernatant eluted by mairein is collected. The absorbance is detected by an ultramicro measuring instrument, and the elution rate is calculated. The results are shown in (Figure 1). Figure 2 In the subsequent SELEX screening process, the final concentration of mairein is gradually reduced from 100 μg / mL to 5 μg / mL, and the other operations are the same as those in the first round.

[0029] (3) PCR amplification. Asymmetric PCR is used in the first to sixth rounds, and symmetric PCR is used in the seventh to tenth rounds to amplify the affinity sequence of mairein. The symmetric PCR amplification conditions are pre-denaturation at 94°C for 3 min; denaturation at 94°C for 30 s, annealing at 51°C for 30 s, and extension at 72°C for 30 s (the cycle number is 18 for the first three rounds, 15 for the fourth to sixth rounds, 12 for the seventh to ninth rounds, and 10 for the tenth round).

[0030] (4) Preparation of ssDNA. After symmetric PCR amplification, the amplification product was verified by 2% agarose gel electrophoresis, precipitated with ice ethanol at -20°C for 3 h, precipitated by centrifugation at 10,000 rpm for 10 min, redissolved, digested with λ-exonuclease at 37°C for 30 min (specifically degrading the P2 chain marked with 5'-PO4), terminated by adding EDTA and inactivated at 80°C for 10 min. The obtained single-stranded DNA was extracted with phenol: chloroform: isoamyl alcohol (25:24:1), precipitated with ethanol again, centrifuged, dissolved in pure water and quantified as an enrichment library for the next round of screening. Finally, the ssDNA of the first 10 rounds was electrophoretically verified, and the results are shown in (Figure 4). Figure 3 ).

[0031] 2. Sequencing and secondary structure prediction of nucleic acid aptamer of sorghum glycoside

[0032] After 10 rounds of SELEX screening, the sorghum glycoside affinity sequences collected were PCR amplified, and the amplification reaction solution was sent to Shanghai Shengong Bioengineering Co., Ltd. for high-throughput sequencing. The top 30 sequences with the highest enrichment degree were selected for sequence analysis, and the results are shown in (Figure 5) Figure 4 ). The 30 sequences were used to make a phylogenetic tree with MEGA11 software (see Figure 6) Figure 5 ).

[0033] 3. Determination of dissociation constant (Kd) of nucleic acid aptamer

[0034] After comprehensive analysis of enrichment degree, homology, conservation and secondary structure, 6 sequences were finally selected for Kd value detection. The 6 affinity sequences were labeled with FAM fluorescence. Then 10 μmol / L of aptamer affinity sequences and 20 μmol / L of biotin-labeled P3 sequences were mixed in the same volume, denatured at 95°C for 5 min, and then incubated at 30°C for 60 min. The magnetic beads were washed with 300 μL of BB buffer for 3-4 times, and then resuspended with 200 μL of BB buffer for standby. The resuspended magnetic beads were mixed with P3 and aptamer affinity sequence hybridization solution for incubation for 60 min. Finally, the affinity sequences and P3 that were not fixed were removed by washing with BB buffer. The fluorescence intensity of the collected eluent was measured, and the amount of fixed affinity sequences was calculated. Finally, the magnetic beads with fixed affinity sequences were dispersed in a certain volume of BB solution, 10 μL of 100 μg / mL target solution was taken and incubated with the magnetic beads with fixed sorghum glycoside affinity sequences at 60°C for 60 min. Finally, 200 μL of supernatant was taken and placed in a black enzyme-labeled plate to measure the fluorescence intensity of the eluent. Curve fitting was performed by Origin 9.0 software, and the fitting formula was Y=Bmax X / (Kd+X), Y: concentration of nucleic acid and target complex; X: nucleic acid concentration; Bmax: constant; Kd: affinity coefficient, and the results are shown in (Figure 7) Figure 6 ).

[0035] 4. Analysis of DHU1 aptamer binding mechanism with dhurrin

[0036] The secondary structure of DHU1 was predicted by RNAstructure (see Figure 7 ), and the secondary structure file of aptamer molecule was saved in Vienna output format (dot bracket notation) for constructing the tertiary (3D) structure of aptamer. The 2D structure of dhurrin small molecule was first drawn by ChemDraw Professional 15.1 software, and then converted into a 3D structure by Chem3D 15.1 software. Finally, the docking input file of aptamer and dhurrin small molecule was generated by AutoDockTools 1.5.6 software, and the interaction and possible binding site between aptamer and dhurrin molecule were analyzed, and visual presentation was performed by PyMoL 1.7.6 software (see Figure 8 ).

[0037] 5. Truncation sequence analysis

[0038] The truncation of aptamer to 23 bases in length was selected based on the retention of DHU1 core binding domain (T-30, C-31, G-51, T-54) and system free energy minimization, and the secondary structure of the truncated molecule is shown in Figure 9 , and the docking results are shown in Figure 10 ): dhurrin is embedded in the hydrophobic cavity composed of C-9, T-13, G-14, A-15 by hydrophobic interaction, which is almost consistent with the position of the original long chain forming the hydrophobic cavity, and forms 5 high-precision hydrogen bonds (C-9 O4'-H1: 2.0 Å, T-13 O4'-H11: 2.2 Å, G-14 O4'-H14: 2.1 Å, G-14 H22-O7: 1.9 Å, A-15 O4-H14: 1.8 Å), and the minimum binding free energy is reduced to -5.53 kcal·mol⁻¹. This reconstruction promotes the cumulative effect of hydrogen bonds and local enrichment of residues, significantly improves the unit binding force density, and realizes the "lock-key" type high specificity recognition.

[0039] 6. Affinity detection of truncated sequence

[0040] To compare the affinity of long sequence DHU1 and truncated sequence to phytosterols, biofilm interference experiment was performed on DHU1 and DHU1-23, and the Kd value was calculated by fitting the binding signal value of a certain amount of nucleic acid aptamer to different concentrations of phytosterols into a kinetic curve. The sensor used in this experiment is SSA sensor, which is fixed on the SSA sensor by using the interaction between streptavidin and biotin. Before the experiment, all the SSA sensors were immersed in HEPES (pH 8, 10 mM) for pre-wetting, and incubated at room temperature for 10 min. After equilibration, the SSA sensors were immersed in biotinylated DHU1 and DHU1-23 aptamer solution for incubation. Phytosterols with concentrations of 65 nM, 12.5 nM, 25 nM, 50 nM, 100 nM and 200 nM were added to the wells of rows A, C and E of the 96-well plate, respectively, and HEPES buffer was used as a control sample. The time of Equilibration, Immobilization and Association steps was set to 60 seconds, respectively. After the blocking and equilibration steps, the SSA sensor was immersed in the phytosterol sample to be tested, and the real-time binding signal value was obtained. After data export, the standard curve was drawn with the response time as the abscissa and the binding signal value of phytosterols and nucleic acid aptamer as the ordinate. The results are shown in (Figures Figure 11 , Figure 12 ), the Kd value of DHU1 is 75.8±3.31 nM, which is basically consistent with the Kd value of 66.06±3.92 nM measured by fluorescence method, and the Kd value of DHU1-23 is 41.2±2.05 nM, which indicates that the affinity of truncated sequence DHU1-23 to phytosterols is stronger than that of long sequence DHU1.

[0041] SEQUENCE LIST INFORMATION:

[0042] DTD Version: V1_3

[0043] File Name: A nucleic acid aptamer specifically binding to phytosterols and its application-01.xml

[0044] Software Name: WIPO Sequence

[0045] Software Version: 2.3.0

[0046] Date of Creation: 2025-09-01

[0047] Basic Information:

[0048] Current Application / Applicant File Name: 12520000429203011T

[0049] Applicant Name or Designation: guizhou University

[0050] Applicant Name or Designation / Language: zh

[0051] Applicant Name or Designation / Latin Name: guizhou University

[0052] Invention Title: A nucleic acid aptamer specifically binding to phlomisoside and application thereof (zh)

[0053] Total Number of Sequences: 2

[0054] Sequences:

[0055] Sequence Number (ID): 1

[0056] Length: 68

[0057] Molecule Type: DNA

[0058] Feature Location / Qualifier:

[0059] - source, 1..68

[0060] > mol_type, other DNA

[0061] > organism, unidentified

[0062] Residues:

[0063] accgaccgtg ctggactctc gagctcatct catcgttgcg agttcaatga gtatgagcga 60

[0064] gcgttgcg 68

[0065] Sequence Number (ID): 2

[0066] Length: 23

[0067] Molecule Type: DNA

[0068] Feature Location / Qualifier:

[0069] - source, 1..23

[0070] > mol_type, other DNA

[0071] > organism, unidentified

[0072] Residues:

[0073] gctcatctca tatgagtatg agc 23

[0074] END.

Claims

1. A nucleic acid aptamer that specifically binds to dhurrin, characterized in that: The nucleic acid aptamer comprises a nucleic acid aptamer DHU1, a nucleic acid aptamer sequence of which is shown as SEQ ID NO. 1, and the sequence of SEQ ID NO. 1 is: 5'-ACCGACCGTGCTGGACTCTCGAGCTCATCTCATCGTTGCGAGTTCAATGAGTATGAGCGAGCGTTGCG-3'.

2. The nucleic acid aptamer of claim 1, wherein: The nucleic acid aptamer further comprises a nucleic acid aptamer DHU1-23, which is obtained by optimizing and truncating the nucleic acid aptamer DHU1, and a sequence of the nucleic acid aptamer DHU1-23 is shown as SEQ ID NO. 2, and the sequence of SEQ ID NO. 2 is: 5'-GCTCATCTCATATGAGTATGAGC-3'.

3. Use of the nucleic acid aptamer according to any one of claims 1-2 in specific recognition of DHU.

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