A nucleoside monomer containing an exogenous functional group chemical modification and a covalent aptamer containing the same

Abstract

This invention belongs to the field of biotechnology and relates to a nucleoside monomer chemically modified with exogenous functional groups and a covalent nucleic acid aptamer containing the same nucleoside monomer. The nucleoside monomer contains exogenous functional group chemical modifications, including deoxyuridine phosphorimide and phosphate thioester modifications; the nucleoside monomer includes Sgc8c; the base sequence of Sgc8c is shown in SEQ ID No. 1. Based on the existing nucleic acid aptamer Sgc8c that binds to PTK7, it is chemically modified into a covalent aptamer. A covalent aptamer is an aptamer modified with a covalently reactive target that can specifically bind to a target protein and undergo a proximity-mediated covalent reaction to form a covalent complex. The covalent aptamer can convert the non-covalent complex of a conventional aptamer with a target protein into a covalent complex, thereby preventing rapid dissociation and withstanding various environmental stresses, thus exerting a long-term and stable biological function.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and relates to a nucleoside monomer containing exogenous functional groups chemically modified and a covalent nucleic acid aptamer containing the nucleoside monomer. Background Technology

[0002] Nucleic acid aptamers are oligonucleotide sequences of approximately 25–80 bases in length, obtained through Systematic Evolution of Ligands with Exponential Enrichment (SELEX) technology. They can fold into a three-dimensional conformation to recognize target molecules, exhibiting high binding affinity and specificity, and are thus sometimes referred to as "chemists' antibodies." Compared to antibodies, nucleic acid aptamers offer advantages such as small size and molecular weight, high tissue penetration, non-immunogenicity, and ease of chemical modification. They can also achieve the same functions as antibodies in disease diagnosis, treatment, and targeted drug delivery.

[0003] Protein tyrosine kinase 7 (PTK7) is a Wnt co-receptor and a crucial regulator of planar cell polarization and directional cell motility in vertebrate development and embryogenesis. Besides its important roles in embryogenesis and stem cell function, PTK7 is also associated with tumorigenesis, development, and invasion, exhibiting high expression in various cancers, including advanced triple-negative breast cancer, non-small cell lung cancer, ovarian cancer, colorectal cancer, gastric cancer, and esophageal cancer. Furthermore, high PTK7 expression is associated with lymph node metastasis and is also highly expressed in tumor-initiating cells or tumor stem cells, as well as stromal cells, which are closely related to tumor recurrence and progression. Therefore, developing high-affinity nucleic acid aptamers targeting PTK7 holds promise for overcoming the challenge of limited efficacy of drugs against malignant tumors such as triple-negative breast cancer and non-small cell lung cancer. Summary of the Invention

[0004] To address the problems of existing high-affinity nucleic acid aptamers targeting PTK7, this invention chemically modifies the existing PTK7-binding nucleic acid aptamer Sgc8c into a covalent aptamer. A covalent aptamer is an aptamer modified with a covalently reactive target that can specifically bind to a target protein and undergo a proximity-mediated covalent reaction to form a covalent complex. Covalent aptamers can transform the non-covalent complexes of conventional aptamers and target proteins into covalent complexes, thereby preventing rapid dissociation and withstanding various environmental stresses, thus exerting a long-lasting and stable biological function.

[0005] Bisacralidine is a three-membered ring structure containing two nitrogen atoms. Initially studied as a carbene precursor in spectroscopic research, it has since been widely used as a photoaffinity probe. Bisacralidine can rapidly generate a carbene structure and nitrogen gas under ultraviolet light irradiation or heating. The resulting carbene intermediate exhibits high reactivity and can selectively label target proteins. Furthermore, the only byproduct of this reaction is nitrogen gas, making it an ideal photocontrolled green cross-linking agent.

[0006] In addition to the bisacrididine group, this invention also employs a sulfonyl fluoride warhead for covalent cross-linking. The sulfonyl fluoride group is relatively stable and not easily hydrolyzed. This allows compounds containing sulfonyl fluoride groups to maintain good stability in humid environments or reaction systems containing water. In the field of chemical biology, sulfonyl fluorides can participate in bioorthogonal reactions, reacting rapidly and efficiently with specific functional groups in biomolecules under physiological conditions with minimal interference to biological systems. This makes sulfonyl fluorides potentially valuable for applications in protein labeling, drug delivery, and bioimaging.

[0007] The technical solution of this application is as follows:

[0008] In a first aspect, this application provides a nucleoside monomer containing exogenous functional group chemical modifications, wherein the exogenous functional group chemical modifications include deoxyuridine phosphoramide and thiophosphate modifications;

[0009] The nucleoside monomer includes Sgc8c; the base sequence of Sgc8c is shown in SEQ ID No.1, specifically: 5'-ATCTAACTGCTGCGCCGCCGGGAAAATACTGTACGGTTAGA-3'.

[0010] According to specific embodiments of this application, the deoxyuridine phosphoridamide class includes at least one of 5-phenylbisacrylidine deoxyuridine phosphoridamide, 5-phenylmethyldeoxyuridine phosphoridamide, and 5-fluorophenyldeoxyuridine phosphoridamide.

[0011] According to a specific embodiment of this application, the 5-phenylbisacrididine deoxyuridine phosphoramide comprises compound 11, with the structural formula shown in Formula I:

[0012]

[0013] According to a specific embodiment of this application, the 5-phenylmethyldeoxyuracil phosphoramidite comprises compound 15, with the structural formula shown in Formula II:

[0014]

[0015]

[0016] According to a specific embodiment of this application, the 5-fluorophenyldeoxyuridine phosphoramide comprises compound 19, with the structural formula shown in Formula III:

[0017]

[0018] This application discloses three modified nucleoside monomers for replacing natural T bases, including compound 11 containing the photo-controlled crosslinking group bis(acrylidine), compound 15 containing benzyl, and compound 19 containing fluorophenyl. These modified nucleosides, along with phosphate thioesters that improve the biostability and pharmacokinetic properties of oligonucleotides (as indicated by the orange asterisks and superscripts in Table 1), are introduced into the sequence of the aptamer Sgc8c via solid-phase synthesis. This invention yields four Sgc compounds with enhanced affinity that can covalently crosslink with the PTK7 protein. The 8c variants, named nucleic acid aptamer 1, nucleic acid aptamer 2, and nucleic acid aptamer 3, have base sequences and structures as shown in the examples for nucleic acid aptamer 1, nucleic acid aptamer 2, and nucleic acid aptamer 3. Based on nucleic acid aptamer 1, this invention further introduces a second covalent target, an aryl sulfonyl fluoride projectile. The introduction of three aryl sulfonyl fluoride groups (as indicated by the blue superscript in Table 1) yields a series of Sgc8c variants with further enhanced covalent cross-linking activity with PTK7, named nucleic acid aptamer 4, with base sequences and structures as shown in the example for nucleic acid aptamer 4.

[0019] Nucleic acid aptamer 1 comprises a sequence of Sgc8c with the base A at position 28 (from the 5' end) replaced by 5-phenylbisacrididinedeoxyuridine phosphoramidamide at positions 11, 27, and 29 (from the 5' end), and thiophosphate independently modified at positions 23, 24, and 26. The specific sequence is shown in Table 1.

[0020] Nucleic acid aptamer 2 comprises the following: subtracting the A at position 28 from the 5' end of the Sgc8c sequence; replacing T at positions 11 and 27 from the 5' end with 5-phenylmethyldeoxyuridine phosphoramidite; replacing T at position 29 with 5-phenylbisacrididinedeoxyuridine phosphoramidite; and independently modifying positions 19, 21, 23, 24, 25, 26, 27, and 29 with thiophosphate. The specific sequence is shown in Table 1.

[0021] Nucleic acid aptamer 3 comprises the following: subtracting the 28th A from the 5' end of the Sgc8c sequence; replacing T with 5-fluorophenyldeoxyuridine phosphoramidite at positions 11 and 27 from the 5' end; replacing T with 5-phenylbisacrididinedeoxyuridine phosphoramidite at position 29; and independently modifying thiophosphate at positions 19, 21, 23, 24, 25, 26, 27, and 29. The specific sequence is shown in Table 1.

[0022] Nucleic acid aptamer 4 comprises a sequence of Sgc8c with the A position minus position 28 modified by 5-phenylbisacrididinedeoxyuridine phosphoramide at positions 11, 27, and 29 (starting from the 5' end). Simultaneously, thiophosphate is independently modified at positions 23, 24, and 26, and an arylsulfonyl fluoride is modified onto the thiophosphate backbone. The specific sequence is shown in Table 1.

[0023] Table 1. Sequence, composition, and affinity constant of covalent Sgc8c variants with PTK7

[0024] Nucleic acid aptamers Sequence and Structure (5' to 3') <![CDATA[K D / nM]]> Nucleic acid aptamer 1 <![CDATA[ATCTAACTGC11GCGCCGCCGGGA # A # AA # 11C11GTACGGTTAGA]]> 4.48 Nucleic acid aptamer 2 <![CDATA[ATCTAACTGC15GCGCCGCC # GG # GA # A # A # A # 15 # C11 # GTACGGTTAGA]]> 1.21 Nucleic acid aptamer 3 <![CDATA[ATCTAACTGC19GCGCCGCC # GG # GA # A # A # A # 19 # C11 # GTACGGTTAGA]]> 1.11 Nucleic acid aptamer 4 <![CDATA[ATCTAACTGC11GCGCCGCCGGGA * A * AA * 11C11GTACGGTTAGA]]> 3.87

[0025] # Modification of the skeletal thiophosphate ester; * Arylsulfonyl fluoride modification.

[0026] According to a preferred embodiment of this application, the synthetic route of modified nucleoside 11 is shown in Formula I, and the preparation method includes the steps described in L1-L6:

[0027]

[0028] L1: 5-Bromo-2′-deoxyuridine (5) was dissolved in N,N-dimethylformamide, and imidazole and tert-butyldimethylchlorosilane were added. The mixture was stirred at room temperature and dried to obtain compound 6.

[0029] L2: Compound 6 was dissolved in ammonia (7M in MeOH), and after the reaction, it was extracted, dried, and separated to obtain compound 7;

[0030] L3: Compound 1 was reacted with 1-hydroxybenzotriazole and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride; after the reaction, compound 7 was added and reacted; after the reaction, the mixture was extracted and separated to obtain compound 8.

[0031] L4: Compound 8 was reacted with hydrochloric acid and methanol and then extracted to obtain a white solid compound 9;

[0032] L5: Compound 9 was added to 4,4'-bismethoxytriphenylmethyl chloride, and after the reaction, it was extracted to obtain a white solid compound 10.

[0033] L6: Compound 10 was activated with tetrazolium, and then bis(diisopropylamino)(2-cyanoethoxy)phosphine was added. The reaction was stirred under nitrogen protection, and after completion, water was added to quench the reaction. The mixture was then extracted and separated to obtain a white solid compound 11.

[0034] According to a preferred embodiment of this application, the synthetic route of modified nucleoside 15 is shown in Formula II, and the preparation method includes the steps described in S1-S4:

[0035]

[0036] Preparation of S1 compound 12

[0037] Compound 2 was added with 1-hydroxybenzotriazole and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride; after the reaction, compound 7 was added and reacted; after the reaction, the mixture was extracted, and the remaining crude product was separated by column chromatography to obtain a white solid compound 12.

[0038] Preparation of S2 compound 13

[0039] Compound 12 was added to hydrochloric acid and methanol, and after the reaction, it was extracted and separated to obtain compound 13.

[0040] Preparation of S3 compound 14

[0041] Compound 13 was added with 4,4'-dimethoxytriphenylmethyl chloride, and after the reaction, it was extracted and separated to obtain compound 14;

[0042] Preparation of S4 compound 15

[0043] Compound 14 was activated with tetrazolium, and then bis(diisopropylamino)(2-cyanoethoxy)phosphine was added. The reaction was stirred under nitrogen protection, and after the reaction was completed, water was added to quench the reaction, and the mixture was extracted and separated to obtain compound 15.

[0044] According to a preferred embodiment of this application, the synthetic route for modified nucleoside 19 is shown in Formula III, and the preparation method includes the steps described in X1-X4:

[0045]

[0046] Preparation of Compound X1 16

[0047] Compound 3 was added with 1-hydroxybenzotriazole and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride; after the reaction, compound 7 was added and reacted; after the reaction, the mixture was extracted, and the remaining crude product was separated by column chromatography to obtain white solid compound 16.

[0048] Preparation of Compound X2 17

[0049] Compound 16 was added to hydrochloric acid and methanol, and after the reaction, it was extracted to obtain compound 17.

[0050] Preparation of Compound X3 18

[0051] Compound 17 was added to 4,4'-bismethoxytriphenylmethyl chloride, and after the reaction, it was extracted to obtain compound 18.

[0052] Preparation of Compound X4 19

[0053] Compound 18 was activated with tetrazolium, and then bis(diisopropylamino)(2-cyanoethoxy)phosphine was added. The reaction was stirred under nitrogen protection, and after the reaction was completed, water was added to quench the reaction, and the mixture was extracted and separated to obtain compound 19. Attached image description:

[0054] Figure 1 This is a flowchart of the solid-phase synthesis process;

[0055] Figure 2 Mass spectrometry results for nucleic acid aptamer 1;

[0056] Figure 3 Mass spectrometry results for nucleic acid aptamer 2;

[0057] Figure 4 Mass spectrometry results for nucleic acid aptamer 13;

[0058] Figure 5 It has a 4-(bromomethyl)benzenesulfonyl fluoride structure;

[0059] Figure 6 Mass spectrometry results for nucleic acid aptamers 4;

[0060] Figure 7 The results are for the affinity assay of aptamer 1 protein.

[0061] Figure 8 The results are for the affinity assay of aptamer 2 protein.

[0062] Figure 9 The results are for the affinity assay of aptamer 3 protein.

[0063] Figure 10 The results are for the affinity assay of aptamer 4 protein;

[0064] Figure 11 The results of the covalent crosslinking experiment for nucleic acid aptamers 1 and 4;

[0065] Figure 12 The results show the covalent cross-linking experiments of nucleic acid aptamers 2 and 3.

[0066] Beneficial effects of this application

[0067] 1) The synthesis of modified nucleosides is a short, mild, and easy-to-operate procedure.

[0068] 2) The raw materials used in the synthesis of modified nucleosides are readily available and easy to produce industrially.

[0069] 3) These modified nucleosides have excellent chemical stability and are easy to store.

[0070] 4) The obtained covalent Sgc8c variant is more stable, can be artificially synthesized, and is easy to preserve and label.

[0071] 5) Covalent Sgc8c variants have promising applications in detection, diagnosis, imaging, treatment, and targeted drug delivery. Detailed Implementation

[0072] Example 1

[0073] The method for synthesizing compound 11 includes the following steps:

[0074]

[0075] ①Preparation of compound 6

[0076] 5-Bromo-2'-deoxyuridine (5) (3.07 g, 10 mmol) was dissolved in N,N-dimethylformamide (DMF) (30 mL), and imidazole (4.08 g, 10 mmol) and tert-butyldimethylchlorosilane (TBDMSCl) (4.5 g, 30 mmol) were added. The reaction mixture was stirred at room temperature for 6 hours to remove DMF. The remaining crude product was extracted with saturated sodium bicarbonate (2 × 50 mL) solution and ethyl acetate (2 × 100 mL). After drying with anhydrous sodium sulfate, the organic phase was desolventized under reduced pressure, and finally dried under vacuum to give a white solid compound 6 (4.4 g, 8.2 mmol, yield 82%).

[0077] ②Preparation of compound 7

[0078] Compound 6 (4.4 g, 8.2 mmol) was dissolved in ammonia (7 M in MeOH) (40 mL), sealed in a tube, heated to 75 °C, and reacted for 48 hours. After removing methanol, the crude product was extracted with water (2 × 50 mL) and ethyl acetate (2 × 100 mL). After drying with anhydrous sodium sulfate, the organic phase was desolventized under reduced pressure. The remaining crude product was separated by column chromatography to obtain a white solid compound 7 (2.79 g, 5.9 mmol, yield 72%).

[0079] ③ Preparation of compound 8

[0080] Compound 1 (1.9 g, 8.3 mmol) was dissolved in N,N-dimethylformamide (DMF) (30 mL), and 1-hydroxybenzotriazole (1.12 g, 8.3 mmol) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (1.7 g, 8.9 mmol) were added. After reacting for 1 hour, compound 7 (2.79 g, 5.9 mmol) was added and reacted for 6 hours. After the reaction was completed, DMF was removed, and the remaining crude product was extracted with saturated sodium bicarbonate (2 × 50 mL) solution and ethyl acetate (2 × 100 mL). After drying with anhydrous sodium sulfate, the organic phase was desolventized under reduced pressure, and the remaining crude product was separated by column chromatography to obtain a white solid compound 8 (2.99 g, 4.4 mmol, yield 74%).

[0081] ④ Preparation of compound 9

[0082] Compound 8 (2.99 g, 4.4 mmol) was dissolved in methanol (20 mL), and hydrochloric acid and methanol were added in a ratio of 1:10 (22 mL). After reacting for 4 hours, the methanol was removed, and the remaining crude product was extracted with saturated sodium bicarbonate (2 × 40 mL) and ethyl acetate (2 × 100 mL). After drying with anhydrous sodium sulfate, the organic phase was desolventized under reduced pressure, and the remaining crude product was separated by column chromatography to obtain a white solid compound 9 (1.29 g, 2.8 mmol, yield 64%).

[0083] ⑤ Preparation of Compound 10

[0084] Compound 9 (1.29 g, 2.8 mmol) was dissolved in pyridine (20 mL), and 4,4'-bismethoxytriphenylmethyl chloride (1.25 g, 3.7 mmol) was added and reacted for 4 hours. After the reaction was completed, the pyridine was removed, and the remaining crude product was extracted with saturated sodium bicarbonate (2 × 50 mL) solution and ethyl acetate (2 × 100 mL). After drying with anhydrous sodium sulfate, the organic phase was desolventized under reduced pressure, and the remaining crude product was separated by column chromatography to obtain a white solid compound 10 (1.85 g, 2.4 mmol, yield 86%).

[0085] ⑥ Preparation of compound 11

[0086] Compound 10 (1.85 g, 2.4 mmol) was dissolved in anhydrous acetonitrile (15 mL), and activated with tetrazolium (85 mg, 1.2 mmol) for 10 minutes. After activation, bis(diisopropylamino)(2-cyanoethoxy)phosphine (4) (1 mL, 3.1 mmol) was added. The reaction was stirred under nitrogen protection for 8 hours. After the reaction was completed, water was added to quench the reaction, and the mixture was extracted with ethyl acetate (2 × 30 mL). After drying with anhydrous sodium sulfate, the organic phase was desolventized under reduced pressure. The remaining crude product was separated by column chromatography to obtain a white solid compound 11 (1.49 g, 1.5 mmol, yield 62.5%).

[0087] Example 2

[0088] The method for synthesizing compound 15 includes the following steps:

[0089]

[0090] ①Preparation of compound 12

[0091] Compound 2 (0.95 g, 7 mmol) was dissolved in N,N-dimethylformamide (DMF) (30 mL), and 1-hydroxybenzotriazole (0.95 g, 7 mmol) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (1.43 g, 7.5 mmol) were added. After reacting for 1 hour, compound 7 (2.36 g, 5 mmol) was added and reacted for 6 hours. After the reaction was completed, DMF was removed, and the remaining crude product was extracted with saturated sodium bicarbonate (2 × 50 mL) solution and ethyl acetate (2 × 100 mL). After drying with anhydrous sodium sulfate, the organic phase was desolventized under reduced pressure, and the remaining crude product was separated by column chromatography to obtain a white solid compound 12 (2.3 g, 3.9 mmol, yield 78%).

[0092] ②Preparation of compound 13

[0093] Compound 12 (2.3 g, 3.9 mmol) was dissolved in methanol (20 mL), and hydrochloric acid and methanol were added in a ratio of 1:10 (22 mL). After reacting for 4 hours, the methanol was removed, and the remaining crude product was extracted with saturated sodium bicarbonate (2 × 40 mL) and ethyl acetate (2 × 100 mL). After drying with anhydrous sodium sulfate, the organic phase was desolventized under reduced pressure, and the remaining crude product was separated by column chromatography to obtain a white solid compound 13 (1.0 g, 2.77 mmol, yield 71%).

[0094] ③Preparation of compound 14

[0095] Compound 13 (1.0 g, 2.77 mmol) was dissolved in pyridine (20 mL), and 4,4'-bismethoxytriphenylmethyl chloride (1.22 g, 3.6 mmol) was added and reacted for 4 hours. After the reaction was completed, the pyridine was removed, and the remaining crude product was extracted with saturated sodium bicarbonate (2 × 50 mL) solution and ethyl acetate (2 × 100 mL). After drying with anhydrous sodium sulfate, the organic phase was desolventized under reduced pressure, and the remaining crude product was separated by column chromatography to obtain a white solid compound 14 (1.56 g, 2.3 mmol, yield 85%).

[0096] ④ Preparation of Compound 15

[0097] Compound 14 (1.56 g, 2.3 mmol) was dissolved in anhydrous acetonitrile (15 mL), and tetrazolium (80.5 mg, 1.15 mmol) was added for activation for 10 minutes. After activation, bis(diisopropylamino)(2-cyanoethoxy)phosphine (4) (0.95 mL, 3 mmol) was added. The reaction was stirred under nitrogen protection for 8 hours. After the reaction was completed, water was added for quenching. The mixture was extracted with ethyl acetate (2 × 30 mL), dried over anhydrous sodium sulfate, and the organic phase was dried under reduced pressure to remove the solvent. The remaining crude product was separated by column chromatography to obtain a white solid compound 15 (1.42 g, 1.6 mmol, yield 70%).

[0098] Example 3

[0099] The method for synthesizing compound 19 includes the following steps:

[0100]

[0101] ①Preparation of compound 16

[0102] Compound 3 (0.98 g, 7 mmol) was dissolved in N,N-dimethylformamide (DMF) (30 mL), and 1-hydroxybenzotriazole (0.95 g, 7 mmol) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (1.43 g, 7.5 mmol) were added. After reacting for 1 hour, compound 7 (2.36 g, 5 mmol) was added and reacted for 6 hours. After the reaction was completed, DMF was removed, and the remaining crude product was extracted with saturated sodium bicarbonate (2 × 50 mL) solution and ethyl acetate (2 × 100 mL). After drying with anhydrous sodium sulfate, the organic phase was desolventized under reduced pressure, and the remaining crude product was separated by column chromatography to obtain a white solid compound 16 (2.1 g, 3.6 mmol, yield 71%).

[0103] ②Preparation of compound 17

[0104] Compound 16 (2.1 g, 3.6 mmol) was dissolved in methanol (20 mL), and hydrochloric acid and methanol were added in a ratio of 1:10 (22 mL). After reacting for 4 hours, the methanol was removed, and the remaining crude product was extracted with saturated sodium bicarbonate (2 × 40 mL) and ethyl acetate (2 × 100 mL). After drying with anhydrous sodium sulfate, the organic phase was desolventized under reduced pressure, and the remaining crude product was separated by column chromatography to obtain a white solid compound 17 (0.83 g, 2.3 mmol, yield 64%).

[0105] ③Preparation of compound 18

[0106] Compound 17 (0.83 g, 2.3 mmol) was dissolved in pyridine (20 mL), and 4,4'-bismethoxytriphenylmethyl chloride (1.01 g, 3.0 mmol) was added and reacted for 4 hours. After the reaction was completed, the pyridine was removed, and the remaining crude product was extracted with saturated sodium bicarbonate (2 × 50 mL) solution and ethyl acetate (2 × 100 mL). After drying with anhydrous sodium sulfate, the organic phase was desolventized under reduced pressure, and the remaining crude product was separated by column chromatography to obtain a white solid compound 18 (1.23 g, 1.9 mmol, yield 2%).

[0107] ④ Preparation of compound 19

[0108] Compound 18 (1.23 g, 1.9 mmol) was dissolved in anhydrous acetonitrile (15 mL), and activated with tetrazolium (66.5 mg, 0.95 mmol) for 10 minutes. After activation, bis(diisopropylamino)(2-cyanoethoxy)phosphine (4) (0.79 mL, 2.5 mmol) was added. The reaction was stirred under nitrogen protection for 8 hours. After the reaction was completed, water was added to quench the reaction, and the mixture was extracted with ethyl acetate (2 × 30 mL). After drying with anhydrous sodium sulfate, the organic phase was desolventized under reduced pressure. The remaining crude product was separated by column chromatography to obtain a white solid compound 19 (1.05 g, 1.25 mmol, yield 66%).

[0109] Example 4

[0110] Solid-phase synthesis method for introducing the modified compounds obtained in Examples 1-3 into the Sgc8c sequence to obtain nucleic acid aptamers 1-4:

[0111] (1) The natural phosphoramidite monomers used, as well as the acetonitrile, deprotecting reagents, activators, blocking agents, and oxidants used in the synthesis, were all from Qingdao Judianer Technology Co., Ltd. All reagents and containers used underwent rigorous drying and dehydration treatment. The magnetic bead carrier was Beijing Haijing Gaochuang Technology Co., Ltd.'s 1000A general-purpose CPG. The DDTT reagent used for phosphate thioester oxidation was from Shanghai Bid Pharmaceutical Technology Co., Ltd. All sequences were synthesized on a K&A H-8 DNA synthesizer according to the instrument instructions, without removing DMT from the last base. The synthesis steps are as follows:

[0112] The single-cycle synthesis scale was 0.5 μmol, with a CPG size of 1000 Å. To improve reaction efficiency, four natural phosphoramidite monomers—dT-CE phosphoramidite, dC-CE phosphoramidite, dA-CE phosphoramidite, and dG-CE phosphoramidite—were used in the coupling reaction step. These were prepared into 0.1 M solutions with anhydrous acetonitrile, and the coupling reaction time was 90 seconds. For the modified monomers obtained in Examples 1-3, a 0.12 M solution was prepared with anhydrous acetonitrile, and the coupling reaction was performed twice, each time for 8 minutes. The synthesis consisted of four steps: deprotection, coupling, capping (blocking), and oxidation.

[0113] Deprotection: Add a deprotection reagent (dichloromethane of 95% trichloroacetic acid) to remove the magnetic bead carrier (if there is a protecting group DMT or MMT) or the hydroxyl protecting group DMT of the nucleoside, and wash with acetonitrile;

[0114] Coupling: 0.25M ethylthiotetrazole (ETT) was added as an activator, and then natural or modified phosphoramide monomers were added to magnetic bead carriers in the synthesis column to extend the DNA strand, followed by washing with acetonitrile;

[0115] Blocking: Add blocking reagents CAPA and CAPB to block unreacted nucleotides to reduce the generation of sequences with missing intermediate bases, and wash with acetonitrile;

[0116] Oxidation: Add 0.05M iodine oxidant and oxidize for 20 seconds to oxidize trivalent phosphorus to pentavalent phosphorus. For thiophosphate modification, replace the oxidant with 0.1M DDTT oxidant and extend the oxidation time to five minutes. Figure 1 This is a schematic diagram of the solid-phase synthesis process.

[0117] (2) Transfer CPG to a 1.5 mL EP tube, add 1.3 mL of 30% concentrated ammonia solution and seal, and incubate at 65 °C with shaking for 2 h; then add 10 times the volume of n-butanol solution, and let stand at -80 °C for 30 minutes; centrifuge at 8000 rpm for 25 minutes to obtain crude DNA product, remove n-butanol and redissolve it with enzyme-free water to a solution of about 0.5 mM.

[0118] (3) The crude DNA product was purified by urea-denatured polyacrylamide gel electrophoresis; the full-length product was recovered by gel excision and electroelution, and the recovered product was desalted and concentrated by solution displacement using a 3K ultrafiltration tube, and the concentration was quantified by Nanodrop. Mass spectrometry results for aptamers 1, 2, and 3 are shown below. Figure 2 , 3 As shown in Figure 4.

[0119] Example 5

[0120] benzenesulfonyl fluoride reaction experiment

[0121] 45 μl of 100 μM aptamer modified with thiophosphate (SEQ ID NO. 1) and 50 μl of 60 mM 4-(bromomethyl)benzenesulfonyl fluoride were added to 10× sodium phosphate buffer (pH 6.0) and incubated at 37 °C for 12 hours. After the reaction, the mixture was extracted with 200 μl of n-butanol and enzyme-free water. The mixture was centrifuged at 150 × 100 rpm for 5 minutes to achieve complete separation. This process was repeated twice. The recovered product was then desalted and concentrated using a 3K ultrafiltration tube via solution displacement, and the concentration was quantified using Nanodrop. The structure of 4-(bromomethyl)benzenesulfonyl fluoride is as follows: Figure 5 As shown, the mass spectrometry results for nucleic acid aptamer 4 are as follows: Figure 6 As shown.

[0122] Example 6

[0123] Surface plasmon resonance (SPR) assay for the affinity of various covalent Sgc8c variants for PTK7 protein

[0124] Aptamer solution preparation: Prepare solutions of Sgc8c standard and various covalent variants in the following gradient dilutions using DPBS buffer: 400 nM, 200 nM, 100 nM, 50 nM, 25 nM.

[0125] Protein conjugation: PTK7 protein was conjugated to channel 2 of the CM5 chip surface using the following method: Equal volumes of EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 0.4M aqueous solution) and NHS (N-hydroxysuccinimide, 0.1M aqueous solution) were mixed and injected in 140 μL to activate the chip at a flow rate of 10 μL / min. PTK7 protein was diluted with 10 mM sodium acetate buffer (pH 4.5) to a final concentration of 25 μg / mL and injected in 200 μL at a flow rate of 10 μL / min, resulting in a PTK7 protein conjugation amount of 6810.5 RU. After injection, ethanolamine (1M) was injected to block the chip at a flow rate of 10 μL / min, with a final injection volume of 140 μL. The same method was used to conjugate the His peptide to channel 1 of the CM5 chip surface as a control channel.

[0126] Affinity activity assay: A surface plasmon resonance (SPR) instrument (GE Healthcare, model: Biacore 8K) was used with the detection parameters set. The diluted nucleic acid aptamer samples were sequentially flowed through channels 1 and 2. The procedure for each aptamer was as follows: injection 30 μL / min, time 2 min; dissociation 30 μL / min, time 7 min; regeneration with 1.5 M NaCl, 30 μL / min, time 30 s. The protein affinity assay results for SEQ ID No. 1, No. 2, No. 3, and No. 4 are shown below. Figure 7 , 8 As shown in 9 and 10.

[0127] Example 7

[0128] Covalent crosslinking experiment

[0129] 0.5 μl of 4 μM CY5-labeled PTK7 protein solution was reacted with 0.5 μl of 25 μM aptamers SEQ ID No. 1, No. 2, No. 3, and No. 4 in 1× reaction buffer (1×DPBS pH 7.4 and 5 mM MgCl2) at room temperature for 30 min. The solution was then irradiated with 365 nm light at room temperature for 10 min. For ligand SEQ ID No. 4, incubation was continued at 37 °C for 6 h. After the reaction, the reaction solution was mixed with 2×SDS-PAGE protein sample buffer (80 mM Tris-HCl, 2% SDS, 0.1 M DTT, 10% glycerol) and heated at 95 °C for 10 min. Gel electrophoresis analysis was then performed at 180 V in 1×MOPS running buffer (50 mM MOPS, 50 mM Tris, 0.1% SDS, 1 mM EDTA, pH 7.7).

Claims

1. A covalent nucleic acid aptamer, characterized in that, The nucleic acid aptamers include nucleoside monomers chemically modified with exogenous functional groups; The nucleoside monomer is Sgc8c; the base sequence of Sgc8c is shown in SEQ ID No.1, specifically: 5'-ATCTAACTGCTGCGCCGCCGGGAAAATACTGTACGGTTAGA-3'; The nucleic acid aptamer is nucleic acid aptamer 2, which includes the following modifications: subtracting the A at position 28 from the 5' end of the Sgc8c sequence; replacing T at positions 11 and 27 from the 5' end with 5-phenylmethyldeoxyuridine phosphoramidite; replacing T at position 29 with 5-phenylbisacrylidinedeoxyuridine phosphoramidite; and independently modifying thiophosphate at positions 19, 21, 23, 24, 25, 26, 27, and 29.

2. The covalent nucleic acid aptamer according to claim 1, characterized in that, The 5-phenylbisacrididine deoxyuridine phosphoramide is compound 11, with the structural formula shown in Formula I: ； And / or, the 5-phenylmethyldeoxyuracil phosphoramidite is compound 15, with the structural formula shown in Formula II: 。 3. The use of the covalent nucleic acid aptamer according to claim 1 or 2 in the preparation of a kit for highly sensitive recognition of PTK7 protein.

4. The use of the covalent nucleic acid aptamer according to claim 1 or 2 in a kit for preparing a highly specific recognition of PTK7 protein.

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