High-coupling-efficiency oligonucleotide synthesis chip and application thereof

Abstract

The invention belongs to the technical field of oligonucleotide synthesis, and particularly relates to a high-coupling-efficiency oligonucleotide synthesis chip and application thereof. The surface treatment method of the high-coupling-efficiency oligonucleotide synthesis chip provided by the invention comprises hydroxylation treatment, amination treatment, long connecting arm connection and the like. Through a specific surface treatment process, the DMT load of the high-coupling-efficiency oligonucleotide synthesis chip is remarkably improved, and when the chip is used for synthesizing oligonucleotides, especially medium-chain oligonucleotides, the coupling efficiency and the product purity can be remarkably improved, and the batch-to-batch difference can be reduced. The invention also provides a synthesis method of the medium-chain oligonucleotide, according to the synthesis method, the high-coupling-efficiency oligonucleotide synthesis chip is used as a solid-phase carrier, and a specific synthesis process is combined, so that the coupling efficiency can be further improved, and by-products and differences between batches can be reduced; the problems of low coupling efficiency, obvious fluctuation, more byproducts, large batch-to-batch difference and the like during synthesis of the medium-chain oligonucleotide in the prior art are effectively solved.

Description

Technical Field

[0001] This invention belongs to the field of oligonucleotide synthesis technology, specifically relating to a high-coupling-efficiency oligonucleotide synthesis chip and its application. Background Technology

[0002] The phosphoramide solid-phase method is the mainstream technique for synthesizing oligonucleotides. The commonly used activator is the ETT (5-ethylthiotetrazole)-acetonitrile system. When used for oligonucleotides ≤30 nt, the coupling efficiency can reach approximately 98%. However, when used for medium-chain (40–60 nt) oligonucleotides, the yield varies greatly between batches (RSD > 20%), and the coupling efficiency fluctuates significantly (single-chain difference > 0.5%). Furthermore, it produces many byproducts (n-1, depurinates), with n-1 > 3%. The DCI-acetonitrile system has been reported, but its use in current techniques is limited to sporadic applications in acetonitrile systems and has not been observed for the synthesis of 40–60 nt medium-chain oligonucleotides. Its coupling efficiency also fluctuates by more than 0.5%. In addition, both methods use acetonitrile solvent, which is volatile, leading to monomer concentration drift.

[0003] In addition, the surface DMT loading of CPG supports commonly used in the phosphorus amide solid-phase method is typically 1.0-1.2 nmol·mm⁻¹. -2 This results in a medium-chain oligonucleotide purity of ≤65%. Summary of the Invention

[0004] To address the above technical problems, this invention provides a high-coupling-efficiency oligonucleotide synthesis chip and its application. The oligonucleotide synthesis chip provided by this invention is suitable for the synthesis of medium-chain (40–60 nt) oligonucleotides, and has the advantages of high and stable coupling efficiency, low batch-to-batch variation, and few byproducts, solving the problems of low and fluctuating coupling efficiency, large batch-to-batch variation, and numerous byproducts in existing synthesis methods.

[0005] To achieve the above-mentioned objectives, the present invention adopts the following technical solution: The first aspect of this invention provides a high-coupling-efficiency oligonucleotide synthesis chip, the preparation method of which includes the following steps: S1. Hydroxylation: The blank substrate is placed in a hydrochloric acid-ethanol mixture at 35-39℃ and soaked for 2.5-3.5h. Then, it is placed in 75%-100%wt ethanol and ultrasonically treated for 3-5min. After drying, it is placed in deionized water at 85-95℃ and soaked for 2.5-3.5h. The hydrochloric acid-ethanol mixture is composed of 36%-38%wt hydrochloric acid and 75%-100%wt ethanol in a volume ratio of (0.6-1):(0.6-1). S2, Aminoation: The hydroxylated substrate is immersed in anhydrous ethanol containing 0.2-1M 3-aminopropyltriethoxysilane and 4-6 drops / L glacial acetic acid at 45-55℃ for 16-24 hours, then removed and dried; S3. Place the aminated substrate in acetonitrile containing 2-8 g / L Unylinker (CAS No. 852684-08-3), 2-8 g / L HBTU (benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate, CAS No. 94790-37-1), and 1-3 mL / L activator and sonicate for 25-35 min. Remove and dry. The activator is N,N-diisopropylethylamine and triethylamine in a volume ratio of (1-20):(1-20). S4. Capping: Place the substrate with the completed long connecting arm connection in the capping agent solution and soak it at room temperature for 10-15 minutes. S5. Clean and dry to obtain the high-coupling-efficiency oligonucleotide synthesis chip.

[0006] The chip surface exhibits a high DMT (dimethoxytriphenylmethyl) loading, reaching 2.2 pmol·mm⁻¹. -2 The above-mentioned components are evenly distributed, and the stability coefficient of DMT loading between chips is <5%. Using this chip to synthesize medium-chain oligonucleotides can significantly improve the purity of the product.

[0007] Preferably, the blank substrate is made of pure silicon substrate with a silicon dioxide oxide layer formed by thermal oxidation on its surface.

[0008] Preferably, the hydrochloric acid-ethanol mixture is composed of 37%wt hydrochloric acid and anhydrous ethanol in a 1:1 volume ratio.

[0009] Preferably, the temperature of the hydrochloric acid-ethanol mixture in S1 is 37°C.

[0010] More preferably, the soaking time in the hydrochloric acid-ethanol mixture in S1 is 3 hours.

[0011] Preferably, the ultrasonic treatment in S1 is performed in anhydrous ethanol.

[0012] Preferably, the temperature of the deionized water in S1 is 90°C.

[0013] More preferably, the soaking time in the deionized water in S1 is 3 hours.

[0014] Preferably, the temperature of anhydrous ethanol containing 0.2~1M 3-aminopropyltriethoxysilane and 4~6 drops / L glacial acetic acid in S2 is 50°C.

[0015] Preferably, the concentration of 3-aminopropyltriethoxysilane in S2 is 0.2~0.4M.

[0016] More preferably, the concentration of 3-aminopropyltriethoxysilane in S2 is 0.2M.

[0017] Preferably, the amount of glacial acetic acid added to the anhydrous ethanol in S2 is 4 drops / L.

[0018] Preferably, the soaking time in S2 is 24 hours.

[0019] Preferably, the concentration of Unylinker in S3 is 2~2.4 g / L, and the concentration of HBTU is 2~2.4 g / L.

[0020] More preferably, the concentration of Unylinker in S3 is 2 g / L, and the concentration of HBTU is 2 g / L.

[0021] Preferably, the ultrasonic treatment time in S3 is 30 minutes.

[0022] Preferably, the ultrasonic power of the ultrasonic treatment in S3 is 20~200W, and the ultrasonic temperature is 15~35℃.

[0023] Preferably, the activator in S3 is N,N-diisopropylethylamine and triethylamine in a volume ratio of 1:1.

[0024] More preferably, the content of the activator in S3 is 2 mL / L.

[0025] Preferably, the capping agent in S4 is Cap A and Cap B in a volume ratio of 1:1.

[0026] More preferably, Cap A consists of acetic anhydride and tetrahydrofuran mixed in a volume ratio of 10:90; and Cap B consists of N-methylimidazolium, pyridine, and tetrahydrofuran mixed in a volume ratio of 16:10:74.

[0027] Preferably, the cleaning operation in S5 is as follows: first clean with acetonitrile, and then clean with anhydrous ethanol.

[0028] More preferably, the cleaning is ultrasonic cleaning.

[0029] The second aspect of this invention provides the application of the above-mentioned high-coupling-efficiency oligonucleotide synthesis chip in the synthesis of medium-chain oligonucleotides.

[0030] A third aspect of this invention provides a method for synthesizing medium-chain oligonucleotides, specifically comprising the following steps: Step a: Rinse the above high-coupling-efficiency oligonucleotide synthesis chip with PC (propylene carbonate) with a water content ≤30ppm, and transfer it into the synthesis column; Step b: Pump the deprotecting agent TCA into the synthesis column; Step c: The phosphorus amide-protected nucleotide monomer is mixed with the activator and fed into a synthesis column containing the high-coupling-efficiency oligonucleotide synthesis chip for coupling reaction. The coupling reaction time is 2-5 min. The activator is a 0.25±0.05M DCI (4,5-dicyanimidazolium) / PC solution. Step d: Add a cap; Step e, oxidation; after the oxidation reaction is completed, the process is repeated starting from step a until the synthesis of the bases of the medium-chain oligonucleotide is completed. Step f: Ammonolysis.

[0031] This synthesis method utilizes the aforementioned high-coupling-efficiency oligonucleotide synthesis chip as a solid-phase carrier and combines it with a specific synthesis process. It can significantly improve the coupling efficiency and stabilize it to ≥99.0%, while reducing by-products (n-1 < 1.5%). Furthermore, it can reduce the batch-to-batch variation in the synthesis of 40-60nt medium-chain oligonucleotides, achieving an RSD < 3%. This effectively solves the problems of low and fluctuating coupling efficiency, numerous by-products, and large batch-to-batch variation in the synthesis of medium-chain oligonucleotides using existing technologies.

[0032] Preferably, the coupling reaction takes 4 minutes.

[0033] The beneficial effects of this invention are as follows: Compared with the prior art, the high-coupling-efficiency oligonucleotide synthesis chip provided by this invention can significantly improve batch consistency and coupling efficiency, reduce n-1 byproducts, and achieve high purity of the synthesized product. The medium-chain oligonucleotide synthesis method provided by this invention utilizes this high-coupling-efficiency oligonucleotide synthesis chip as a solid-phase carrier, combined with a specific synthesis process, to further reduce batch-to-batch variability and byproducts, further improve coupling efficiency and purity of the synthesized product, and achieve a single target peak in the 40–60 nt mass spectrometry, meeting the quality requirements of nucleic acid drugs without additional purification. Attached Figure Description

[0034] Figure 1 This is a schematic diagram of the process steps S1 to S3 in the chip manufacturing of the present invention; Figure 2 This is the concentration-DMT loading curve of the long connecting arm in Example 1 of the present invention; Figure 3 This is the amination concentration-DMT loading curve in Example 1 of the present invention; Figure 4 This is the hydroxylation time-DMT loading curve in Example 1 of this invention; Figure 5HPLC chromatograms of 10 oligonucleotides (40nt) were obtained for this invention (main peak 17.2 min, 300 dpi). Figure 6 MS deconvolution peaks of 10 oligonucleotides (40 nt) were performed for this invention; Figure 7 HPLC chromatograms of 11 oligonucleotides (60nt) were obtained for this invention (main peak 19.5 min, 300 dpi). Figure 8 This is a scatter plot of purity-coupling efficiency in Comparative Example 9 of the present invention; Figure 9 This is a scatter plot of purity-coupling efficiency in Comparative Example 10 of the present invention. Detailed Implementation

[0035] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention. All other embodiments obtained by those skilled in the art based on the implementation methods of this invention without inventive effort fall within the protection scope of this invention.

[0036] In the existing technology, the commonly used activator for the solid-phase synthesis of oligonucleotides using phosphoramidite is mainly the ETT-acetonitrile system, which has problems such as low coupling efficiency, large batch-to-batch variation, and many by-products in the synthesis of medium-chain (40–60 nt) oligonucleotides.

[0037] To address the above problems, embodiments of the present invention provide a high-coupling-efficiency oligonucleotide synthesis chip, the preparation method of which includes the following steps: S1. Hydroxylation: The blank substrate is placed in a hydrochloric acid-ethanol mixture at 35-39℃ and soaked for 2.5-3.5h. Then, it is placed in 75%-100%wt ethanol and ultrasonically treated for 3-5min. After drying, it is placed in deionized water at 85-95℃ and soaked for 2.5-3.5h. The hydrochloric acid-ethanol mixture is composed of 36%-38%wt hydrochloric acid and 75%-100%wt ethanol in a volume ratio of (0.6-1):(0.6-1). S2, Aminoation: The hydroxylated substrate is immersed in anhydrous ethanol containing 0.2-1M 3-aminopropyltriethoxysilane and 4-6 drops / L glacial acetic acid at 45-55℃ for 16-24 hours, then removed and dried; S3. Place the aminated substrate in acetonitrile containing 2-8 g / L Unylinker, 2-8 g / L HBTU, and 1-3 mL / L activator, and sonicate for 25-35 min. Remove and dry. The activator is N,N-diisopropylethylamine and triethylamine in a volume ratio of (1-20):(1-20). The flowcharts for S1-S3 are as follows: Figure 1 As shown; S4. Capping: Place the substrate with the completed long connecting arm connection in the capping agent solution and soak it at room temperature for 10-15 minutes. S5. Clean and dry to obtain the high-coupling-efficiency oligonucleotide synthesis chip.

[0038] This invention also provides the application of the above-mentioned high-coupling-efficiency oligonucleotide synthesis chip in the synthesis of medium-chain oligonucleotides.

[0039] This invention also provides a method for synthesizing medium-chain oligonucleotides, specifically including the following steps: Step a: Rinse the above high-coupling-efficiency oligonucleotide synthesis chip with PC containing ≤30ppm water, and then transfer it into the synthesis column; Step b: Pump the deprotecting agent TCA into the synthesis column; Step c: The phosphorus amide-protected nucleotide monomer is mixed with an activator and fed into a synthesis column containing the above-mentioned high-coupling-efficiency oligonucleotide synthesis chip for coupling reaction. The coupling reaction time is 2-5 min. The activator is a 0.25±0.05M DCI / PC solution. Step d: Add a cap; Step e, oxidation; after the oxidation reaction is completed, the process is repeated starting from step a until the synthesis of the bases of the medium-chain oligonucleotide is completed. Step f: Ammonolysis.

[0040] The 0.25±0.05M DCI / PC solution indicates that the concentration of DCI in PC is 0.25±0.05M, that is, each 1L of PC contains 0.25±0.05M DCI.

[0041] The present invention will be described below through specific embodiments.

[0042] Unless otherwise specified, the experimental methods used in the following examples are conventional methods in the art. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0043] Example 1 This embodiment provides a high-coupling-efficiency oligonucleotide synthesis chip, and the specific steps of its preparation method are as follows: S1, Hydroxylation: A blank substrate (single-layer polished silicon dioxide wafer, 60×20×0.6mm, effective reaction area ≈2400mm²) is prepared. 2 The substrate was immersed in a 37°C hydrochloric acid-ethanol mixture (composed of 37%wt hydrochloric acid and anhydrous ethanol in a 1:1 volume ratio) for 3 hours, then ultrasonically treated in anhydrous ethanol for 5 minutes. After drying with nitrogen, it was immersed in 90°C deionized water for 3 hours. After immersion, the thickness of the water-containing layer on the substrate surface was approximately 1.2 nm, and the contact angle was ≤10°. S2, Aminoation: The hydroxylated substrate was immersed in 500 mL of anhydrous ethanol containing 0.60 M 3-aminopropyltriethoxysilane and 2 drops of glacial acetic acid at 50 °C for 24 h, then removed and dried with nitrogen. S3. Connecting the long connecting arm: Place the amination-completed substrate in 500 mL of acetonitrile containing 1.0 g Unylinker + 1.0 g HBTU + 1 mL activator, and sonicate (ultrasonic power of 150 W, ultrasonic temperature of 30 °C) for 30 min, then remove and dry with nitrogen gas; the activator is N,N-diisopropylethylamine and triethylamine in a volume ratio of 1:1.

[0044] S4. Capping: Place the substrate with the completed long connecting arm in a capping agent (Cap A:Cap B=1:1) solution and soak at room temperature for 15 min; Cap A consists of acetic anhydride and tetrahydrofuran, mixed in a volume ratio of 10:90; Cap B consists of N-methylimidazolium, pyridine and tetrahydrofuran, mixed in a volume ratio of 16:10:74. S5. Cleaning: First, ultrasonic cleaning is performed with acetonitrile, followed by ultrasonic cleaning with anhydrous ethanol, and then drying with nitrogen gas to obtain the oligonucleotide synthesis chip. The surface modification density of this oligonucleotide synthesis chip is 2.36 ± 0.07 pmol·mm⁻¹. -2 (n=5, RSD=2.9%).

[0045] Example 2 This embodiment provides a high-coupling-efficiency oligonucleotide synthesis chip, and the specific steps of its preparation method are as follows: S1, Hydroxylation: Same as in Example 1; S2, Amination: Same as in Example 1; S3. Connecting the long connecting arm: Place the amination-completed substrate in 500 mL of acetonitrile containing 2.0 g Unylinker + 2.0 g HBTU + 1 mL activator, and sonicate (ultrasonic power 150 W, ultrasonic temperature 30 °C) for 30 min, then remove and dry with nitrogen gas; the activator is N,N-diisopropylethylamine and triethylamine in a volume ratio of 1:1. S4, Cap: Same as in Example 1; S5. Cleaning: Same as in Example 1.

[0046] Example 3 This embodiment provides a high-coupling-efficiency oligonucleotide synthesis chip, and the specific steps of its preparation method are as follows: S1, Hydroxylation: Same as in Example 1; S2, Amination: Same as in Example 1; S3. Connecting the long connecting arm: Place the amination-completed substrate in 500 mL of acetonitrile containing 3.0 g Unylinker + 3.0 g HBTU + 1 mL activator, and sonicate (ultrasonic power 150 W, ultrasonic temperature 30 °C) for 30 min, then remove and dry with nitrogen gas; the activator is N,N-diisopropylethylamine and triethylamine in a volume ratio of 1:1. S4, Cap: Same as in Example 1; S5. Cleaning: Same as in Example 1.

[0047] Example 4 This embodiment provides a high-coupling-efficiency oligonucleotide synthesis chip, and the specific steps of its preparation method are as follows: S1, Hydroxylation: Same as in Example 1; S2, Amination: Same as in Example 1; S3. Connecting the long connecting arm: Place the amination-completed substrate in 500 mL of acetonitrile containing 4.0 g Unylinker + 4.0 g HBTU + 1 mL activator, and sonicate (ultrasonic power 150 W, ultrasonic temperature 30 °C) for 30 min, then remove and dry with nitrogen gas; the activator is N,N-diisopropylethylamine and triethylamine in a volume ratio of 1:1. S4, Cap: Same as in Example 1; S5. Cleaning: Same as in Example 1.

[0048] Example 5 This embodiment provides a high-coupling-efficiency oligonucleotide synthesis chip, and the specific steps of its preparation method are as follows: S1, Hydroxylation: Same as in Example 1; S2, Aminoation: The hydroxylated substrate was immersed in 500 mL of anhydrous ethanol containing 0.20 M 3-aminopropyltriethoxysilane and 2 drops of glacial acetic acid at 50 °C for 24 h, then removed and dried with nitrogen. S3. Connecting the long connecting arm: Same as in Example 3; S4, Cap: Same as in Example 1; S5. Cleaning: Same as in Example 1.

[0049] Example 6 This embodiment provides a high-coupling-efficiency oligonucleotide synthesis chip, and the specific steps of its preparation method are as follows: S1, Hydroxylation: Same as in Example 1; S2, Aminoation: The hydroxylated substrate was immersed in 500 mL of anhydrous ethanol containing 0.40 M 3-aminopropyltriethoxysilane and 2 drops of glacial acetic acid at 50 °C for 24 h, then removed and dried with nitrogen. S3. Connecting the long connecting arm: Same as in Example 3; S4, Cap: Same as in Example 1; S5. Cleaning: Same as in Example 1.

[0050] Example 7 This embodiment provides a high-coupling-efficiency oligonucleotide synthesis chip, and the specific steps of its preparation method are as follows: S1, Hydroxylation: Same as in Example 1; S2, Aminoation: The hydroxylated substrate was immersed in 500 mL of anhydrous ethanol containing 1.00 M 3-aminopropyltriethoxysilane and 2 drops of glacial acetic acid at 50 °C for 24 h, then removed and dried with nitrogen. S3. Connecting the long connecting arm: Same as in Example 3; S4, Cap: Same as in Example 1; S5. Cleaning: Same as in Example 1.

[0051] Example 8 This embodiment provides a high-coupling-efficiency oligonucleotide synthesis chip, and the specific steps of its preparation method are as follows: S1, Hydroxylation: A blank substrate (single-layer polished silicon dioxide wafer, 60×20×0.6mm, effective reaction area ≈2400mm²) is prepared. 2 The sample was immersed in a 37°C hydrochloric acid-ethanol mixture (composed of 37%wt hydrochloric acid and anhydrous ethanol in a 1:1 volume ratio) for 4 hours, then ultrasonically treated in anhydrous ethanol for 5 minutes, dried with nitrogen, and then immersed in 90°C deionized water for 4 hours. S2, Amination: Same as in Example 1; S3. Connecting the long connecting arm: Same as in Example 3; S4, Cap: Same as in Example 1; S5. Cleaning: Same as in Example 1.

[0052] Example 9 This embodiment provides a high-coupling-efficiency oligonucleotide synthesis chip, and the specific steps of its preparation method are as follows: S1, Hydroxylation: A blank substrate (single-layer polished silicon dioxide wafer, 60×20×0.6mm, effective reaction area ≈2400mm²) is prepared. 2The sample was immersed in a 37°C hydrochloric acid-ethanol mixture (composed of 37%wt hydrochloric acid and anhydrous ethanol in a 1:1 volume ratio) for 5 hours, then ultrasonically treated in anhydrous ethanol for 5 minutes, dried with nitrogen, and then immersed in 90°C deionized water for 5 hours. S2, Amination: Same as in Example 1; S3. Connecting the long connecting arm: Same as in Example 3; S4, Cap: Same as in Example 1; S5. Cleaning: Same as in Example 1.

[0053] Comparative Examples 1-3 Comparative Examples 1-3 each provided an oligonucleotide synthesis chip, and the specific steps of its preparation method were the same as in Example 1, except that the amounts of Unylinker and HBTU in 500 mL of acetonitrile in S3 were 0.05 g, 0.1 g, and 0.5 g, respectively. As shown in Table 1.

[0054] Table 1. Concentrations of long connecting arms in Comparative Examples 1-3

[0055] Comparative Examples 4-6 Comparative Examples 4-6 each provide an oligonucleotide synthesis chip, and the specific steps of its preparation method are the same as those in Example 1, except that the concentration of 3-aminopropyltriethoxysilane in 500 mL of anhydrous ethanol in S2 is 0.005 M, 0.01 M, and 0.05 M, respectively.

[0056] Comparative Examples 7 and 8 Comparative Examples 7 and 8 respectively provide an oligonucleotide synthesis chip. The specific steps of its preparation method are the same as those in Example 1. The difference is that in S1, the blank substrate is soaked in hydrochloric acid-ethanol mixture for 1 h and 2 h, respectively. After being ultrasonically treated with anhydrous ethanol and dried with nitrogen, it is soaked in deionized water at 90°C for 1 h and 2 h, respectively.

[0057] Test Example 1 Oligonucleotide synthesis chips were prepared according to the preparation methods of Examples 1-9 and Comparative Examples 1-8, and their DMT loading was detected (n=5).

[0058] The DMT loading results of the oligonucleotide synthesis chips in Examples 1-4 and Comparative Examples 1-3 are as follows: Figure 2 As shown, the DMT loading increases with increasing long linker reagent concentration, but the DMT loading no longer increases after the long linker reagent concentration reaches 2 g / L (i.e., 1 g Unylinker and 1 g HBTU in 500 mL of acetonitrile).

[0059] The DMT loading results of the oligonucleotide synthesis chips in Examples 5-7 and Comparative Examples 4-6 are as follows: Figure 3 As shown, DMT loading increases with increasing amination reagent concentration, but after the amination reagent concentration reaches 0.2 M, DMT loading no longer increases but instead decreases, and then increases slightly after 0.6 M.

[0060] The DMT loading results of the oligonucleotide synthesis chips in Examples 8 and 9 and Comparative Examples 7 and 8 are as follows: Figure 4 As shown, DMT loading increases with prolonged hydroxylation time, but the hydroxylation time begins to decrease after 3 hours.

[0061] Example 10 This embodiment provides a method for synthesizing a medium-chain oligonucleotide (40nt).

[0062] The sequence of the oligonucleotide is shown in SEQ ID NO.1: 5'-TTCGGTCCACTGTTTCGGTCGAGTTTGTGCGCGAGTTTGC-3'. The synthesis scale was 200 nmol.

[0063] The synthesis steps are as follows: (1) Rinse the high coupling efficiency oligonucleotide synthesis chip of Example 1 with PC (propylene carbonate) with a water content of 25 ppm, and transfer it into the synthesis column; (2) Pump 200 μL of 3% TCA (trichloroacetic acid) / dichloromethane solution into the synthesis column as a deprotecting agent; (3) The phosphorus amide-protected nucleotide monomer was mixed with 0.25M DCI / PC activator solution and fed into a synthesis column equipped with the above-mentioned high-coupling-efficiency oligonucleotide synthesis chip for coupling reaction. The concentration of nucleotide monomer solution was 0.04M, the volume ratio of nucleotide monomer solution to activator solution was 1:1.44, and the coupling time was 4min. (4) Capping: Add capping reagents CapA and CapB. CapA consists of acetic anhydride and tetrahydrofuran, mixed in a volume ratio of 10:90; CapB consists of N-methylimidazolium, pyridine and tetrahydrofuran, mixed in a volume ratio of 16:10:74. (5) Oxidation: Add iodine solution with an iodine concentration of 0.025 mol / L and a solvent of pyridine, water and tetrahydrofuran in a volume ratio of 20:10:70; (6) Steps (1) to (5) constitute a synthetic cycle reaction. Each time a synthetic cycle reaction is completed, the oligonucleotide chain on the carrier is increased by one base, thus completing the synthesis of the above oligonucleotide. (7) Ammoniacide is prepared by mixing methylamine and ammonia in equal volume ratio at 65°C for 2 hours.

[0064] Testing showed that the coupling efficiency of the oligonucleotide synthesized using the high coupling efficiency oligonucleotide synthesis chip of Example 1 was 99.06%. This was confirmed by HPLC (e.g., [missing information - likely a HPLC method]). Figure 5 The purity of the obtained oligonucleotides (as shown in Table 2) was determined, and the product purity was 68.8%. MS 12323.02 Da (as shown in Table 2) Figure 6 (as shown in SEQ ID NO. 1), n-1=1.1%. Three batches of oligonucleotides with sequences as shown in SEQ ID NO. 1 were synthesized using the above method, with a coupling efficiency RSD=2.8%.

[0065] Table 2 Figure 5 chromatographic peaks

[0066] Example 11 This embodiment provides a method for synthesizing oligonucleotides (60nt).

[0067] The sequence of the oligonucleotide is shown in SEQ ID NO.2: 5'-TTCGGTCCACTGTTTCGGTCTTCGGTCCACTGTTTCGGTCGAGTTTGTGCGCGAGTTTGC-3'.

[0068] The synthesis steps are the same as in Example 10.

[0069] Testing showed that the coupling efficiency of the oligonucleotide synthesized using the high coupling efficiency oligonucleotide synthesis chip of Example 1 was 99.26%. This was confirmed by HPLC (e.g., [missing information - likely a HPLC method]). Figure 7 The purity of the obtained oligonucleotides (as shown in Table 3) was determined, and the product purity was 64.25%. MS 18450.96 Da, n-1=1.3%. Three batches of oligonucleotides with sequences as shown in SEQ ID NO.2 were synthesized according to the above method, with a coupling efficiency RSD=3.0%.

[0070] Table 3 Figure 7 chromatographic peaks

[0071] Comparative Example 9 This comparative example provides a method for synthesizing an oligonucleotide (40nt). The sequence of the oligonucleotide is shown in SEQ ID NO.1.

[0072] The synthesis steps are basically the same as in Example 10, except that the oligonucleotide synthesis chip is the same as that in Comparative Example 3 (DMT 1.516 pmol·mm). -2 ).

[0073] The coupling efficiency of the oligonucleotide synthesized using the oligonucleotide synthesis chip in Comparative Example 3 was 97.2%, n-1=2.8%, and the RSD of the coupling efficiency of the three batches of oligonucleotides synthesized was 16.4%, which was significantly different from that in Example 10 (t test, P<0.01).

[0074] Purity-coupling efficiency scatter plot as follows Figure 8 As shown (n=5, linear regression R), 2 (≥0.95). This result demonstrates that the high coupling efficiency oligonucleotide synthesis chip provided by this invention can achieve higher and more stable coupling efficiency.

[0075] Comparative Example 10 This comparative example provides a method for synthesizing an oligonucleotide (40nt). The sequence of the oligonucleotide is shown in SEQ ID NO.1.

[0076] The synthesis steps are basically the same as in Example 10, except that the activator is a 0.25M ETT-acetonitrile solution.

[0077] The coupling efficiency of the oligonucleotide synthesized using 0.25M ETT-acetonitrile solution as an activator was 98.47%, with RSD of 18.5% and n-1 of 3.1%, which was significantly higher than that in Example 10.

[0078] Purity-coupling efficiency scatter plot as follows Figure 9 As shown (n=5, linear regression R), 2 (≥0.95). This result demonstrates that the oligonucleotide synthesis method of the present invention can achieve higher and more stable coupling efficiency.

[0079] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A high coupling efficiency oligonucleotide synthesis chip, characterized in that, The preparation method comprises the following steps: S1, hydroxylation: placing a blank substrate in a 35-39℃ hydrochloric acid-ethanol mixed solution, soaking for 2.5-3.5h, then placing in 75%-100%wt ethanol for ultrasonic treatment for 3-5min, drying, and then placing in 85-95℃ deionized water for soaking for 2.5-3.5h; the hydrochloric acid-ethanol mixed solution is composed of 36%-38%wt hydrochloric acid and 75%-100%wt ethanol at a volume ratio of (0.6-1):(0.6-1); S2, amination: placing the substrate after hydroxylation in 45-55℃ anhydrous ethanol containing 0.2-1M 3-aminopropyl triethoxysilane and 4-6 drops / L glacial acetic acid for 16-24h, taking out, and drying; S3, placing the substrate after amination in acetonitrile containing 2-8g / L Unylinker, 2-8g / L HBTU and 1-3mL / L activating agent for ultrasonic treatment for 25-35min, taking out, and drying; wherein the activating agent is N,N-diisopropylethylamine and triethylamine at a volume ratio of (1-20):(1-20); S4, capping: placing the substrate after long arm connection in a capping agent solution, soaking for 10-15min at room temperature; S5, washing and drying, to obtain the high-coupling-efficiency oligonucleotide synthesis chip.

2. The high coupling efficiency oligonucleotide synthesis chip of claim 1, wherein, The blank substrate is made of pure silicon substrate, and an oxide layer of silicon dioxide generated by thermal oxidation exists on the surface; and / or The hydrochloric acid-ethanol mixed solution is composed of 37%wt hydrochloric acid and anhydrous ethanol at a volume ratio of 1:1; and / or The temperature of the hydrochloric acid-ethanol mixed solution in S1 is 37℃; and / or The ultrasonic treatment in S1 is performed in anhydrous ethanol; and / or The temperature of the deionized water in S1 is 90℃.

3. The high coupling efficiency oligonucleotide synthesis chip of claim 2, wherein, The soaking time in the hydrochloric acid-ethanol mixed solution in S1 is 3h; and / or The soaking time in the deionized water in S1 is 3h.

4. The high coupling efficiency oligonucleotide synthesis chip of claim 1, wherein, The temperature of the anhydrous ethanol containing 0.2-1M 3-aminopropyl triethoxysilane and 4-6 drops / L glacial acetic acid in S2 is 50℃; and / or The concentration of the 3-aminopropyl triethoxysilane in S2 is 0.2-0.4M; and / or The addition amount of the glacial acetic acid in the anhydrous ethanol in S2 is 4 drops / L; and / or The soaking time in S2 is 24h.

5. The high coupling efficiency oligonucleotide synthesis chip of claim 4, wherein, The concentration of the 3-aminopropyl triethoxysilane in S2 is 0.2M.

6. The high coupling efficiency oligonucleotide synthesis chip of claim 1, wherein, The concentration of the Unylinker in S3 is 2-2.4g / L, and the concentration of the HBTU is 2-2.4g / L; and / or The ultrasonic treatment time in S3 is 30min; and / or The ultrasonic treatment power in S3 is 20-200W, and the ultrasonic temperature is 15-35℃; and / or The activating agent in S3 is N,N-diisopropylethylamine and triethylamine at a volume ratio of 1:

1.

7. The high coupling efficiency oligonucleotide synthesis chip of claim 6, wherein, The concentration of the Unylinker in S3 is 2g / L, and the concentration of the HBTU is 2g / L; and / or The content of the activating agent in S3 is 2mL / L.

8. The high coupling efficiency oligonucleotide synthesis chip of claim 1, wherein, The capping agent in S4 is CapA and Cap B at a volume ratio of 1:1; and / or The washing operation in S5 is: first washing with acetonitrile, and then washing with ethanol.

9. Use of the high coupling efficiency oligonucleotide synthesis chip according to any one of claims 1-8 in the synthesis of middle-chain oligonucleotides.

10. A method of synthesis of a medium chain oligonucleotide, characterized in that, Specifically comprising the following steps: Step a, flushing the high coupling efficiency oligonucleotide synthesis chip according to any one of claims 1-8 with PC with water content ≤30 ppm, and transferring into a synthesis column; Step b, pumping a deprotection agent TCA into the synthesis column; Step c, mixing the phosphoramidite-protected nucleotide monomer with an activating agent and entering the synthesis column with the high coupling efficiency oligonucleotide synthesis chip to perform a coupling reaction, the coupling reaction time being 2-5 min; the activating agent being a 0.25±0.05 M DCI / PC solution; Step d, capping; Step e, oxidation; after the oxidation reaction is completed, repeating from step a until the synthesis of the bases of the middle-chain oligonucleotide is completed; Step f, ammonolysis.

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