An oligonucleotide cleavage reagent

Abstract

This invention provides an oligonucleotide cleavage reagent (JS2 & S4 reagent) and its preparation method, as well as a method for cleaving oligonucleotides. The oligonucleotide cleavage reagent comprises NaOH, KOH, methanol, and a cleavage buffer. The oligonucleotide cleavage reagent JS2 & S4 and the cleavage method using it provided by this invention can efficiently cleave oligonucleotides, especially those synthesized using solid-phase phosphorous acid cleavage. This reduces chain breakage and side reactions caused by high-temperature cleavage, and also reduces equipment costs. While shortening the cleavage time, it can effectively reduce oligonucleotide quality problems caused by cleavage.

Description

[0001] Cross-reference to related applications

[0002] This application claims priority to PCT patent application No. PCT / CN2020 / 124821, filed on October 29, 2020, entitled “An Oligonucleotide Cleavage Reagent”, the entire contents of which are incorporated herein by reference. Technical Field

[0003] This invention belongs to the field of nucleotide cleavage, and more specifically, relates to an oligonucleotide cleavage reagent and its preparation method, as well as a method for cleaving oligonucleotides, especially solid-phase synthesized oligonucleotides. Background Technology

[0004] Oligonucleotides, also known as oligomeric nucleotides, are often synthesized using the solid-phase phosphoramidite method. This method involves immobilizing oligonucleotides on a solid support and synthesizing the oligonucleotide chain through four cyclic steps: deprotection, coupling, capping, and oxidation. For the cleavage of crude oligonucleotides synthesized in the solid phase, the most widely used cleavage methods include ammonia oven cleavage, gas-phase cleavage, and microwave cleavage, among others. However, each method has its own advantages and disadvantages in terms of cleavage efficiency, as detailed in Table 1 below.

[0005] Table 1

[0006]

[0007]

[0008] Although the oligonucleotides lysed by the ammonia oven lysis method are of acceptable quality, they have the following drawbacks: First, the lysis method is a single-tube operation, which cannot be adapted to the current large-scale quantitative production experiments; second, the lysed products need to be desalted by a C18 column before they can be detected and applied, which is a relatively cumbersome operation; third, the lysis time is long, requiring 2-3 hours.

[0009] As an evolution of the ammonia oven pyrolysis method, the gas-phase pyrolysis method has greatly simplified the operation and improved the product quality. However, its pyrolysis time remains at 2-3 hours and requires a dedicated high-pressure device for pyrolysis. The improvement in throughput is limited and still cannot meet the increasing production and experimental needs.

[0010] Microwave lysis is currently the fastest method for cleaving oligonucleotides, requiring only 12 minutes. However, it also has significant drawbacks. First, the quality of the cleaved products is relatively low, failing to meet customers' increasingly demanding quality requirements for oligonucleotides. Second, microwave reactors suffer from poor temperature control, which can easily lead to high-temperature breakage of oligonucleotide chains. Third, the cleavage reagents, primarily composed of organic amines, commonly exhibit side reactions during oligonucleotide cleavage, affecting the quality of the oligonucleotides.

[0011] In conclusion, to meet customers' demand for high-quality oligonucleotides, it is urgent to explore an efficient and reasonable oligonucleotide cleavage method that can shorten the cleavage time while effectively reducing oligonucleotide quality problems caused by cleavage. Summary of the Invention

[0012] The purpose of this invention is to address the shortcomings of existing technologies in oligonucleotide cleavage, which make it difficult to simultaneously consider operation time, product quality, and equipment cost. The invention optimizes the cleavage reagent used in oligonucleotide cleavage and, more specifically, provides an oligonucleotide cleavage reagent that is more suitable for cleaving oligonucleotides synthesized in solid phase, thereby effectively reducing oligonucleotide quality problems caused by cleavage while shortening the cleavage time.

[0013] The inventors have discovered and confirmed that by using the oligonucleotide cleavage reagent provided in this application (also referred to herein as JS2 & S4 reagent) as the cleavage reagent to separate crude oligonucleotide products from the solid synthesis support, and then heating in an oven at approximately 100°C for about 1 hour, batch deprotection cleavage of oligonucleotides synthesized on 96-well plates can be achieved. After washing with acetonitrile, the oligonucleotides dissolved and eluted using TE buffer can be used in subsequent experimental production. Based on the above findings, the inventors have thus completed this invention.

[0014] In one aspect, the present invention provides an oligonucleotide cleavage reagent (JS2 & S4 reagent), which comprises NaOH, KOH, methanol and cleavage buffer.

[0015] In one embodiment of the invention, the methanol content is 60-95% by volume, preferably 90% by volume, based on the total volume of the oligonucleotide cleavage reagent. In one embodiment, the methanol content is 90%.

[0016] In one embodiment of the present invention, the concentrations of NaOH and KOH in the oligonucleotide cleavage reagent are each independently 0.4-2 mol / L, preferably 1 mol / L. In one embodiment, the concentrations of NaOH and KOH in the oligonucleotide cleavage reagent are each independently 0.8-1.2 mol / L. In another embodiment, the concentrations of NaOH and KOH in the oligonucleotide cleavage reagent are each independently 0.4 mol / L, 0.6 mol / L, 0.8 mol / L, 1 mol / L, 1.2 mol / L, 1.5 mol / L, 1.8 mol / L, or 2 mol / L. In one specific embodiment, the concentrations of NaOH and KOH in the oligonucleotide cleavage reagent are each independently 1 mol / L. In another specific embodiment, the concentrations of NaOH and KOH in the oligonucleotide cleavage reagent are each independently 0.4 mol / L. In one specific embodiment, the concentrations of NaOH and KOH in the oligonucleotide cleavage reagent are each independently 2 mol / L.

[0017] In one embodiment of the invention, the lysis buffer is an aqueous ethanol solution, wherein the volume ratio of ethanol to water is 1:9 to 5:5, preferably 2:8. In a specific embodiment, the volume ratio of ethanol to water is 2:8.

[0018] In one embodiment of the present invention, the oligonucleotide cleavage reagent is used to cleave oligonucleotides synthesized on a solid support.

[0019] In another aspect, the present invention also provides a method for preparing the oligonucleotide cleavage reagent as described above, the method comprising contacting NaOH, KOH, methanol and cleavage buffer with each other.

[0020] In one embodiment of the present invention, NaOH, KOH and lysis buffer are first brought into contact with each other, and then the resulting solution is brought into contact with methanol.

[0021] In another aspect, the present invention also provides a method for cleaving oligonucleotides, the method comprising contacting a solid-phase synthesized oligonucleotide with an oligonucleotide cleavage reagent, wherein the oligonucleotide cleavage reagent comprises NaOH, KOH, methanol and a cleavage buffer.

[0022] The definitions and all characteristics of the oligonucleotide cleavage reagents mentioned above also apply to the oligonucleotide cleavage reagents used in this method, and will not be elaborated upon here.

[0023] In one embodiment of the present invention, the amount of the oligonucleotide cleavage reagent used is 0.1-10 μL / nmol oligonucleotide, preferably 0.8 μL / nmol, based on the molar amount of the oligonucleotide.

[0024] In one embodiment of the invention, the contact is carried out at a temperature of 80-120°C for 0.5-2 hours. In another specific embodiment, the contact is carried out under constant temperature conditions of 80-120°C. In yet another specific embodiment, the contact is carried out under constant temperature conditions of 100°C for 1 hour. The constant temperature conditions can be achieved by any heating and heat preservation device, such as an oven. Maintaining a constant temperature is beneficial for the stable progress of the reaction and reduces the occurrence of side reactions.

[0025] In one embodiment of the present invention, the method further includes washing the cleaved oligonucleotides with acetonitrile and dissolving the eluted oligonucleotides with Tris-EDTA.

[0026] Another aspect of the present invention provides a method for cleavage solid-phase synthesis of oligonucleotides, comprising the following steps:

[0027] Step 1: Add oligonucleotide lysis reagent to the synthesis column used for solid-phase synthesis of oligonucleotides, wherein the oligonucleotide lysis reagent comprises NaOH, KOH, methanol and lysis buffer;

[0028] Step 2: Place the synthesis column in a sealable container containing lysis buffer and react at 80-120℃ for 0.5-1.5h;

[0029] Step 3: Remove the synthesis column to remove the water. Wash the synthesis column with washing solution, then treat the synthesis column with elution solution and collect the effluent.

[0030] In some embodiments of the present invention, in step 1, the methanol content is 60-95% by volume, preferably 90% by volume, based on the total volume of the oligonucleotide cleavage reagent.

[0031] In some embodiments of the present invention, the concentrations of NaOH and KOH in the oligonucleotide cleavage reagent in step 1 are each independently 0.4-2 mol / L, preferably each independently 1 mol / L.

[0032] In some embodiments of the present invention, the lysis buffer is an aqueous ethanol solution, wherein the volume ratio of ethanol to water is 1:9-5:5, preferably 2:8.

[0033] In some embodiments of the present invention, the synthesis column in step 1 is one or more, preferably a 96-hole synthesis plate.

[0034] In some embodiments of the present invention, step 2 is carried out under constant temperature conditions of 80-120°C for 0.5-1.5 hours, preferably under constant temperature conditions of 100°C for 1 hour.

[0035] In some embodiments of the present invention, the method for removing moisture from the synthesis column in step 3 includes centrifugation and drying.

[0036] In some embodiments of the present invention, the washing solution in step 3 contains acetonitrile, and the eluent contains Tris-EDTA.

[0037] Beneficial technical effects of the present invention:

[0038] The oligonucleotide cleavage reagents JS2 & S4 and the cleavage method using them provided by this invention can efficiently cleave oligonucleotides, especially those synthesized by solid-phase phosphorous amide. This reduces chain breakage and side reactions caused by high-temperature cleavage, and also reduces equipment costs. While shortening the cleavage time, it can effectively reduce oligonucleotide quality problems caused by cleavage. Attached Figure Description

[0039] The accompanying drawings are provided to further illustrate the invention and form part of the specification. They are used together with the following detailed description to explain the invention, but do not constitute a limitation thereof. In the drawings:

[0040] Figure 1 An analytical plate image of an experimental sample treated with the pyrolysis method according to an embodiment of the present invention is shown, wherein... Figure 1 a and 1d are the analysis plates of the experimental samples treated with the JS2 & S4 pyrolysis method in three experiments. Figure 1 b and 1c are the analytical gel images of the experimental samples treated by microwave pyrolysis in three experiments; and

[0041] Figure 2 The mass spectrometry results of the experimental sample treated by the pyrolysis method shown in the embodiment of the present invention are illustrated, wherein... Figure 2 a is the mass spectrum of the product obtained by microwave pyrolysis of CPR-01-T-seqR; Figure 2 b is the mass spectrum of the product obtained by the JS2&S4 pyrolysis method of CPR-01-T-seqR. Detailed Implementation

[0042] The following provides a detailed description of specific embodiments of the present invention. It should be understood that the specific embodiments described herein are for illustrative and explanatory purposes only and are not intended to limit the scope of the invention.

[0043] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0044] Before describing the invention in detail, it should be understood that the terminology used herein is for describing particular embodiments only and is not intended to limit the scope of the invention, which is defined only by the appended claims. For a more complete understanding of the invention described herein, the following terms are used, and their definitions are as follows. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as understood by one of ordinary skill in the art to which this invention pertains.

[0045] As used herein, the terms "DNA solid-phase synthesis" or "oligonucleotide solid-phase synthesis" refer to a method commonly used in the art for synthesizing DNA or oligonucleotide chains, comprising: pre-fixing the terminal nucleotide (e.g., the 3' terminal nucleotide) of the DNA chain to be synthesized onto an insoluble solid support, and then sequentially attaching other nucleotides one by one from this end in a predetermined order until the entire DNA chain is synthesized. Each attachment of a nucleotide residue involves one round of the same operation (see the Background section), and the extending DNA chain is kept fixed on the solid support throughout. Excess unreacted material or reaction byproducts can be removed by filtration or washing. The synthesized DNA chain to the desired length can be cleaved from the solid support and deprotected, and then purified to obtain the final product. DNA chains synthesized in this manner are typically tens of bases, or up to hundreds of bases, and can be used, for example, as PCR primers, adapters, or probes.

[0046] The term "synthesis column" as used in this article typically comprises three parts: a solid support, a sieve plate, and an empty column tube. The empty column tube is usually injection-molded from polypropylene, with an upper opening larger than the lower opening, and the solid support and sieve plate are filled inside. "Porous synthesis column" includes a synthesis plate for one or more synthesis columns, such as a 96-well synthesis plate or a 384-well synthesis plate.

[0047] As used in this article, “oligonucleotide cleavage” or “oligonucleotide cleavage reaction” refers to the chemical process, in the final stage of oligonucleotide (DNA) solid-phase synthesis, in which the synthesized DNA strand is cleaved from the solid-phase support, the amino protecting groups on the bases are removed, and / or the phosphate groups at the 3' end are removed, using oligonucleotide cleavage reagents, such as cleavage reagents containing NaOH, KOH, methanol, and cleavage buffer.

[0048] As used herein, the term "sealable container" refers to a container in which oligonucleotide cleavage reactions are performed, which can be sealed, for example, by capping. The sealable container is heatable and, for example, made of glass, ceramic, etc. During the cleavage reaction, a cleavage buffer is pre-added and vaporized under microwave treatment. The vapor enters the pores of the solid-phase support through the top and bottom openings of the synthesis column (i.e., placing the synthesis column in a "vapor atmosphere"), promoting the cleavage reaction between the cleavage reagent and the oligonucleotides synthesized in the solid phase.

[0049] In one aspect, the present invention provides an oligonucleotide cleavage reagent (also referred to herein as the JS2&S4 reagent), which comprises NaOH (JS2), KOH (JS3), methanol (JS4) and a cleavage buffer.

[0050] According to the present invention, based on the physicochemical properties of each component, the JS2 & S4 reagents can generally be in the form of a solution of JS2 and JS4 dissolved in JS3 and lysis buffer, and therefore their volume will be substantially dependent on the volumes of JS3 and lysis buffer. As a key reagent in the oligonucleotide lysis reagent according to the present invention, the volume percentage of JS3 reagent can generally be higher than that of the lysis buffer. For example, in a preferred embodiment of the present invention, based on the total volume of the oligonucleotide lysis reagent (approximately equal to or even equal to the sum of the volumes of JS3 reagent and lysis buffer), the content of JS3 reagent (methanol) can be 60-95% by volume, such as 70%, 80%, or 90% by volume.

[0051] Accordingly, in the JS2 & S4 reagent according to the present invention, JS2 and JS4 can generally be in the form of solutes dissolved in the aforementioned JS4 and lysis buffer, wherein the contents of the JS2 and JS4 as solutes can be the same or different from each other. For example, in a preferred embodiment of the present invention, the concentrations of JS2 (NaOH) and JS4 (KOH) in the oligonucleotide lysis reagent can each be independently 0.4-2 mol / L (i.e., 0.4-2 M) without being affected by each other. For example, in a preferred embodiment, the concentration of NaOH in the oligonucleotide lysis reagent can be independently 0.5 mol / L, 1 mol / L, or 1.5 mol / L, etc. Similarly, the concentration of KOH in the oligonucleotide lysis reagent can also be independently 0.5 mol / L, 1 mol / L, or 1.5 mol / L, etc.

[0052] According to the present invention, there is no particular limitation on the type of lysis buffer used in the present invention, and it can be any lysis buffer commonly used in the art. For example, in one embodiment of the present invention, the lysis buffer can be an aqueous ethanol solution, wherein the volume ratio of ethanol to water is 1:9-5:5 (e.g., 2:8, 3:7 or 4:6, etc.), but the lysis buffer of the present invention is not limited to this.

[0053] In another aspect, the present invention also provides a method for preparing the oligonucleotide cleavage reagent as described above, the method comprising contacting NaOH, KOH, methanol and cleavage buffer with each other.

[0054] According to the present invention, the oligonucleotide cleavage reagent of the present invention can be obtained by directly contacting its components (i.e., NaOH, KOH, methanol, and cleavage buffer) with each other. More specifically, oligonucleotide cleavage reagents with different specific compositions can be prepared as needed, based on the desired proportions of the components in the oligonucleotide cleavage reagent. The contacting process of the above components can be carried out in any order, for example, the components can be contacted simultaneously, sequentially, or separately, without any limitation.

[0055] Furthermore, for the sake of accurate final volume, methanol or lysis buffer can be used for final volume adjustment. For example, in a preferred embodiment of the invention, NaOH, KOH, and lysis buffer can be contacted with each other first (which can be considered a dissolution process), and then the resulting solution can be contacted with methanol (which can be considered a volume adjustment process). In another preferred embodiment of the invention, NaOH, KOH, and methanol can also be contacted with each other first, and then the resulting solution can be contacted with lysis buffer.

[0056] In another aspect, the present invention also provides a method for cleaving oligonucleotides, the method comprising contacting a solid-phase synthesized oligonucleotide with an oligonucleotide cleavage reagent, wherein the oligonucleotide cleavage reagent comprises NaOH, KOH, methanol and a cleavage buffer.

[0057] According to the present invention, there is no particular limitation on the amount of the lysis reagent (JS2 & S4 lysis reagent) used in the lysis method of the present invention, and it can be determined based on the experience of those skilled in the art. To achieve better lysis results, in a preferred embodiment of the present invention, based on the molar amount of the oligonucleotide, the amount of the oligonucleotide lysis reagent can be 0.1-10 μL / nmol (e.g., 0.5 μL / nmol, 1 μL / nmol, or 5 μL / nmol) of oligonucleotide.

[0058] Based on the beneficial effects of the present invention mentioned above, the lysis method using the oligonucleotide lysis reagent provided by the present invention can achieve excellent oligonucleotide lysis effects and quality under relatively low temperature conditions and relatively short time. For example, in a preferred embodiment of the present invention, the contact can be carried out at a temperature of 80-120°C (e.g., 90°C, 100°C, or 110°C, etc.) for 0.5-2 hours (0.8 hours, 1 hour, or 1.5 hours, etc.). More preferably, the contact can be carried out under constant temperature conditions of 80-120°C (e.g., 90°C, 100°C, or 110°C, etc.), and the temperature of the contact reaction can be maintained at 80-120°C by any technique commonly used in the art, such as oven heating to maintain a stable contact reaction (lysis reaction) temperature. In addition, the lysis method provided by the present invention can realize the batch deprotection lysis of oligonucleotides synthesized in porous synthesis columns, such as the batch deprotection lysis of oligonucleotides in porous synthesis plates such as 96-well synthesis plates (or 96-well synthesis columns) and 384-well synthesis plates (or 384-well synthesis columns). Simultaneously, oligonucleotides synthesized in one or more well-dwelling synthesis plates (such as 96-well or 384-well plates) can be cleaved in batches under a constant temperature environment of 80-120°C (e.g., 100°C). The cleavage method of this invention can maintain a stable reaction temperature, which is more conducive to the smooth progress of the reaction. At the same time, it is suitable for the cleavage of oligonucleotides synthesized in large batches on a solid-phase basis, such as the cleavage of primers synthesized in batches on a solid-phase basis.

[0059] Furthermore, the lysis method according to the present invention may also include a subsequent process for extracting the lysis products. Due to the lysis method of the present invention, the product extraction process available under this invention is simpler to operate compared to existing ammonia oven lysis and gas-phase lysis methods. For example, in a preferred embodiment of the present invention, the method may further include washing the lysed oligonucleotides with acetonitrile and dissolving the eluted oligonucleotides with Tris-EDTA. The oligonucleotides thus extracted can be directly used in subsequent experimental production.

[0060] The inventors have discovered that the oligonucleotide cleavage reagents JS2 & S4 and the cleavage method using them provided by this invention can efficiently cleave oligonucleotides, especially those synthesized by solid-phase phosphorous amide. This reduces chain breakage and side reactions caused by high-temperature cleavage, and also reduces equipment costs. While shortening the cleavage time, it can effectively reduce oligonucleotide quality problems caused by cleavage.

[0061] The effects of the specific oligonucleotide synthesis catalyst of the present invention will be described in detail below through examples.

[0062] Example

[0063] Example 1: Preparation of Instruments and Materials

[0064] 1. The lysis reagent was prepared as follows:

[0065] 1) The microwave pyrolysis reagent of the published patent application CN109956987A was used as the control group pyrolysis reagent. The content of each component of the microwave pyrolysis reagent is shown in Table 2 below.

[0066] Table 2

[0067] reagents content Organic amines (n-butylamine, hexamethylenediamine, methylamine, etc.) 30ml Lysis buffer (ethanol:water = 1:9 to 5:5) 70ml

[0068] Note: The concentration of the microwave lysis reagent is determined by the proportion of organic amines: 20-90% by volume is acceptable.

[0069] 2) JS2 (NaOH) and JS4 (KOH) cleavage reagents with concentrations of 0.4 mol / L, 1 mol / L and 2 mol / L, respectively, were selected as the cleavage reagents for the experimental group. The contents of each component of the JS2 (NaOH) cleavage reagent are shown in Table 3 below.

[0070] Table 3

[0071] reagents 1M 0.4M 2M quantity JS2(NaOH) 4g 1.6g 8g Lysis buffer (ethanol:water = 1:9 to 5:5) 10ml 10ml 10ml JS4(KOH) 5.6g 2.24g 11.2g JS3 (Methanol) 90ml 90ml 90ml

[0072] The JS2&S4 lysis reagent of this application is prepared using the following method:

[0073] First, dissolve the weighed JS2 and JS4 reagents in lysis buffer; then dilute to 100 mL using JS3 reagent; JS3 reagent is the key reagent, and its proportion can be 60%-95% of the total solution volume.

[0074] 2. Oligonucleotide sample preparation shall be carried out as follows:

[0075] 1) The experimental group and the control group synthesized the same oligonucleotides using the solid-phase phosphorous amide method. The oligonucleotide sequence information and molecular weight are shown in Table 4 below.

[0076] 2) Eight samples are tested in each batch, and each experimental sample corresponds to one control. A total of 24 oligonucleotides are needed for the experimental group and 24 oligonucleotides for the control group.

[0077] 3) The oligonucleotides used in this experiment were synthesized on a 50 nmol carrier scale using the solid-phase phosphoramide method.

[0078] Table 4

[0079]

[0080]

[0081] 3. The names and sources of the remaining materials and instruments are shown in Table 5 below.

[0082] Table 5

[0083]

[0084]

[0085] Example 2: Cleavage of oligonucleotides using JS2 & S4 cleavage reagents

[0086] The experimental samples were processed using the JS2 & S4 lysis method (experimental group), and the specific steps are as follows:

[0087] 1) Add 20 μL / well of JS2 & S4 lysis reagent to the 96-well primer synthesis column;

[0088] 2) Place the 96-well plate holder face up, centrifuge at 300 rpm for 1 minute, and add 20 μL of JS2 & S4 lysis reagent.

[0089] 3) Add 40 mL of lysis buffer to the ammonia hydrolysis glass tank (17 cm × 12 cm × 6 cm), and invert the 96-well plate holder onto the ammonia hydrolysis holder. Tighten the lid of the ammonia hydrolysis tank and place it in an oven at 100 °C for 1 hour.

[0090] 4) After the reaction is complete, invert the 96-well plate holder in the centrifuge and centrifuge at 300 rpm for 1 minute;

[0091] 5) Place the 96-well plate holder on its side in a microwave oven and heat for 1 minute, then cool in a fume hood for 5 minutes;

[0092] 6) Add 200 μL of acetonitrile to each synthesis column, centrifuge at 1600 rpm / min for 5-10 seconds, discard the effluent, and repeat once;

[0093] 7) Add 200 μL of acetonitrile to each synthesis column, centrifuge at 1600 rpm / min for 1 minute, and discard the eluent; and

[0094] 8) Place the 96-well plate holder on the 96-well deep well plate, add 200 μL of TE buffer to each synthesis column, let stand for 1 minute, centrifuge at 1600 rpm / min for 1 minute, and collect the eluent.

[0095] Example 3: Cleavage of oligonucleotides using microwave lysis reagents

[0096] The experimental samples were treated using the microwave pyrolysis method (control group), and the specific steps are as follows (see also the description in patent application CN109956987A):

[0097] 1) Transfer the primer synthesis column to a 96-well plate holder and add 20 μL of microwave ammonolysis reagent into the synthesis column;

[0098] 2) Place the 96-well plate holder face up and centrifuge at 300 rpm for 1 minute (centrifuge purchased from Shanghai Luxiangyi Centrifuge Instrument Co., Ltd., model L-550), then add 20 μL of microwave ammonia hydrolysis reagent and let it stand for 3 minutes.

[0099] 3) Add 80 mL of microwave buffer solution to the microwave ammonia hydrolysis glass tank (17 cm × 12 cm × 6 cm), and place the 96-well plate holder upside down on the ammonia hydrolysis holder. Tightly close the lid of the ammonia hydrolysis tank and place it in a 700 W microwave oven on high for 12 minutes.

[0100] 4) After the reaction is complete, invert the 96-well plate holder in the centrifuge and centrifuge at 300 rpm for 1 minute;

[0101] 5) Place the 96-well plate holder on its side in a microwave oven and heat for 1 minute, then cool in a fume hood for 5 minutes;

[0102] 6) Add 200 μL of acetonitrile to each synthesis column, centrifuge at 1600 rpm / min for 5-10 seconds, discard the effluent, and repeat once;

[0103] 7) Add 200 μL of acetonitrile to each synthesis column, centrifuge at 1600 rpm / min for 1 minute, and discard the eluent; and

[0104] 8) Place the 96-well plate holder on the 96-well deep well plate, add 200 μL of sterile water to each synthesis column, let stand for 1 minute, centrifuge at 1600 rpm / min for 1 minute, and collect the effluent.

[0105] Test case

[0106] The eluents collected in the examples were subjected to QC-MS detection and OD measurement using an enzyme-linked immunosorbent assay (ELISA) reader. 260 The values ​​and 10% urea denaturation PAGE glue control analysis plate were tested, and the corresponding acceptance criteria for the three different testing methods are shown in Table 6 below.

[0107] Table 6

[0108]

[0109]

[0110] Note: 1) During the cleavage of oligonucleotides, there is a phenomenon of incomplete removal of protecting groups. Among them, the molecular weight of the oligonucleotide produced by incomplete removal of protecting groups is defined as impurities caused by incomplete cleavage.

[0111] 2) When using lysis reagents with organic amines as the main component, lysis side reactions may occur during the lysis process. Specifically, this manifests as the appearance of impurity peaks after the target molecule's mass spectrometry peak during detection, for example... Figure 2 As shown in a.

[0112] Test Example 1: OD measured using an ELISA reader 260 Value detection

[0113] The eluted experimental samples were quantitatively analyzed using a TECAN Infinite M201PRO microplate reader, and their OD values ​​were measured. 260 The results are shown in Table 7 (which shows the quantitative data of the experimental samples) and Table 8 (which shows the OD). 260 The quantitative values ​​are summarized as shown in the figure.

[0114] Table 7

[0115]

[0116] Table 8

[0117]

[0118] As shown in the table, the quantitative values ​​of the experimental samples treated by the JS2&S4 lysis method and the control group treated by the microwave lysis method were not significantly different, both meeting the current qualified standards for oligonucleotides. Moreover, the recovery of the experimental samples treated by the JS2&S4 lysis method was slightly higher than that of the control group.

[0119] Test Example 2: PAGE Electrophoresis Control Panel Detection

[0120] The three batches of experimental samples were combined and analyzed using an analytical plate. The results are as follows: Figure 1 As shown, where Figure 1 a and 1d show the analysis plates of experimental samples treated with the JS2&S4 pyrolysis method in three experiments (i.e., three different batches from the first to the third batch); Figure 1 Figures b and 1c show the analytical plate images of experimental samples treated with microwave lysis in three experiments (i.e., three different batches from the first to the third batch). The results show that the analytical plate images of samples treated by both methods are bright and clear, with no obvious impurities, meeting the release criteria for oligonucleotide analytical plates.

[0121] Test Example 3: Molecular Weight Mass Spectrometry Detection

[0122] The experimental samples were subjected to mass spectrometry detection, and the results are shown in Table 9 (showing the peak area percentage in terms of abundance percentage) and Table 10 (showing the summary of mass spectrometry detection results of the experimental samples).

[0123] Table 9

[0124]

[0125]

[0126] Note: N-1 and NX (X can be 1, 2, 3, 4, 5) represent oligonucleotide impurities that differ from the target synthesized oligonucleotide by one or more bases (bp or nt), i.e., the so-called base deficiency phenomenon. Their molecular weight differs from the target by approximately 300 or X*300, for example... Figure 2 The target mass spectrometry peak and the pyrolysis side reaction impurity peak are shown in figure a.

[0127] Table 10

[0128]

[0129] Note: The qualified group refers to the number of groups that meet the mass spectrometry fragmentation detection method, that is, the number of experimental groups that meet the standard of incomplete fragmentation and ammonolysis side reaction impurities ≤10%. The NX unqualified number refers to the number of groups that do not meet the qualified standard of the mass spectrometry detection method. The qualified standard for NX is N-1 to N-5 ≤4%, and NX ≤10%.

[0130] As shown in Table 10, which summarizes the results, excluding base deficiency caused by the difficulty of oligonucleotide synthesis, the mass spectrometry results of the experimental group samples treated by the JS2&S4 lysis method all meet the current oligonucleotide release standard of ≤10% incomplete lysis impurities. Furthermore, compared to the control group samples treated by the microwave lysis method, the results are significantly better. Figure 2 As shown in a and 2b, it has the following three advantages:

[0131] 1) No impurities caused by pyrolysis side reactions, such as Figure 2 As shown in b;

[0132] 2) The phenomenon of incomplete pyrolysis was significantly reduced; and

[0133] 3) The NX base deficiency phenomenon is slightly reduced.

[0134] In summary, through experimental comparison, the newly developed JS2&S4 oligonucleotide cleavage method has several advantages over the commonly used microwave cleavage method in terms of oligonucleotide cleavage quality: First, this cleavage method does not cause side reactions that affect oligonucleotide quality; second, although the cleavage time is longer, the phenomenon of incomplete cleavage is weaker; third, the JS2&S4 cleavage method uses an oven for heating, which can effectively reduce the phenomenon of high-temperature chain breakage through temperature control; fourth, this cleavage method does not require special heating equipment, and one oven can simultaneously cleave 18 synthesis plates. Therefore, the cleavage method provided by this invention is a highly effective oligonucleotide cleavage method that can be applied in the laboratory and in large-scale oligonucleotide production.

[0135] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

[0136] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not describe the various possible combinations separately.

[0137] Furthermore, various different embodiments of the present invention can be combined in any way, as long as they do not violate the spirit of the present invention, they should also be regarded as the content disclosed by the present invention.

[0138] References

[0139] [1]MPREDDY,NBHANNA,&F.FAROOQUI.(2010).Cheminform abstract:fastcleavage and deprotection of oligonucleotides.Cheminform,25(25),no-no.

[0140] [2]MPReddy and NBHanna and Firdous Farooqui.(1994).Fast cleavageand deprotection of oligonucleotides.Tetrahedron Letters.

[0141] [3] Pon, RT. (2001). Solid-phase supports for oligonucleotide synthesis. Methods in Molecular Biology, 20, 465-496.

[0142] [4] Chinese patent application CN109956987A.

Claims

1. An oligonucleotide cleavage reagent consisting of NaOH, KOH, methanol, and a cleavage buffer; wherein, The content of the methanol is 60-95% by volume based on the total volume of the oligonucleotide cleavage reagent; the concentration of NaOH and KOH in the oligonucleotide cleavage reagent is independently 0.4-2 mol / L; the cleavage buffer is an ethanol aqueous solution, and the volume ratio of ethanol to water is 1:9-5:

5.

2. The oligonucleotide cleavage reagent of claim 1, wherein, The content of the methanol is 90% by volume based on the total volume of the oligonucleotide cleavage reagent.

3. The oligonucleotide cleavage reagent of claim 1, wherein, The concentration of NaOH and KOH in the oligonucleotide cleavage reagent is independently 1 mol / L.

4. The oligonucleotide cleavage reagent of claim 1, wherein, The volume ratio of ethanol to water in the ethanol aqueous solution is 2:

8.

5. Use of the oligonucleotide cleavage reagent according to any one of claims 1-4 for cleaving oligonucleotides synthesized on a solid support.

6. A method for preparing the oligonucleotide cleavage reagent according to any one of claims 1-4, comprising contacting NaOH, KOH, methanol and a cleavage buffer with each other.

7. The method of claim 6, wherein, NaOH, KOH and the cleavage buffer are contacted with each other first, and then the obtained solution is contacted with methanol.

8. A method for cleaving an oligonucleotide comprising contacting a solid phase synthesized oligonucleotide with an oligonucleotide cleaving reagent, wherein, The oligonucleotide cleavage reagent is the oligonucleotide cleavage reagent according to any one of claims 1-4, and the contacting is performed at a temperature of 80-120℃ for 0.5-2 h.

9. The method of claim 8, wherein, The amount of the oligonucleotide cleavage reagent is 0.1-10 μL per nmol of oligonucleotide based on the molar amount of the oligonucleotide.

10. The method of claim 9, wherein, The amount of the oligonucleotide cleavage reagent is 0.8 μL per nmol of oligonucleotide based on the molar amount of the oligonucleotide.

11. The method according to claim 8, wherein the contacting is performed under constant temperature conditions.

12. The method according to claim 8 or 11, wherein the contacting is performed under constant temperature conditions at 100℃ for 1 h.

13. The method according to claim 8, further comprising washing the cleaved oligonucleotide with acetonitrile and eluting the oligonucleotide with a Tris-EDTA solution.

14. A method for cleaving a solid-phase synthesized oligonucleotide, comprising the following steps: Step 1 : adding an oligonucleotide cleavage reagent into a synthesis column for solid phase synthesis of an oligonucleotide, wherein the oligonucleotide cleavage reagent consists of NaOH, KOH, methanol, and a cleavage buffer, wherein, The content of the methanol is 60-95% by volume based on the total volume of the oligonucleotide cleavage reagent; the concentration of NaOH and KOH in the oligonucleotide cleavage reagent is independently 0.4-2 mol / L; the cleavage buffer is an ethanol aqueous solution, and the volume ratio of ethanol to water is 1:9-5:

5. Step 2: placing the synthesis column in a sealable container containing a cleavage buffer, and reacting at 80-120℃ for 0.5-1.5 h; Step 3: removing the water in the synthesis column, washing the synthesis column with a washing solution, and then treating the synthesis column with an elution solution to collect the effluent.

15. The method according to claim 14, wherein the content of the methanol is 90% by volume based on the total volume of the oligonucleotide cleavage reagent in step 1.

16. The method according to claim 14 or 15, wherein the concentration of NaOH and KOH in the oligonucleotide cleavage reagent is independently 1 mol / L in step 1.

17. The method according to claim 14, wherein the volume ratio of ethanol to water in the ethanol aqueous solution is 2:

8.

18. The method of claim 14, wherein the synthesis column in step 1 is one or more.

19. The method of claim 14, wherein the synthesis column in step 1 is a 96-well synthesis plate.

20. The method of claim 14, wherein the step 2 is reacted at a constant temperature of 80-120 °C for 0.5-1.5 h.

21. The method of claim 14, wherein the step 2 is reacted at a constant temperature of 100 °C for 1 h.

22. The method of claim 14, wherein the method of removing water from the synthesis column in step 3 comprises centrifugation and oven drying.

23. The method of claim 14, wherein the washing solution in step 3 comprises acetonitrile and the elution solution comprises Tris-EDTA.

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