A histamine aptamer and application thereof

Abstract

The application discloses a histamine nucleic acid aptamer and application thereof, and belongs to the technical field of nucleic acid aptamer.The histamine nucleic acid aptamer is aptamer His_AB, and the nucleotide sequence is shown as SEQ ID NO.4.The application of the histamine nucleic acid aptamer in detecting, separating or enriching histamine, and the application of the histamine nucleic acid aptamer in preparing products for detecting, separating or enriching histamine.Based on two original histamine aptamers L2 and HIS-apt, the aptamer His_AB with higher affinity to histamine is obtained through key binding domain modular splicing and end locking strategy.The affinity of the aptamer His_AB to histamine is as low as 1.27*10 ‑6 M, which is 9.29 times higher than that of the original aptamer L2 and 12.05 times higher than that of the original aptamer HIS-apt.The research of the application has important significance for the detection of histamine and the improvement of small molecule target nucleic acid aptamer.

Description

Technical Field

[0001] This invention relates to a histamine nucleic acid aptamer and its application, belonging to the field of nucleic acid aptamer technology. Background Technology

[0002] Aquatic products are highly susceptible to spoilage during storage. The autolysis process, which degrades the product's own muscle tissue, releases large amounts of free peptides and amino acids. Among these, free histidine is decarboxylated by histidine decarboxylases in microorganisms, producing histamine. Excessive intake of histamine can cause symptoms such as decreased blood pressure, skin redness and swelling, headache, nausea, and vomiting. Therefore, histamine content is an important indicator for evaluating the degree of spoilage and food safety of aquatic products.

[0003] Nucleic acid aptamers are single-stranded nucleotides with specific recognition capabilities, enabling them to bind specifically to targets. Utilizing nucleic acid aptamers for the detection of small molecules such as histamine is currently a hot research topic. However, nucleic acid aptamers targeting small molecules often suffer from insufficient affinity, necessitating modification to construct new aptamers and improve their performance. Traditional truncation strategies are unsuitable for short nucleic acid aptamers; rational site-directed mutations and chemical modifications rely heavily on trial and error, lacking systematic design rules; the recognition mechanism of split aptamers is still unclear, and the number of high-performance aptamers obtained through splitting strategies remains limited. Therefore, there is an urgent need to explore novel and universally applicable strategies for improving and constructing nucleic acid aptamers targeting small molecules to overcome the problems of low affinity and structural instability of original aptamers. Summary of the Invention

[0004] In view of the above-mentioned prior art, the present invention provides a histamine nucleic acid aptamer and its application, belonging to the field of nucleic acid aptamer technology.

[0005] This invention is achieved through the following technical solution: A histamine nucleic acid aptamer, aptamer His_AB, has the nucleotide sequence shown in SEQ ID NO.4.

[0006] The application of the histamine nucleic acid aptamer in the detection, isolation or enrichment of histamine.

[0007] The application of the histamine nucleic acid aptamer in the preparation of products for the detection, separation or enrichment of histamine.

[0008] This invention, based on two original histamine aptamers, L2 and HIS-apt, utilizes a modular splicing strategy of key binding domains and an end-locking technique to obtain the aptamer His_AB, which exhibits a higher affinity for histamine. The affinity of aptamer His_AB for histamine is as low as 1.27 × 10⁻⁶. -6Compared to the original aptamer L2, M showed a 9.29-fold improvement, and compared to the original aptamer HIS-apt, a 12.05-fold improvement. Furthermore, the aptamer His_AB exhibited no significant binding to histamine structural analogs (L-histidine, 5-hydroxytryptamine, and spermidine), demonstrating a more stable structure and higher specificity. This invention provides a novel biorecognition element with high affinity and high specificity for rapid histamine detection and verifies the high feasibility of a novel construction strategy for improved nucleic acid aptamers. This has significant implications for histamine detection and the improvement of nucleic acid aptamers targeting small molecules.

[0009] The various terms and phrases used in this invention have their general meanings known to those skilled in the art. Attached Figure Description

[0010] Figure 1 : Schematic diagram of the secondary structure of the original aptamer L2.

[0011] Figure 2 : Schematic diagram of the secondary structure of the original aptamer HIS-apt.

[0012] Figure 3 Schematic diagram of the molecular docking results between the original aptamer L2 and histamine.

[0013] Figure 4 Schematic diagram of the molecular docking results between the original aptamer HIS-apt and histamine.

[0014] Figure 5 : Schematic diagram of the improved aptamer construction strategy.

[0015] Figure 6 : Schematic diagram of the secondary structure of the improved aptamer His_AB.

[0016] Figure 7 : Schematic diagram of the secondary structure of the improved aptamer His_BA.

[0017] Figure 8 : Graph showing the thermal changes in the binding of the primitive aptamer L2 to histamine.

[0018] Figure 9 : Graph showing the thermal changes in the binding of the primitive aptamer HIS-apt to histamine.

[0019] Figure 10 Graph showing the thermal changes in the binding of the modified aptamer His_AB to histamine.

[0020] Figure 11 Graph showing the thermal changes in the binding of the modified aptamer His_BA to histamine.

[0021] Figure 12 Schematic diagram of the thermodynamic parameters of the binding of each aptamer to histamine.

[0022] Figure 13 : Combine with curve comparison chart.

[0023] Figure 14 The binding status of the aptamer His_AB-nolock to histamine.

[0024] Figure 15 The binding status of the aptamer L2-lock to histamine.

[0025] Figure 16 The binding status of the aptamer HIS-apt-lock to histamine.

[0026] Figure 17 : Improve the binding of the aptamer His_AB to L-histidine.

[0027] Figure 18 : Improve the binding of the aptamer His_AB to 5-hydroxytryptamine.

[0028] Figure 19 : Improve the binding of the aptamer His_AB to spermidine.

[0029] Figure 20 : Results of characterization of the specificity of the modified aptamer His_AB by nano-gold colorimetric method.

[0030] Figure 21 Schematic diagram of the molecular docking results between the modified aptamer His_AB and histamine.

[0031] Figure 22 Structural changes of the original aptamer L2 after binding with histamine.

[0032] Figure 23 Structural changes of the original aptamer HIS-apt after binding with histamine.

[0033] Figure 24 : Structural changes of the modified aptamer His_AB after binding with histamine. Detailed Implementation

[0034] The present invention will be further described below with reference to embodiments. However, the scope of the present invention is not limited to the following embodiments. Those skilled in the art will understand that various changes and modifications can be made to the present invention without departing from the spirit and scope thereof.

[0035] Unless otherwise specified, the instruments, reagents, and materials used in the following embodiments are all conventional instruments, reagents, and materials already available in the prior art and can be obtained through legitimate commercial channels. Unless otherwise specified, the experimental methods and detection methods used in the following embodiments are all conventional experimental methods and detection methods already available in the prior art.

[0036] This invention employs isothermal titration calorimetry to determine the affinity of nucleic acid aptamers for their targets. Isothermal titration calorimetry (ITC) is a biophysical analytical technique that directly measures the heat flow changes during molecular binding at an isothermal temperature, thereby obtaining complete thermodynamic parameters in a single experiment without the need for labeling. Isothermal titration calorimetry is a common analytical instrument used to quantitatively characterize the thermodynamic parameters and affinities of intermolecular interactions. By monitoring the heat changes generated by interactions between samples, thermodynamic parameters such as the equilibrium dissociation constant (Kd), enthalpy change (ΔH), and entropy change (ΔS) are obtained, which is an important means of characterizing intermolecular interactions. The specific determination method is as follows: a target solution with a concentration of 500 μM and an aptamer solution with a concentration of 10 μM are prepared in advance; 300 μL of aptamer solution is taken, and after denaturation annealing treatment, it is injected into the sample cell of the isothermal titration calorimeter. During the titration, the temperature is kept constant at 25℃. 2 μL of target solution is added to the sample cell each time, with a titration interval of 120 s. The titration is repeated 20 times, and the stirring speed is 350 r / min.

[0037] This invention verifies the affinity differences of aptamers through a competitive adsorption experiment based on the colorimetric properties of gold nanoparticles. The preparation method of the gold nanoparticles is as follows: A round-bottomed three-necked flask and rotor are rinsed with aqua regia (prepared by mixing concentrated hydrochloric acid and concentrated nitric acid in a volume ratio of 3:1), and then rinsed thoroughly with ultrapure water. 95 mL of ultrapure water and 1 mL of a 1% chloroauric acid solution are added to the three-necked flask, and the mixture is heated while stirring vigorously. After the solution boils, 4 mL of a 1% trisodium citrate solution is added to the container, and heating continues until the solution turns a uniform and stable wine-red color.

[0038] Example 1: Analysis of the molecular recognition mechanism of the primitive aptamer The nucleotide sequence of histamine primitive aptamer L2 is shown in SEQ ID NO.1, and the nucleotide sequence of histamine primitive aptamer HIS-apt is shown in SEQ ID NO.2.

[0039] The secondary structures of the primitive aptamer L2 and HIS-apt were predicted using the mFold website (http: / / www.unafold.org / ). Results: A schematic diagram of the secondary structure of the primitive aptamer L2 is shown below. Figure 1As shown, the Gibbs free energy (ΔG) is -3.87 kcal / mol, indicating a multi-stem-ring structure. The stem may work in conjunction with surrounding bases to recognize the target. A schematic diagram of the secondary structure of the primitive aptamer HIS-apt is shown below. Figure 2 As shown, ΔG = -5.13 kcal / mol, which belongs to a single stem-ring structure. The flexible ring portion may be the main region where this aptamer binds to the target.

[0040] The binding mechanism between the original aptamer and histamine was analyzed using molecular docking. The aptamer's PDB file was downloaded from the RNAComposer website, and a histamine mol2 file was prepared in advance. The histamine mol2 file and the aptamer's PDB file were imported into AutodockTools software. The docking box parameter was set to Spacing=1.000, and the center parameter and size of the docking box were adjusted to ensure complete coverage of the aptamer. The docking parameters were set as follows: Number of GARuns = 100, Population Size = 150, Maximum Numbers of Evals = 25,000,000, with other parameters set to default. The Lamarckian Genetic Algorithm (GA) was selected. After docking, the docking result with the lowest binding free energy was selected and visualized using PyMOL software.

[0041] A schematic diagram of the molecular docking results between the original aptamer L2 and histamine is shown below. Figure 3 As shown, the key binding sites for the primitive aptamer L2 with histamine are A31 and T32, located in the region adjacent to the stem. A schematic diagram of the molecular docking results between the primitive aptamer HIS-apt and histamine is shown below. Figure 4 As shown, the key binding sites of the original aptamer HIS-apt to histamine are G15, T18, C21, and G22, located in the flexible ring region.

[0042] Example 2: Design and Construction of Improved Aptamers This embodiment employs a construction method based on modular splicing of key binding domains and end-locking to improve the original histamine aptamers L2 and HIS-apt, thereby constructing improved aptamers with higher affinity. This construction method is proposed for the first time in this invention.

[0043] A schematic diagram of the improved aptamer construction strategy is shown below. Figure 5 As shown, based on the results of computer molecular docking simulations, key base fragments of the original aptamer are extracted. Two to five additional bases are extracted before the first key base and after the last key base, respectively, to maintain the three-dimensional structure of the extracted fragments as much as possible.

[0044] For the original aptamer L2, cleavage was performed between T27 and C28, and between T34 and G35, to obtain a 7-base fragment A, which corresponds to positions 28 to 34 of the original aptamer L2. For the original aptamer HIS-apt, cleavage was performed between G13 and C14, and between T23 and T24, to obtain a 10-base fragment B, which corresponds to positions 14 to 23 of the original aptamer HIS-apt. The nucleotide sequence of fragment B is shown in SEQ ID NO. 3.

[0045] By arranging two base fragments in different orders and adding a 9-length terminal locking sequence A at the 5' end and a 9-length terminal locking sequence B at the 3' end, the histamine modified aptamers His_AB and His_BA were constructed.

[0046] The nucleotide sequence of terminal locking sequence A is shown below (direction 5'-3'): gcgcgcgtt.

[0047] The nucleotide sequence of terminal locking sequence B is shown below (direction 5'-3'): ttcgcgcgc.

[0048] The nucleotide sequence of the modified aptamer His_AB is shown in SEQ ID NO.4, and the nucleotide sequence of the modified aptamer His_BA is shown in SEQ ID NO.5.

[0049] The secondary structure diagram of the modified aptamer His_AB is shown below. Figure 6 As shown, ΔG = -10.26 kcal / mol. A schematic diagram of the secondary structure of the modified aptamer His_BA is shown below. Figure 7 As shown, ΔG = -10.26 kcal / mol. Both improved aptamers have a single stem-ring structure, and ΔG is reduced by 6.39 kcal / mol compared to the original aptamer L2 and by 5.13 kcal / mol compared to the original aptamer HIS-apt, indicating significantly enhanced stability.

[0050] Example 3: Characterization of affinity and thermodynamic parameters by isothermal titration calorimetry The thermal changes during the binding of each aptamer to histamine were obtained by ITC characterization. The thermal change of the original aptamer L2 during histamine binding is shown in the figure below. Figure 8 As shown in the figure, the thermal changes in the binding of the primitive aptamer HIS-apt to histamine are as follows: Figure 9 As shown in the figure, the thermal change of the modified aptamer His_AB binding to histamine is as follows: Figure 10 As shown in the figure, the thermal change of the modified aptamer His_BA binding to histamine is as follows: Figure 11 As shown in the figure, the data fit is good, indicating that all four aptamers can bind to the target. The affinity of the original aptamer L2 for histamine is 1.18 × 10⁻⁶.-5 M, the primitive aptamer HIS-apt has an affinity of 1.53 × 10⁻⁶ for histamine. -5 M. The affinity of the modified aptamer His_AB for histamine is 1.27 × 10⁻⁶. -6 M showed a 9.29-fold increase in affinity compared to the original aptamer L2 and a 12.05-fold increase compared to the original aptamer HIS-apt. The modified aptamer His_BA exhibited an affinity of 5.42 × 10⁻⁶ for histamine. -6 M is 2.18 times higher than the original aptamer L2 and 2.82 times higher than the original aptamer HIS-apt.

[0051] Thermodynamic parameters of the binding of each aptamer to histamine are shown in the diagram below. Figure 12 As shown, the binding of the original aptamer to histamine is an entropy-decreasing and enthalpy-decreasing process. The ΔG after binding of the original aptamer L2 to histamine is -28.81 kJ / mol, and the ΔG after binding of the original aptamer HIS-apt to histamine is -27.49 kJ / mol. The binding of the modified aptamer to histamine is an entropy-increasing and enthalpy-decreasing process, which is conducive to obtaining a lower ΔG. The ΔG after binding of the modified aptamer His_AB to histamine is -33.65 kJ / mol, which is 5.52 kJ / mol lower than that of the original aptamer L2 and 6.16 kJ / mol lower than that of the original aptamer HIS-apt. The ΔG after binding of the modified aptamer His_BA to histamine is -30.06 kJ / mol, which is 1.93 kJ / mol lower than that of the original aptamer L2 and 2.57 kJ / mol lower than that of the original aptamer HIS-apt. This indicates that the modified aptamer can bind with histamine to form a more stable complex. Taking into account both affinity and stability, the modified aptamer His_AB was selected for subsequent embodiments.

[0052] Example 4: Competitive Adsorption Experiment of Gold Nanoparticles to Cross-validate Affinity In Na + In the presence of the target molecule, gold nanoparticles aggregate, leading to a decrease in absorbance at 520 nm and an increase in absorbance at 650 nm. The absorbance ratio A650 / A520 increases with the degree of aggregation of gold nanoparticles. The aptamer can adsorb onto the surface of the gold nanoparticles via electrostatic interactions, constructing the AuNPs-Apt complex, which significantly improves the dispersibility of gold nanoparticles in salt solution. Under the induction of the target molecule, the aptamer on the AuNPs-Apt surface desorbs and then binds to the target. The gold nanoparticles, deprived of aptamer protection, re-aggregate in the salt solution, causing a change in the A650 / A520 signal of the system. Therefore, by detecting the colorimetric signal of the system at different target concentrations, binding curves of different aptamers can be constructed, allowing for the evaluation of the affinity differences of different aptamers from a kinetic perspective.

[0053] 10 μL of 65 nM aptamer solution was added to the microwells, followed by 150 μL of AuNPs solution, and incubated in the dark for 15 min. Subsequently, 5 μL of target solutions with concentrations of 0 nM, 25 nM, 50 nM, 100 nM, 200 nM, 400 nM, 800 nM, and 1600 nM were added, and incubated in the dark for 15 min. After incubation, 90 μL of the mixed solution was taken, and 10 μL of 10× buffer (1 M NaCl, 200 mM Tris, 50 mM KCl, 20 mM MgCl2, 10 mM CaCl2, 0.2% Tween-20, pH 7.6) was added, and the absorbance values ​​at 520 nm and 650 nm were measured. Binding curves of different aptamers (original aptamer L2, original aptamer HIS-apt, and modified aptamer His_AB) were constructed to compare the affinity of different aptamers. The method for constructing the curve is as follows: The A650 / A520 ratio of the system at different target concentrations is measured, and a nonlinear regression equation is fitted. The specific formula is: Y = B max · X / ( K d + X ),in, Y A650 / A520: The absorbance ratio of the system at 650 nm and 520 nm; X Target concentration; B max Maximum signal response value, i.e., the signal value reached by the system when all aptamers bind to the target; K d : Dissociation constant, which is the target concentration required to reach half of the maximum signal.

[0054] Binding curves for each aptamer were constructed using competitive adsorption experiments with gold nanoparticles. A comparison of the binding curves is shown in the figure below. Figure 13 As shown, in the low concentration range, with the increase of target concentration, the aptamers rapidly desorb from the gold nanoparticles and bind to the target molecules, and the A650 / A520 ratio of the system increases rapidly. With the continuous increase of target concentration, the change in the A650 / A520 ratio gradually flattens out and approaches a certain plateau value, indicating that most of the aptamers in the system have completed desorption from the gold nanoparticles and binding to the target molecules. Among them, the modified aptamer His_AB exhibits a steeper binding curve slope, proving that the modified aptamer His_AB has a stronger affinity for histamine and can desorb from the surface of the gold nanoparticles more rapidly under the induction of histamine molecules; at the plateau phase, the modified aptamer His_AB system... B maxThe larger size indicates that, under the induction of histamine molecules, more aptamers in the system desorb from the surface of the gold nanoparticles, showing a more significant tendency to bind to the target.

[0055] The binding curve equation for the primitive aptamer L2 is: Y= 0.8603 X / (221.4681) +X () R 2 =0.9858), K d It is 221.4681 nM. B max It is 0.8603.

[0056] The binding curve equation for the primitive aptamer HIS-apt is: Y= 0.7962 X / (252.2780) +X () R 2 =0.9879), K d It is 252.2780 nM. B max It is 0.7962.

[0057] The binding curve equation for the modified aptamer His_AB is: Y= 0.9076 X / (119.9584) +X () R 2 =0.9897), K d It is 119.9584 nM. B max It is 0.9076.

[0058] The above results confirm that the modified aptamer His_AB has higher affinity, with significant differences.

[0059] Example 5: Verification of the necessity of the end-lock strategy using isothermal titration calorimetry First, by splicing only base fragments A and B and adding two T-base spacers, the non-terminal-locked aptamer His_AB-nolock was constructed. Terminal-locking sequences were then added to the 5' and 3' of the original aptamers L2 and HIS-apt, respectively, to construct the terminal-locked aptamers L2-lock and HIS-apt-lock.

[0060] The nucleotide sequence of aptamer His_AB-nolock is shown in SEQ ID NO.6, the nucleotide sequence of aptamer L2-lock is shown in SEQ ID NO.7, and the nucleotide sequence of aptamer HIS-apt-lock is shown in SEQ ID NO.8.

[0061] The binding of aptamers His_AB-nolock, L2-lock, and HIS-apt-lock to histamine was determined using the ITC method to verify the necessity of the terminal locking strategy in this invention. Results: The binding of aptamer His_AB-nolock to histamine is as follows: Figure 14 As shown, the binding of the aptamer L2-lock to histamine is as follows: Figure 15 As shown, the binding of the aptamer HIS-apt-lock to histamine is as follows: Figure 16 As shown, the aptamer His_AB-nolock cannot bind to histamine, indicating that simply splicing base fragments does not directly improve aptamer performance. Instead, it may lead to changes in the three-dimensional structure due to excessively short sequences, resulting in a loss of target recognition and binding. In contrast, the modified aptamer His_AB with the added terminal locking sequence exhibits significantly improved performance, indicating that the terminal locking sequence enhances the stability of the aptamer structure, helps maintain the three-dimensional structure of the base fragment, and thus maintains and improves the aptamer's ability to recognize and capture targets. The affinity of aptamer L2-lock for histamine is 1.18 × 10⁻⁶. -5 M, the aptamer HIS-apt-lock has an affinity of 1.48 × 10⁻⁶ for histamine. -5 M showed no significant difference from the case without the added end-locking sequence, indicating that the end-locking strategy cannot directly improve the performance of the aptamer.

[0062] The above results indicate that the end-locking strategy alone cannot improve the affinity of the original aptamer for histamine, while in this invention, the end-locking strategy is necessary to ensure that the base fragment maintains its original three-dimensional structure. The end-locking strategy can form a unique synergistic effect with the key binding domain modular splicing strategy, improving aptamer stability while helping to maintain the three-dimensional structure of the base fragment, thereby significantly improving the performance of the modified aptamer.

[0063] Example 6: Specificity evaluation of the modified aptamer The binding of the modified aptamer His_AB to histamine structural analogs (L-histidine, 5-hydroxytryptamine, and spermidine) was determined using the ITC method. Results: The binding of the modified aptamer His_AB to L-histidine was as follows: Figure 17 As shown, the binding of the modified aptamer His_AB to 5-hydroxytryptamine is as follows: Figure 18 As shown, the binding of the modified aptamer His_AB to spermidine is as follows: Figure 19 As shown, the modified aptamer His_AB cannot bind to L-histidine. Although binding curves can be fitted for both 5-hydroxytryptamine and spermidine with the modified aptamer His_AB, their slopes are significantly lower than those for histamine. The affinity of the modified aptamer His_AB for 5-hydroxytryptamine is 3.07 × 10⁻⁶. -4 M has a much lower affinity for histamine (241.73 times lower than histamine); the modified aptamer His_AB has an affinity for spermidine of 2.66 × 10⁻⁶. -4 M has a much lower affinity for histamine (209.45 times lower than histamine). Furthermore, ITC characterization revealed that the ΔG of the modified aptamer His_AB after binding to 5-hydroxytryptamine was -20.06 kJ / mol, an increase of 13.59 kJ / mol compared to histamine; the ΔG of the modified aptamer His_AB after binding to spermidine was -20.41 kJ / mol, an increase of 13.24 kJ / mol compared to histamine.

[0064] The specificity of the modified aptamer His_AB was verified by competitive adsorption experiments using gold nanoparticles. Specifically, 10 μL of a 65 nM aptamer solution and 150 μL of AuNPs solution were added to micropores and incubated in the dark for 15 min. Subsequently, 5 μL of each of the following solutions were added: 800 nM histamine, L-histidine, 5-hydroxytryptamine, and spermidine, respectively, and incubated in the dark for 15 min. The absorbance values ​​at 520 nm and 650 nm were measured.

[0065] The results of the nano-gold colorimetric method for characterizing the specificity of the modified aptamer His_AB are as follows: Figure 20 As shown, the results indicate that, under histamine induction, the aptamers on the AuNPs-Apt surface rapidly desorb, leading to a decrease in absorbance at 520 nm and an increase in absorbance at 650 nm, with a sharp increase in the A650 / A520 ratio. In contrast, the inductive effect of histamine structural analogs is extremely limited, and the A650 / A520 ratio of the system remains at a low level.

[0066] The above results indicate that the modified aptamer His_AB does not bind with significant high affinity to histamine structural analogs L-histidine, 5-hydroxytryptamine, and spermidine, and has high specificity.

[0067] Example 7 Molecular docking analysis of modified aptamers Download the modified aptamer His_AB pdb file from the RNAComposer website and perform a computer-aided molecular docking simulation using AutoDockTools software. The specific operating parameters are the same as in Example 1. A schematic diagram of the molecular docking results between the modified aptamer His_AB and histamine is shown below. Figure 21As shown, the key binding sites of the modified aptamer His_AB and histamine are located at T19 and A20, on base fragment B. This indicates that during the recognition process, base fragment B, derived from the original aptamer HIS-apt, plays a dominant role, while base fragment A, derived from the original aptamer L2, plays a supporting role. This division of labor mechanism may be due to the similar secondary structures of the modified aptamer His_AB and the original aptamer HIS-apt, both of which achieve target recognition and capture through conformational changes in the flexible loop. The original aptamer L2, however, relies on the stem adjacent to the binding site for collaborative target recognition and capture. When the modified aptamer lacks the stem structure, base fragment A cannot effectively perform its original recognition function.

[0068] Example 8: Characterization of conformational changes during the binding process by circular dichroism chromatography A 25 μM histamine solution and a 2.5 μM aptamer solution were prepared. The aptamer solution was pre-denatured and annealed, then mixed with the histamine solution and incubated in the dark for 40 min. A 1× buffer solution without histamine was used as a control. The operating parameters for the circular dichroism chromatograph were: CD / FL scale 200 mdeg / 0.1 d OD, Bandwidth 1 nm, DIT 1 s, spectral range 225 nm–320 nm, data acquisition interval 0.5 nm, and scan rate 50 nm / min. The test was repeated three times to obtain automatically fitted data.

[0069] Type B DNA exhibits a positive peak at around 280 nm and a negative peak at around 250 nm. The positive peak reflects the strength of DNA base stacking, while the negative peak reflects the helicity of the DNA.

[0070] The structural changes of the original aptamer L2 after binding with histamine are as follows: Figure 22 As shown, after binding to histamine molecules, the negative peak amplitude of the original aptamer L2 decreases, while the positive peak remains almost unchanged, indicating that the original aptamer L2 achieves target recognition and capture by fine-tuning the helicity of the nucleic acid backbone.

[0071] The structural changes of the primitive aptamer HIS-apt after binding with histamine are as follows: Figure 23 As shown, after binding to histamine molecules, the negative peak of the original aptamer HIS-apt remains almost unchanged, while the positive peak decreases significantly, indicating that the original aptamer HIS-apt recognizes and captures the target through local unwinding of the flexible ring.

[0072] The structural changes of the modified aptamer His_AB after binding with histamine are as follows: Figure 24As shown, after binding to histamine, the positive peak of the modified aptamer His_AB remained almost unchanged, while the amplitude of the negative peak increased, indicating that the aptamer-target complex formed a more ordered helical structure after binding to histamine. This reveals a different recognition and binding mode between the modified aptamer His_AB and the original aptamer. Before binding, the conformation at the binding pocket of the modified aptamer His_AB may still be in a relatively flexible state, while under the induction of the target molecule, the binding pocket region of the aptamer folds into a more stable helical structure. This result explains why the modified aptamer His_AB has a lower ΔG and a higher affinity for histamine after binding.

[0073] The above embodiments are provided to those skilled in the art to fully disclose and describe how the claimed implementations can be carried out and used, and are not intended to limit the scope of the disclosure herein. Modifications that will be obvious to those skilled in the art will be within the scope of the appended claims.

Claims

1. A histamine nucleic acid aptamer, characterized in that: The aptamer is His_AB, and its nucleotide sequence is shown in SEQ ID NO.

4.

2. The use of the histamine nucleic acid aptamer according to claim 1 in the preparation of products for detecting, separating or enriching histamine.

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