Oligonucleotide separation and recovery method and application thereof

Abstract

The invention provides an oligonucleotide separation and recovery method and application thereof, and the method comprises the following steps: mixing an oligonucleotide-containing test solution with an acetone solution of a lithium salt, then centrifuging the mixed solution, and reserving the precipitate, wherein the lithium salt is selected from any one or two of lithium chloride and lithium sulfate. The method has the advantages of simple operation, high separation efficiency, high recovery rate of oligonucleotide, high product purity, and good industrial application prospect.

Description

Technical Field

[0001] The invention belongs to the technical field of oligonucleotide production, and specifically relates to a method for separating and recovering oligonucleotides and applications thereof. Background Art

[0002] Oligonucleotides, also known as short-chain nucleotides, include nucleotides in deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). Oligonucleotides can easily link with their complementary pairs, so they are often used as primers or probes to determine the structure of DNA or RNA. They are often used in gene chips, electrophoresis, fluorescence in situ hybridization and other processes. Primer is a short segment of single-stranded DNA or RNA, which serves as the starting point for DNA replication and the starting point for the extension of each polynucleotide chain during nucleic acid synthesis. At present, primer synthesis is mainly carried out by solid-phase phosphoramidite triester method. Solid-phase phosphoramidite triester method synthesizes DNA fragments, which has the characteristics of high efficiency, rapid coupling and relatively stable starting reactants; this method completes the synthesis of DNA chains on a solid phase carrier. Unlike the enzymatic DNA synthesis process, which extends from the 5→3 direction, DNA chemical synthesis starts from the 3' end, and adjacent nucleotides are connected by 3→5 phosphodiester bonds.

[0003] The precipitation separation method is a separation method based on the solubility product principle and utilizing precipitation reaction. In the test solution to be separated, an appropriate precipitant is added, and under certain conditions, the predicted component is precipitated, or the interfering component is analyzed and precipitated to achieve the purpose of removing interference. In the coupling reaction of oligonucleotides, due to the complex components in the water sample, there are many interfering factors, and the organic reaction is a hydrophobic reaction; therefore, the main components in the water sample must be separated and enriched before the reaction to eliminate interference and inhibition during the coupling process, improve the efficiency of the coupling reaction, and meet the synthesis quality of the final product. In the prior art, alcohol precipitation is generally used to remove excess free modification dyes and buffer salts in the synthesized primer probes, because for specific application fields, these free modification dyes and salts need to be removed in advance in subsequent production, otherwise they will affect subsequent reactions.

[0004] However, the alcohol precipitation method cannot completely precipitate the DNA / RNA modified probes. The DNA / RNA modified probes are lost during the precipitation process, and the general precipitation loss rate is about 20-40%. In addition, a large amount of organic solvents are required to repeatedly precipitate and wash during the treatment process, and the material cost is too high. At the same time, the alcohol reagent used in the precipitation washing process needs to be pre-cooled at 20°C for 1-2 hours, which has high process requirements and cumbersome process.

[0005] Patent CN114317521A also proposes a method for purifying oligoribonucleotides (RNA) using a perchlorate solution. However, the perchlorate used is an explosive substance and is subject to national control. In addition, the method requires mixing the perchlorate solution with the oligoribonucleotide solution and then keeping it at 20°C to -40°C for 10 to 60 minutes. The purification time is long and the conditions are relatively harsh. Therefore, this method is limited in industrial applications. Moreover, this method is mainly used for the purification of oligoribonucleotides with short sequences (less than 10 nt in length) and high U content sequences.

[0006] In summary, there is an urgent need to provide a method for purifying oligoribonucleotides that can be applied in industrialization, especially a method that is simple to operate, low in cost, safe, and has high purification efficiency. Summary of the invention

[0007] The purpose of the present invention is to provide a method for separating and recovering oligonucleotides and application thereof. The method is simple to operate, has high separation efficiency, high oligonucleotide recovery rate, high product purity, and has good industrial application prospects.

[0008] The above objects of the present invention are achieved through the following technical solutions:

[0009] In a first aspect, the present invention provides a method for separating and recovering oligonucleotides, the method comprising mixing a test solution containing the oligonucleotides with an acetone solution of a lithium salt, and then centrifuging the mixed solution to retain the precipitate; wherein the lithium salt is selected from any one or both of lithium chloride and lithium sulfate.

[0010] In some embodiments of the present invention, the concentration of the oligonucleotide in the test solution containing the oligonucleotide is ≤1 Mm, preferably 0.1 to 1 Mm, and more preferably 0.5 to 0.9 Mm.

[0011] In some embodiments of the present invention, the concentration of the oligonucleotide in the test solution containing the oligonucleotide is 0.1-1Mm, which means that the concentration of the oligonucleotide in the test solution containing the oligonucleotide can be selected from any value of the following concentrations or any value between any two of them: 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1Mm.

[0012] In some embodiments of the present invention, the test solution containing oligonucleotides contains water; preferably, the test solution containing oligonucleotides is an aqueous solution of oligonucleotides.

[0013] In some embodiments of the present invention, the mass fraction of the lithium salt in the acetone solution of the lithium salt is ≥2%, preferably 2% to 5%, and more preferably 2% to 3%.

[0014] In some embodiments of the present invention, the mass fraction of the lithium salt in the acetone solution of the lithium salt is 2% to 5%, which means that the mass fraction of the lithium salt in the acetone solution of the lithium salt can be selected from any of the following mass fractions or any value between any two of them: 2%, 2.1%, 2.2%, 2.3%, 2.4%, 2.5%, 2.6%, 2.7%, 2.8%, 2.9%, 3%, 3.1%, 3.2%, 3.3%, 3.4%, 3.5%, 3.6%, 3.7%, 3.8%, 3.9%, 4%, 4.1%, 4.2%, 4.3%, 4.4%, 4.5%, 4.6%, 4.7%, 4.8%, 4.9%, 5%.

[0015] In some embodiments of the present invention, the mixing volume ratio of the test solution containing the oligonucleotide to the acetone solution of the lithium salt is 1:(5-15), preferably 1:(5-10), and more preferably 1:(5-8).

[0016] In some embodiments of the present invention, the mixing volume ratio of the test solution containing oligonucleotides to the acetone solution of lithium salt is 1:(5-15), which means that the mixing volume ratio can be selected from any value in the following ratios or any value between any two of them: 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15.

[0017] In some embodiments of the present invention, the centrifugal rotation speed is ≥5000 rpm, preferably 8000-15000 rpm, and more preferably 10000-15000 rpm.

[0018] In some embodiments of the present invention, the centrifugal speed of 8000-15000 rpm means that the centrifugal speed can be selected from any value of the following speeds or any value between any two of them: 8000, 8500, 9000, 9500, 10000, 10500, 11000, 11500, 12000, 12500, 13000, 13100, 13200, 13300, 13400, 13500, 13600, 13800, 14000, 14500, 15000 rpm.

[0019] In some embodiments of the present invention, the centrifugation time is 1 to 9 min, preferably 3 to 7 min, and more preferably 4 to 6 min.

[0020] In some embodiments of the present invention, the centrifugation time of 1 to 9 min means that the centrifugation time can be selected from any value of the following time or any value between any two of them: 1, 2, 3, 4, 5, 6, 7, 8, 9 min.

[0021] In some embodiments of the present invention, the method further comprises washing the precipitate with an ethanol aqueous solution for 1 to 5 times, preferably 2 to 4 times.

[0022] In some embodiments of the present invention, the volume percentage of ethanol in the ethanol aqueous solution is ≥ 60%, preferably 70% to 90%, and more preferably 70% to 80%.

[0023] In some embodiments of the present invention, the volume percentage of ethanol is 70% to 90%, which means that the volume percentage of ethanol can be selected from any value of the following volume percentages or any value between any two of them: 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%.

[0024] In some embodiments of the present invention, the temperature of the ethanol aqueous solution is 2 to 30°C, preferably 2 to 25°C, and more preferably 4 to 10°C.

[0025] In some embodiments of the present invention, the temperature of the ethanol aqueous solution is 2 to 30°C, which means that the temperature of the ethanol aqueous solution can be selected from any value of the following temperatures or any value between any two of them: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30°C.

[0026] In some embodiments of the present invention, the method further comprises drying the obtained precipitate; preferably, the drying method comprises any one or any combination of two or more of centrifugal concentration drying, freeze drying, vacuum drying, oven drying, and natural air drying.

[0027] In some embodiments of the present invention, the method further comprises shaking the mixed solution before centrifugation; preferably, the mixed solution is shaken until flocculent precipitation occurs before centrifugation, wherein the shaking is performed using an oscillator for about 30 to 90 seconds.

[0028] In some embodiments of the invention, the method comprises the following steps:

[0029] (1) mixing a test solution containing oligonucleotides with a concentration of oligonucleotides ≤ 1 mM and an acetone solution of lithium salt with a mass fraction of lithium salt ≥ 2% at a volume ratio of 1: (5-15), and then shaking the mixture until flocculent precipitation occurs;

[0030] (2) centrifuging the shaken mixture at a speed of 5000 rpm or more for 1 to 9 minutes, discarding the supernatant and retaining the precipitate;

[0031] (3) washing the precipitate obtained in step (2) 1 to 5 times with an ethanol aqueous solution having an ethanol volume fraction of ≥60% and a temperature of 2 to 30° C., and then drying the precipitate; thus, a recovered product is obtained.

[0032] In a second aspect, the present invention provides the use of the method described in the first aspect in the production of oligonucleotides.

[0033] In some embodiments of the present invention, the sequence length of the oligonucleotide is ≥9; preferably, the sequence length of the oligonucleotide is 9-99.

[0034] In some embodiments of the present invention, the oligonucleotide sequence length of 9 to 99 means that the oligonucleotide may include any one or more of the following oligonucleotide sequence lengths: 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43 , 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99.

[0035] The beneficial effects of the present invention are as follows:

[0036] The method of the present invention accelerates the sedimentation of oligonucleotides by adding lithium salts to a test solution containing oligonucleotides. The negatively charged phosphate groups on the oligonucleotide skeleton can adsorb lithium ions and make the lithium ions evenly bonded to the molecular surface of the oligonucleotides, thereby reducing the repulsion of like charges between oligonucleotide molecules, making them easy to polymerize and precipitate; then centrifugation is used to accelerate the polymerization and sedimentation of oligonucleotide molecules, and the oligonucleotide precipitates after polymerization are adhered to the bottom of the centrifugal container, so as to prevent the precipitates from being poured out with the supernatant during solid-liquid separation. In addition, the present invention allows oligonucleotide molecules to be combined with lithium ions under the condition of acetone and water as the medium and centrifuged; water-soluble impurities are dissolved in water and appear in the supernatant after centrifugation and are separated out as the supernatant is decanted; oligonucleotide molecules are insoluble in acetone and can exist in the form of solid precipitation in acetone and are well separated from impurities; and lithium chloride and lithium sulfate have high solubility in acetone and can effectively ionize lithium ions and quickly combine with negatively charged phosphate groups on the oligonucleotide skeleton, thereby ensuring that the negatively charged phosphate groups can have enough lithium ions combined with them to accelerate the sedimentation of the oligonucleotide, and at the same time, can avoid the introduction of new impurities in the separation and recovery process.

[0037] The present invention also proposes to use an ethanol aqueous solution with an ethanol volume fraction of ≥60% and a temperature of 2-30°C to wash the precipitate obtained by centrifugation, which can further wash away water-soluble impurities on the surface of the precipitate and avoid unnecessary loss due to precipitate dissolution as much as possible, thereby further improving the purity of the product while ensuring a high recovery rate.

[0038] The method of the present invention is simple to operate and has high separation efficiency. The entire separation and recovery process can be completed within 5 to 15 minutes, and the recovery rate can reach more than 80% or even 95%. After separation and recovery, the purity of the product can be effectively improved. The method has good industrial application prospects for the separation and recovery of oligonucleotides with a sequence length of ≥9. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 It is a display diagram of a lithium sulfate acetone solution with a lithium sulfate mass fraction of 2% and a lithium carbonate acetone solution with a lithium carbonate mass fraction of 2%.

[0040] Figure 2 This is the state after sample 1 and sample 2 were left to stand for 10 minutes and sample 1 was centrifuged at 13200 rpm for 5 minutes.

[0041] Figure 3 This is the state of samples 1-3 after centrifugation using a lithium chloride tetrahydrofuran solution with a lithium chloride mass fraction of 2% to separate oligonucleotides (from left to right in the figure are samples 1, 2, and 3 respectively).

[0042] Figure 4 This is the precipitation state of Sample 1 after washing with ethanol aqueous solutions having ethanol volume fractions of 50% and 70%.

[0043] Figure 5 This is the state of sample ① and sample ② after centrifugation. DETAILED DESCRIPTION

[0044] The following examples further illustrate the technology of the present invention. These examples are for illustration and example of the present invention and are not intended to limit the scope of the present invention in any form.

[0045] Example 1

[0046] A method for separating and recovering oligonucleotides, the method comprising the following steps:

[0047] (1) mixing an aqueous solution containing oligonucleotides at a concentration of 1 mM with an acetone solution of lithium chloride having a mass fraction of 2% lithium chloride at a volume ratio of 1:5, and then shaking the mixture on an oscillator until flocculent precipitation occurs;

[0048] (2) Centrifuge the shaken mixture at 132,000 rpm for 5 min, discard the supernatant and retain the precipitate;

[0049] (3) The precipitate obtained in step (2) is washed twice with an ethanol aqueous solution having an ethanol volume fraction of 70% and a temperature of 4° C., and then the precipitate is placed in a centrifugal dryer and centrifuged at 4000 rpm to obtain a recovered product.

[0050] Example 2

[0051] A method for separating and recovering oligonucleotides, the method comprising the following steps:

[0052] (1) mixing an aqueous solution containing oligonucleotides at a concentration of 1 mM with an acetone solution of lithium sulfate having a mass fraction of 2% lithium sulfate at a volume ratio of 1:5, and then shaking the mixture on an oscillator until flocculent precipitation occurs;

[0053] (2) Centrifuge the shaken mixture at 132,000 rpm for 5 min, discard the supernatant and retain the precipitate;

[0054] (3) The precipitate obtained in step (2) is washed twice with an ethanol aqueous solution having an ethanol volume fraction of 70% and a temperature of 4° C., and then the precipitate is placed in a centrifugal dryer and centrifuged at 4000 rpm to obtain a recovered product.

[0055] Conditional investigation of the method

[0056] Prepare the test solution containing oligonucleotides for condition investigation as follows:

[0057] Ⅰ. Take an appropriate amount of 200 nmol of different types of solid phase carrier CPG, load it into a synthesis column tube to make a solid phase synthesis column, and load it onto the column base of the oligonucleotide automatic synthesizer.

[0058] Ⅱ. Prepare the reagents needed for synthesizing oligonucleotides, and load all reagents onto the automatic oligonucleotide synthesizer. The reagents include the following: the deprotection agent is a 3% (w / v) trichloroacetic acid solution in dichloromethane, and the amount used each time is 180ul; the activation coupling agent is a 0.25M ethylthiotetrazolyl solution in acetonitrile, and the amount used each time is 80ul; the capping agent CAP A is a 10% (v / v) acetic anhydride solution in tetrahydrofuran, and the amount used each time is 80ul; the capping agent CAP B is a 16% (v / v) 1-methylimidazole solution in tetrahydrofuran, and the amount used each time is 80ul. The capping agent CAP A and the capping agent CAP B are automatically added to the solid phase synthesis column by the machine at the same time; the oxidant is a 0.05M iodine reagent, and the amount used each time is 150ul; the solvent is 20g of DNA phosphoramidite monomer dissolved in 450ml of anhydrous acetonitrile under nitrogen protection and loaded onto the automatic oligonucleotide synthesizer under nitrogen protection, and the amount used each time is 60ul.

[0059] III. Review the sequence and start synthesis; during the synthesis process, pay attention to parameters such as moisture control and instrument pressure.

[0060] IV. After the synthesis is completed, deprotection is carried out by gas phase ammonolysis. 200ul of DEA is added to the synthesis column to control the formation of cyanoethyl groups. Ammonolysis is carried out in an ammonolysis pot at 95°C for 1h. After the ammonolysis is completed, acetonitrile is used for washing. After completion, 500ul of 5% TEA is used for elution, and the eluent is retained.

[0061] V. The eluate was tested for purity using Thermo U3000 high performance liquid chromatography, and the mass spectrometry relative purity was tested using LC-MS; the sequences of the synthesized samples of 6 different sequences and their purity test results are shown in Table 1 below.

[0062] Table 1 Sequences and purity test results of 6 samples with different sequences

[0063]

[0064] Ⅵ. Measure the OD value of the eluate and divide it into 10OD / tube. Then add each tube of sample into a 2ml sample centrifuge tube and place it in a Lanbconco refrigerated centrifuge to dry. Then add 100ul of dd H 2 O The sample was completely dissolved to obtain a test solution containing oligonucleotide for the following examination.

[0065] 1. Investigation of lithium salts

[0066] (I) Investigation of types of lithium salts

[0067] The test solution containing the oligonucleotide is mixed with a lithium chloride acetone solution with a mass fraction of 2% lithium chloride, a lithium sulfate acetone solution with a mass fraction of 2% lithium sulfate, and a lithium carbonate acetone solution with a mass fraction of 2% lithium carbonate at a volume ratio of 1:5, and then the mixed solution is placed on an oscillator for shaking until flocculent precipitation appears; the shaken mixed solution is centrifuged at 132000rpm for 5min, the supernatant is poured off and the precipitate is retained; then the obtained precipitate is washed twice with an ethanol aqueous solution with a volume fraction of 70% ethanol and a temperature of 4°C, and then the precipitate is placed in a centrifugal dryer and centrifuged and dried at 4000rpm; the recovered product is obtained.

[0068] Oligonucleotides were separated and recovered from 6 samples according to the above method, and then the obtained products were subjected to OD measurement and mass spectrometry detection. The OD measurement results are shown in Table 2 below. All products passed the mass spectrometry detection, indicating that the separation and recovery process had no negative impact on the oligonucleotides.

[0069] Table 2 Effect of lithium salt types on separation and recovery effects

[0070]

[0071] As can be seen from Table 2, the recovery rate of oligonucleotides recovered by using lithium chloride acetone solution is the highest, and the recovery rate of oligonucleotides recovered by using lithium sulfate acetone solution can also reach 84.7%. In addition, the acetone solution of lithium sulfate with a mass fraction of 2% and the acetone solution of lithium carbonate with a mass fraction of 2% are as follows: Figure 1 As shown, from Figure 1 It can be seen that the solubility of lithium carbonate in acetone is lower than that of lithium sulfate; combined with the results in Table 1, it can be seen that it is not suitable to use an acetone solution of lithium carbonate to provide lithium ions to bind to oligonucleotides to separate and recover oligonucleotides.

[0072] (II) Investigation of the mass fraction of lithium salt

[0073] The test solution containing oligonucleotides is mixed with lithium chloride acetone solutions with a mass fraction of 1%, 2%, 3%, 4% and 5% of lithium chloride respectively at a volume ratio of 1:5, and then the mixed solution is placed on an oscillator for shaking until flocculent precipitation appears; the shaken mixed solution is centrifuged at 132000rpm for 5min, the supernatant is poured off and the precipitate is retained; then the obtained precipitate is washed twice with an ethanol aqueous solution with a volume fraction of 70% ethanol and a temperature of 4°C, and then the precipitate is placed in a centrifugal dryer and centrifuged and dried at 4000rpm; the recovered product is obtained.

[0074] Oligonucleotides were separated and recovered from 6 samples according to the above method, and then the obtained products were subjected to OD measurement, purity detection and mass spectrometry detection. The results of OD measurement and purity detection are shown in Table 3 below. All products passed the mass spectrometry detection, indicating that the separation and recovery process had no negative impact on the oligonucleotides.

[0075] Table 3 Effect of the mass fraction of lithium chloride on separation and recovery effect

[0076]

[0077] It can be seen from Table 3 that when the mass fraction of lithium chloride in the acetone solution is ≥2%, the recovery rate of the oligonucleotide can be guaranteed to exceed 90%, and as the mass fraction of lithium chloride in the acetone solution increases, the recovery rate will not increase significantly.

[0078] 2. Investigation of centrifugal conditions for separation of precipitates

[0079] (I) Investigation of the number of centrifugal revolutions

[0080] The test solution containing the oligonucleotide is mixed with a lithium chloride acetone solution with a mass fraction of 2% of lithium chloride in a volume ratio of 1:5, and then the mixed solution is placed on an oscillator for shaking until flocculent precipitation appears; the shaken mixed solution is centrifuged at 2000 rpm, 5000 rpm, 8000 rpm, 13200 rpm, and 15000 rpm for 5 minutes, respectively, the supernatant is poured off and the precipitate is retained; then the obtained precipitate is washed twice with an ethanol aqueous solution with a volume fraction of 70% ethanol and a temperature of 4°C, and then the precipitate is placed in a centrifugal dryer and centrifuged and dried at 4000 rpm; the recovered product is obtained.

[0081] Oligonucleotides were separated and recovered from 6 samples according to the above method, and then the obtained products were subjected to OD measurement and mass spectrometry detection. The OD measurement results are shown in Table 4 below. All products were qualified in mass spectrometry detection, indicating that the separation and recovery process had no negative impact on the oligonucleotides.

[0082] Table 4 Effect of the number of rotations of the mixed solution centrifuge on the separation and recovery effect

[0083]

[0084] It can be seen from Table 4 that a centrifugal speed of 5000 rpm or above can ensure that the recovery rate of oligonucleotides exceeds 85%; the recovery rate is the highest when the centrifugal speed is 13200 rpm.

[0085] (II) Centrifugal time investigation

[0086] The test solution containing oligonucleotides was mixed with a lithium chloride acetone solution having a mass fraction of 2% lithium chloride at a volume ratio of 1:5, and then the mixture was placed on an oscillator and shaken until flocculent precipitation appeared; the shaken mixture was allowed to stand for 10 minutes or centrifuged at 13200 rpm for 1 minute, 3 minutes, 5 minutes, 7 minutes, and 9 minutes, respectively. The conditions of samples 1 and 2 standing for 10 minutes and sample 1 being centrifuged at 13200 rpm for 5 minutes were as follows: Figure 2 After centrifugation, the supernatant of the sample is removed and the precipitate is retained; the precipitate is then washed twice with an ethanol aqueous solution having an ethanol volume fraction of 70% and a temperature of 4°C, and then the precipitate is placed in a centrifugal dryer and centrifuged at 4000 rpm to obtain a recovered product.

[0087] Oligonucleotides were separated and recovered from 6 samples according to the above method, and then the obtained products were subjected to OD measurement, purity detection and mass spectrometry detection. The results of OD measurement and purity detection are shown in Table 5 below. All product mass spectrometry detections were qualified, indicating that the separation and recovery process had no negative impact on the oligonucleotides.

[0088] Table 5 Effect of centrifugation time of mixed solution on separation and recovery effect

[0089]

[0090] As can be seen from Table 5, centrifugation at 132000rpm for more than 3min can ensure that the recovery rate of oligonucleotides exceeds 85%; the recovery rate is the highest when the centrifugation time is 5min. In addition, the test shows that after adding a lithium chloride acetone solution with a mass fraction of 2% lithium chloride to the test solution containing oligonucleotides, the oligonucleotides begin to settle. After standing for about 5min, the oligonucleotides settle to the bottom of the container and gather into a ball. Therefore, sedimentation will also occur without centrifugation, but because the oligonucleotides are suspended at the bottom of the container at this time, an extraction device is required to achieve effective recovery of the oligonucleotides, which is more cumbersome and time-consuming.

[0091] 3. Investigation of solvents for lithium salts

[0092] The test solution containing oligonucleotides is respectively mixed with a lithium chloride acetone solution with a lithium chloride mass fraction of 2%, a lithium chloride alcohol solution with a lithium chloride mass fraction of 2%, and a lithium chloride tetrahydrofuran solution with a lithium chloride mass fraction of 2% at a volume ratio of 1:5, and then the mixed solution is placed on an oscillator for shaking until flocculent precipitation appears; the shaken mixed solution is centrifuged at 132000rpm for 5min, the supernatant is poured off and the precipitate is retained; then the obtained precipitate is washed twice with an ethanol aqueous solution with a volume fraction of 70% ethanol and a temperature of 4°C, and then the precipitate is placed in a centrifugal dryer and centrifuged and dried at 4000rpm; the recovered product is obtained.

[0093] Oligonucleotides were separated and recovered from the six samples according to the above method, and then the obtained products were subjected to OD measurement, purity test and mass spectrometry test. Among them, the oligonucleotides separated from sample 1-6 using a lithium chloride tetrahydrofuran solution with a mass fraction of 2% lithium chloride could not be effectively precipitated after centrifugation, and the state of sample 1-3 after centrifugation was as follows Figure 3 As shown; the OD measurement and purity test results of the products obtained by separating and recovering oligonucleotides using a lithium chloride acetone solution with a lithium chloride mass fraction of 2% and a lithium chloride alcohol solution with a lithium chloride mass fraction of 2% are shown in Table 6 below, and the mass spectrometry test of 12 products were all qualified, indicating that the separation and recovery process has no negative impact on oligonucleotides.

[0094] Table 6 Effect of LiCl Solvent on Separation and Recovery Effect

[0095]

[0096] As can be seen from Table 6, compared with using lithium chloride alcohol solution, using lithium chloride acetone solution to separate and recover oligonucleotides has a higher recovery rate and higher product purity, and can separate and recover oligonucleotides with shorter sequence lengths.

[0097] 4. Investigation of solvents for washing precipitates

[0098] (I) Investigation of the volume fraction of ethanol in ethanol-water solution

[0099] The test solution containing the oligonucleotide and the acetone solution of lithium chloride with a mass fraction of 2% of lithium chloride are mixed in a volume ratio of 1:5, and then the mixed solution is placed on an oscillator for shaking until flocculent precipitation appears; the shaken mixed solution is centrifuged at 132000rpm for 5min, the supernatant is poured off and the precipitate is retained; then the obtained precipitate is washed twice with ethanol aqueous solutions with a volume fraction of 50%, 60%, 70%, 80%, and 90% of ethanol (temperature is 4°C), and then the precipitate is placed in a centrifugal dryer and centrifuged and dried at 4000rpm; the recovered product is obtained.

[0100] Oligonucleotides were separated and recovered from 6 samples according to the above method. The precipitate obtained from sample 1 was washed with 50% and 70% ethanol aqueous solutions as shown in the following figure. Figure 4 The final product was subjected to OD measurement and mass spectrometry detection. The OD measurement results are shown in Table 7 below. The mass spectrometry detection of the products was qualified, indicating that the separation and recovery process had no negative impact on the oligonucleotides.

[0101] Table 7 Effect of the volume fraction of ethanol in ethanol-water solution on separation and recovery effect

[0102]

[0103] from Figure 4 It can be seen that when the precipitate is washed with an ethanol aqueous solution having a volume fraction of 50%, part of the precipitate is visibly dissolved, while when the precipitate is washed with an ethanol aqueous solution having a volume fraction of 70% or more, the precipitate is not visibly dissolved. As can be seen from Table 7, when the precipitate is washed with an ethanol aqueous solution having a volume fraction of 70% or more, the loss is small and the yield is high. However, in order to more effectively remove the salt that is highly soluble in water, it is better to wash the precipitate with an ethanol aqueous solution having a volume fraction of 70% or more.

[0104] (II) Temperature investigation of ethanol aqueous solution

[0105] The test solution containing the oligonucleotide and the acetone solution of lithium chloride with a mass fraction of 2% of lithium chloride are mixed in a volume ratio of 1:5, and then the mixed solution is placed on an oscillator for shaking until flocculent precipitation appears; the shaken mixed solution is centrifuged at 132000rpm for 5min, the supernatant is poured off and the precipitate is retained; then the obtained precipitate is washed twice with ethanol aqueous solution with a temperature of 4°C, 10°C, 15°C, 20°C, and 25°C and a volume fraction of ethanol of 70%, and then the precipitate is placed in a centrifugal dryer and centrifuged and dried at 4000rpm; the recovered product is obtained.

[0106] Oligonucleotides were separated and recovered from 6 samples according to the above method, and the final products were subjected to OD measurement and mass spectrometry detection. The OD measurement results are shown in Table 8 below, and the mass spectrometry detection of the products was qualified, indicating that the separation and recovery process had no negative impact on the oligonucleotides.

[0107] Table 8 Effect of the temperature of ethanol aqueous solution on separation and recovery effect

[0108]

[0109]

[0110] It can be seen from Table 8 that the lower the temperature of the ethanol aqueous solution, the smaller the loss and the higher the yield.

[0111] Investigation of the scope of application of the method

[0112] Prepare the test solution containing oligonucleotides for application scope investigation as follows:

[0113] (I) Take an appropriate amount of 200 nmol of different types of solid phase carrier CPG, load it into a synthesis column tube to make a solid phase synthesis column, and load it onto the column base of an automatic oligonucleotide synthesizer.

[0114] (II), prepare the reagents needed for synthesizing oligonucleotides, and load all the reagents onto the oligonucleotide automatic synthesizer. The reagents include the following: the deprotection agent is a 3% (w / v) trichloroacetic acid in dichloromethane solution, and the amount used each time is 180ul; the activation coupling agent is a 0.25M ethylthiotetrazolyl acetonitrile solution, and the amount used each time is 80ul; the capping agent CAP A is a 10% (v / v) acetic anhydride in tetrahydrofuran solution, and the amount used each time is 80ul; the capping agent CAP B is a 16% (v / v) 1-methylimidazole in tetrahydrofuran solution, and the amount used each time is 80ul. The capping agent CAP A and the capping agent CAP B are automatically added to the solid phase synthesis column by the machine at the same time; the oxidant is a 0.05M iodine reagent, and the amount used each time is 150ul; the solvent is 20g of DNA phosphoramidite monomer dissolved in 450ml of anhydrous acetonitrile under nitrogen protection and loaded onto the oligonucleotide automatic synthesizer under nitrogen protection, and the amount used each time is 60ul.

[0115] (III) Review the sequence and start synthesis; pay attention to parameters such as moisture control and instrument pressure during the synthesis.

[0116] (IV) After the synthesis is completed, deprotection is carried out by gas phase ammonolysis. 200ul of DEA is added to the synthesis column to control the formation of cyanoethyl groups. Ammonolysis is carried out in an ammonolysis pot at 95°C for 1h. After the ammonolysis is completed, acetonitrile is used for washing. After completion, 500ul of 5% TEA is used for elution, and the eluent is retained.

[0117] (V) Measure the OD value of the eluate and divide it into 10OD / tube. Then add each tube of sample into a 2ml sample centrifuge tube and place it in a Lanbconco refrigerated centrifuge to dry. Then add 100ul of ddH 2 O The sample was completely dissolved to obtain a test solution containing oligonucleotides for range investigation. The obtained samples of 7 sequences of different lengths are shown in Table 9 below.

[0118] Table 9 Samples of 7 sequences of different lengths

[0119]

[0120] The above 7 kinds of oligonucleotide-containing test solutions were mixed with a lithium chloride acetone solution having a mass fraction of 2% lithium chloride at a volume ratio of 1:5, and then the mixed solution was placed on an oscillator and shaken until flocculent precipitation appeared; the shaken mixed solution was centrifuged at 132000rpm for 5min, wherein the states of sample ① and sample ② after centrifugation were as follows Figure 5 After centrifugation, the supernatant of the sample is removed and the precipitate is retained; the precipitate is then washed twice with an ethanol aqueous solution having an ethanol volume fraction of 70% and a temperature of 4°C, and then the precipitate is placed in a centrifugal dryer and centrifuged at 4000 rpm to obtain a recovered product.

[0121] The obtained products were subjected to OD measurement and mass spectrometry detection, wherein the OD measurement results are shown in Table 10 below. All products were qualified in mass spectrometry detection, indicating that the separation and recovery process had no negative impact on the oligonucleotides.

[0122] Table 10 Application effect of the method of the present invention in separating and recovering oligonucleotides of different lengths

[0123] Product ① Product ② Product ③ Product ④ Product ⑤ Product ⑥ Product ⑦ OD value - 8.58 8.86 9.03 9.25 9.31 9.65

[0124] It can be seen from Table 10 that the method of the present invention has a higher recovery rate when used for separation and recovery of oligonucleotides with a sequence length ≥ 9.

[0125] It should be noted that the embodiments described above are only used to explain the present invention and do not constitute any limitation of the present invention. The present invention has been described with reference to the embodiments, but it should be understood that the words used therein are descriptive and explanatory words, rather than restrictive words. The present invention may be modified as specified within the scope of the claims of the present invention, and the present invention may be revised without departing from the scope and spirit of the present invention. Although the present invention described therein relates to specific methods, materials and embodiments, it does not mean that the present invention is limited to the specific examples disclosed therein, on the contrary, the present invention can be extended to all other methods and applications with the same functions.

Claims

1. A method for separating and recovering oligonucleotides, characterized in that: The method comprises mixing a test solution containing oligonucleotides with an acetone solution of a lithium salt, and then centrifuging the mixed solution to retain a precipitate; wherein the lithium salt is selected from any one or both of lithium chloride and lithium sulfate.

2. The method according to claim 1, characterized in that The concentration of the oligonucleotide in the test solution containing the oligonucleotide is ≤1Mm, preferably 0.1 to 1Mm, more preferably 0.5 to 0.9Mm; And / or, the mass fraction of the lithium salt in the acetone solution of the lithium salt is ≥ 2%, preferably 2% to 5%, more preferably 2% to 3%; And / or, the mixing volume ratio of the test solution containing the oligonucleotide to the acetone solution of the lithium salt is 1:(5-15), preferably 1:(5-10), and more preferably 1:(5-8).

3. The method according to claim 1 or 2, characterized in that: The centrifugal speed is ≥5000 rpm, preferably 8000-15000 rpm, more preferably 10000-15000 rpm; And / or, the centrifugation time is 1 to 9 min, preferably 3 to 7 min, more preferably 4 to 6 min.

4. The method according to any one of claims 1 to 3, characterized in that The method further comprises washing the precipitate with ethanol aqueous solution for 1 to 5 times.

5. The method according to claim 4, characterized in that The volume percentage of ethanol in the ethanol aqueous solution is ≥ 60%, preferably 70% to 90%, more preferably 70% to 80; And / or, the temperature of the ethanol aqueous solution is 2-30°C, preferably 2-25°C, more preferably 4-10°C.

6. The method according to any one of claims 1 to 5, characterized in that: The method further comprises drying the obtained precipitate; preferably, the drying method comprises any one or any combination of two or more of centrifugal concentration drying, freeze drying, vacuum drying, oven drying, and natural air drying.

7. The method according to any one of claims 1 to 6, characterized in that: The method further comprises shaking the mixed solution before centrifugation; preferably, the mixed solution is shaken until flocculent precipitation occurs before centrifugation.

8. The method according to any one of claims 1 to 7, characterized in that: The method comprises the following steps: (1) mixing a test solution containing oligonucleotides with a concentration of oligonucleotides ≤ 1 mM and an acetone solution of lithium salt with a mass fraction of lithium salt ≥ 2% at a volume ratio of 1: (5-15), and then shaking the mixture until flocculent precipitation occurs; (2) centrifuging the shaken mixture at a speed of 5000 rpm or more for 1 to 9 minutes, discarding the supernatant and retaining the precipitate; (3) washing the precipitate obtained in step (2) 1 to 5 times with an ethanol aqueous solution having an ethanol volume fraction of ≥60% and a temperature of 2 to 30° C., and then drying the precipitate; thus, a recovered product is obtained.

9. Use of the method according to any one of claims 1 to 8 in the production of oligonucleotides.

10. The use according to claim 9, characterized in that: The sequence length of the oligonucleotide is ≥9; preferably, the sequence length of the oligonucleotide is 9-99.

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