A 5-HMF aptamer, its optimization method and application

Abstract

This invention provides a 5-HMF aptamer, an optimization method, and its application. The 5-HMF aptamer is one of the following sequences: H1-21m: the sequence of SEQ ID NO1; H1-21m-5Fu-d: replacing the T at position 20 of the SEQ ID NO1 sequence with 5Fu; or H1-21mdUb: replacing the T at position 13 of the SEQ ID NO1 sequence with U. 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 key sites of the truncated aptamer with structurally similar uracil and 5-fluorouracil, and constructs a 5-HMF detection method based on the label-free aptamer.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of assays or test methods comprising enzymes, nucleic acids or microorganisms, in particular to a 5-HMF aptamer, optimization method and application. BACKGROUND

[0002] 5-Hydroxymethylfurfural (5-HMF) and furfural (FF) are characteristic products of Maillard reaction and caramelization, which are widely present in heat-processed food systems. Studies have shown that 5-HMF presents a significant accumulation effect during food storage, which makes it an important indicator for evaluating the degree of heat treatment and storage quality of food. In contrast, our previous studies have found that the content of FF presents a decreasing trend with the extension of storage time. The Chinese Pharmacopoeia stipulates that the determination absorbance value of 5-HMF content in glucose injection at 284 nm wavelength should not exceed 0.32; according to the evaluation report of the European Union's 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. The detection and analysis of commercially available dairy products show that the pollution level of 5-HMF is significantly higher than that of FF, among which the 5-HMF content (0.22-1.70 mg / 100 g protein) in milk powder samples is about 10 times that of FF, and the accumulation of 5-HMF in fermented dairy products (such as charcoal yogurt) is more significant. These data show that the dietary exposure risk of 5-HMF in dairy products deserves special attention.

[0003] From the perspective of toxicology, 5-HMF shows a typical dose-effect relationship: it can exert antioxidant and neuroprotective effects within the physiological concentration range, but when the threshold concentration is exceeded, it may induce oxidative stress and exhibit kidney toxicity and potential genetic toxicity. Given the important position of dairy products in the human dietary structure, especially their special nutritional significance for the infant and young child population in the critical period of growth and development, it is of great public health value to establish an effective 5-HMF monitoring system. Without effective regulatory measures, this substance may be included in the list of priority-controlled food contaminants due to its potential health risks.

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

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

[0006] In a first aspect, the present application provides a 5-HMF aptamer, which is one of the following sequences:

[0007] H1-21m: SEQ ID NO 1 sequence,

[0008] H1-21m-5Fu-d: replacing T at the 20th position of the SEQ ID NO 1 sequence with 5Fu or

[0009] H1-21mdUb: replacing T at the 13th position of the SEQ ID NO 1 sequence with U.

[0010] In a second aspect, the present application provides an optimization method of a 5-HMF aptamer, which is used for optimizing the 5-HMF aptamer described in the first aspect of the present application, comprising the following steps:

[0011] S1: constructing a plurality of truncated sequences of a 5-HMF original aptamer, performing binding property characterization, and screening a truncated body with excellent ligand binding capacity;

[0012] S2: using a systematic site-directed mutation strategy to analyze base substitution of key nucleotide sites for the truncated body, replacing the identified key bases with pyrimidine nucleotide analogs with similar spatial configurations and hydrogen bond formation abilities in turn, and determining the binding capacity to screen a mutant with excellent binding capacity;

[0013] S3: evaluating the molecular recognition specificity of the mutant, screening a mutant with significant selective recognition ability for the target molecule 5-HMF, and obtaining a 5-HMF aptamer.

[0014] In combination with the second aspect and the above implementation manners, in some implementation manners of the second aspect, the step of screening a truncated body with excellent ligand binding capacity comprises:

[0015] Performing binding property characterization by using a ThT fluorescence probe method, incubating each truncated sequence with ThT and different gradient concentrations of 5-HMF in SELEX Buffer at room temperature, detecting fluorescence spectrum by an enzyme marker, taking fluorescence intensity at 485 nm as an analysis index, setting multiple groups of independent repeats in the experiment, constructing a binding curve by calculating fluorescence intensity ratio, performing nonlinear regression fitting, calculating dissociation constant K d according to the nonlinear regression equation, and the lower the K d value, the higher the binding capacity.

[0016] In combination with the second aspect and the above implementation manners, in some implementation manners of the second aspect, the step of screening a mutant with excellent binding capacity comprises:

[0017] The mutant sequence is subjected to secondary structure prediction by using a prediction platform, a plurality of possible conformations are obtained, the mutant is modified by 2AP fluorescent labeling, the change of fluorescent intensity after the mutant binds with 5-HMF is monitored, unreasonable conformations are excluded, a candidate conformation is obtained, the predicted conformation is combined with 5-HMF by using a molecular docking technology, and the conformation with the lowest docking score value is obtained by statistical analysis of docking score data, which indicates that the combination ability of the conformation with 5-HMF is optimal, and the most possible secondary structure conformation of the mutant is determined by comprehensively considering the fluorescent labeling experiment and the molecular docking result.

[0018] In combination with the second aspect and the above implementation manners, in some implementation manners of the second aspect, in the step of identifying the key bases, the method of identifying the key bases comprises: after mutating the bases at specific sites, it is found by binding property characterization that the mutant loses binding activity, which proves that these sites play a key role in ligand recognition.

[0019] In combination with the second aspect and the above implementation manners, in some implementation manners of the second aspect, in S1, the original aptamer is aptamer H1, and the truncated body with the optimal ligand binding ability screened out is named H1-21, and the nucleic acid sequence of H1-21 is 5´-3´ GTAGCGGCGGTGTGGGCATTTTGGGCTAA.

[0020] In combination with the second aspect and the above implementation manners, in some implementation manners of the second aspect, in S2, the H1-21 aptamer is subjected to site-directed mutation modification: the guanosine at the 5' end is mutated into thymidine, and is named H1-21m; the adenosine at the 3' end is mutated into cytidine, and is named H1-21m3C; further, specific T bases are mutated into A, to obtain mutants H1-21m1 and H1-21m4; thymine has structural similarity with five fluorouracil 5Fu and uracil dU, which are selected as modification groups for site-directed modification of the aptamer, and the obtained modification products are named H1-21m-5Fu-a and H1-21m-dUa respectively; then, modification is sequentially performed along the 5' end direction, and the obtained modification products are named H1-21m-5Fu-(a-f) and H1-21m-dU(a-f) respectively.

[0021] In combination with the second aspect and the above implementation manners, in some implementation manners of the second aspect, in S3, the structural analog of 5-HMF, i.e., furfural, is selected as a control ligand for fluorescent binding analysis.

[0022] In a third aspect, the application further provides an application of the 5-HMF aptamer, and the 5-HMF aptamer described in the first aspect of the application is applied to detection of 5-HMF.

[0023] The technical solution provided in the embodiments of the application has the following beneficial technical effects:

[0024] 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.

[0025] 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

[0026] Figure 1 This is a flowchart illustrating the optimization method for the 5-HMF aptamer of the present invention;

[0027] Figure 2 The ThT staining fluorescence values ​​of the aptamer of this invention in different ionic solutions;

[0028] Figure 3 The aptamer of this invention is K under different ionic conditions d Value measurement analysis Figure 1 ;

[0029] Figure 4 The aptamer of this invention is K under different ionic conditions d Value measurement analysis Figure 2 ;

[0030] Figure 5 It is the K of H1-21H1-21m / H1-213C of the present invention. d Value measurement analysis chart;

[0031] 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 );

[0032] Figure 7 This is a schematic diagram of the molecular chemical structures of thymine, pentafluorouracil, and uracil.

[0033] Figure 8 This is a schematic diagram of the 5FU and dU modified aptamer of the present invention;

[0034] Figure 9 shows the K of the 5FU-modified aptamer af of the present invention. d Value measurement analysis (including) Figure 9-1 andFigure 9-2 );

[0035] 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 );

[0036] Figure 11 The aptamers H1-21mdU-b, H1-21m5Fu-d, and 5HMF and FF of the present invention are K d Value measurement and analysis;

[0037] 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;

[0038] Figure 13 This is a spectral image of the H1-21mdU-b detection of 5-HMF content in glucose solution according to the present invention;

[0039] 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 );

[0040] Figure 15 This is a comparison chart of the affinity data of H1-21, H1-21m, and H1-21m3C in this invention. Detailed Implementation

[0041] 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.

[0042] Those skilled in the art will appreciate that unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art in the field of the application. It will be further understood that terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the relevant art and will not be interpreted in an idealized or overly formal sense unless expressly so defined herein.

[0043] Those skilled in the art will appreciate that unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art in the field of the application. It will be further understood that terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the relevant art and will not be interpreted in an idealized or overly formal sense unless expressly so defined herein.

[0044] The technical solutions of the present application and how the technical solutions solve the above technical problems will be described in detail below with specific examples.

[0045] Embodiments of the first aspect of the present application provide a 5-HMF aptamer, which is one of the following sequences:

[0046] H1-21m: SEQ ID NO 1 sequence,

[0047] H1-21m-5Fu-d: replacing T at the 20th position of the sequence of SEQ ID NO 1 with 5Fu or

[0048] H1-21mdUb: replacing T at the 13th position of the sequence of SEQ ID NO 1 with U.

[0049] Wherein T is thymine, U is uracil, and 5Fu is 5-fluorouracil.

[0050] The 5-HMF aptamer provided by the present application significantly enhances the binding affinity of the aptamer to 5-HMF by systematically replacing thymine at the key site of the truncated body with structurally similar uracil and 5-fluorouracil.

[0051] Embodiments of the second aspect of the present application provide an optimization method for a 5-HMF aptamer, for optimizing the 5-HMF aptamer as described in the first aspect of the present application, as shown in Figure 1 The method comprises the following steps:

[0052] S1: Construct multiple truncated sequences of 5-HMF original aptamer, perform binding property characterization, and screen truncated bodies with excellent ligand binding capacity.

[0053] S2: Perform base substitution analysis on key nucleotide sites of the truncated body using a systematic site-directed mutation strategy, replace the identified key bases with pyrimidine nucleotide analogs with similar spatial configuration and hydrogen bond formation ability, and determine the binding capacity to screen mutants with excellent binding capacity.

[0054] S3: Evaluate the molecular recognition specificity of the mutant, screen mutants with significant selective recognition ability for the target molecule 5-HMF, and obtain 5-HMF aptamer.

[0055] Specifically, in combination with the embodiments of the second aspect, in the specific implementation manner of the embodiments of the second aspect, the step of screening truncated bodies with excellent ligand binding capacity in S1 includes:

[0056] The binding property characterization is performed using the ThT fluorescence probe method. Each truncated sequence is incubated with ThT and different gradient concentrations of 5-HMF in SELEX Buffer at room temperature. The fluorescence spectrum is detected by an enzyme marker. The fluorescence intensity at 485 nm wavelength is used as an analysis index. Multiple independent repeats are set in the experiment. The binding curve is constructed by calculating the fluorescence intensity ratio. Nonlinear regression fitting is performed. The dissociation constant K d is calculated according to the nonlinear regression equation. The lower the K d value, the higher the binding capacity.

[0057] In combination with the embodiments of the second aspect and the above implementation manners, in some other specific implementation manners of the embodiments of the second aspect, screening mutants with excellent binding capacity in S2 includes:

[0058] The secondary structure of the mutant sequence is predicted using a prediction platform to obtain multiple possible conformations. The mutant is modified with 2AP fluorescence labeling. The change in fluorescence intensity after binding with 5-HMF is monitored. Unreasonable conformations are excluded. Candidate conformations are obtained. The predicted conformations are combined with 5-HMF for binding simulation using molecular docking technology. Statistical analysis of docking score data shows that the conformation with the lowest docking score value indicates the best binding capacity with 5-HMF. The most possible secondary structure conformation of the mutant is determined by combining the results of fluorescence labeling experiments and molecular docking.

[0059] In combination with the embodiments of the second aspect and the above implementation manners, in some other specific implementation manners of the embodiments of the second aspect, in the step of identifying the key bases, the method of identifying the key bases includes: after mutating the specific site base, it is found that the mutant loses binding activity through binding property characterization, and it is confirmed that these sites play a key role in ligand recognition.

[0060] In some specific implementations of the second aspect, the original aptamer in S1 is aptamer H1, and the optimal aptamer binding ability of the truncated body screened is named H1-21, and the nucleic acid sequence of H1-21 is 5'-3' GTAGCGGCGGTGTGGGCATTTTGGGCTAA.

[0061] In some specific implementations of the second aspect, the H1-21 aptamer is subjected to site-directed mutation modification 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; five fluorouracil 5Fu and uracil dU, which have structural similarity with thymine, are selected as modification groups to modify the aptamer, and the modified products are named H1-21m-5Fu-a and H1-21m-dUa, respectively; then, modification is sequentially performed in the 5' direction, and the modified products are named H1-21m-5Fu-(a-f) and H1-21m-dU(a-f), respectively.

[0062] More specifically, the H1-21 aptamer is subjected to site-directed mutation modification: the guanosine (G) at the 5' end is mutated to thymidine (T), named H1-21m; the adenosine (A) at the 3' end is mutated to cytidine (C), named

[0063] H1-21m3C. By comparing the affinities of H1-21, H1-21m and H1-21m3C, it is found that H1-21m has the optimal target molecule binding performance; first, the aptamer structure is changed by deleting part of the T base, to obtain mutants H1-21m2 and H1-21m3; H1-21m1 and H1-21m4 are obtained by mutating part of the T base; mutant H1-21m5 is obtained by deleting the 16th G base; mutant H1-21m6 is obtained by mutating the bases at both ends, as shown in Table 1 below:

[0064] Table 1 H1-21m mutants: H1-21m1~H1-21m6 sequences

[0065]

[0066] It is found that the binding activity is lost by the binding property characterization, indicating that the integrity of the aptamer structure plays an important role in regulating the binding affinity of the aptamer; further, the specific T base is mutated to A to obtain mutants H1-21m1 and H1-21m4, and it is found that the binding activity is lost, confirming that the T at positions 11, 13, 19-22 plays a key role in the aptamer-ligand recognition; five fluorouracil 5Fu and uracil dU with structural similarity to thymine are selected as modification groups for site-specific modification of the aptamer, specifically, starting from the 3' end of the H1-21m aptamer, the T at position 11 is modified and replaced by 5Fu or dU respectively, and the obtained modified products are named as H1-21m-5Fu-a and H1-21m-dUa respectively; then, the modification is sequentially performed along the 5' end direction, and is correspondingly named, and the series of modified products H1-21m-5Fu-(a-f) and H1-21m-dU(a-f) are subjected to the binding property characterization experiment with 5HMF to evaluate the binding affinity, and the experimental data show that the binding affinities of the H1-21m-5Fu-(a-f) series of compounds are 6.22 μM, 9.97 μM, 10.12 μM, 4.103 μM, 8.367 μM and 15.08 μM respectively; and the H1-21m-dU(a-f) exhibits K d values of 5.195 μM, 1.95 μM, 3.447 μM, 14.61 μM, 6.338 μM and 9.263 μM respectively. d To verify these results, the gold standard isothermal titration calorimetry (ITC) is used for retesting, and the results show that the actual K d values of H1-21m-5Fu-(a-f) are 20.6 μM, 5.578 μM, 17.7 μM, 0.708 μM, 15.9 μM and 2.19 μM respectively; and the ITC measured values of H1-21m-dU(a-f) are 16.6 μM, 5.96 μM, 8.08 μM, 7.39 μM, 3.96 μM and 1.08 μM respectively, and after systematic analysis and comparison, the two aptamers H1-21m-5Fu-d and H1-21m-dUb exhibit the most excellent molecular recognition characteristics.

[0067] In combination with the second aspect and the above implementation manners, in certain implementation manners of the second aspect, in S3, the structural analog of 5-HMF, i.e., furfural, is selected as a control ligand for fluorescence binding analysis. The experimental data show that neither the candidate aptamer H1-21m-5Fu-d nor the H1-21m-dUb exhibits detectable binding activity with FF, indicating that the two aptamers have significant selective recognition ability for the target molecule 5-HMF.

[0068] Based on the same technical concept, the embodiments of the third aspect of the present application also provide an application of the 5-HMF aptamer. The 5-HMF aptamer described in the first aspect of the present application is applied to the detection of 5-HMF.

[0069] The 5-HMF aptamer provided by the application significantly enhances the binding affinity of the aptamer to 5-HMF by systematically replacing the thymine at the key site of the truncation body with structurally similar uracil and 5-fluorouracil. The detection system constructed by using the 5-HMF aptamer has excellent sensitivity and specificity, and provides a new efficient detection technology platform for food quality and safety monitoring. Not only does it expand 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.

[0070] The following is a specific embodiment, with reference to Figures 2-15 :

[0071] An optimization method of a 5-HMF aptamer, comprising the following contents,

[0072] 1. Experimental materials

[0073] Aptamer sequence: The original aptamer, the truncation body, and the mutant were synthesized by Shenguo Bioengineering (Shanghai) Co., Ltd.; The modified body (pentafluorouracil, uracil, and diamino purine labeled body) was synthesized by Huzhou HeMa Biological Technology Co., Ltd.

[0074] Reagents and instruments

[0075] 5-HMF: purchased from Shenguo Bioengineering (Shanghai) Co., Ltd.;

[0076] K⁺ buffer: 500 mM KCl, 10 mM MgCl2, and 50 mM HEPES at pH 7.5;

[0077] Na⁺ buffer (SELEX Buffer): 500 mM NaCl, 10 mM MgCl2, and 50 mM HEPES at pH 7.5;

[0078] Li⁺ buffer: 500 mM LiCl, 10 mM MgCl2, and 50 mM HEPES at pH 7.5;

[0079] PBS buffer, microplate reader, isothermal titration calorimeter, ultraviolet spectrophotometer, and molecular docking software (AutoDock, PyMOL).

[0080] 2. Experiment

[0081] 2.1. Truncation optimization of aptamer H1

[0082] As shown in Table 2, based on the truncated sequence screening experiment of aptamer H1, the present application constructs five gradient truncated sequences of H1-8, H1-12, H1-14, H1-21 and H1-27. Referring to Figure 2 , the binding characteristics are characterized by using thioflavin T (ThT) fluorescence probe method: each truncated body (final concentration 1 μM) is incubated with 3 μM ThT and gradient concentration of 5-HMF of 0, 10 μM, 20 μM, 40 μM, 80 μM, 160 μM, 320 μM and 640 μM in SELEX Buffer at room temperature for 20 minutes. The fluorescence spectrum is detected by the enzyme marker (excitation wavelength 415 nm, emission wavelength scanning range 440-600 nm, step 5 nm), and the fluorescence intensity at 485 nm is used as the analysis index. Three groups of independent repeats are set in the experiment, the binding curve is constructed by calculating the fluorescence intensity ratio (F / F0, F0 is the average of the fluorescence values of the three parallel experiments when the 5HMF concentration is 0), and the nonlinear regression fitting is carried out, and the dissociation constant (K d ) is calculated according to the nonlinear regression equation. Finally, the core functional domain of the aptamer is determined by comparing the affinities. The experimental system effectively characterizes the interaction strength between the aptamer and the ligand through the change of fluorescence signal response, and provides a quantitative basis for the structure optimization of the aptamer.

[0083] Table 2 H1, H1-8, H1-12, H1-14, H1-21, H1-27 sequences

[0084]

[0085] The affinity determination results show that, referring to Figure 3 Figure 6, the dissociation constants of different truncated bodies are significantly different: 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 shows the best ligand binding capacity (K d = 11.36 μM), indicating that when the core sequence is shortened to 23 nucleotides (nt), the structure optimization of the aptamer significantly enhances its affinity to the target molecule.

[0086] 2.2, Effect of G4 polymer on affinity

[0087] In order to study the mechanism of G4 polymer regulating the binding of aptamer and 5HMF: the aptamer sequence is introduced into the GC content analysis website to calculate the GC content of the aptamer, and then the fraction of G4 polymer formed by the aptamer is predicted on the QGRS website; the stability of G4 polymer in K + environment > Na + environment > 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.

[0088] 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.

[0089] 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.

[0090] 2.3 Mutation and Modification

[0091] 2.3.1 3' and 5' mutations in H1-21

[0092] 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.

[0093] Specifically, during the molecular structure optimization process, site-directed mutagenesis was performed on the H1-21 aptamer: the guanosine (G) at the 5' end was mutated to thymidine (T), designated as H1-21m; the adenosine (A) at the 3' end was mutated to cytidine (C), designated as 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 structure optimization was not only effective in a specific ionic environment, but also had stable and efficient binding capacity under various ionic conditions.

[0094] 2.3.2, Secondary structure prediction of aptamer

[0095] 2-Aminopurine (2-AP) is a fluorescent nucleoside analogue that can replace adenine (A) or guanine (G) and be inserted into the nucleic acid chain. Its fluorescence characteristics are highly sensitive to the surrounding microenvironment, so it is often used to study the 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, and when the aptamer binds to the target, the nucleic acid chain forms a stable secondary structure (such as G-quadruplex, stem-loop structure) through base stacking, hydrogen bonding or hydrophobic interaction, resulting in fluorescence quenching. Therefore, the secondary structure of the aptamer can be predicted by changes in fluorescence intensity.

[0096] Specifically, the UNAFold online prediction platform (http: / / www.unafold.org / ) was used to predict the secondary structure of the aptamer sequence under standard simulation conditions (25°C, 500mM Na⁺, 10 mM Mg²⁺), and five possible conformations were obtained. The Gibbs free energy (ΔG) of each conformation was: H1-21m2d1 (-2.13 kcal / mol), H1-21m2d2 (-2.01 kcal / mol), H1-21m2d3 (-1.97 kcal / mol), H1-21m2d4 (-1.95 kcal / mol), H1-21m2d5 (-1.21 kcal / mol). Based on the thermodynamic stability, H1-21m2d1 was initially selected as the representative conformation for subsequent analysis.

[0097] Specifically, the secondary structure of the aptamer sequence is predicted under standard simulation conditions (25℃, 500mM Na⁺, 10 mM Mg²⁺) by using the UNAFold online prediction platform (http: / / www.unafold.org / ), and five possible conformations are obtained. The Gibbs free energy (ΔG) of each conformation is: 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 the thermodynamic stability, H1-21m2d1 is selected as the representative conformation for subsequent analysis.

[0098] 2.3.3, molecular docking further confirms the secondary structure of the aptamer

[0099] The present application uses molecular docking technology to systematically analyze the interaction mechanism of the aptamer and 5HMF. First, based on the nucleotide sequence of the aptamer, the tertiary structure is predicted by the 3D RNA / DNA online platform. Then the tertiary structure of the aptamer and the 5HMF small molecule are introduced into the HDOCK molecular docking platform. The platform uses fast Fourier transform correlation algorithm to realize global conformation search, and calculates the comprehensive binding score (docking score) by evaluating the energy terms such as van der Waals force, electrostatic interaction, hydrogen bond formation, etc. The lower the score value, the more negative the binding free energy (ΔG) of the complex system, and the more stable the interaction between molecules. By systematically comparing the docking scores of different aptamer conformations, the secondary structure corresponding to the optimal binding conformation is selected.

[0100] Specifically, the molecular docking technology (HDOCK Server) is further used to combine and simulate the five predicted conformations with 5HMF. Statistical analysis of the docking score (docking score) data shows that the H1-21m2d4 conformation has the lowest docking score value (-1.95 kcal / mol), indicating that it has the optimal binding affinity with 5HMF. Combining the results of fluorescence labeling experiment and molecular docking, H1-21m2d4 is determined as the most possible secondary structure conformation.

[0101] 2.3.4, 6 mutants of H1-21m - identification of key base sites

[0102] After identifying the key nucleotide sites of the aptamer that specifically bind to the target molecule 5-HMF, the present application uses systematic site-directed mutagenesis strategy to analyze base substitution at the key sites. Specifically, for the identified functional bases, they are replaced with pyrimidine nucleotide analogs (including but not limited to uracil, thymine and its methylated derivatives) with similar spatial configuration and hydrogen bond formation ability, and the aptamer affinity is determined.

[0103] Specifically, to analyze the functional influence of the key base sites of H1-21m aptamer, systematic site-directed mutagenesis experiments were performed. First, by deleting part of the T bases (H1-21m2, H1-21m3, H1-21m5, H1-21m6), the structure of the aptamer was changed, resulting in K d d values decreased or lost binding activity, indicating that the integrity of the aptamer structure plays an important regulatory role in the affinity of the aptamer. Further mutation of specific T bases to A (H1-21m1, H1-21m4) found that K d d values further increased or even lost binding activity, confirming that these sites (T at positions 11, 13, 19-22) play a key role in aptamer-ligand recognition.

[0104] 2.3.5, five-fluorouracil modification and uracil modification of H1-21m

[0105] As shown in Figure 7 and Figure 8 , after identifying the key base as thymine (T), the present application selected five-fluorouracil (5-fluorouracil, 5Fu) and uracil (uracil, U) with structural similarity to thymine as modification groups for site-directed modification of the aptamer. Specifically, starting from the 3' end of the H1-21m aptamer, the 11th T was modified by replacing it with 5Fu or U, and the resulting modified products were named H1-21m-5Fu-a and H1-21m-dUa, respectively. Subsequently, modification was performed in the 5' direction, and the corresponding names were given. The series of modified products H1-21m-5Fu-(a-f) and H1-21m-dU(a-f) were subjected to binding experiments with 5HMF to evaluate their affinity. Referring to FIG. 9 and FIG. 10, the experimental data showed that the binding affinity of the H1-21m-5Fu-(a-f) series of compounds was 6.22 μM, 9.97 μM, 10.12 μM, 4.103 μM, 8.367 μM and 15.08 μM, respectively; while H1-21m-dU(a-f) showed K dValues. As shown in Figure 14, to verify these results, the gold standard isothermal titration calorimetry (ITC) was used for retesting, and the results showed that the actual Kd values of H1-21m-5Fu-(a-f) were 20.6 μM, 5.578 μM, 17.7 μM, 0.708 μM, 15.9 μM, and 2.19 μM, respectively; and the ITC-determined values of H1-21mdU (a-f) were 16.6 μM, 5.96 μM, 8.08 μM, 7.39 μM, 3.96 μM, and 1.08 μM, respectively. The principles of ITC and fluorescence methods for detecting the affinity of aptamers are different, and the results are different, which is a normal phenomenon. After systematic analysis and comparison, it was found that the two aptamers H1-21m-5Fu-d and H1-21mdUb exhibited the best molecular recognition characteristics.

[0106] 2.4, Verification of aptamer specificity experiment

[0107] To evaluate the molecular recognition specificity of the high-affinity aptamer obtained by screening, the present application selected the structural analog of 5-HMF, furfural (FF), as a control ligand for fluorescence binding analysis. The experimental data showed that neither the candidate aptamer H1-21m-5Fu-d nor H1-21mdUb exhibited detectable binding activity with FF, indicating that these two aptamers have significant selective recognition ability for the target molecule 5HMF.

[0108] Development of a non-labeled detection method

[0109] Precisely weigh the 5-HMF standard, dilute it with 10% glucose solution as a solvent 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-visible spectrophotometer, take the blank 10% glucose solution as a reference, and measure the absorbance (A) at 284 nm for each concentration. Take the 5-HMF concentration (μg / mL) as the abscissa (x) and the absorbance as the ordinate (y), and perform linear regression by the least squares method to obtain the standard curve equation. Similarly, measure the absorbance (A) at 284 nM using 10% glucose injection, and calculate the 5HMF content in the glucose injection by using the standard curve;

[0110] After completing the optimization of the aptamer affinity and selecting the aptamer with the best binding performance, the optimized aptamer was subjected to specific binding reaction with different concentrations of 5HMF in glucose injection, and a standard curve was drawn by quantitative analysis. Based on this, a non-labeled detection method for 5HMF was constructed, which relies on the specific recognition ability of the aptamer to achieve sensitive detection and quantitative analysis of 5HMF.

[0111] Performance verification of non-labeled detection method: After screening the aptamer with the optimal affinity, the present application applies H1-21m-dUb to the detection of 5-hydroxymethylfurfural (5HMF) in glucose injection to establish a 5-HMF analysis method based on non-labeled aptamer. The experimental results show that the limit of detection (LOD) of the method is 0.22 μg / mL.

[0112] Data analysis method: The analysis of fluorescence intensity data, the dissociation constant (K d In the molecular docking simulation, the molecular structure optimization of the single-point mutation modification of the aptamer was performed by AutoDock and PyMOL, and the molecular docking binding site prediction of the binding mode of the aptamer and 5HMF was performed by the HDOCK platform. The experimental data is based on three independent repeated experiments, and the statistical analysis results are expressed in the form of mean ± standard deviation (Mean ± SD), and the error bar is used to represent the dispersion degree of the experimental data.

[0113] Those skilled in the art can understand that the steps, measures and schemes in various operations, methods and processes discussed in the present application can be alternated, changed, combined or deleted. Further, other steps, measures and schemes in various operations, methods and processes discussed in the present application can also be alternated, changed, rearranged, decomposed, combined or deleted. Further, the steps, measures and schemes in the prior art with the various operations, methods and processes disclosed in the present application can also be alternated, changed, rearranged, decomposed, combined or deleted.

[0114] The terms "first", "second" are only for descriptive purposes, and cannot be understood as indicating or implying relative importance or implicitly indicating the number of indicated technical features. Therefore, the features defined with "first", "second" can explicitly or implicitly include one or more of the features. In the description of the present application, unless otherwise stated, the meaning of "a plurality of" is two or more.

[0115] In the description of the present application, specific features, structures, materials or characteristics can be combined in any one or more embodiments or examples in a suitable manner.

[0116] It should be understood that although each step in the flowchart of the accompanying drawings is displayed in sequence according to the direction of the arrow, these steps are not necessarily executed in sequence according to the direction of the arrow. Unless otherwise stated herein, the execution of these steps is not strictly limited in sequence, and they can be executed in other order.

[0117] The above merely describes some embodiments of the present application, and it should be pointed out that, for those skilled in the art, some improvements and refinements can be made without departing from the principles of the present application, and these improvements and refinements should also be considered as the protection scope of the present application.

Claims

1. A 5-HMF aptamer, characterized in that, For one of the following sequences: H1-21m: SEQ ID NO 1 sequence, H1-21m-5Fu-d: replacing T at position 20 of the SEQ ID NO 1 sequence with 5Fu or H1-21mdUb: replacing T at position 13 of the SEQ ID NO 1 sequence with U.

2. Use of a 5-HMF aptamer, characterized in that, The 5-HMF aptamer of claim 1 is applied to the detection of 5-HMF, which is not for the purpose of disease diagnosis.

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