Nucleoside monomer containing exogenous functional group chemical modification and covalent nucleic acid aptamer containing nucleoside monomer

Abstract

The invention belongs to the technical field of biology, and relates to a nucleoside monomer containing exogenous functional group chemical modification and a covalent nucleic acid aptamer containing the nucleoside monomer. The invention relates to a nucleoside monomer containing exogenous functional group chemical modification. The exogenous functional group chemical modification comprises deoxyuracil phosphoramidite modification and phosphorothioate modification; the nucleoside monomer comprises Sgc8c; the base sequence of the Sgc8c is as shown in SEQ ID No. 1 (sequence identifier number 1). On the basis of the existing nucleic acid aptamer Sgc8c combined with PTK7, the nucleic acid aptamer Sgc8c is transformed into a covalent aptamer through chemical modification. The covalent aptamer is an aptamer modified with a covalent reaction target head, and the covalent aptamer can be subjected to proximity effect mediated covalent reaction after being specifically combined with target protein so as to generate a covalent complex. According to the covalent aptamer, a non-covalent complex of a conventional aptamer and target protein can be converted into a covalent complex, so that rapid dissociation is prevented, various environmental pressures are borne, and a long-acting and stable biological function is achieved.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology and relates to a chemically modified nucleoside monomer containing an exogenous functional group and a covalent nucleic acid aptamer containing the nucleoside monomer. Background Art

[0002] Nucleic acid aptamers are oligonucleotide sequences of about 25 to 80 bases in length obtained through the systematic evolution of ligands by exponential enrichment (SELEX). They can recognize target molecules by folding into a three-dimensional conformation and have high binding affinity and specificity. They are also called chemists' antibodies. Compared with antibodies, nucleic acid aptamers have the advantages of small size and molecular weight, high tissue penetration, no immunogenicity, and easy chemical modification. They can also achieve the same functions of antibodies in disease diagnosis, treatment, and targeted drug delivery.

[0003] Protein tyrosine kinase 7 (PTK7) is a Wnt co-receptor and an important regulator of planar cell polarization and directional cell movement in vertebrate development and embryogenesis. In addition to its important role in embryogenesis and stem cell function, PTK7 is also associated with tumor occurrence, development and invasion, and is highly expressed in a variety of cancers, including advanced triple-negative breast cancer, non-small cell lung cancer, ovarian cancer, colorectal cancer, gastric cancer and esophageal cancer. In addition, high expression of PTK7 is associated with lymph node metastasis, and is highly expressed in tumor-initiating cells or tumor stem cells and stromal cells that are closely related to tumor recurrence and progression. Therefore, the development of high-affinity nucleic acid aptamers targeting PTK7 is expected to overcome the problem of limited efficacy of drugs for malignant tumors such as triple-negative breast cancer and non-small cell lung cancer. Summary of the invention

[0004] In view of the problems existing in the existing high-affinity nucleic acid aptamers targeting PTK7, the present invention transforms the existing nucleic acid aptamer Sgc8c binding to PTK7 into a covalent aptamer through chemical modification. A covalent aptamer refers to an aptamer modified with a covalent reaction target head that can specifically bind to a target protein and undergo a covalent reaction mediated by a proximity effect to generate a covalent complex. Covalent aptamers can convert the non-covalent complex of a conventional aptamer and a target protein into a covalent complex, thereby preventing rapid dissociation and withstanding various environmental pressures, thereby exerting a long-lasting and stable biological function.

[0005] Diaziridine is a three-membered ring structure containing two nitrogen atoms. It was originally studied as a spectral precursor of carbenes and later widely used as a photoaffinity probe. Diaziridine can quickly generate carbene structures and nitrogen gas under ultraviolet light irradiation or heating conditions. The generated carbene intermediate has high reactivity and can highly selectively label the target protein. At the same time, the byproduct of this reaction is only nitrogen gas, which is an ideal light-controlled green cross-linking agent.

[0006] In addition to the diaziridine group, the present invention also uses a sulfonyl fluoride warhead for covalent cross-linking reaction. The sulfonyl fluoride group is relatively stable and not easily hydrolyzed. This allows compounds containing sulfonyl fluoride groups to maintain good stability in a humid environment or in a reaction system with water. In the field of chemical biology, sulfonyl fluoride can participate in bioorthogonal reactions and react with specific functional groups in biomolecules under physiological conditions in a fast, efficient and less disturbing reaction to the biological system. This makes sulfonyl fluoride have potential application value in protein labeling, drug delivery, bioimaging, etc.

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

[0008] In a first aspect of the present application, a nucleoside monomer containing an exogenous functional group chemical modification is provided, wherein the exogenous functional group chemical modification includes deoxyuracil phosphoramidite and thiophosphate modification;

[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 a specific embodiment of the present application, the deoxyuracil phosphoramidite includes at least one of 5-phenylbis(aziridine) deoxyuracil phosphoramidite, 5-benzyl deoxyuracil phosphoramidite, and 5-fluorophenyl deoxyuracil phosphoramidite.

[0011] According to a specific embodiment of the present application, the 5-phenylbis(aziridine) deoxyuracil phosphoramidite includes compound 11, the structural formula of which is shown in Formula I:

[0012]

[0013] According to a specific embodiment of the present application, the 5-benzyl deoxyuracil phosphoramidite includes compound 15, the structural formula of which is shown in Formula II:

[0014]

[0015]

[0016] According to a specific embodiment of the present application, the 5-fluorophenyl deoxyuracil phosphoramidite includes compound 19, the structural formula of which is shown in Formula III:

[0017]

[0018] The present application invents three modified nucleoside monomers for replacing natural T bases, including compound 11 containing a photo-controlled cross-linking group diaziridine, compound 15 containing a benzyl group, and compound 19 containing a fluorophenyl group; these modified nucleosides and backbone phosphorothioate modifications (as indicated by the orange asterisk and the well sign superscript in Table 1) that can improve the biological stability and pharmacokinetic properties of oligonucleotides are introduced into the sequence of the aptamer Sgc8c by solid phase synthesis, and the present invention obtains four Sgc8c monomers with enhanced affinity and capable of covalent cross-linking reaction with PTK7 protein. 8c variants, named nucleic acid aptamer 1, nucleic acid aptamer 2, and nucleic acid aptamer 3, whose base sequences and compositions are as shown in nucleic acid aptamer 1, nucleic acid aptamer 2, and nucleic acid aptamer 3; on the basis of nucleic acid aptamer 1, the present invention further introduces a second covalent target head, an aryl sulfonyl fluoride warhead, and the introduction of three aryl sulfonyl fluoride groups (as shown by the blue asterisk superscripts in Table 1), obtains a series of Sgc8c variants with further improved covalent cross-linking activity with PTK7, named nucleic acid aptamer 4, whose base sequences and compositions are as shown in nucleic acid aptamer 4.

[0019] Nucleic acid aptamer 1 includes the sequence of Sgc8c minus the base A at the 28th position from the 5' end, and the 11th, 27th and 29th positions from the 5' end are modified with 5-phenylbis(aziridine) deoxyuracil phosphoramidite to replace T, and the 23rd, 24th and 26th positions are independently modified with phosphorothioate. The specific sequence is shown in Table 1.

[0020] Nucleic acid aptamer 2 includes the sequence of Sgc8c minus the A at position 28 from the 5' end, 5-benzyl deoxyuracil phosphoramidite is used to modify and replace T at position 11 and position 27 from the 5' end, 5-phenyl diaziridine deoxyuracil phosphoramidite is used to modify and replace T at position 29, and phosphorothioate is independently modified at positions 19, 21, 23, 24, 25, 26, 27, and 29. The specific sequence is shown in Table 1.

[0021] Nucleic acid aptamer 3, which includes the sequence of Sgc8c minus the A at position 28 from the 5' end, 5-fluorophenyl deoxyuracil phosphoramidite modification and substitution of T at position 11 and position 27 from the 5' end of the sequence, 5-phenyl diaziridine deoxyuracil phosphoramidite modification and substitution of T at position 29, and phosphorothioate modification at positions 19, 21, 23, 24, 25, 26, 27, and 29. The specific sequence is shown in Table 1.

[0022] Nucleic acid aptamer 4 includes the sequence of Sgc8c minus the A at position 28, and the 11th, 27th, and 29th positions from the 5' end of the sequence are modified with 5-phenylbis(aziridine) deoxyuracil phosphoramidite to replace T, and the 23rd, 24th, and 26th positions are independently modified with phosphorothioate and the arylsulfonyl fluoride is modified on the phosphorothioate backbone. The specific sequence is shown in Table 1.

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

[0024] Aptamers Sequence and structure (5'to3') <![CDATA[K D / nM]]> Aptamer 1 <![CDATA[ATCTAACTGC11GCGCCGCCGGGA # A # AA # 11C11GTACGGTTAGA]]> 4.48 Aptamer 2 <![CDATA[ATCTAACTGC15GCGCCGCC # GG # GA # A # A # A # 15 # C11 # GTACGGTTAGA]]> 1.21 Aptamer 3 <![CDATA[ATCTAACTGC19GCGCCGCC # GG # GA # A # A # A # 19 # C11 # GTACGGTTAGA]]> 1.11 Aptamer 4 <![CDATA[ATCTAACTGC11GCGCCGCCGGGA * A * AA * 11C11GTACGGTTAGA]]> 3.87

[0025] # Backbone phosphorothioate modification; * Arylsulfonyl fluoride modification.

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

[0027]

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

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

[0030] L3: Compound 1, 1-hydroxybenzotriazole, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride added; after the reaction, compound 7 was added for reaction, and after the reaction, compound 8 was separated by extraction;

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

[0032] L5: Compound 9 was added with 4,4'-bismethoxytrityl chloride, and the mixture was reacted and extracted to obtain a white solid compound 10;

[0033] L6: Compound 10 was activated by adding tetrazole, and then bis(diisopropylamino)(2-cyanoethoxy)phosphine was added. The reaction was stirred under nitrogen protection. After completion, water was added to quench the reaction. Compound 11 was obtained by extraction and separation to obtain a white solid.

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

[0035]

[0036] S1 Preparation of compound 12

[0037] Compound 2 was added with 1-hydroxybenzotriazole and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride; after the reaction, compound 7 was added for reaction, and after the reaction, extraction was performed, 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 with hydrochloric acid and methanol, and after reaction, it was extracted and separated to obtain compound 13;

[0040] Preparation of S3 compound 14

[0041] Compound 13 was added with 4,4'-bismethoxytrityl chloride, and after reaction, extraction was performed to separate compound 14;

[0042] Preparation of S4 compound 15

[0043] Compound 14 was activated by adding tetrazole, and after activation, bis(diisopropylamino)(2-cyanoethoxy)phosphine was added, and the reaction was stirred under nitrogen protection. After completion, water was added to quench, and compound 15 was obtained by extraction and separation.

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

[0045]

[0046] Preparation of X1 compound 16

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

[0048] Preparation of X2 compound 17

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

[0050] Preparation of X3 compound 18

[0051] Compound 17 was added with 4,4'-bismethoxytrityl chloride, and after reaction, extraction was performed to separate compound 18;

[0052] Preparation of X4 compound 19

[0053] Compound 18 was activated by adding tetrazole, and after activation, bis(diisopropylamino)(2-cyanoethoxy)phosphine was added, and the reaction was stirred under nitrogen protection. After completion, water was added to quench, and compound 19 was obtained by extraction and separation. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] Figure 1 is a solid phase synthesis flow chart;

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

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

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

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

[0059] Figure 6 Mass spectrometry results of nucleic acid aptamer 4;

[0060] Figure 7 This is the result of affinity test of nucleic acid aptamer 1 protein;

[0061] Figure 8 This is the result of affinity test of nucleic acid aptamer 2 protein;

[0062] Fig. 9 This is the result of affinity test of nucleic acid aptamer 3 protein;

[0063] Fig.10 This is the result of affinity test of nucleic acid aptamer 4 protein;

[0064] Fig.11 The covalent cross-linking experimental results of nucleic acid aptamers 1 and 4;

[0065] Fig.12 The covalent cross-linking experimental results of nucleic acid aptamers 2 and 3.

[0066] Beneficial effects of this application

[0067] 1) The synthesis steps of modified nucleosides are short, the conditions are mild, and the operation is simple.

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

[0069] 3) This type of modified nucleoside has good chemical stability and is easy to store.

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

[0071] 5) The covalent Sgc8c variant has application prospects in detection, diagnosis, imaging, therapy, and targeted drug delivery. DETAILED DESCRIPTION

[0072] Example 1

[0073] The synthesis method of compound 11 comprises the following steps:

[0074]

[0075] ① Preparation 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, 10 mmol) and tert-butyldimethylsilyl chloride (TBDMSCl) (4.5 g, 30 mmol) were added. The reaction solution was stirred at room temperature for 6 hours and DMF was removed. The remaining crude product was extracted with saturated sodium bicarbonate (2×50 mL) solution and ethyl acetate (2×100 mL). After drying over anhydrous sodium sulfate, the organic phase was freed from the solvent under reduced pressure to obtain white solid compound 6 (4.4 g, 8.2 mmol, yield 82%) under vacuum drying.

[0077] ② Preparation of compound 7

[0078] Compound 6 (4.4 g, 8.2 mmol) was dissolved in ammonia (7 M in MeOH) (40 mL), sealed and heated to 75 ° C. After reacting for 48 hours, methanol was removed, and the crude product was extracted with water (2×50 mL) and ethyl acetate (2×100 mL). After drying over anhydrous sodium sulfate, the organic phase was freed of the solvent under reduced pressure, and the remaining crude product was separated by column chromatography to obtain 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, 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 over anhydrous sodium sulfate, the organic phase was freed from the solvent under reduced pressure, and the remaining crude product was separated by column chromatography to obtain 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 = 1:10 (22 mL) were added to react for 4 hours, and then methanol was removed. The remaining crude product was extracted with saturated sodium bicarbonate (2×40 mL) and ethyl acetate (2×100 mL). After drying over anhydrous sodium sulfate, the organic phase was freed of the solvent under reduced pressure, and the remaining crude product was separated by column chromatography to obtain 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'-bismethoxytrityl chloride (1.25 g, 3.7 mmol) was added to react for 4 hours. After the reaction, 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 over anhydrous sodium sulfate, the organic phase was freed of the solvent under reduced pressure, and the remaining crude product was separated by column chromatography to obtain 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 tetrazole (85 mg, 1.2 mmol) was added for activation for 10 minutes. After activation, bis(diisopropylamino)(2-cyanoethoxy)phosphine (4) (1 mL, 3.1 mmol) was added, and the reaction was stirred for 8 hours under nitrogen protection. After completion, water was added to quench the reaction, and the reaction mixture was extracted with ethyl acetate (2×30 mL). After drying over anhydrous sodium sulfate, the organic phase was freed from the solvent under reduced pressure, and the remaining crude product was separated by column chromatography to obtain white solid compound 11 (1.49 g, 1.5 mmol, yield 62.5%).

[0087] Example 2

[0088] The synthesis method of compound 15 comprises the following steps:

[0089]

[0090] ① Preparation 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, 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 over anhydrous sodium sulfate, the organic phase was freed from the solvent under reduced pressure, and the remaining crude product was separated by column chromatography to obtain white solid compound 12 (2.3 g, 3.9 mmol, yield 78%).

[0092] ② Preparation of compound 13

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

[0094] ③ Preparation of compound 14

[0095] Compound 13 (1.0 g, 2.77 mmol) was dissolved in pyridine (20 mL), and 4,4'-bismethoxytrityl chloride (1.22 g, 3.6 mmol) was added to react for 4 hours. After the reaction, 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 over anhydrous sodium sulfate, the organic phase was freed of the solvent under reduced pressure, and the remaining crude product was separated by column chromatography to obtain 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 tetrazole (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 for 8 hours under nitrogen protection. After completion, water was added to quench the reaction, and the reaction mixture was extracted with ethyl acetate (2×30 mL). After drying over anhydrous sodium sulfate, the organic phase was freed from the solvent under reduced pressure, and the remaining crude product was separated by column chromatography to obtain white solid compound 15 (1.42 g, 1.6 mmol, yield 70%).

[0098] Example 3

[0099] The synthesis method of compound 19 comprises the following steps:

[0100]

[0101] ① Preparation 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, 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 over anhydrous sodium sulfate, the organic phase was freed from the solvent under reduced pressure, and the remaining crude product was separated by column chromatography to obtain white solid compound 16 (2.1 g, 3.6 mmol, yield 71%).

[0103] ② Preparation of compound 17

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

[0105] ③ Preparation of compound 18

[0106] Compound 17 (0.83 g, 2.3 mmol) was dissolved in pyridine (20 mL), and 4,4'-bismethoxytrityl chloride (1.01 g, 3.0 mmol) was added to react for 4 hours. After the reaction, 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 over anhydrous sodium sulfate, the organic phase was freed of the solvent under reduced pressure, and the remaining crude product was separated by column chromatography to obtain 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 tetrazole (66.5 mg, 0.95 mmol) was added for activation for 10 minutes. After activation, bis(diisopropylamino)(2-cyanoethoxy)phosphine (4) (0.79 ml, 2.5 mmol) was added. The reaction was stirred for 8 hours under nitrogen protection. After completion, water was added to quench the reaction, and the reaction mixture was extracted with ethyl acetate (2×30 mL). After drying over anhydrous sodium sulfate, the organic phase was freed from the solvent under reduced pressure, and the remaining crude product was separated by column chromatography to obtain white solid compound 19 (1.05 g, 1.25 mmol, yield 66%).

[0109] Example 4

[0110] Solid phase synthesis method of introducing the modified compound obtained in Example 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, deprotection reagents, activators, blocking agents, and oxidants used in the synthesis were all from Qingdao Judianer Technology Co., Ltd., and the reagents and containers used were strictly dried and dehydrated. The magnetic bead carrier used was 1000A general CPG from Beijing Haijing Gaochuang Technology Co., Ltd. The DDTT reagent used for thiophosphate oxidation was from Shanghai Bid Pharmaceutical Technology Co., Ltd. All sequences were synthesized on K&A's H-8 DNA synthesizer according to the instrument instructions, and the DMT on the last base was not removed. The synthesis steps are as follows:

[0112] The single synthesis scale is 0.5 μmol, the CPG size is 1000A, and in order to improve the reaction efficiency, in the coupling reaction step, the natural phosphoramidite monomers use dT-CE phosphoramidite, dC-CE phosphoramidite, dA-CE phosphoramidite, and dG-CE phosphoramidite, and are respectively prepared into 0.1M concentration solutions with anhydrous acetonitrile, and the coupling reaction time is 90 seconds. For the modified monomers obtained by Examples 1-3, anhydrous acetonitrile is used to prepare a 0.12M concentration solution, and the coupling reaction time is 8 minutes each time for two couplings. The synthesis is divided into four steps, including deprotection, coupling, capping (sealing) and oxidation.

[0113] Deprotection: Add a deprotection reagent (95% trichloroacetic acid in dichloromethane) 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: 0.25 M concentration of ethylthiotetrazolyl (ETT) was added as an activator, and then the natural phosphoramidite monomer or modified phosphoramidite monomer was added to the magnetic bead carrier in the synthesis column, the DNA chain was extended, and washed with acetonitrile;

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

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

[0117] (2) CPG was transferred to a 1.5 mL EP tube, and 1.3 mL of 30% concentrated ammonia water was added and sealed. The tube was incubated at 65°C for 2 h with shaking. Subsequently, 10 times the volume of n-butanol solution was added and the tube was allowed to stand at -80°C for 30 min. The tube was centrifuged at 8000 rpm for 25 min to obtain a crude DNA product. After removing the n-butanol, the product was redissolved in 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 replacement using a 3K ultrafiltration tube, and the concentration was quantified using Nanodrop. The mass spectrometry results of nucleic acid aptamers 1, 2, and 3 are shown in Figure 2 , 3 , as shown in Figure 4.

[0119] Example 5

[0120] Benzenesulfonyl fluoride reaction experiment

[0121] 45 μl of 100 μM phosphorothioate-modified aptamer 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, 200 μl of n-butanol and enzyme-free water were used for extraction. The mixture was centrifuged at 150×100 rpm for 5 minutes to completely separate the layers. The above steps were repeated twice. After the reaction, the recovered product was desalted and concentrated by solution replacement using a 3K ultrafiltration tube, and the concentration was quantified using Nanodrop. The structure of 4-(bromomethyl)benzenesulfonyl fluoride is shown in FIG. Figure 5 As shown, the mass spectrometry results of nucleic acid aptamer 4 are as follows Figure 6 shown.

[0122] Example 6

[0123] Surface plasmon resonance (SPR) detection of the affinity of covalent Sgc8c variants to PTK7 protein

[0124] Preparation of aptamer solution: Prepare the following serial dilution solutions of Sgc8c standard and each covalent variant using DPBS buffer: 400 nM, 200 nM, 100 nM, 50 nM, and 25 nM.

[0125] Protein coupling: PTK7 protein was coupled to the second channel of the CM5 chip surface. The specific method was as follows: 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 140 μL was injected to activate the chip at a flow rate of 10 μL / min. PTK7 protein was diluted to a final concentration of 25 μg / mL with 10 mM sodium acetate buffer at pH 4.5 and then injected with an injection volume of 200 μL, a flow rate of 10 μL / min, and a PTK7 protein coupling amount of 6810.5 RU. After the injection was completed, ethanolamine (1M) was injected to close the chip at a flow rate of 10 μL / min and 140 μL was injected. The His small peptide was coupled to the first channel of the CM5 chip surface using the same method as the control channel.

[0126] Affinity activity detection: Use a surface plasmon resonance instrument (GE Healthcare, model: Biacore 8K) to set the detection parameters, and pass the diluted nucleic acid aptamer samples through channels 1 and 2 in turn. The procedure for each aptamer is as follows: injection 30 μL / min, time 2 minutes; dissociation 30 μL / min, time 7 minutes; 1.5M NaCl regeneration, 30 μL / min, time 30 seconds. The affinity detection results of SEQID No.1, No.2, No.3 and No.4 proteins are shown in Figure 2. Figure 7 , 8 , 9, and 10.

[0127] Example 7

[0128] Covalent cross-linking experiments

[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 minutes. The solution was then irradiated with light at a wavelength of 365 nm for 10 minutes at room temperature. For ligand SEQ ID No.4, the incubation was continued at 37°C for 6 hours. 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), heated at 95°C for 10 minutes, and then analyzed by gel electrophoresis at a voltage of 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 nucleoside monomer chemically modified with an exogenous functional group, characterized in that: The chemical modification containing exogenous functional groups includes deoxyuracil phosphoramidite and phosphorothioate modification; The nucleoside monomer includes Sgc8c; the base sequence of Sgc8c is shown in SEQ ID No. 1, specifically: 5'-ATCTAACTGCTGCGCCGCCGGGAAAATACTGTACGGTTAGA-3'.

2. The nucleoside monomer containing chemical modification of exogenous functional groups according to claim 1, characterized in that: The deoxyuracil phosphoramidites include at least one of 5-phenylbis(aziridine) deoxyuracil phosphoramidite, 5-benzyl deoxyuracil phosphoramidite, and 5-fluorophenyl deoxyuracil phosphoramidite.

3. The nucleoside monomer containing chemical modification of exogenous functional groups according to claim 2, characterized in that: The 5-phenylbis(aziridine) deoxyuracil phosphoramidite includes compound 11, the structural formula of which is shown in Formula I: And / or, the 5-benzyldeoxyuracil phosphoramidite includes compound 15, the structural formula of which is shown in Formula II: And / or, the 5-fluorophenyl deoxyuracil phosphoramidite includes compound 19, the structural formula of which is shown in formula III:

4. A covalent nucleic acid aptamer, characterized in that: The nucleic acid aptamer comprises the chemically modified nucleoside monomer containing exogenous functional groups as described in any one of claims 1-3.

5. The covalent nucleic acid aptamer according to claim 4, characterized in that The nucleic acid aptamer includes nucleic acid aptamer 1, which includes the sequence of Sgc8c minus the base A at the 28th position from the 5' end, and the 11th, 27th and 29th positions from the 5' end are modified with 5-phenylbis(aziridine)deoxyuracil phosphoramidite to replace T, and the 23rd, 24th and 26th positions are independently modified with thiophosphate.

6. The covalent nucleic acid aptamer according to claim 4 or 5, characterized in that The nucleic acid aptamer also includes nucleic acid aptamer 2, which includes the sequence of Sgc8c minus the A at the 28th position from the 5' end, the 11th and 27th positions from the 5' end are modified with 5-benzyl deoxyuracil phosphoramidite to replace T, the 29th position is modified with 5-phenyl diaziridine deoxyuracil phosphoramidite to replace T, and the 19th, 21st, 23rd, 24th, 25th, 26th, 27th and 29th positions are each independently modified with thiophosphate.

7. The covalent nucleic acid aptamer according to any one of claims 4 to 6, characterized in that: The nucleic acid aptamer also includes nucleic acid aptamer 3, which includes the sequence of Sgc8c minus the A at the 28th position from the 5' end, the 11th and 27th positions from the 5' end of the sequence are modified with 5-fluorophenyl deoxyuracil phosphoramidite to replace T, the 29th position is modified with 5-phenyl diaziridine deoxyuracil phosphoramidite to replace T, and the 19th, 21st, 23rd, 24th, 25th, 26th, 27th and 29th positions are each independently modified with thiophosphate.

8. The covalent nucleic acid aptamer according to any one of claims 4 to 7, characterized in that: The nucleic acid aptamer also includes nucleic acid aptamer 4, which includes subtracting the 28th position A from the sequence of Sgc8c, and replacing T with 5-phenylbis(aziridine) deoxyuracil phosphoramidite at the 11th, 27th and 29th positions from the 5' end of the sequence, and independently modifying the thiophosphate at the 23rd, 24th and 26th positions and modifying the aryl sulfonyl fluoride on the thiophosphate backbone.

9. Use of the nucleic acid aptamer according to any one of claims 4 to 8 in high-sensitivity recognition of PTK7 protein.

10. Use of the nucleic acid aptamer according to any one of claims 4 to 8 for high-specific recognition of PTK7 protein.

Images