5-HMF aptamer, optimization method and application

Abstract

The invention provides a 5-HMF aptamer, an optimization method and application, the 5-HMF aptamer is one of the following sequences: H1-21m: SEQ ID NO1 sequence, H1-21m-5Fu-d: replacing T at the 20th site in the SEQ ID NO1 sequence with 5Fu, or H1-21mdUb: replacing T at the 13th site in the SEQ ID NO1 sequence with U, and the 5-HMF aptamer is one of the following sequences: H1-21m: SEQ ID NO1 sequence, H1-21m-5Fu-d: replacing T at the 20th site in the SEQ ID NO1 sequence with 5Fu, or H1-21mdUb: replacing T at the 13th site in the SEQ ID NO1 sequence with U. According to the 5-HMF aptamer provided by the invention, thymine at a key site of a truncated body is systematically replaced with uracil and 5-fluorouracil which are similar in structure, so that the binding affinity of the aptamer and the 5-HMF is remarkably enhanced, and a 5-HMF detection method based on a non-labeled aptamer is constructed.

Description

Technical Field

[0001] This invention relates to the field of assay or testing methods involving enzymes, nucleic acids or microorganisms, and particularly to a 5-HMF aptamer, an optimized method and its application. Background Technology

[0002] 5-Hydroxymethylfurfural (5-HMF) and furfural (FF), as characteristic products of Maillard reaction and caramelization, are widely present in heat-processed food systems. Studies have shown that 5-HMF exhibits a significant accumulation effect during food storage, making it an important indicator for evaluating the degree of heat treatment and storage quality of food. Conversely, previous studies by our research group found that FF content decreases with prolonged storage time. The Chinese Pharmacopoeia stipulates that the absorbance value of 5-HMF in glucose injection at a wavelength of 284 nm should not exceed 0.32; according to the assessment report of the European Union Scientific Committee on Food Additives and Processing Aids (ANS Panel), the acceptable daily intake (ADI) of 5-HMF is set at 1.6 mg / person. Analysis of commercially available dairy products showed that the contamination level of 5-HMF was significantly higher than that of FF (fermented milk powder). Specifically, the 5-HMF content (0.22-1.70 mg / 100g protein) in milk powder samples was approximately 10 times that of FF, while the accumulation of 5-HMF was even more pronounced in fermented dairy products (such as charcoal-roasted yogurt). These data indicate that the dietary exposure risk of 5-HMF from dairy products warrants close attention.

[0003] From a toxicological perspective, 5-HMF exhibits a typical dose-response relationship: within physiological concentration ranges, it exerts antioxidant and neuroprotective effects; however, exceeding threshold concentrations may induce oxidative stress and demonstrate nephrotoxicity and potential genotoxicity. Given the important role of dairy products in the human diet, especially their special nutritional significance for infants and young children during their critical growth and development, establishing an effective 5-HMF monitoring system has significant public health value. Without effective regulatory measures, this substance may be included in the list of priority food contaminants due to its potential health risks.

[0004] In the field of analytical detection technology, various 5-HMF detection methods have been established, including ultra-high performance liquid chromatography (UPLC), gas chromatography (GC), and enzyme-linked immunosorbent assay (ELISA). Although these traditional analytical methods have good accuracy and reproducibility, their reliance on expensive instruments, complex sample pretreatment procedures, and low detection throughput limit their practical application. Summary of the Invention

[0005] Based on this, it is necessary to provide a 5-HMF aptamer, optimization method, and application to address at least one of the problems mentioned above.

[0006] In the first aspect, this application provides a 5-HMF aptamer, which is one of the following sequences: H1-21m: SEQ ID NO1 sequence, H1-21m-5Fu-d: Replace the T at position 20 of the SEQ ID NO1 sequence with 5Fu or H1-21mdUb: Replace the T in the 13th position of the SEQ ID NO1 sequence with U.

[0007] Secondly, this application provides a method for optimizing 5-HMF aptamers, which optimizes 5-HMF aptamers as described in the first aspect of this application, comprising the following steps: S1: Construct multiple truncated sequences of the original 5-HMF aptamer, characterize their binding properties, and screen out truncated sequences with superior ligand binding ability; S2: The truncated body is subjected to a systematic site-directed mutagenesis strategy to perform base substitution analysis on key nucleotide sites. For the identified key bases, they are replaced sequentially with pyrimidine nucleotide analogs with similar spatial configuration and hydrogen bonding ability. The binding ability is measured and mutants with excellent binding ability are screened out. S3: Evaluate the molecular recognition specificity of the mutants, screen out mutants that have significant selective recognition ability for the target molecule 5-HMF, and obtain 5-HMF aptamers.

[0008] In conjunction with the second aspect and the above implementation methods, in some implementation methods of the second aspect, the step of screening for truncated forms with superior ligand binding ability includes: Binding characteristics were characterized using the ThT fluorescent probe method. Each truncated sequence was incubated with ThT and different gradient concentrations of 5-HMF in SELEX Buffer at room temperature. Fluorescence spectra were detected using a microplate reader, with fluorescence intensity at 485 nm as the analytical index. Multiple independent replicates were set up for the experiment. Binding curves were constructed by calculating the fluorescence intensity ratios, and nonlinear regression fitting was performed. The dissociation constant K was calculated based on the nonlinear regression equation. d K d The lower the value, the higher the binding ability.

[0009] In conjunction with the second aspect and the above implementation methods, in some implementation methods of the second aspect, the screening of mutants with superior binding ability includes: The secondary structure of the mutant sequence was predicted using a prediction platform, yielding multiple possible conformations. The mutant was modified with 2AP fluorescent labeling, and the fluorescence intensity changes after binding with 5-HMF were monitored. Unreasonable conformations were eliminated, and candidate conformations were obtained. Molecular docking technology was used to simulate the binding of the predicted conformations with 5-HMF. Statistical analysis of the docking score data revealed that the conformation with the lowest docking score value had the best binding ability with 5-HMF. Combining the results of the fluorescent labeling experiment and molecular docking, the most likely secondary structure conformation of the mutant was determined.

[0010] In conjunction with the second aspect and the above implementation methods, in some implementation methods of the second aspect, the method for identifying key bases in the step of targeting the identified key bases includes: after mutating the bases at specific sites, characterizing the binding properties to find that the mutants lose binding activity, thus confirming that these sites play a key role in ligand recognition.

[0011] In combination with the second aspect and the above implementation methods, in some implementation methods of the second aspect, in S1, the original aptamer is aptamer H1, and the truncated version with the best ligand binding ability selected is named H1-21, and the nucleic acid sequence 5´-3´ of H1-21 is GTAGCGGCGGTGTGGGCATTTTGGGCTAA.

[0012] In conjunction with the second aspect and the above implementation methods, in some implementation methods of the second aspect, in S2, the H1-21 aptamer is modified by site-directed mutagenesis: the guanosine at the 5' end is mutated to thymidine, named H1-21m; the adenosine at the 3' end is mutated to cytidine, named H1-21m3C; further, a specific T base is mutated to A to obtain mutants H1-21m1 and H1-21m4; pentafluorouracil 5Fu and uracil dU, which have structural similarity with thymine, are selected as modifying groups to perform site-directed modification on the aptamer, and the resulting modified products are named H1-21m-5Fu-a and H1-21m-dUa, respectively; subsequently, modification is performed sequentially along the 5' end, and the resulting modified products are named H1-21m-5Fu-(af) and H1-21m-dU(af), respectively.

[0013] In combination with the second aspect and the above implementation methods, in some implementation methods of the second aspect, in step S3, furfural, a structural analog of 5-HMF, is selected as a control ligand for fluorescence binding analysis.

[0014] Thirdly, this application also provides an application of the 5-HMF aptamer, which is used to detect 5-HMF.

[0015] The technical solutions provided in the embodiments of the present invention bring the following beneficial technical effects: The 5-HMF aptamer provided by this invention significantly enhances the binding affinity between the aptamer and 5-HMF by systematically replacing the thymine at the key site of the truncated form with structurally similar uracil and 5-fluorouracil. The detection system constructed using the 5-HMF aptamer exhibits excellent sensitivity and specificity, providing a novel and efficient detection technology platform for food quality and safety monitoring. This not only expands the application of aptamers in the field of food safety detection but also provides an important reference for the development of rapid detection methods for other small molecule pollutants.

[0016] Additional aspects and advantages of the invention will be set forth in the following sections and will be understood in detail from the following description, or may be learned by specific practice of the invention. Attached Figure Description

[0017] Figure 1 This is a flowchart illustrating the optimization method for the 5-HMF aptamer of the present invention; Figure 2 The ThT staining fluorescence values ​​of the aptamer of this invention in different ionic solutions; Figure 3 The aptamer of this invention is K under different ionic conditions d Value measurement analysis Figure 1 ; Figure 4 The aptamer of this invention is K under different ionic conditions d Value measurement analysis Figure 2 ; Figure 5 It is the K of H1-21H1-21m / H1-213C of the present invention. d Value measurement analysis chart; Figure 6 is a graph showing the measurement and analysis of Kd values ​​of H1-21H1-21m / H1-213C under different ionic conditions (including...). Figure 6-1 and Figure 6-2 ); Figure 7 This is a schematic diagram of the molecular chemical structures of thymine, pentafluorouracil, and uracil. Figure 8 This is a schematic diagram of the 5FU and dU modified aptamer of the present invention; Figure 9 shows the K of the 5FU-modified aptamer af of the present invention. d Value measurement analysis (including) Figure 9-1 and Figure 9-2 ); Figure 10 shows the K of the dU-modified aptamer af of the present invention. d Value measurement analysis (including) Figure 10-1 and Figure 10-2 ); Figure 11The aptamers H1-21mdU-b, H1-21m5Fu-d, and 5HMF and FF of the present invention are K d Value measurement and analysis; Figure 12 This is a linear relationship graph of the H1-21mdU-b detection of 5-HMF content in glucose solution according to the present invention; Figure 13 This is the spectrum of the H1-21mdU-b detection of 5-HMF content in glucose solution according to the present invention; Figure 14 is a graph showing the Kd values ​​of H1-21m-5Fu-(af) / H1-21mdU(af) of the present invention measured by the ITC method (including...). Figure 14-1 , Figure 14-2 , Figure 14-3 , Figure 14-4 , Figure 14-5 and Figure 14-6 ); Figure 15 This is a comparison chart of the affinity data of H1-21, H1-21m, and H1-21m3C in this invention. Detailed Implementation

[0018] To facilitate understanding of the present invention, a more complete description will be given below with reference to the accompanying drawings. Possible embodiments of the invention are shown in the drawings. However, the invention can be implemented in many different forms and is not limited to the embodiments described herein with reference to the drawings. The embodiments described with reference to the drawings are exemplary and intended to provide a more thorough and complete understanding of the disclosure of the invention, and should not be construed as limiting the invention. Furthermore, detailed descriptions of known techniques may be omitted where such details are not essential to the features of the illustrated invention.

[0019] Those skilled in the art will understand that, unless otherwise defined, all terms used herein (including technical and scientific terms) have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. It should also be understood that terms such as those defined in general dictionaries should be understood to have the same meaning as in the prior art and should not be interpreted in an idealized or overly formal sense unless specifically defined as herein.

[0020] Those skilled in the art will understand that, unless specifically stated otherwise, the singular forms “a,” “an,” “the,” and “the” used herein may also include the plural forms. It should be further understood that the term “comprising” as used in the specification of this application means the presence of the stated features, integers, steps, operations, elements, and / or components, but does not exclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and / or groups thereof. It should be understood that the term “and / or” as used herein includes all or any units and all combinations of one or more associated listed items.

[0021] The technical solution of the present invention and how the technical solution solves the above-mentioned technical problems will be described in detail below with specific embodiments.

[0022] An embodiment of the first aspect of this application provides a 5-HMF aptamer, which is one of the following sequences: H1-21m: SEQ ID NO1 sequence, H1-21m-5Fu-d: Replace the T at position 20 of SEQ ID NO1 with 5Fu or H1-21mdUb: Replace the T in the 13th position of the SEQ ID NO1 sequence with U.

[0023] Where T stands for thymine, U for uracil, and 5Fu for 5-fluorouracil.

[0024] The 5-HMF aptamer provided by this invention significantly enhances the binding affinity of the aptamer to 5-HMF by systematically replacing the thymine at the key site of the truncated body with structurally similar uracil and 5-fluorouracil.

[0025] A second aspect of the present invention provides a method for optimizing 5-HMF aptamers, for optimizing 5-HMF aptamers as described in the first aspect of the present invention, such as... Figure 1 As shown, it includes the following steps: S1: Construct multiple truncated sequences of the original 5-HMF aptamer, characterize their binding properties, and screen out truncated sequences with superior ligand binding ability.

[0026] S2: A systematic site-directed mutagenesis strategy was used to perform base substitution analysis on key nucleotide sites in the truncated variant. For the identified key bases, they were replaced sequentially with pyrimidine nucleotide analogs with similar spatial configurations and hydrogen bonding capabilities. The binding ability was measured, and mutants with superior binding ability were screened out.

[0027] S3: Evaluate the molecular recognition specificity of mutants, screen for mutants that have significant selective recognition ability for the target molecule 5-HMF, and obtain 5-HMF aptamers.

[0028] Specifically, in conjunction with the embodiments of the second aspect, in the specific implementation of the second aspect embodiment, the step of screening out the truncated form with superior ligand binding ability in S1 includes: Binding characteristics were characterized using the ThT fluorescent probe method. Each truncated sequence was incubated with ThT and different gradient concentrations of 5-HMF in SELEX Buffer at room temperature. Fluorescence spectra were detected using a microplate reader, with fluorescence intensity at 485 nm as the analytical index. Multiple independent replicates were set up for the experiment. Binding curves were constructed by calculating the fluorescence intensity ratios, and nonlinear regression fitting was performed. The dissociation constant K was calculated based on the nonlinear regression equation. d K d The lower the value, the higher the binding ability.

[0029] In conjunction with the embodiments of the second aspect and the above implementation methods, in some other specific implementation methods of the embodiments of the second aspect, the mutants with superior binding ability selected in S2 include: The secondary structure of the mutant sequence was predicted using a prediction platform, yielding multiple possible conformations. The mutant was modified with 2AP fluorescent labeling, and the fluorescence intensity changes after binding with 5-HMF were monitored. Unreasonable conformations were eliminated, and candidate conformations were obtained. Molecular docking technology was used to simulate the binding of the predicted conformations with 5-HMF. Statistical analysis of the docking score data revealed that the conformation with the lowest docking score value had the best binding ability with 5-HMF. Combining the results of the fluorescent labeling experiment and molecular docking, the most likely secondary structure conformation of the mutant was determined.

[0030] In conjunction with the embodiments of the second aspect and the above implementation methods, in some further specific implementations of the embodiments of the second aspect, in the step of identifying key bases, the method for identifying key bases includes: after mutating the bases at specific sites, characterizing the binding properties to find that the mutants lose binding activity, thus confirming that these sites play a key role in ligand recognition.

[0031] In conjunction with the embodiments of the second aspect and the above implementation methods, in some specific implementations of the embodiments of the second aspect, the original aptamer in S1 is aptamer H1, and the truncated version with the best ligand binding ability is named H1-21, and the nucleic acid sequence 5´-3´ of H1-21 is GTAGCGGCGGTGTGGGCATTTTGGGCTAA.

[0032] In conjunction with the embodiments of the second aspect and the above implementation methods, in some other specific implementation methods of the embodiments of the second aspect, the H1-21 aptamer is modified by site-directed mutagenesis in S2: the guanosine at the 5' end is mutated to thymidine, named H1-21m; the adenosine at the 3' end is mutated to cytidine, named H1-21m3C; further, a specific T base is mutated to A to obtain mutants H1-21m1 and H1-21m4; pentafluorouracil 5Fu and uracil dU, which have structural similarity with thymine, are selected as modifying groups to perform site-directed modification on the aptamer, and the resulting modified products are named H1-21m-5Fu-a and H1-21m-dUa, respectively; subsequently, modification is performed sequentially along the 5' end, and the resulting modified products are named H1-21m-5Fu-(af) and H1-21m-dU(af), respectively.

[0033] More specifically, the H1-21 aptamer underwent site-directed mutagenesis modification: the 5' end guanosine (G) was mutated to thymidine (T), named H1-21m; the 3' end adenosine (A) was mutated to cytidine (C), named H1-21m. The aptamer was H1-21m3C. By comparing the affinities of H1-21, H1-21m, and H1-21m3C, the results showed that H1-21m exhibited the best target molecule binding performance. First, by deleting some T bases to change the aptamer structure, mutants H1-21m2 and H1-21m3 were obtained. By mutating some T bases, H1-21m1 and H1-21m4 were obtained. By deleting the G base at position 16, mutant H1-21m5 was obtained. By mutating the bases at both ends, mutant H1-21m6 was obtained, as shown in Table 1 below. Table 1. H1-21m mutant: H1-21m1~H1-21m6 sequences

[0034] Characterization of binding properties revealed loss of binding activity, indicating that the integrity of the aptamer structure plays a crucial regulatory role in aptamer binding affinity. Further mutation of specific T bases to A yielded mutants H1-21m1 and H1-21m4, which also showed loss of binding activity, confirming that T at positions 11, 13, and 19-22 plays a key role in aptamer-ligand recognition. Pentafluorouracil (5Fu) and uracil (dU), which have structural similarities to thymine, were selected as modifying groups for site-specific modification of the aptamer. Specifically, starting from the 3' end of the H1-21m aptamer, modification was performed at position 11 (T), replacing it with 5Fu or dU, respectively. The resulting modified products were named H1-21m-5Fu-a. H1-21m-dUa was then modified sequentially along the 5' end and named accordingly. The binding characteristics of the series of modified products H1-21m-5Fu-(af) and H1-21m-dU(af) with 5HMF were characterized to evaluate their binding affinity. Experimental data showed that the binding affinities of the H1-21m-5Fu-(af) series compounds were 6.22 μM, 9.97 μM, 10.12 μM, 4.103 μM, 8.367 μM, and 15.08 μM, respectively; while H1-21mdU(af) exhibited K0.05 values ​​of 5.195 μM, 1.95 μM, 3.447 μM, 14.61 μM, 6.338 μM, and 9.263 μM. d To verify these results, the gold standard isothermal titration calorimetry (ITC) was used for retesting, and the results showed that the actual K of H1-21m-5Fu-(af) was... d The values ​​were 20.6 μM, 5.578 μM, 17.7 μM, 0.708 μM, 15.9 μM, and 2.19 μM, respectively; the ITC values ​​of H1-21mdU(af) were 16.6 μM, 5.96 μM, 8.08 μM, 7.39 μM, 3.96 μM, and 1.08 μM. Through systematic analysis and comparison, the two aptamers H1-21m-5Fu-d and H1-21mdUb exhibited the best molecular recognition characteristics.

[0035] Combining the second aspect and the above implementation methods, in some implementations of the second aspect, specifically in S3, furfural, a structural analog of 5-HMF, is selected as a control ligand for fluorescence binding analysis. Experimental data show that neither the candidate aptamers H1-21m-5Fu-d nor H1-21mdUb exhibited detectable binding activity with FF, indicating that these two aptamers have a significant selective recognition ability for the target molecule 5-HMF.

[0036] Based on the same technical concept, an embodiment of the third aspect of the present invention also provides an application of the 5-HMF aptamer, which applies the 5-HMF aptamer described in the first aspect of this application to the detection of 5-HMF.

[0037] The 5-HMF aptamer provided by this invention significantly enhances the binding affinity between the aptamer and 5-HMF by systematically replacing the thymine at the key site of the truncated form with structurally similar uracil and 5-fluorouracil. The detection system constructed using the 5-HMF aptamer exhibits excellent sensitivity and specificity, providing a novel and efficient detection technology platform for food quality and safety monitoring. This not only expands the application of aptamers in the field of food safety detection but also provides an important reference for the development of rapid detection methods for other small molecule pollutants.

[0038] The following are specific embodiments, for reference. Figures 2-15 : An optimization method for 5-HMF aptamers includes the following: 1. Experimental materials Aptamer sequences: The original aptamer, truncated aptamer, and mutant were synthesized by Sangon Biotech (Shanghai) Co., Ltd.; the modified aptamers (pentafluorouracil, uracil, and diaminopurine markers) were synthesized by Huzhou Hippo Biotechnology Co., Ltd.

[0039] Reagents and Instruments 5-HMF: Purchased from Sangon Biotech (Shanghai) Co., Ltd.; K⁺ buffer: 500 mM KCl, 10 mM MgCl₂, and 50 mM HEPES at pH 7.5; Na⁺ buffer (SELEX Buffer): 500 mM NaCl, 10 mM MgCl2, and 50 mM HEPES atpH 7.5; Li⁺ buffer: 500 mM LiCl, 10 mM MgCl₂, and 50 mM HEPES at pH 7.5; PBS buffer, microplate reader, isothermal titration calorimeter, UV spectrophotometer, molecular docking software (AutoDock, PyMOL).

[0040] 2. Experiment 2.1 Truncation Optimization of Aptamer H1 As shown in Table 2 below, based on the truncated sequence screening experiment using aptamer H1, this invention constructed five gradient truncated sequences: H1-8, H1-12, H1-14, H1-21, and H1-27. (Reference) Figure 2The binding characteristics were characterized using a thioflavin T (ThT) fluorescent probe method: Each truncated form (final concentration 1 μM) was incubated with 3 μM ThT and gradient concentrations of 0, 10 μM, 20 μM, 40 μM, 80 μM, 160 μM, 320 μM, and 640 μM 5-HMF in SELEX Buffer at room temperature for 20 minutes. Fluorescence spectra were detected using a microplate reader (excitation wavelength 415 nm, emission wavelength scan range 440-600 nm, step size 5 nm), with fluorescence intensity at 485 nm as the analytical index. The experiment was conducted in three independent replicates. Binding curves were constructed by calculating the fluorescence intensity ratio (F / F0, where F0 is the average fluorescence value of the three parallel experiments at a 5HMF concentration of 0). Nonlinear regression fitting was performed, and the dissociation constant (K0) was calculated based on the nonlinear regression equation. d Ultimately, the core functional domains of the aptamer were determined through affinity comparison. This experimental system effectively characterizes the strength of aptamer-ligand interactions through changes in fluorescence signal response, providing a quantitative basis for aptamer structure optimization.

[0041] Table 2. Sequences H1, H1-8, H1-12, H1-14, H1-21, and H1-27

[0042] Affinity test results show that, reference Figure 3 Figure 6 shows that the dissociation constants of different truncated forms differ significantly: H1-8 (24.03 μM), H1-12 (15.96 μM), H1-14 (11.42 μM), H1-21 (11.36 μM), and H1-27 (105.1 μM). Among them, H1-21 exhibits the best ligand binding capacity (K0). d =11.36 μM), indicating that when the core sequence was shortened to 23 nucleotides (nt), the structural optimization of the aptamer significantly enhanced its affinity for the target molecule.

[0043] 2.2 Effect of G4 polymer on affinity To investigate the regulatory mechanism of G4 polymers on the binding of aptamers to 5HMF: aptamer sequences were imported into a GC content analysis website to calculate the GC content of the aptamers, and then the fraction of aptamers forming G4 polymers was predicted on a QGRS website; G4 polymers are located at: K + Stability in the environment > Na + Environmental stability > Li + The stability of G4 polymers in the environment is moderate, so the stability of G4 polymers is assessed by the fluorescence intensity of ThT (G4 probe) in K⁺, Na⁺, and Li⁺ buffers.

[0044] GC content analysis of five aptamer sequences (H1, H1-8, H1-12, H1-14, H1-21, H1-27, and H-29) was performed using the NovoPro online tool (https: / / www.novopro.cn / tools / gc-content.html). The results showed that their GC contents were 60.00%, 59.52%, 60.53%, 58.33%, 58.62%, 69.57%, and 66.67%, respectively, all significantly higher than 50%. The G contents were as high as 38.00%, 40.48%, 42.11%, 41.67%, 44.83%, 52.17%, and 52.38%, respectively, indicating that these sequences have high thermal stability potential. Further evaluation of their G-quadruplex (G4) formation tendency was conducted using QGRS Mapper (a G-quadruplex specific analysis tool), with a maximum G-score of 16. This parameter is positively correlated with the probability of forming G-quadruplex structures; a higher value indicates a greater likelihood that the sequence will form an intramolecular G-quadruplex secondary structure through Hoogsteen hydrogen bonds.

[0045] Experimental results show that different ionic environments have a significant impact on the structural stability of the truncated G-quadruplex and its affinity for target molecules. For example... Figure 15 As shown, in K⁺ buffer, the truncated G4 aggregate forms the most stable G4 polymer (confirmed by the highest ThT fluorescence intensity), but it has the lowest affinity for the target molecule. In Na⁺ buffer, the G4 structure partially unwinds, at which point the aptamer exhibits the best affinity. However, in Li⁺ buffer, G4 polymers cannot form. This indicates that the stability of the G4 structure is negatively correlated with aptamer affinity, and the degree of G4 polymer formation needs to be maintained at an appropriate level—an overly stable G4 structure weakens affinity, while the complete absence of the G4 structure is also detrimental to function. Therefore, the existence of G4 polymers requires a dynamic balance between stability and affinity.

[0046] 2.3 Mutation and Modification 2.3.1 3' and 5' mutations in H1-21 Based on the understanding that the structure of G-quadruplex (G4) regulates the function of aptamers, this invention uses a systematic truncation strategy to screen out truncated aptamers with optimal affinity, and employs site-directed base mutation technology to explore the affinity of the mutated aptamers and their key binding sites.

[0047] Specifically, during the molecular structure optimization process, the H1-21 aptamer underwent site-directed mutagenesis modification: the 5' end guanosine (G) was mutated to thymidine (T), named H1-21m; and the 3' end adenosine (A) was mutated to cytidine (C), named H1-21m3C. By comparing the affinities of H1-21, H1-21m, and H1-21m3C, the results showed that H1-21m exhibited the best target molecule binding performance. Further analysis showed that H1-21m exhibited the best affinity in K⁺, Na⁺, and Li⁺ buffers, indicating that its structural optimization is not only effective under specific ionic environments but also possesses stable and efficient binding capabilities under various ionic conditions.

[0048] 2.3.2. Prediction of aptamer secondary structure 2-Aminopurine (2-AP) is a fluorescent nucleoside analog that can replace adenine (A) or guanine (G) in the insertion of nucleic acid chains. Its fluorescence properties are highly sensitive to the surrounding microenvironment, and therefore it is often used to study conformational changes, binding kinetics, and molecular recognition mechanisms of nucleic acids. When the 2-AP-labeled aptamer is in a free state, it emits strong fluorescence. When the aptamer binds to the target, the nucleic acid chain forms a stable secondary structure (such as a G-quadruplex or stem-loop structure) through base stacking, hydrogen bonding, or hydrophobic interactions, resulting in fluorescence quenching. Therefore, changes in fluorescence intensity can be used to predict the secondary structure of the aptamer.

[0049] Specifically, the secondary structure of the aptamer sequence was predicted using the UNAFold online prediction platform (http: / / www.unafold.org / ) under standard simulation conditions (25℃, 500mM Na⁺, 10 mM Mg²⁺), yielding five possible conformations. The Gibbs free energies (ΔG) for each conformation are: H1-21m2d1 (-2.13 kcal / mol), H1-21m2d2 (-2.01 kcal / mol), H1-21m2d3 (-1.97 kcal / mol), H1-21m2d4 (-1.95 kcal / mol), and H1-21m2d5 (-1.21 kcal / mol). Based on thermodynamic stability, H1-21m2d1 was initially selected as the representative conformation for further analysis.

[0050] Specifically, the secondary structure of the aptamer sequence was predicted using the UNAFold online prediction platform (http: / / www.unafold.org / ) under standard simulation conditions (25℃, 500mM Na⁺, 10 mM Mg²⁺), yielding five possible conformations. The Gibbs free energies (ΔG) for each conformation are: H1-21m2d1 (-2.13 kcal / mol), H1-21m2d2 (-2.01 kcal / mol), H1-21m2d3 (-1.97 kcal / mol), H1-21m2d4 (-1.95 kcal / mol), and H1-21m2d5 (-1.21 kcal / mol). Based on thermodynamic stability, H1-21m2d1 was initially selected as the representative conformation for further analysis.

[0051] 2.3.3 Molecular docking further confirmed the secondary structure of the aptamer. This invention employs molecular docking technology to systematically analyze the interaction mechanism between aptamers and 5HMF. First, based on the aptamer nucleotide sequence, tertiary structure prediction is performed using a 3D RNA / DNA online platform. Then, the tertiary structure of the aptamer and the 5HMF small molecule are jointly imported into the HDOCK molecular docking platform. This platform uses a fast Fourier transform correlation algorithm to achieve global conformational search, calculating a comprehensive binding score by evaluating energy terms such as van der Waals forces, electrostatic interactions, and hydrogen bond formation. A lower score indicates a more negative binding free energy (ΔG) in the complex system, indicating more stable intermolecular interactions. By systematically comparing the docking scores of different aptamer conformations, the secondary structure corresponding to the optimal binding configuration is selected.

[0052] Specifically, molecular docking technology (HDOCK Server) was used to simulate the binding of the five predicted conformations with 5HMF. Statistical analysis of the docking score data showed that the H1-21m2d4 conformation had the lowest docking score value (-1.95 kcal / mol), indicating that it had the best binding affinity with 5HMF. Combining the results of the fluorescence labeling experiment and molecular docking, H1-21m2d4 was determined to be the most likely secondary structure conformation.

[0053] 2.3.4 Identification of key base sites in six mutants of H1-21m The aptamer modification optimization strategy, after identifying the key nucleotide sites for the specific binding of the aptamer to the target molecule 5-HMF, employs a systematic site-directed mutagenesis strategy to perform base substitution analysis on these key sites. Specifically, for the identified functional bases, they are sequentially replaced with pyrimidine nucleotide analogs (including but not limited to uracil, thymine, and their methylated derivatives) with similar spatial configurations and hydrogen bonding capabilities, and the aptamer affinity is then determined.

[0054] Specifically, to analyze the functional impact of key base sites in the H1-21m aptamer, we conducted a systematic site-directed mutagenesis experiment. First, by deleting some T bases (H1-21m2, H1-21m3, H1-21m5, H1-21m6), we altered the aptamer structure, leading to the K... d The decrease or loss of binding activity indicates that the integrity of the aptamer structure plays an important regulatory role in aptamer affinity. Further mutation of specific T bases to A (H1-21m1, H1-21m4) revealed K... d The value further increased or even lost binding activity, confirming that these sites (T at positions 11, 13, and 19-22) play a key role in aptamer-ligand recognition.

[0055] 2.3.5. Modification of H1-21m with pentafluorouracil and uracil like Figure 7 and Figure 8 As shown, after identifying the key base as thymine (T), this invention selected 5-fluorouracil (5Fu) and uracil (U), which have structural similarities to thymine, as modifying groups to perform site-specific modifications on the aptamer. Specifically, starting from the 3' end of the H1-21m aptamer, modification was performed at position 11 (T), replacing it with 5Fu or U. The resulting modified products were named H1-21m-5Fu-a and H1-21m-dUa, respectively. Subsequently, modifications were performed sequentially along the 5' end, and named accordingly. The series of modified products H1-21m-5Fu-(af) and H1-21m-dU(af) were subjected to binding experiments with 5HMF to evaluate their affinity. Referring to Figures 9 and 10, the experimental data show that the binding affinities of the H1-21m-5Fu-(af) series compounds are 6.22 μM, 9.97 μM, 10.12 μM, 4.103 μM, 8.367 μM, and 15.08 μM, respectively; while H1-21mdU(af) exhibits Kb values ​​of 5.195 μM, 1.95 μM, 3.447 μM, 14.61 μM, 6.338 μM, and 9.263 μM. dAs shown in Figure 14, to verify these results, the gold standard isothermal titration calorimetry (ITC) was used for retesting. The results showed that the actual Kd values ​​of H1-21m-5Fu-(af) were 20.6 μM, 5.578 μM, 17.7 μM, 0.708 μM, 15.9 μM, and 2.19 μM, respectively; while the ITC values ​​of H1-21mdU(af) were 16.6 μM, 5.96 μM, 8.08 μM, 7.39 μM, 3.96 μM, and 1.08 μM. The principles of ITC and fluorescence methods for detecting aptamer affinity are different, so the differences in results are normal. After systematic analysis and comparison, H1-21m-5Fu-d and H1-21mdUb aptamers exhibited the best molecular recognition characteristics.

[0056] 2.4 Aptamer Specificity Verification Experiment To evaluate the molecular recognition specificity of the selected high-affinity aptamers, this invention selected furfural (FF), a structural analog of 5-HMF, as a control ligand for fluorescence binding analysis. Experimental data showed that neither the candidate aptamers H1-21m-5Fu-d nor H1-21mdUb exhibited detectable binding activity with FF, indicating that these two aptamers possess significant selective recognition ability for the target molecule 5HMF.

[0057] Development of label-free detection methods Accurately weigh 5-HMF standard and serially dilute it with 10% glucose solution to obtain a series of standard solutions with concentrations of 0, 0.3125, 0.625, 1.25, 2.5, 5, and 10 μg / mL. Using a UV-Vis spectrophotometer and with a blank 10% glucose solution as a reference, measure the absorbance (A) at 284 nm for each concentration. Plot the 5-HMF concentration (μg / mL) on the x-axis and absorbance on the y-axis, and perform linear regression using the least squares method to obtain the standard curve equation. Similarly, measure the absorbance (A) of 10% glucose injection at 284 nm and substitute it into the standard curve to calculate the 5-HMF content in the glucose injection. After optimizing aptamer affinity and screening for aptamers with optimal binding performance, the optimized aptamers were subjected to specific binding reactions with different concentrations of 5HMF in glucose injection, and standard curves were plotted through quantitative analysis. Based on this, a label-free 5HMF detection method was constructed. This method relies on the specific recognition ability of aptamers to achieve sensitive detection and quantitative analysis of 5HMF.

[0058] Performance validation of the label-free detection method: After screening and obtaining the aptamer with optimal affinity, this invention applied H1-21m-dUb to the detection of 5-hydroxymethylfurfural (5HMF) in glucose injection to establish a label-free aptamer-based analytical method for 5-HMF. Experimental results show that the limit of detection (LOD) of this method is 0.22 μg / mL.

[0059] Data analysis methods: Analysis of fluorescence intensity data, and calculation of the dissociation constant (Ki) between the aptamer and 5-HMF through nonlinear regression fitting. d (Value). In molecular docking simulations, the molecular structure of the aptamer was optimized by single-site mutation modification using AutoDock and PyMOL. Simultaneously, the HDOCK platform was used to predict the molecular docking binding site for the aptamer's binding mode with 5HMF. Experimental data were based on three independent replicate experiments. Statistical analysis results are expressed as mean ± standard deviation (Mean ± SD), and error bars were used to characterize the dispersion of the experimental data.

[0060] Those skilled in the art will understand that the steps, measures, and solutions in the various operations, methods, and processes discussed in this application can be alternated, modified, combined, or deleted. Furthermore, other steps, measures, and solutions in the various operations, methods, and processes discussed in this application can also be alternated, modified, rearranged, decomposed, combined, or deleted. Furthermore, steps, measures, and solutions in the prior art that are similar to those disclosed in this application can also be alternated, modified, rearranged, decomposed, combined, or deleted.

[0061] The terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. Therefore, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. In the description of this application, unless otherwise stated, "a plurality of" means two or more.

[0062] In the description of this specification, specific features, structures, materials, or characteristics may be combined in any suitable manner in one or more embodiments or examples.

[0063] It should be understood that although the steps in the flowcharts in the accompanying drawings are shown sequentially as indicated by the arrows, these steps are not necessarily performed in the order indicated by the arrows. Unless otherwise expressly stated herein, there is no strict order in which these steps are performed, and they may be performed in other orders.

[0064] The above description is only a partial embodiment of this application. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of this application, and these improvements and modifications should also be considered within the scope of protection of this application.

Claims

1. A 5-HMF aptamer, characterized in that, It is one of the following sequences: H1-21m: SEQ ID NO1 sequence, H1-21m-5Fu-d: Replace the T at position 20 of the SEQ ID NO1 sequence with 5Fu or H1-21mdUb: Replace the T in the 13th position of the SEQ ID NO1 sequence with U.

2. A method for optimizing 5-HMF aptamers, characterized in that, Optimizing the 5-HMF aptamer as described in claim 1 includes the following steps: S1: Construct multiple truncated sequences of the original 5-HMF aptamer, characterize their binding properties, and screen out truncated sequences with superior ligand binding ability; S2: The truncated body is subjected to a systematic site-directed mutagenesis strategy to perform base substitution analysis on key nucleotide sites. For the identified key bases, they are replaced sequentially with pyrimidine nucleotide analogs with similar spatial configuration and hydrogen bonding ability. The binding ability is measured and mutants with excellent binding ability are screened out. S3: Evaluate the molecular recognition specificity of the mutants, screen out mutants that have significant selective recognition ability for the target molecule 5-HMF, and obtain 5-HMF aptamers.

3. The optimization method for the 5-HMF aptamer according to claim 2, characterized in that, The step of screening for truncated variants with superior ligand binding ability includes: Binding characteristics were characterized using the ThT fluorescent probe method. Each truncated sequence was incubated with ThT and different gradient concentrations of 5-HMF in SELEX Buffer at room temperature. Fluorescence spectra were detected using a microplate reader, with fluorescence intensity at 485 nm as the analytical index. Multiple independent replicates were set up for the experiment. Binding curves were constructed by calculating the fluorescence intensity ratios, and nonlinear regression fitting was performed. The dissociation constant K was calculated based on the nonlinear regression equation. d K d The lower the value, the higher the binding ability.

4. The optimization method for the 5-HMF aptamer according to claim 2, characterized in that, The mutants selected for superior binding ability include: The secondary structure of the mutant sequence was predicted using a prediction platform, yielding multiple possible conformations. The mutant was modified with 2AP fluorescent labeling, and the fluorescence intensity changes after binding with 5-HMF were monitored. Unreasonable conformations were eliminated, and candidate conformations were obtained. Molecular docking technology was used to simulate the binding of the predicted conformations with 5-HMF. Statistical analysis of the docking score data revealed that the conformation with the lowest docking score value had the best binding ability with 5-HMF. Combining the results of the fluorescent labeling experiment and molecular docking, the most likely secondary structure conformation of the mutant was determined.

5. The optimization method for the 5-HMF aptamer according to claim 2, characterized in that, In the step of identifying key bases, the method for identifying key bases includes: after mutating the bases at specific sites, the binding characteristics are characterized to find that the mutants lose binding activity, thus confirming that these sites play a key role in ligand recognition.

6. The optimization method for the 5-HMF aptamer according to claim 2, characterized in that, In S1, the original aptamer is aptamer H1, and the truncated version with the best ligand binding ability is named H1-21. The 5´-3´ nucleic acid sequence of H1-21 is GTAGCGGCGGTGTGGGCATTTTGGGCTAA.

7. The optimization method for the 5-HMF aptamer according to claim 6, characterized in that, In S2, the H1-21 aptamer was modified by site-directed mutagenesis: the guanosine at the 5' end was mutated to thymidine, named H1-21m; the adenosine at the 3' end was mutated to cytidine, named H1-21m3C; further, a specific T base was mutated to A to obtain mutants H1-21m1 and H1-21m4; pentafluorouracil 5Fu and uracil dU, which have structural similarity with thymine, were selected as modifying groups to perform site-directed modification on the aptamer, and the resulting modified products were named H1-21m-5Fu-a and H1-21m-dUa, respectively; subsequently, modification was performed sequentially along the 5' end, and the resulting modified products were named H1-21m-5Fu-(af) and H1-21m-dU(af), respectively.

8. The optimization method for a 5-HMF aptamer according to claim 7, characterized in that, In step S3, furfural, a structural analog of 5-HMF, was selected as a control ligand for fluorescence binding analysis.

9. An application of a 5-HMF aptamer, characterized in that, The 5-HMF aptamer described in claim 1 is applied to the detection of 5-HMF.

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