CN114426565A - Oligonucleotide cracking reagent - Google Patents

Abstract

The present invention provides an oligonucleotide cleavage reagent (JS2&S4 reagent) and a preparation method thereof, as well as a method for cleaving oligonucleotides, characterized in that the oligonucleotide cleavage reagent comprises NaOH, KOH , methanol, and lysis buffer. The oligonucleotide cleavage reagent JS2&S4 and the cleavage method using the oligonucleotide cleavage reagents provided by the present invention can efficiently cleavage oligonucleotides, especially oligonucleotides synthesized by solid-phase phosphoramidite, which not only reduces the chain breaking caused by cleaving high temperature And the occurrence of side reactions also reduces equipment costs; while shortening the cleavage time, it can effectively reduce the quality problems of oligonucleotides caused by cleavage.

Description

An oligonucleotide cleavage reagent

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to the PCT patent application with the application number PCT/CN2020/124821 and the invention titled "An oligonucleotide cleavage reagent" filed on October 29, 2020, the entire contents of which are incorporated herein by reference .

technical field

The present invention belongs to the field of nucleotide cleavage, and more particularly, relates to an oligonucleotide cleavage reagent and a preparation method thereof, as well as a method for cleaving oligonucleotides, especially solid-phase synthesized oligonucleotides .

Background technique

Oligonucleotides, also known as oligonucleotides, are mostly synthesized by the solid-phase phosphoramidite triester method; this method is to immobilize oligonucleotides on a solid-phase carrier through cycles of deprotection, coupling, capping, and oxidation. Four steps complete the synthesis of the oligonucleotide chain. For the cleavage of crude oligonucleotides synthesized by solid-phase synthesis, currently widely used oligonucleotide cleavage methods mainly include ammonia water oven cleavage method, gas phase cleavage method and microwave cleavage method, etc., but their cleavage effects have their own advantages and disadvantages. , as shown in Table 1 below.

Table 1

Although the quality of the oligonucleotides cracked by the ammonia water oven cracking method is acceptable, it has the following defects: First, the cracking method is a single-tube operation, which cannot be adapted to today's large-scale production experiments; Second, the cracked oligonucleotides The product needs to be desalted by a C18 column before it can be detected and applied, and the operation is cumbersome; third, the cracking time is long, and it takes 2-3h.

The gas-phase cracking method is an evolutionary version of the ammonia-water oven cracking method. Although it greatly simplifies the operation and improves the product quality, the cracking time remains at 2-3h, and special high-pressure equipment is required for cracking. The increase in throughput is limited, and it is still unable to meet the increasing demand for production experiments.

Microwave cleavage is the fastest oligonucleotide cleavage method at present, and it only takes 12 minutes; but its disadvantages are also obvious. First, the quality of the products obtained from cleavage is low, which cannot meet the needs of customers for more oligonucleotides. Second, the microwave reactor has the problem of poor temperature control, which can easily cause the phenomenon of high temperature cleavage of oligonucleotide chains; When cleaving oligonucleotides, cleavage side reactions are common, which affects the quality of oligonucleotides.

In summary, in order to meet the high-quality requirements of customers for oligonucleotides, it is urgent to explore an efficient and reasonable oligonucleotide cleavage method, which can effectively reduce the oligonucleotide caused by cleavage while shortening the cleavage time. Nucleotide quality issues.

SUMMARY OF THE INVENTION

The purpose of the present invention is to optimize the cleavage reagent used in the oligonucleotide cleavage process in order to solve the defect that it is difficult to take into account the operation time, product quality and equipment cost in the oligonucleotide cleavage process in the prior art, and more specifically Therefore, an oligonucleotide cleavage reagent that is more suitable for cleaving solid-phase synthetic oligonucleotides is provided, so as to shorten the cleavage time and effectively reduce the quality problems of oligonucleotides caused by cleavage.

The inventors have found and confirmed that the oligonucleotide cleavage reagent (also referred to herein as JS2&S4 reagent) provided by the present application is used as the cleavage reagent for separating crude oligonucleotide products from the solid synthetic support, and then used in an oven The method of heating at about 100 °C for about 1 hour can realize the batch deprotection and cleavage of the oligonucleotides synthesized on the 96-well plate. After washing with acetonitrile, the eluted oligonucleotides can be used for subsequent experimental production. middle. Based on the above findings, the present inventors have thus completed the present invention.

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

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

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 independently 0.4mol/L, 0.6mol/L, 0.8mol/L, 1mol/L, 1.2mol/L, 1.5 mol/L, 1.8mol/L or 2mol/L. In a 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 a specific embodiment, the concentrations of NaOH and KOH in the oligonucleotide cleavage reagent are each independently 2 mol/L.

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

In one embodiment of the present invention, the oligonucleotide cleavage reagent is used for cleaving oligonucleotides synthesized on a solid support.

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

In one embodiment of the present invention, the NaOH, KOH and lysis buffer are contacted with each other first, and then the resulting solution is contacted with methanol.

In another aspect, the present invention also provides a method for cleaving an oligonucleotide, the method comprising contacting a solid-phase synthesized oligonucleotide with an oligonucleotide cleavage reagent, wherein the oligonucleotide Glyide cleavage reagents include NaOH, KOH, methanol, and lysis buffer.

The definitions and all features of the oligonucleotide cleavage reagent described above are applicable to the oligonucleotide cleavage reagent in this method, and will not be repeated here.

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

In one embodiment of the present invention, the contacting is carried out at a temperature of 80-120°C for 0.5-2 h. In a specific embodiment, the contacting is performed under a constant temperature of 80-120°C. In another specific embodiment, the contacting is performed at a constant temperature of 100°C for 1 h. The constant temperature condition can be achieved by any heating and heat preservation device, such as an oven, and maintaining a constant temperature is conducive to the stable progress of the reaction, so as to reduce the occurrence of side reactions.

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

Another aspect of the present invention provides a method for cleaving solid-phase synthetic oligonucleotides, comprising the steps of:

Step 1: adding an oligonucleotide cleavage reagent to the synthesis column for solid-phase synthesis of oligonucleotides, wherein the oligonucleotide cleavage reagent comprises NaOH, KOH, methanol and a lysis buffer;

Step 2: place the synthesis column in a sealable container with lysis buffer, and react at 80-120°C for 0.5-1.5h;

Step 3: Take out the synthesis column to remove the water therein, wash the synthesis column with the washing solution, and then treat the synthesis column with the eluent to collect the effluent.

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

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.

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

In some embodiments of the present invention, the number of synthesis columns in step 1 is one or more, preferably a 96-well synthesis plate.

In some embodiments of the present invention, the step 2 is to react at a constant temperature of 80-120 °C for 0.5-1.5 h, preferably at a constant temperature of 100 °C for 1 h.

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

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

Beneficial technical effects of the present invention:

The oligonucleotide cleavage reagents JS2&S4 and the cleavage method using the oligonucleotide cleavage reagents provided by the present invention can efficiently cleavage oligonucleotides, especially oligonucleotides synthesized by solid-phase phosphoramidite, which not only reduces the chain breakage caused by high temperature cleavage And the occurrence of side reactions also reduces equipment costs; while shortening the cleavage time, it can effectively reduce the quality problems of oligonucleotides caused by cleavage.

Description of drawings

The accompanying drawings are used to provide a further understanding of the present invention, and constitute a part of the specification, and together with the following specific embodiments, are used to explain the present invention, but do not constitute a limitation to the present invention. In the attached image:

Fig. 1 shows the analysis plate gel image of the experimental sample processed according to the cleavage method shown in the embodiment of the present invention, wherein Figs. 1a and 1d are the analysis plate image of the experimental sample processed by the JS2&S4 cleavage method of three experiments, Fig. 1b and 1c Plate gel maps were analyzed for the microwave-lysed experimental samples of the three experiments; and

Fig. 2 shows the mass spectrometry detection result of the experimental sample processed according to the cracking method shown in the embodiment of the present invention, wherein Fig. 2a is the mass spectrum peak diagram of the product obtained by the microwave cracking method of CPR-01-T-seqR; Fig. 2b is the CPR -01-T-seqR JS2&S4 cleavage method obtained product mass spectrum peak map.

Detailed ways

Specific embodiments of the present invention will be described in detail below. It should be understood that the specific embodiments described herein are only used to illustrate and explain the present invention, but not to limit the present invention.

The endpoints of ranges and any values disclosed herein are not limited to the precise ranges or values, which are to be understood to encompass values proximate to those ranges or values. For ranges of values, the endpoints of each range, the endpoints of each range and the individual point values, and the individual point values can be combined with each other to yield one or more new ranges of values that Ranges should be considered as specifically disclosed herein.

Before describing the present invention in detail, it is to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the invention, which is defined solely by the appended claims. For a more complete understanding of the invention described herein, the following terms are employed, the definitions of which are shown below. 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 belongs.

The term "DNA solid-phase synthesis" or "oligonucleotide solid-phase synthesis" as used herein refers to a method commonly used in the art for synthesizing DNA strands or oligonucleotide strands, including: combining the ends of the DNA strands to be synthesized Nucleotides (eg, 3'-terminal nucleotides) are pre-immobilized on an insoluble solid support, and other nucleotides are added one by one in a predetermined sequence from this end until the entire DNA chain is synthesized. Each additional nucleotide residue undergoes one round of the same operation (see the Background section), and keeps the extending DNA strand always immobilized on the solid support. Excess unreacted material or reaction by-products can be removed by filtration or washing. The DNA chain synthesized to the desired length can be cut off from the solid support and various protecting groups can be removed, and then purified to obtain the final product. DNA strands synthesized in this way are usually several tens of bases, or as many as several hundreds of bases, which can be used, for example, as PCR primers, linkers or probes, and the like.

The term "synthesis column" as used herein generally includes three parts: solid support, frit and empty column tube. The empty column tube is usually made of polypropylene injection molding, the upper opening is larger than the lower opening, and the solid phase carrier and the sieve plate are filled in it. "Porous synthesis column" includes synthesis plate of one or more synthesis columns, such as 96-well synthesis plate, 384-well synthesis plate.

The term "oligonucleotide cleavage" or "oligonucleotide cleavage reaction" as used herein refers to the final stage of oligonucleotide (DNA) solid phase synthesis by oligonucleotide cleavage reagents such as NaOH, KOH , methanol and cleavage buffer cleavage reagents, the chemical process of cleaving the synthesized DNA strand from the solid support, cleaving the amino protecting group on the base and/or cleaving the phosphate group at the 3' end.

The term "sealable container" as used herein refers to a container in which the oligonucleotide cleavage reaction is carried out, which container may be sealed, eg, by capping. The sealable container is heatable, eg, made of glass, ceramic, or the like. When the lysis reaction is performed, the lysis buffer is added in advance to generate steam under microwave treatment. The steam enters the pore size of the solid-phase carrier through the upper and lower ports of the synthesis column (ie, the synthesis column is placed in a "steam atmosphere") to promote the cleavage reaction between the cleavage reagent and the solid-phase synthesized oligonucleotide.

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

According to the present invention, based on the physicochemical properties of each component, the JS2&S4 reagent can generally be in the form of a solution in which JS2 and JS4 are dissolved in JS3 and lysis buffer, so the volume will basically depend on JS3 and lysis buffer volume of. As the key reagent of the oligonucleotide cleavage reagent according to the present invention, the JS3 reagent can generally have a higher volume content ratio than the lysis buffer. For example, in a preferred embodiment of the present invention, based on the total volume of the oligonucleotide cleavage reagent (approximately equal to or even equal to the sum of the volumes of the JS3 reagent and the lysis buffer), the volume of the JS3 reagent (methanol) The content may be 60-95% by volume, such as 70% by volume, 80% by volume, or 90% by volume, and the like.

Accordingly, in the JS2&S4 reagent according to the present invention, JS2 and JS4 may generally appear in the form of solutes dissolved in the above-mentioned JS4 and lysis buffer, wherein the contents of said JS2 and JS4 as solutes may 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 cleavage reagent may be independently 0.4-2mol/L (ie 0.4-2M), while not affected by each other. For example, in a preferred embodiment, the concentration of NaOH in the oligonucleotide cleavage reagent can be independently 0.5mol/L, 1mol/L or 1.5mol/L, etc. Similarly, the oligonucleotide cleavage reagent The concentration of KOH in the reagent can also be independently 0.5 mol/L, 1 mol/L or 1.5 mol/L, etc.

According to the present invention, the type of lysis buffer used in the present invention is not particularly limited, and can be a common lysis buffer in the art. For example, in one embodiment of the present invention, the lysis buffer may be an aqueous ethanol solution, and wherein the volume ratio of ethanol to water is 1:9-5:5 (eg, 2:8, 3:7, or 4:6). etc.), but the lysis buffer of the present invention is not limited thereto.

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

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

Further, methanol or lysis buffer can be used for final volume for the sake of accurate final volume. For example, in a preferred embodiment of the present invention, NaOH, KOH and lysis buffer can be contacted with each other first (which can be regarded as a dissolution process), and then the resulting solution is contacted with methanol (which can be regarded as a constant volume process). In another preferred embodiment of the present invention, NaOH, KOH and methanol can also be contacted with each other first, and then the resulting solution can be contacted with the lysis buffer.

In another aspect, the present invention also provides a method for cleaving an oligonucleotide, the method comprising contacting a solid-phase synthesized oligonucleotide with an oligonucleotide cleavage reagent, wherein the oligonucleotide Glyide cleavage reagents include NaOH, KOH, methanol, and lysis buffer.

According to the present invention, in the cleavage method of the present invention, the amount of the cleavage reagent (JS2&S4 cleavage reagent) is not particularly limited, and can be judged according to the experience of those skilled in the art. In order to achieve a better cleavage effect, in a preferred embodiment of the present invention, based on the molar amount of the oligonucleotide, the amount of the oligonucleotide cleavage reagent may be 0.1-10 μL/nmol (for example, 0.5 μL /nmol, 1 μL/nmol or 5 μL/nmol) oligonucleotides.

Based on the beneficial effects of the present invention mentioned above, the cleavage method using the oligonucleotide cleavage reagent provided by the present invention can achieve an excellent oligonucleotide cleavage effect in a relatively short time under relatively low temperature conditions and quality. For example, in a preferred embodiment of the present invention, the contacting can be carried out at a temperature of 80-120°C (eg, 90°C, 100°C or 110°C, etc.) for 0.5-2h (0.8h, 1h or 1.5h, etc.) . More preferably, the contacting can be carried out at a constant temperature of 80-120°C (for example, 90°C, 100°C or 110°C, etc.), and the temperature of the contact reaction can be maintained at 80-120°C by any technical means common in the art. ℃, such as oven heating to maintain a stable contact reaction (cracking reaction) temperature. In addition, the cleavage method provided by the present invention can realize the batch deprotection cleavage of the oligonucleotides synthesized in the porous synthesis column, such as through a 96-well synthesis plate (or a 96-well synthesis column), a 384-well synthesis plate ( Or 384-well synthesis column) and other multi-well synthesis plate oligonucleotide batch deprotection and cleavage. At the same time, in a constant temperature environment (such as an oven) maintained at 80-120 °C (such as 100 °C), one or more multi-well synthesis plates (such as 96-well synthesis plates, 384-well synthesis plates) synthesized oligonuclei can be cleaved in batches. Glycosides. The cleavage method of the present invention can maintain a stable reaction temperature, which is more conducive to the smooth progress of the reaction, and at the same time, is suitable for the cleavage of oligonucleotides synthesized by solid phase synthesis in large quantities, such as the cleavage of solid phase synthesis primers in batches.

In addition, the cleavage method according to the present invention may also include a subsequent process of extracting the cleavage product. Due to the cracking method of the present invention, the process of extracting the product available in the present invention can be operated more simply than the existing ammonia water oven cracking method and gas phase cracking method. For example, in a preferred embodiment of the present invention, the method may further comprise washing the cleaved oligonucleotides with acetonitrile, and dissolving the eluted oligonucleotides with Tris-EDTA. The oligonucleotides thus extracted can be directly used in subsequent experimental production.

The inventors have found that the oligonucleotide cleavage reagents JS2&S4 provided by the present invention and the cleavage method using the same can efficiently cleavage oligonucleotides, especially oligonucleotides synthesized by solid-phase phosphoramidite, which not only reduces The occurrence of chain scission and side reactions caused by high temperature cleavage also reduces equipment costs; while shortening the cleavage time, it can effectively reduce the quality problems of oligonucleotides caused by cleavage.

Hereinafter, the effects of the specific oligonucleotide synthesis catalyst of the present invention will be described in detail by way of examples.

Example

Example 1: Preparation of Instruments and Materials

1. The preparation of the lysis reagent is as follows:

1) Referring to the microwave cleavage reagent of the published patent application CN109956987A as the cleavage reagent of the control group, the content of each component of the microwave cleavage reagent is shown in Table 2 below.

Table 2

reagent content Organic amines (n-butylamine, hexamethylenediamine, methylamine, etc.) 30ml Lysis buffer (ethanol:water=1:9～5:5) 70ml

Note: The concentration of microwave cracking reagent is determined by the proportion of organic amine: 20-90% by volume.

2) The JS2&S4 cleavage reagents with the concentrations of JS2(NaOH) and JS4(KOH) being respectively 0.4mol/L, 1mol/L and 2mol/L were selected as the cleavage reagents of the experimental group, and the content of each component of the JS2&S4 cleavage reagents was as follows shown in Table 3.

table 3

reagent 1M amount 0.4M amount 2M amount JS2(NaOH) 4g 1.6g 8g Lysis buffer (ethanol:water=1:9～5:5) 10ml 10ml 10ml JS4(KOH) 5.6g 2.24g 11.2g JS3 (methanol) 90ml 90ml 90ml

Wherein, the JS2&S4 cleavage reagent of the present application is prepared by the following method:

First dissolve the weighed JS2 and JS4 reagents with lysis buffer; use JS3 reagent to dilute to 100mL; JS3 reagent is the key reagent, and its proportion can account for 60%-95% of the total volume of the solution.

2. The oligonucleotide sample preparation is carried out as follows:

1) The same oligonucleotides synthesized by the solid-phase phosphoramidite method were used in the experimental group and the control group. The sequence information and molecular weight of the oligonucleotides are shown in Table 4 below.

2) 8 samples were tested in each batch, and each experimental sample corresponds to a reference substance, a total of 24 oligonucleotides in the experimental group and 24 oligonucleotides in the control group are required.

3) The oligonucleotides of this experiment were synthesized using the solid-phase phosphoramidite method at a scale of 50 nmol carrier.

Table 4

3. The names and sources of other materials and instruments used are shown in Table 5 below.

table 5

Example 2: Cleavage of oligonucleotides by JS2&S4 cleavage reagents

The experimental samples were treated by JS2&S4 cracking method (experimental group) respectively. The specific steps are as follows:

1) Add 20 μL/well JS2&S4 cleavage reagent to the primer 96-well synthesis column;

2) Centrifuge the 96-well plate with the front side up, 300 rpm/min for 1 minute, and add 20 μL of JS2&S4 lysis reagent.

3) Add 40 mL of lysis buffer to the aminolysis glass tank (size 17cm×12cm×6cm), place the 96-well plate holder upside down on the aminolysis holder, close the lid of the ammonialysis tank, and place it in an oven at 100°C for 1 hour ;

4) After the reaction, the 96-well plate holder was inverted in a centrifuge, and centrifuged at 300 rpm/min for 1 minute;

5) Heat the 96-well plate holder side in a microwave oven for 1 minute, then cool in a fume hood for 5 minutes;

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;

7) Add 200 μL of acetonitrile to each synthesis column, centrifuge at 1600 rpm/min for 1 minute, and discard the flow-through; and

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

Example 3: Cleavage of oligonucleotides by microwave cleavage reagents

The experimental samples were treated by the microwave cracking method (control group) respectively, and the specific steps were as follows (see the description in the patent application CN109956987A for details):

1) Transfer the primer synthesis column to the 96-well plate holder, and add 20 μL of microwave aminolysis reagent to the synthesis column;

2) The 96-well plate holder was turned upside down, centrifuged at 300rpm/min for 1 minute (the centrifuge was purchased from Shanghai Luxiangyi Centrifuge Instrument Co., Ltd., model L-550), and 20 μL of microwave ammonolysis reagent was added, and then allowed to stand. 3 minutes;.

3) Add 80 mL of microwave buffer to the microwave ammonia solution glass tank (size 17cm×12cm×6cm), place the 96-well plate holder upside down on the ammonia solution holder, close the lid of the ammonia solution tank, and put it in a 700W microwave oven on high heat 12 minutes of reaction;

4) After the reaction, the 96-well plate holder was inverted in a centrifuge, and centrifuged at 300 rpm/min for 1 minute;

5) Heat the 96-well plate holder side in a microwave oven for 1 minute, then cool in a fume hood for 5 minutes;

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;

7) Add 200 μL of acetonitrile to each synthesis column, centrifuge at 1600 rpm/min for 1 minute, and discard the flow-through; and

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

test case

Take the eluate collected in the examples for QC-MS detection, use a microplate reader to determine its OD ₂₆₀ value and 10% urea denaturing PAGE gel in the control analysis plate detection, according to three different detection methods and confirmed qualified The criteria are shown in Table 6 below.

Table 6

Note: 1) During the cleavage process of oligonucleotides, the protective group is not completely removed, and the molecular weight produced by the incomplete removal of the protective group is greater than the molecular weight of the target oligonucleotide, which is defined as an impurity caused by incomplete cleavage.

2) When the cleavage reagent with organic amine as the main component is selected, the cleavage side reaction phenomenon will occur during the cleavage process, and the specific manifestation is that the target molecule mass spectrum peak appears after the mass spectrometry detection, for example, as shown in Figure 2a.

Test Example 1: Detection of OD ₂₆₀ value using a microplate reader

Use the TECAN infinite M201PRO microplate reader to quantitatively detect the eluted experimental samples and measure their OD ₂₆₀ . The results are as follows in Table 7 (showing the quantitative data of the experimental samples) and Table 8 (showing the summary of the OD ₂₆₀ quantitative values) shown.

Table 7

Table 8

As shown in the data in the table, the quantitative values of the experimental samples treated by the JS2&S4 cleavage method and the control group treated by the microwave cleavage method have no significant difference, and both meet the current oligonucleotide qualification standards, and the recovery of the experimental samples treated by the JS2&S4 cleavage method The amount was slightly higher than that of the control group.

Test example 2: PAGE electrophoresis control analysis plate detection

Three batches of experimental samples were combined for assay plate detection, and the detection results are shown in Figure 1, wherein Figures 1a and 1d show the JS2&S4 lysis method for the three experiments (that is, three different batches from the first to the third batch). The experimental sample analysis plate gel map of ; Figures 1b and 1c show the analysis plate gel map of the experimental samples processed by the microwave lysis method for three experiments (ie, three different batches from the first batch to the third batch). The results showed that the gel images of the samples treated by the two methods were bright and clear, with no obvious stray bands, which met the criteria for judging the release of oligonucleotide analysis plates.

Test Example 3: Molecular Weight Mass Spectrometry Detection

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

Table 9

Note: N-1 and N-X (X can be 1, 2, 3, 4, 5) represent oligonucleotide impurities that differ by one or more bases (bp or nt) from the oligonucleotide obtained by the target synthesis, namely The so-called lack of base, the molecular weight of the target substance differs by about 300 or X*300, such as the target substance spectrum detection peak and the cleavage side reaction impurity peak shown in Figure 2a.

Table 10

Note: Qualified group refers to the number of groups that meet the mass spectrometry cracking detection method, that is, the number of experimental groups that meet the standard of incomplete cracking and ammonolysis side reaction impurities ≤ 10%. The number of unqualified N-X refers to the number of groups that do not meet the qualification standard of the mass spectrometry detection method. The qualified standard of N-X is N-1～N-5≤4%, and N-X≤10%.

It can be seen from Table 10 of the summary of the results that, excluding the lack of bases caused by the difficulty of synthesizing some oligonucleotides, the mass spectrometry detection results of the samples in the experimental group treated by the JS2&S4 cleavage method are in line with the current oligonucleotide release standards. Incomplete impurities are less than or equal to 10%, and compared with the control samples treated by microwave pyrolysis, as shown in Figures 2a and 2b, it has the following three advantages:

1) No impurities caused by cracking side reactions, as shown in Figure 2b;

2) The phenomenon of incomplete cracking is greatly reduced; and

3) The phenomenon of lack of base N-X is relatively slightly weakened.

In summary, through experimental comparison, the newly developed JS2&S4 oligonucleotide cleavage method has some advantages in the quality of oligonucleotide cleavage compared with the commonly used microwave cleavage method: First, this cleavage method does not cause side effects. The reaction affects the quality of oligonucleotides; secondly, although the cleavage time is longer, the incomplete cleavage phenomenon is weak; thirdly, the JS2&S4 cleavage method uses oven heating, which can effectively reduce the phenomenon of high temperature chain scission through temperature control; Fourth, this cracking method does not require special heating equipment, and one oven can satisfy the simultaneous cracking of 18 synthetic plates. Therefore, the cleavage method provided by the present invention is a quite effective oligonucleotide cleavage method, which can be applied in laboratory and large-scale oligonucleotide production.

The preferred embodiments of the present invention are described in detail above, but the present invention is not limited to the specific details of the above-mentioned embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solutions of the present invention. These simple modifications All belong to the protection scope of the present invention.

In addition, it should be noted that the specific technical features described in the above-mentioned specific embodiments can be combined in any suitable manner under the condition of no contradiction. In order to avoid unnecessary repetition, the present invention has The combination method will not be specified otherwise.

In addition, the various embodiments of the present invention can also be combined arbitrarily, as long as they do not violate the spirit of the present invention, they should also be regarded as the contents disclosed in the present invention.

references

[1] M.P.REDDY, N.B.HANNA, & F. FAROOQUI. (2010). Cheminform abstract: fastcleavage and deprotection of oligonucleotides. Cheminform, 25(25), no-no.

[2] M.P.Reddy and N.B.Hanna and Firdous Farooqui. (1994). Fast cleavage and deprotection of oligonucleotides. Tetrahedron Letters.

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

[4] Chinese patent application CN109956987A.

Claims (21)

1. An oligonucleotide lysis reagent comprising NaOH, KOH, methanol, and a lysis buffer.

2. The oligonucleotide cleaving reagent according to claim 1, wherein the methanol is present in an amount of 60-95 vol%, preferably 90 vol%, based on the total volume of the oligonucleotide cleaving reagent.

3. The oligonucleotide cleaving reagent of claim 1, wherein the concentration of NaOH and KOH in the oligonucleotide cleaving reagent is each independently 0.4-2mol/L, preferably each independently 1 mol/L.

4. The oligonucleotide lysis reagent according to claim 1, wherein the lysis buffer is aqueous ethanol and wherein the volume ratio of ethanol to water is 1:9-5:5, preferably 2: 8.

5. An oligonucleotide cleavage reagent according to any one of claims 1 to 4 for use in cleaving an oligonucleotide synthesized on a solid support.

6. A method of preparing the oligonucleotide cleavage reagent of any one of claims 1-5, comprising contacting NaOH, KOH, methanol, and a cleavage buffer with one another.

7. The process of claim 6, wherein NaOH, KOH and lysis buffer are contacted with each other before contacting the resulting solution with methanol.

8. A method for cleaving an oligonucleotide comprising contacting a solid phase synthesized oligonucleotide with an oligonucleotide cleaving reagent, wherein the oligonucleotide cleaving reagent comprises NaOH, KOH, methanol, and a cleaving buffer.

9. The method according to claim 8, wherein the oligonucleotide cleaving reagent is used in an amount of 0.1-10 μ L/nmol oligonucleotide, preferably 0.8 μ L/nmol, based on the molar amount of the oligonucleotide.

10. The method of claim 8, wherein the contacting is performed at a temperature of 80-120 ℃ for 0.5-2 h.

11. The method of claim 10, wherein the contacting is performed under isothermal conditions.

12. The method of claim 10 or 11, wherein the contacting is performed at a constant temperature of 100 ℃ for 1 hour.

13. The method of claim 8, further comprising washing the cleaved oligonucleotide with acetonitrile and solubilizing the eluted oligonucleotide with Tris-EDTA.

14. A method for cleaving a solid phase synthetic oligonucleotide comprising the steps of:

step 1: adding an oligonucleotide lysis reagent to a synthesis column for solid phase synthesis of oligonucleotides, wherein the oligonucleotide lysis reagent comprises NaOH, KOH, methanol, and a lysis buffer;

step 2: placing the synthetic column in a sealable container filled with lysis buffer solution, and reacting for 0.5-1.5h at 80-120 ℃;

and step 3: taking out the synthetic column to remove water, washing the synthetic column with washing liquid, treating the synthetic column with eluent, and collecting effluent.

15. The method according to claim 14, wherein in step 1, the methanol is contained in an amount of 60-95 vol%, preferably 90 vol%, based on the total volume of the oligonucleotide cleaving reagent.

16. The method of claim 14 or 15, wherein the concentration of NaOH and KOH in the oligonucleotide cleaving reagent in step 1 is independently 0.4-2mol/L, preferably independently 1 mol/L.

17. The method according to claim 14, wherein the lysis buffer is an aqueous ethanol solution and wherein the volume ratio of ethanol to water is 1:9 to 5:5, preferably 2: 8.

18. The method according to claim 14, wherein the synthesis column in step 1 is one or more, preferably a 96-well synthesis plate.

19. The method according to claim 14, wherein the step 2 is carried out at 80-120 ℃ for 0.5-1.5h, preferably at 100 ℃ for 1 h.

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

21. The method of claim 14, wherein in step 3 the wash solution comprises acetonitrile and the eluent comprises Tris-EDTA.

Images