RU2157377C1 - Method of immobilization of oligonucleotides modified with unsaturated fragments by copolymerization - Google Patents

Abstract

FIELD: molecular biology, biotechnology. SUBSTANCE: invention relates to method of immobilization of oligonucleotides in organic polymeric gels. Invention describes method of immobilization of oligonucleotides in organic polymeric gels by copolymerization of oligonucleotides modified with unsaturated fragments with unsaturated monomers. Method involves use one or some groups as unsaturated fragment of oligonucleotide of the general formula: R¹R²C=CR³R⁴ where R¹, R⁴ are H or C₁-C₃-alkyl; R² is (CH₂)_n-O-X; R³ is (CH₂)_n- -O-Y; n = 1-6; X and Y are phosphodiester groups binding unsaturated fragments with adjacent nucleotide links or adjacent unsaturated fragments, or one of X or Y groups is hydrogen atom. These groups are components of oligonucleotide in the process of standard phosphoramidite oligonucleotide synthesis using phosphoramidites of the general formula: R⁵R⁶C=CR⁷R⁸ where R⁵, R⁸ are H or C₁-C₃-alkyl; R⁶ is (CH₂)_n-O-P(OCH₂CH₂CN)(N(C₃H₇)₂)₂; n = 1-6; R⁷ is -(CH₂)_n-O-DMT where n = 1-6 and DMT is 4,4'-dimethoxytrityl. Invention can be used in DNA sequenation and mapping, genetic analysis, mutations detection, in medicine and for other aims. Method provides increase of resistance of oligonucleotides modified with acrylamide and N,N'-methylene-bis-acrylamide and realization of incorporation of these reactive groups in any required position of oligonucleotide chain. EFFECT: improved method of immobilization. 4 cl, 4 dwg, 3 ex

Description

 The invention relates to molecular biology and biotechnology, and more specifically to a method for immobilizing oligonucleotides in organic polymer gels obtained by copolymerization of unsaturated monomers, where oligonucleotides modified with unsaturated fragments are used as one of the monomers. The invention may find application in DNA sequencing and mapping, in genetic analysis, mutation detection, in medicine, and for other applications.

A known method of immobilizing oligonucleotides in organic gels [Rehman FN, Audeh M., Abrams ES, Hammond PW, Boles TC, Nucleic Acids Res., 27 (1999) 649-655] consists in copolymerizing oligonucleotides modified with unsaturated groups with acrylamide and N, N '- methylene bisacrylamide. The synthesis of the corresponding oligonucleotides is carried out using Acrydite ^™ phosphoramidite manufactured by Mosaic Technologies. This product allows you to enter at the 5'-position of the oligonucleotide acrylamide fragment.

In accordance with this method, the immobilization of oligonucleotides modified with Acrydite ^TM phosphoramidite in a polyacrylamide gel is carried out by copolymerization of acrylamide, N, N'-methylenebisacrylamide and a modified oligonucleotide under the action of ammonium persulfate and tetramethylethylenediamine catalysts.

 The disadvantages of this method of immobilization are as follows.

 The stability of acrylamide derivatives of oligonucleotides, determined by the stability of the acrylamide fragment, is low. At the same time, for the production of oligonucleotide microarrays, it is necessary to contain a library of modified oligonucleotides and often use it. This means carrying out numerous cycles of freezing - thawing of oligonucleotide solutions and a noticeable time of their holding at room temperature. It is known that solutions of acrylamide in buffer solutions easily undergo spontaneous polymerization at room temperature. Thus, the presence of a reactive and unstable acrylamide fragment in the composition of oligonucleotides significantly narrows the scope of possible applications of this method of immobilization.

Another disadvantage of this method is the limitation in the position of modification of oligonucleotides. The introduction of an acrylamide unit using the Acrydite ^TM reagent is possible only at the 5'-end position, while in a large number of cases it is necessary to modify this position by some other group, for example, a fluorescent dye.

 The basis of the present invention is the task of increasing the stability of oligonucleotides modified with unsaturated groups capable of copolymerization with acrylamide and N, N'-methylenebisacrylamide, and the implementation of the possibility of introducing such reactive groups at any predetermined position of the oligonucleotide chain.

The problem is solved in that in the claimed method of immobilizing oligonucleotides in organic polymer gels by copolymerizing oligonucleotides modified with unsaturated fragments with unsaturated monomers as unsaturated fragments, groups of the general formula R ¹ R ² C = CR ³ R ^{4 are used} , where R ¹ and R ⁴ are H or C ₁ -C ₃ alkyl; R ² - (CH ₂ ) _n -O-X and R ³ - (CH ₂ ) _n -OY, n = 1-6; X and Y are phosphodiester groups linking unsaturated fragments to adjacent nucleotide units or adjacent unsaturated fragments, or one of the X or Y groups is a hydrogen atom that is introduced into a synthetic oligonucleotide using the standard phosphoramidite method using phosphoramidites of the general formula R ⁵ R ⁶ C = CR ⁷ R ⁸ where R ⁵ - R ⁸ - H or C ₁ -C ₃ alkyl; R ⁶ - (CH ₂ ) _n —O — P (OCH ₂ CH ₂ CN) (N (C ₃ H ₇ ) ₂ ) ₂ ,
n is 1-6; R ⁷ - - (CH ₂ ) _n -O-DMT, n = 1-6 (DMT = 4,4'-dimethoxytrityl).

 These phosphoramidites can be used as “building blocks” at any stage of oligonucleotide synthesis, i.e. to introduce unsaturated fragments at any predetermined position of the oligonucleotide chain. These “blocks” are prepared from unsaturated compounds containing a hydroxyl group by reaction with 2-cyanoethyl-N, N, N ′, N′-tetraisopropylphosphoramidite in acetonitrile in the presence of 1H-tetrazole.

 To prevent undesirable reactions during the oligonucleotide synthesis by double bond of an unsaturated fragment, the standard oxidizing solution is replaced with a 0.1 M solution of tert-butyl hydroperoxide in tetrahydrofuran.

 It is advisable to use acrylamide as one of the monomers in the copolymerization.

The method of immobilization of oligonucleotides can be implemented in two ways:
1) by photoinitiated copolymerization, in which a mixture of unsaturated monomers, including a modified oligonucleotide containing all the necessary additives, such as TEMED, glycerin and other components, is exposed to UV or visible light, resulting in polymerization.

When using this method of immobilization, for example, for the manufacture of oligonucleotide matrices, photoinitiated copolymerization is carried out between two glasses, one of which serves as a mask. The mask can be made of ordinary or quartz glass. The formation of a gel cell with an immobilized oligonucleotide occurs only in areas corresponding to transparent windows in the mask. The topology of the microarray corresponds to the location of the windows on the mask. The immobilization of an individual oligonucleotide in each individual cell is achieved either by physically separating the polymerization zones, or by sequentially conducting copolymerization cycles of individual oligonucleotides;
2) by chemically induced copolymerization of a mixture of unsaturated monomers under the action of the ammonium persulfate - TEMED system.

 The manufacture of microarrays using chemically induced copolymerization is as follows. Mixtures of unsaturated monomers containing individual oligonucleotides and other necessary components, except for one (for example, TEMED), are distributed in the form of microdroplets on the surface of a glass or other surface using any application device. Such a device may be a micropipette, a pin carrier, an inkjet printer, or other device. After the matrix of monomer solutions is formed, the glass is immersed in an organic solvent solution containing the component necessary to initiate polymerization (TEMED). The organic solvent does not mix with the solutions of monomers, thus preventing the possible mixing of solutions with various oligonucleotides.

 The inventive method of immobilization is suitable for use in the manufacture of polyacrylamide oligonucleotide microarrays with cell sizes of 5 microns or more.

The invention is illustrated by the following figures and examples, where
in FIG. 1 shows a synthesis scheme for one of the phosphoramidites used as a “building block” for the synthesis of oligonucleotides modified with unsaturated groups,
in FIG. 2 is a diagram of photoinitiated or chemically induced immobilization of oligonucleotides,
in FIG. 3 - the use of immobilized using photoinitial copolymerization of oligonucleotides for hybridization,
in FIG. 4 shows the use of an oligonucleotide immobilized by chemically induced copolymerization for hybridization.

 Example 1. Synthesis of phosphoramidite O-dimethoxytritylbut-2-en-1,4-diol.

 To a solution of 5 mmol of but-2-en-1,4-diol in 50 ml of pyridine was added 1 mmol of 4,4'-dimethoxytrityl chloride with stirring. The progress of the reaction is monitored by TLC in a chloroform-methanol-hexane system (9: 1: 10). Upon completion of the reaction, pyridine was evaporated, toluene was added to the residue and evaporated again. The viscous mass is dissolved in chloroform and washed with water. The chloroform solution was dried with sodium sulfate and evaporated. The residue was purified by silica gel chromatography. The product yield after purification is 80%. Monoprotected diol (0.5 mmol) is dissolved in 2 ml of anhydrous acetonitrile and 1 equiv. 1H-tetrazole and 1.1 equiv. 2-cyanethyl-N, N, N'N'-tetraisopropylphosphoramidite. The progress of the reaction is monitored by TLC in a chloroform-methanol-hexane system (9: 1:10). Upon completion of the reaction, the reaction mixture was diluted with ethyl acetate to 100 ml and washed successively with an aqueous solution of sodium bicarbonate and water. The organic layer was dried with sodium sulfate and evaporated. The yield of the target product is 90%. The synthesis scheme is shown in FIG. 1.

 Example 2. Photoinitiated copolymerization of modified oligonucleotides.

 The monomer solution for copolymerization contains a 5% mixture of acrylamide-N, N'-methylenebisacrylamide (19: 1), 40% glycerol, 0.1 M sodium phosphate buffer, pH 7.0, 1.2% TEMED, 0.002% methylene blue and 0.3 mm solution an oligonucleotide modified with a unsaturated group (0.2 μl). The polymerization is carried out in a UV oven (Stratalinker 1800-UV) by irradiation with 254 nm light (Fig. 2).

 Copolymerization is used to make microarrays with a mesh size of 500 microns. After each copolymerization cycle, the glass with the polymerized cells is washed with water for 5 minutes. Then the mask and oligonucleotide solution are changed and the polymerization cycle is repeated. The mask has only one square window in different positions with respect to the matrix. Immobilization control is carried out by observing the binding of complementary fluorescently labeled oligonucleotides (Fig. 3).

 Example 3. Chemically induced copolymerization of modified oligonucleotides.

Drops of a solution containing a 5% mixture of acrylamide-N, N'-methylene bisacrylamide (19: 1), 0.1 M sodium phosphate buffer, pH 7.0, 40% glycerol, 0.05% ammonium persulfate and 0.3 mm oligonucleotide modified with unsaturated fragments (0.2 μl ), applied with a microdoser on the glass surface. Glass is pretreated with 13- (triethoxysilyl) propyl] methacrylate. The formed matrix of solutions is immersed in 80 ml of a 0.5% solution of TEMED in hexane. Polymerization usually takes 1 hour at room temperature. After that, the microarray is washed with water for 1 hour at 60 ^o C and dried (Fig. 2). Immobilization control is carried out by observing the binding of a complementary fluorescently labeled oligonucleotide (Fig. 4).

Claims (4)

1. A method of immobilizing oligonucleotides in organic polymer gels by copolymerizing oligonucleotides modified with unsaturated fragments with unsaturated monomers, characterized in that one or more groups of the general formula R ¹ R ² C = CR ³ R ⁴ are used as an unsaturated fragment of the oligonucleotide, where R ¹ and R ⁴ is H or C ₁ -C ₃ alkyl; R ² - (CH ₂ ) _n —O — X and R ³ - (CH ₂ ) _n —O — Y, n = 1-6, X and Y are phosphordiamide groups linking unsaturated fragments to adjacent nucleotide units or adjacent unsaturated fragments or one of the groups X or Y represents a hydrogen atom that is introduced into the oligonucleotide during standard phosphoramidite oligonucleotide synthesis using phosphoramidites of the general formula R ⁵ R ⁶ C = CR ⁷ R ⁸ , where R ⁵ and R ⁸ are H or alkyl C ₁ -C ₃ ; R ⁶ - (CH ₂ ) _n —O — P (OCH ₂ CH ₂ CN) (N (C ₃ H ₇ ) ₂ ) ₂ , n = 1-6; R ⁷ - - (CH ₂ ) _n -O-DMT, n = 1-6 (DMT = 4,4'-dimethoxytrityl).  2. The method according to claim 1, characterized in that acrylamide is used as one of the unsaturated monomers.  3. The method according to claim 1, characterized in that the immobilization is carried out by photo-initiated copolymerization.  4. The method according to claim 1, characterized in that the immobilization is carried out by chemically induced copolymerization.

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