RU2501802C1 - Method of purifying g-rich oligodeoxyribonucleotides - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: method of purifying G-rich oligodeoxyribonucleotides with size of 20-50 bases from silicic acid derivative impurities comprises the following consecutive steps: using oligodeoxyribonucleotides synthesised on controlled pore glass (CPG), where the last dimethoxytrityl protection is not removed; preparing an oligodeoxyribonucleotide solution by mixing 300 mcl of oligodeoxyribonucleotide with concentration of 0.5 mcm/ml with 25 ml of water and 0.5 ml of 2.0 M solution of an ion-pair reagent of triethylammonium acetate, hereinafter TEAA; the solutions are deposited on Glen Research Poly-Pak Cartridges, 60-1100-10, which contain a polymer carrier having dimethoxytrityl affinity; 50 ml Millipore, Plastic syringes, XX1105005, without plungers are connected to the cartridges; the cartridges with syringes are connected to a vacuum manifold CHROMBOND 730151; a 50 kPa vacuum is formed using a water-jet pump; the cartridges are washed with 2 ml aqueous solution of ammonia and TEAA of the following composition; 1/40 volume of 2.0 M TEAA solution is added to 20-fold diluted 25% ammonia solution; the cartridges are washed with 2 ml of water, 2 ml of 2% aqueous trifluoroacetic acid solution and 4 ml deionised water; the purified oligodeoxyribonucleotide is removed from the cartridges by washing the cartridges with 2 ml of a mixture of 95% ethyl alcohol with 25% ammonia solution; the purified oligodeoxyribonucleotide is separated from the solution by placing a test-tube with the solution of purified oligodeoxyribonucleotide into a concentrator, evaporating the solution for 10 hours at temperature of 60°C.

EFFECT: efficient method of purifying oligodeoxyribonucleotides with high output and high degree of purification from silicic acid derivatives suitable for large-scale synthesis.

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Description

The invention relates to the field of biochemistry and molecular biology, and in particular to a method for the purification of synthetic oligodeoxyribonucleotides.

The synthesis of oligodeoxyribonucleotides and oligoribonucleotides is an object of both intensive research and commercial use (hereinafter, the term “oligonucleotides” is used when the nature of sugar does not matter). Various methods of automatic synthesis have been developed; there is a wide range of equipment for automatic synthesis, including installations for large-scale synthesis. Since the main market for synthetic oligonucleotides is concentrated in the field of diagnostics, namely, the use of the polymerase chain reaction, most methods are designed to produce small amounts of oligodeoxyribonucleotides. In this case, the quantity and quality of the oligodeoxyribonucleotides obtained satisfy most research and commercial purposes. In contrast, oligodeoxyribonucleotides intended for therapeutic purposes (therapeutic oligonucleotides) are subject to more stringent requirements, in particular, regarding the absence of both low molecular weight impurities and “spelling errors” in the nucleotide sequence due to synthesis errors. Large-scale synthesis of oligonucleotides has become of great importance due to the high therapeutic potential of aptamers and small interfering RNAs (siRNAs, siRNAs) [1].

The main difficulty in promoting the practical application of therapeutic oligonucleotides is the achievement of a high degree of purification of automatic synthesis products. For the purification of oligonucleotides on a small scale (mg), a convenient method is reverse phase high performance liquid chromatography (of HPLC); it has an optimal ratio of time costs and efficiency.

Earlier, Glen Research (USA) developed a standard solid-phase extraction technique that is used to purify synthetic oligodeoxyribonucleotides protected by a dimethoxytrityl group [2].

Also known source 2007132440/04 (01/30/2006), which describes a method for purification of synthetic oligodeoxyribonucleotides with a length of from 2 to 30 nucleotides having dimethoxytrityl protection. Cleaning includes the following steps:

1) obtaining a solution of a protected oligodeoxyribonucleotide in solvent A (having a boiling point lower than the boiling point of solvent B);

2) heating the solution to 30 ° C, but below the boiling point of solvent A;

3) the addition of solvent B to the visible precipitation of the substance. Solvent B is an alcohol containing from 1 to 6 C atoms; or a diol containing from 2 to 6 C atoms;

4) then solvent B is heated to a temperature of 30 ° C, but below the boiling point of solvent B;

5) a solution of the protected oligodeoxyribonucleotide in solvent A is added to the heated solvent B until the substance precipitates in solution, then the solution is cooled with stirring until a precipitate forms and the supernatant is removed.

The main objective of the present invention is to eliminate the disadvantages of the purification methods known from the prior art and the development of an effective method for purification of oligodeoxyribonucleotides suitable for large-scale synthesis.

The technical result is to obtain a product with a high yield and a high degree of purification from derivatives of silicic acid.

Currently, there is an urgent need for effective methods for purification of oligodeoxyribonucleotides with a high content of G residues. They have a complex and stable tertiary structure, often in the form of so-called G-quartets and G-quadruplexes, which causes interest in them from both researchers and pharmacologists. At the same time, the presence of a large number of G residues so specifically changes the physicochemical properties of oligonucleotides, which makes it impossible to simply use previously developed standard purification methods.

The stated technical problem and the obtained technical result are achieved by the new proposed method for purification of G-rich oligodeoxyribonucleotides of 20-50 bases in size from impurities of silicic acid derivatives, including the following sequential operations:

- use oligodeoxyribonucleotides oligodeoxyribonucleotides synthesized on a carrier CPG (controlled pore glass, SCRP - glass with controlled pore size)

- prepare a solution of oligodeoxyribonucleotide, for which 25 ml of water and 0.5 ml of a 2.0 M solution of ion-pair reagent of triethylammonium acetate (hereinafter TEAA) are added to 300 μl of oligodeoxyribonucleotide with a concentration of 0.5 μm / ml

- solutions are applied to Glen Research Poly-Pak Cartridge 60-1100-10 cartridges containing a polymeric carrier having an affinity for dimethoxytrityl, syringes without plungers Millipore, Plastic syringe 50 ml, XX1105005 are attached to the cartridges, cartridges with syringes are attached to the CHROMBOND vacuum manifold 730151, and with the help of a water-jet pump, a vacuum of about 50 kPa is created

- the cartridge is washed with 2 ml of an aqueous solution of ammonia and TEAA of the following composition: 1/40 volume of a 2.0 M TEAA solution is added to a diluted 20 times 25% ammonia solution

- the cartridge is washed with 2 ml of water

- the cartridge is washed with 2 ml of a 2% aqueous solution of trifluoroacetic acid

- the cartridge is washed with 4 ml of deionized water

- emit purified oligodeoxyribonucleotide from the cartridge, for which the cartridge is washed with 2 ml of a mixture of 95% ethyl alcohol with 25% aqueous ammonia

- isolated purified oligodeoxyribonucleotide is isolated from the solution, for which test tubes with a solution of purified oligodeoxyribonucleotide are placed in a concentrator, the solution is evaporated for 10 hours at a temperature of 60 ° C

So, the essence of the invention consists in the use of mixtures of a certain composition of certain solvents, namely:

1) in the addition of an ion-pair reagent of triethylammonium acetate to the ammonia solution used to wash the derivatives of silicic acid

2) in the use of aqueous ammonia and ethyl alcohol to remove the purified oligodeoxyribonucleotide from the Poly Pak sorbent. Both solutions have good volatility, which determines the ease of their subsequent removal by evaporation in vacuum.

The yield of the product is pure oligonucleotide, ranges from 30 to 70% with a silicate content of less than 10 ^-6 % by weight.

A detailed description of the invention and the rationale for its advantages.

For the synthesis of oligodeoxyribonucleotides, two types of carriers are mainly used: a polystyrene carrier and a glass carrier with a controlled pore size (SCRP; controlled pore glass, CPG). In the case of using SCRP, upon completion of the synthesis, the removal of oligodeoxyribonucleotide from the carrier is carried out in an alkaline medium (most often aqueous ammonia or a mixture of ammonia with methylamine); therefore, the glass is leached and the product, oligodeoxyribonucleotide, is contaminated with silicic acid derivatives [3]. The presence of silicic acid derivatives makes the clinical use of oligodeoxyribonucleotide impossible without special purification and development of quality control methods.

The question of purification of oligodeoxyribonucleotides from derivatives of silicic acid is poorly understood [4].

The standard method of purification on a polymer sorbent Poly Pak, proposed by Glen Research [2], is the binding of dimethoxytrityl-oligodeoxyribonucleotide to the Poly Pak sorbent due to hydrophobic interactions, and in the presence of an ion-pair reagent triethylammonium acetate, and the oligonucleotide itself. Such stable sorption allows multiple washing with ammonia without significant desorption of the oligonucleotide.

However, the described standard method is not suitable for purification of G-rich oligodeoxyribonucleotides, since such oligonucleotides are washed off the carrier by washing with diluted ammonia, which is associated with poor sorption, which is a feature of G-rich oligonucleotides.

The claimed invention relates to the following purification method for G-rich oligonucleotides. The oligodeoxyribonucleotide is purified using 0.5 ml Glen Research cartridges (Poly-Pak Cartridge 60-1100-10) with 50 ml Millipore syringes attached (XX1105005) without plungers that are mounted on a 24 column CHROMABOND 730151 vacuum manifold Macherey-Nagel (Germany). Using a water-jet pump, a vacuum of 50-60 kPa is created. Solutions in accordance with the following scheme are applied to cartridges.

Used deionized water obtained by purification on Milli-Q Advantage A10 Ultrapure Water Purification System (USA).

The first stage of cleaning is application to the cartridge. Upon completion of the solid-phase automatic synthesis using SKRP (Mermade 48 synthesizer) and after removing 300 μl of an ammonia solution of oligodeoxyribonucleotide (concentration - 0.5 μm / ml; 0.5 mmol) from the carrier, dilute with 25 ml of water and add 0.5 ml of 2, 0 M solution of triethylammonium acetate (TEAA), the solution is applied to a 0.5 ml cartridge. In this case, the oligonucleotide is adsorbed onto the cartridge carrier.

The second stage of purification is washing with a solution of ammonia and TEAA. The cartridge is washed with 2 ml of aqueous ammonia and TEAA of the following composition: 1/40 volume of a 2.0 M TEAA solution is added to a 20% diluted 25% ammonia solution. The derivatives of silicic acid and other soluble impurities are removed.

The third stage of purification is washing with water (2 ml). Excess ammonia and TEAA are removed.

The fourth stage of purification is the removal of the 5'-terminal protective group, washing with 2 ml of a 2% aqueous solution of trifluoroacetic acid (TFA). The cartridge is washed with 2 ml of a 2% solution of TFA, while the dimethoxytrityl group is removed.

The fifth stage of purification is washing with 4 ml of deionized water. The cartridge is washed with 4 ml of deionized water; this removes excess TFU, which is detected using universal indicator paper.

The sixth stage of purification is elution from the carrier. The cartridge is washed with 2 ml of a mixture of 95% alcohol with 25% ammonia (3: 7 by volume). Oligodeoxyribonucleotide, washed from impurities, passes into the solution.

Cartridge regeneration for subsequent cleaning. To prepare for the next cleaning, the cartridge is washed successively with 2 ml of acetonitrile and 2 ml of deionized water to remove possible sorbed impurities.

The resulting solution was transferred to a 2 ml tube. A tube with a solution of purified oligodeoxyribonucleotide is placed in an Eppendorf concentrator (Eppendorf Concentrator plus). The solution was evaporated for 10 hours at 60 ° C.

After evaporation, the purified oligodeoxyribonucleotide is dissolved in water. The yield of oligodeoxyribonucleotide, determined by the UV spectrum from the difference in absorbance values at wavelengths of 260 and 320 nanometers, ranges from 30 to 70%. The extinction coefficient is calculated by the formula:

□ = N ₁ * 15200 + N ₂ * 12010 + N ₃ * 7050 + N ₄ * 8400,

where N ₁ is the number of adenines, N ₂ is the number of cytosines, N ₃ is the number of guanines, N ₄ is the number of thymines in the sequence.

In solutions of the obtained oligodeoxyribonucleotide with a concentration of 100 nmol / ml (100 μm), silicon is not detected, i.e. the concentration of silicon in the oligonucleotide solution is below 10 ^-6 % by weight.

The cleaning method is illustrated by the following examples, not limiting the claimed invention.

Example 1. Purification of the oligodeoxyribonucleotide dGGTTGGTGTGGTTGGTGGTTGGTGTGGTTGG.

The indicated oligodeoxyribonucleotide was synthesized on an MM48 DNA-RNA synthesizer (BioAutomation, USA) [5] using the standard protocol for 1 μmol of the product. The protocol included in the instrument software is designed for synthesis on columns with a glass carrier SKRP.

To 300 μl of a solution of oligodeoxyribonucleotide dGGTTGGTGTGGTTGGTGGTTGGTGTGGTTGG (concentration - 0.5 μm / ml; 0.5 mmol) add 25 ml of water and 0.5 ml of a 2.0 M solution of triethylammonium acetate. The solution is applied to the cartridge; the cartridge is washed with 2 ml of an aqueous solution of ammonia and TEAA of the following composition: 1/40 volume of a 2.0 M TEAA solution is added to a diluted 20 times 25% ammonia solution. This removes the dissolved derivatives of silicic acid. Next, the cartridge is washed with 2 ml of water, which removes excess ammonia and TEAA. The cartridge is washed with 2 ml of a 2% solution of TFU, while the dimethoxytrityl protection is removed. Then the cartridge is washed with 4 ml of deionized water, while the excess of TFA is removed. To elute the purified oligodeoxyribonucleotide, the cartridge is washed with 2 ml of a mixture of 95% ethyl alcohol with 25% aqueous ammonia (3: 7 by volume).

The resulting solution was transferred to a 2 ml tube. A tube with a solution of purified oligodeoxyribonucleotide is placed in an Eppendorf concentrator (Eppendorf Concentrator plus). The solution was evaporated for 10 hours at 60 ° C.

After evaporation, the purified oligodeoxyribonucleotide is dissolved in water. The yield of oligodeoxyribonucleotide is determined by the UV spectrum by the difference in absorbance values at wavelengths of 260 and 320 nanometers.

In solutions of the obtained oligodeoxyribonucleotide with a concentration of 100 nmol / ml silicon is not detected.

Example 2. Purification of the oligodeoxyribonucleotide dGTTGGTGTGGTTGGTGT.

The indicated oligodeoxyribonucleotide was synthesized on an MM48 DNA-RNA synthesizer [5] using the standard protocol for 1 μmol of product. The protocol included in the instrument software is designed for synthesis on columns with SCRP media.

To 300 μl of a solution of oligodeoxyribonucleotide GTTGGTGTGGTTGGTGT (concentration - 0.5 μm / ml), 25 ml of water and 0.5 ml of a 2.0 M solution of ion-pair reagent-triethylammonium acetate are added. The oligodeoxyribonucleotide solution thus prepared is applied to the cartridge. A dimethoxytrityl group having an affinity for the Poly Pak sorbent provides sorption of the oligodeoxyribonucleotide on the sorbent. The cartridge is washed with 2 ml of aqueous ammonia and TEAA of the following composition: 1/40 volume of a 2.0 M TEAA solution is added to a 20% diluted 25% ammonia solution. This removes the dissolved silicic acid. Rinse with 2 ml of water (excess ammonia and TEAA are removed). The cartridge is washed with 2 ml of a 2% solution of TFU, while the dimethoxytrityl protection is removed. The cartridge is washed with 4 ml of deionized water, while the excess of TFA is removed. Purified oligodeoxyribonucleotide is isolated. The cartridge is washed with 2 ml of a mixture of ethyl alcohol 95% with aqueous ammonia 25% (3: 7 by volume). The resulting solution was transferred to a 2 ml tube. A tube with a solution of purified oligodeoxyribonucleotide is placed in an Eppendorf concentrator (Eppendorf Concentrator plus). The solution was evaporated for 10 hours at 60 ° C.

After evaporation, the purified oligodeoxyribonucleotide is dissolved in water. The yield of oligodeoxyribonucleotide is determined by the UV spectrum by the difference in absorbance values at wavelengths of 260 and 320 nanometers.

In solutions of the obtained oligodeoxyribonucleotide with a concentration of 100 nmol / ml silicon is not detected.

Example 3. Purification of the oligodeoxyribonucleotide dGGTTGGTGTGGTTGGTGGTTGGTGTGGTTGGGTTGGTGTGGTTGGTG.

The indicated oligodeoxyribonucleotide was synthesized on an MM48 DNA-RNA synthesizer [5] using the standard protocol for 1 μmol of product. The protocol included in the instrument software is designed for synthesis on columns with SCRP media.

To 300 μl of a solution of oligodeoxyribonucleotide GGTTGGTGTGGTTGGTGGTTGGTGTGGTTGGGTTGGTGTGGTTGGTG (concentration - 0.5 μm / ml) add 25 ml of water and 0.5 ml of a 2.0 M solution of ion-pair reagent-triethylammonium acetate. The oligodeoxyribonucleotide solution thus prepared is applied to the cartridge. A dimethoxytrityl group having an affinity for the Poly Pak sorbent provides oligodeoxyribonucleotide on the sorbent. The cartridge is washed with 2 ml of aqueous ammonia and TEAA of the following composition: 1/40 volume of a 2.0 M TEAA solution is added to a 20% diluted 25% ammonia solution. This removes the dissolved silicic acid. Rinse with 2 ml of water (excess ammonia and TEAA are removed). The cartridge is washed with 2 ml of a 2% solution of TFA, while dimethoxytrityl. The cartridge is washed with 4 ml of deionized water, while the excess of TFA is removed. The cartridge is washed with 2 ml of a mixture of ethyl alcohol 95% with aqueous ammonia 25% (3: 7 by volume). The resulting solution was transferred to a 2 ml tube. A tube with a solution of purified oligodeoxyribonucleotide is placed in an Eppendorf concentrator (Eppendorf Concentrator plus). The solution was evaporated for 10 hours at 60 ° C.

After evaporation, the purified oligodeoxyribonucleotide is dissolved in water. The yield of oligodeoxyribonucleotide is determined by the UV spectrum by the difference in absorbance values at wavelengths of 260 and 320 nanometers.

In solutions of the obtained oligodeoxyribonucleotide with a concentration of 100 nmol / ml silicon is not detected.

Table 1 shows the results of experiments on the purification of oligodeoxyribonucleotides.

Table 1 The outputs of oligodeoxyribonucleotides purified by the proposed method. Sample Number The number of nucleotides in the sequence Primary structure Exit, % one twenty GTTGGTGTGGTTGGTGT 48 2 31 GGTTGGTGTGGTTGGTGGTTGGTGTGGTTGG 43 3 fifty GGTTGGTGTGGTTGGTGGTTGGTGTGGTTGGG TTGGTGTGGTTGGTG 32

Thus, the described method is applicable for the purification of G-rich oligodeoxyribonucleotides. Using TEAA allows reliable sorption of the G-rich oligodeoxyribonucleotide on Poly Pak columns, which allows them to be washed effectively to remove impurities of silicic acid derivatives. The method is applicable for the purification of therapeutic oligodeoxyribonucleotides.

Literature

1. Wuming Gong, Yongliang Ren, Qiqi Xu, Yejun Wang, Dong Lin, Haiyan Zhou and Tongbin Li. (2006) BMC Bioinformatics, 7, 516

2. McCarthy, S., Gilar, M., Gebler J. (2009) Analytical Biochemistry, 390, 181-188

3. Makino, K., Ozaki, H., Matsumoto, T., Imaishi, H., Takeuchi, T. (1987) Journal of Chromatography, 400, 211-277

4. Gilar M., Bouvier, E. (2000) Journal of Chromatography A, 890, 167-177

5. http://www.glenresearch.com//Technical/PolvPakBooklet.pdf

6. http://www.glenresearch.com/GlenReports/GR19-2.pdf

7. Syrzhikov, S., Timofeev, E., Chernov, B., Golova, J., Mirzabekov, A. (2000) Nucl. Acids Res., 28, e29

8.http: //www.bioautomation.com/Synthesizers-MerMade%2048.html

Claims (1)

The method of purification of G-rich oligodeoxyribonucleotides of 20-50 bases in size from impurities of derivatives of silicic acid, comprising the following successive stages:
- use synthesized (on a CPG substrate (SCRP, controlled pore glass)) oligodeoxyribonucleotides, where the terminal dimethoxytrityl protection is not removed,
- prepare a solution of oligodeoxyribonucleotide, for which to 300 μl of oligodeoxyribonucleotide with a concentration of 0.5 μm / ml add 25 ml of water and 0.5 ml of a 2.0 M solution of ion-pair reagent of triethylammonium acetate, then TEAA,
- solutions are applied to Glen Research Poly-Pak Cartridge 60-1100-10 cartridges containing a polymeric carrier having an affinity for dimethoxytrityl, syringes without plungers Millipore, Plastic syringe 50 ml, XXI 105005 are attached to the cartridges, cartridges with syringes are attached to the vacuum manifold CHROMBOND 730151, and using a water-jet pump, a vacuum of about 50 kPa is created,
- the cartridge is washed with 2 ml of an aqueous solution of ammonia and TEAA of the following composition:
1/40 volume of a 2.0 M TEAA solution is added to a diluted 20 times 25% ammonia solution,
- the cartridge is washed with 2 ml of water,
- the cartridge is washed with 2 ml of a 2% aqueous solution of trifluoroacetic acid,
- the cartridge is washed with 4 ml of deionized water,
- emit purified oligodeoxyribonucleotide from the cartridge, for which the cartridge is washed with 2 ml of a mixture of 95% ethyl alcohol with 25% aqueous ammonia,
- isolate the purified oligodeoxyribonucleotide from the solution, for which tubes with a solution of purified oligodeoxyribonucleotide are placed in a concentrator, and the solution is evaporated for 10 hours at a temperature of 60 ° C.