RU2175972C2 - Method of immobilization of oligonucleotides containing unsaturated groups in polymeric hydrogels in forming microchip - Google Patents

Abstract

FIELD: biochemistry. SUBSTANCE: described is method of immobilization of oligonucleotides in polymeric hydrogels including polyacrylamide gel. Said method comprises copolymerizing oligonucleotides modified by unsaturated groups of general formula I:

with unsaturated monomers which represent basic constituents of the resulting hydrogel. Also described are modified oligonucleotides of formula I wherein R¹, R², R³ are H, C₁-C₆ alkyl, Ph, PhCH_2-; Z is (CH₂)_nCH(CH₂OH)CH₂OX wherein n is 1-6; or (CH₂)_n-OX, wherein n is 2-6; and X is phosphodiether group binding unsaturated fragment with 5-5'and/or 3'-oligonucleotide end; R⁴ is H, (CH₂)_n-OH, wherein m is 2-6; Y is p-C₆H₄)_n wherein n is 0-2. Oligonucleotides are modified by phosphoamidite of general formula II:

wherein R¹, R² and Y are as defined in formula I, R³ is C₁-C₆ alkyl, R⁴ is H, (CH₂)_n-ОДМТ, wherein n is 2-6, or by acylation of oligonucleotide containing aminolinic by activated unsaturated acid ester. EFFECT: high degree of immobilization of oligonucleotides in polymeric hydrogel in forming microchip. 19 cl, 5 dwg, 10 ex, 2 tbl

Description

 The invention relates to molecular biology and bioorganic chemistry and contemplates oligonucleotides modified with unsaturated groups capable of undergoing a copolymerization reaction with unsaturated monomers during the formation of polymer hydrogels, as well as methods for their synthesis and method for copolymerization. The invention also relates to a method for immobilizing oligonucleotides in hydrogels obtained by polymerization of unsaturated monomers of acrylamide and bis-acrylamide, or any other monomers based on them, which is used in the manufacture of biological microarrays used in molecular biology for sequencing and mapping of DNA, detection of mutations and a number of medical applications.

State of the art
Two methods are known for immobilizing oligonucleotides modified with unsaturated groups in a hydrogel.

The first method consists in copolymerizing an oligonucleotide with an N-alkyl-substituted methacrylamide residue (Acrydite ^TM ) attached via a phosphoamide bond to acrylamide and bisacrylamide. This reagent is manufactured by Mosaic Technologies [FN Rehman, M. Audeh, ES Abrams, PW Hammond, M. Kenney and TC Boles, Nucleic Acids Research, 1999. V.27, N 15, P. 649-655] and allows the introduction of methacrylamide group into the oligonucleotide at the 5'-end in the automatic synthesis mode.

This method has several limitations. Despite the fact that the methacrylamide group is stable both in the acidic and alkaline regions, the phosphoamide bond chosen by the authors for binding the methacrylamide fragment to the oligonucleotide limits the scope of use of oligonucleotides with this modification only to the framework of a neutral and slightly alkaline medium. It is known that the phosphoamide bond is unstable in an acidic environment [NN Preobrazhenskaya, Russ. Chem. Rev., 1972, 41, P.54; Chanley JD, Feageson E., J. Am. Chem. Soc., 1965, 87, P.3199; Glark VM, Kirby GW, Todd AR, J. Chem. Soc., 1957, P.1497]. Since oligonucleotides are polyacids, the storage of aqueous solutions of such oligonucleotides leads, as a rule, to cleavage of the unsaturated group and, as a result, to the loss of the ability to enter into the copolymerization reaction. In this regard, the manufacturer suggests storing oligonucleotides in dry form at -20 ^o C, which greatly limits the scope of use of these derivatives. So, in the manufacture of oligonucleotide microarrays, the problem of storage and reuse of the library of oligonucleotides will inevitably arise.

Thus, the acid lability of the phosphoamide bond in modified oligonucleotides sold under the Acrydite ^™ brand sharply limits their field of application.

 Another disadvantage of this method is the introduction of unsaturated groups only at the 5'-end of the synthesized oligonucleotide.

 The second method described [Vasiliskov AV, Timofeev EN, Surzhikov SA, Drobyshev AL, Shick VV, Mirzabekov AD, BioTechniques 1999,27, P. 592-606] eliminates some of the disadvantages of the first, namely, suggests the use of allylic derivatives of oligonucleotides, which increases the stability of the modified oligonucleotides to changes in pH of solutions and, thereby, removes the restrictions imposed on the first method.

 The disadvantage of this method is the low affinity of the allyl fragment for acrylamide and bisacrylamide in the copolymerization reaction when the oligonucleotide is immobilized in an acrylamide hydrogel. So, for example, in the manufacture of oligonucleotide microarrays, in comparison with the first method, a huge excess of oligonucleotide is required. This method is certainly much more expensive.

SUMMARY OF THE INVENTION
The essence of the invention lies in the fact that oligonucleotides are modified by various unsaturated groups having a high affinity for the unsaturated monomer that forms the basis of the hydrogel formed (for example, acrylamide, methacrylamide or any other monomer based on them), which allows for the efficient immobilization of the obtained modified oligonucleotides into a gel when directly making a microchip.

где R¹, R², R³ представляет собой H, алкил С₁-C₆, Ph, PhCH₂-;
Z представляет собой (CH₂)_nCH(CH₂OH)CH₂OX, где n=1-6; или (CH₂)_n-OX, где n= 2-6; и X представляет собой фосфодиэфирную группу, связывающую непредельный фрагмент с 5'- и/или 3'-концом олигонуклеотида;
R⁴ представляет собой H, (CH_2 nОН, где n=2-6;
Y представляет собой (p-C₆H₄)_n, где n= 0-2;
DNA/RNA представляет собой одноцепочечный фрагмент олигонуклеотидов дезокси- и риборяда, полученный на автоматическом синтезаторе по стандартной фосфорамидитной химии; двухцепочечный фрагмент DNA, полученный в условиях полимеразной цепной реакции (PCR) с использованием синтетических праймеров с концевой непредельной группой; одноцепочечный фрагмент DNA, полученный в условиях ассиметрической полимеразной цепной реакции с использованием синтетических праймеров с концевой непредельной группой, получают с помощью соответствующего фосфоамидитного твердофазного синтеза или ацилирования синтезированного олигонуклеотида, содержащего аминолинк, активированным эфиром непредельной кислоты в поставтоматическом режиме. Модифицированные таким образом олигонуклеотиды иммобилизуют в акриламидном геле с помощью реакции радикальной сополимеризации.Synthetic oligonucleotides of the general formula [1]:

where R ¹ , R ² , R ³ represents H, alkyl C ₁ -C ₆ , Ph, PhCH ₂ -;
Z represents (CH ₂ ) _n CH (CH ₂ OH) CH ₂ OX, where n = 1-6; or (CH ₂ ) _n —OX, where n = 2-6; and X represents a phosphodiester group linking the unsaturated fragment to the 5 ′ and / or 3 ′ end of the oligonucleotide;
R ⁴ represents H, (CH _{2 n} OH, where n = 2-6;
Y represents (pC ₆ H ₄ ) _n , where n = 0-2;
DNA / RNA is a single-chain fragment of oligonucleotides of deoxy and riboride obtained on an automatic synthesizer using standard phosphoramidite chemistry; a double-stranded DNA fragment obtained under the conditions of polymerase chain reaction (PCR) using synthetic primers with terminal unsaturated group; a single-stranded DNA fragment obtained under conditions of an asymmetric polymerase chain reaction using synthetic primers with a terminal unsaturated group is obtained using the corresponding phosphoamidite solid-phase synthesis or acylation of the synthesized oligonucleotide containing aminoline with activated unsaturated ester in a post-automatic mode. The oligonucleotides so modified are immobilized in an acrylamide gel using a radical copolymerization reaction.

 The objective of the present invention is to develop a method for the immobilization of oligonucleotides containing various unsaturated groups having high affinity for the unsaturated monomer that forms the basis of the hydrogel formed (e.g., acrylamide, methacrylamide or any other monomer based on them) and stable under the conditions of their use and storage.

The present invention proposes the use of a number of alkyl (aryl) substituted acrylamide and styrene fragments having high affinity for acrylamide in the copolymerization reaction and the method of their introduction into the oligonucleotide:
the use in the automatic mode of synthesis of phosphoamidite, which differs from commercial Acrydite ^{TM by the} type of bond of phosphorus with an unsaturated group (in this case P-O), which leads to significant stability of the obtained products in the acidic pH range.

 the introduction of a method of activated esters of unsaturated groups in a post-automatic mode.

 In the automatic mode, the introduction of unsaturated compounds into a synthetic oligonucleotide is carried out using the corresponding phosphoamidite [Froehler B.C., Matteucci M.D., Nucleic Acids Res. 1983, 11, P. 8031-8036]. For example, substituted N-hydroxyalkylmethacrylamide can be converted to the corresponding phosphoamidite in the reaction of 2-O-cyanoethyl-N, N, N ', N'-tetraisopropylphosphodiamidite in acetonitrile in the presence of tetrazole. The resulting reagent in the form of a 0.1 M solution in acetonitrile is used without isolation under standard conditions.

где R¹, R² представляет собой H, алкил С₁-С₆, Ph-,PhCH₂-;
R³ представляет собой алкил С₁-С₆;
R⁴ представляет собой H, (CH₂)_n-ОДМТ, где n=2-6;
Y представляет собой (р-C₆H_4 n где n=0-2; n=2-6;
с типом связи фосфора с непредельной группой Р-О.In the General case, for the modification of oligonucleotides can be used phosphoamidite (formula [II]:

where R ¹ , R ² represents H, alkyl C ₁ -C ₆ , Ph-, PhCH ₂ -;
R ³ is C ₁ -C ₆ alkyl;
R ⁴ represents H, (CH ₂ ) _n -ODMT, where n = 2-6;
Y represents (p-C ₆ H _{4 n} where n = 0-2; n = 2-6;
with a type of bond of phosphorus to the unsaturated group P-O.

 In the post-automatic mode, the introduction of the unsaturated residue into the oligonucleotide is carried out as a result of the reaction of 4-nitrophenyl ether of the corresponding acid with the synthesized oligonucleotide containing aminolink at the 5'- and (or) 3'-end. 4-Nitrophenyl esters of unsaturated acids are obtained by reaction with 4-nitrophenol in the presence of dicyclohexylcarbodiimide.

 The method of immobilization of oligonucleotides with unsaturated fragments is implemented in the variant of photopolymerization under the influence of UV radiation. Application of the polymerization mixture is carried out by the drip method: drops are applied to pre-modified glass and polymerization is carried out. The application is carried out either using a microsyringe or using an automatic robot for applying microdrops [Froehler B. C., Matteucci M.D., Nucleic Acids Res. 1983, 11, P. 8031-8036, G. Yershov, V. Barsky, A. Belgovskiy, E. Kirilov, E. Kreindlin, l. lvanov, S. Parinov, D. Guschin, A. Drobishev, S. Dubiley, A. Mirzabekov. Proc.Natl.AcadSci.USA. 1996, V.93, P.4913-4918], which makes it possible to apply droplets with a volume of the order of 1 nl.

 The inventive method allows to obtain gel matrices containing immobilized oligonucleotides with a high degree of immobilization, depending on the unsaturated group used.

Information confirming the possibility of carrying out the invention
The following examples are preferred, intended only to confirm the feasibility of the invention and should not become the basis for limiting the scope of the applicant's claims. A person skilled in the art will easily find the possibilities of other embodiments of the invention, which certainly fall within the scope of the Applicant’s claims, as set forth in the claims below.

 Example 1. The synthesis of phosphoamidite containing methacrylamide residue.

 As an example of the synthesis of phosphoamidite containing a methacrylamide residue, the synthesis of N-methacryl-O- (2-O-cyanoethyl-N, N'-diisopropylaminophosphite) ethanolamine according to Scheme 1 is given. (Given at the end of the description).

 Synthesis of N-hydroxyethyl methacrylamide.

Methacrylic acid 4-nitrophenyl ether (0.300 g, 1.45 mmol) was dissolved in acetonitrile (20 ml). Ethanolamine (0.182 g, 2.98 mmol) was added to the solution and left at room temperature for 12 hours. The precipitate was filtered off and the filtrate was evaporated. The product was purified by silica gel column chromatography (eluent: 3: 2 acetone-petroleum ether). Yield 83%. Oil. R _f = 0.41 (in the acetone-petroleum ether 3: 2 system).

Synthesis of N-methacryl-O- (2-O-cyanoethyl-N, N-diisopropylamino-phosphite) ethanolamine
N-Hydroxyethyl methacrylamide (0.013 g, 0.101 mmol) was dissolved in acetonitrile (0.266 ml). To the solution were added 2-cyanoethyl-N, N, N ', N'-tetraisopropylphosphoamidite (0.030 g, 0.101 mmol) and a 0.4 M solution of tetrazole in acetonitrile (0.255 ml). The solution was stirred for 30 minutes and filtered. Acetonitrile (0.479 ml) was added to the filtrate. The resulting 0.1 M phosphoamidite solution was directly used in the automatic synthesis of oligonucleotides.

 Example 2. Synthesis of oligonucleotide-CAACTGAT with a 3'-terminal acrylamide group.

As an example of the synthesis of an oligonucleotide with a 3'-terminal acrylamide group, the synthesis of 5'-CAASTGAT-3'- (CH ₂ CH (CH ₂ OH) (CH ₂ ) ₃ CH ₂ NHC (O) - CH = CH ₂ ) according to the scheme 2. (Given at the end of the description).

Synthesis of 4-nitrophenyl Acrylic Acid
Acrylic acid (0.850 g, 11.79 mmol) was dissolved in ethyl acetate (7 ml) and a solution of 4-nitrophenol (1.804 g, 12.97 mmol) and dicyclohexylcarbodiimide (2.680 g, 12.97 mmol) in ethyl acetate (14 ml) was poured with stirring. The resulting solution was stirred at room temperature for 12 hours. The dicyclohexylurea precipitate was filtered off, the filtrate was cooled to -70 ^° C, and the precipitated nitrophenyl ether crystals were filtered off. The filtrate was evaporated twice and cooled again to -70 ^° C. The crystals obtained in two portions were dried at room temperature and a pressure of 3 mm Hg.

Yield 87%. _Mp = 142-143 ^o C.

Synthesis of 5'-CAACTGAT-3 '- (CH ₂ CH (CH ₂ OH (CH ₂ ) ₃ CH ₂ NHC (O) -CH = CH ₂ )
A solution of 4-nitrophenyl ether was added to a solution of 5'-CAASTGAT-3 '- oligonucleotide - (CH ₂ CH- (CH ₂ OH) (CH ₂ ) ₃ CH ₂ NH ₂ (21.7 nmol) in borate buffer (0.050 ml) with a pH of 9.53. acrylic acid (0.40 mg, 2170 nmol) in dimethylformamide (0.100 ml). The resulting homogeneous solution was kept at 35 ^° C. for 12 hours. Then, the oligonucleotide fraction was separated from the low molecular weight fraction by gel filtration on a Sephadex-G25 sorbent using deionized water as an eluent. components.

The oligonucleotide was purified by HPLC on a Gilson instrument (USA), Supelco column 4.6 × 250 mm, in the eluent system: buffer A — 0.05 M triethylammonium acetate, buffer B — 50% acetonitrile in buffer A, gradient from 0 to 50% buffer B in 30 min, detection at a wavelength of λ = 260 nm. Retention time R _t 19 min. The yield of the modified oligonucleotide was 72%.

 Example 3. Photoinitiated copolymerization of modified oligonucleotides.

The percent immobilization was assessed by the radioactive label P ³² introduced into the oligonucleotide.

The composition of the polymerization mixture corresponded to 5% polyacrylamide gel: 5% acrylamide-bisacrylamide (19: 1), 40% glycerol, 2% acetone, 1.2% TEMED, 0.1 M sodium phosphate buffer, pH 7.0. The concentration of labeled P ³² oligonucleotide, 5'-P ³² -GAAAGGTGGTGTTCTCTACGC- (CH ₂ CH (CH ₂ OH) (CH ₂ ) ₃ CH ₂ NHC (O) - C (CH ₃ ) = CH ₂ ) -3 'was 1 • 10 ^-7 M. Drops were applied using a microsyringe onto glass pretreated with Bind-Silan (3- (trimethoxysilyl) -propylmethacrylate). Drop volume 0.2 μl. The polymerization was carried out in a UV oven (Stratolinker 1800-UV). The exposure time is 20 minutes at a wavelength of 254 nm. After copolymerization, the total radioactive background of the glass with the deposited drops was measured, which was taken as 100%. Next, a series of washes was carried out under various conditions until a constant background radioactive background was measured and the average% immobilization of the oligonucleotide, which was 65-70%, was evaluated.

 Example 4. Evaluation of the percentage of immobilization by the fluorescence intensity of the fluorescent label introduced into the copolymerizable oligonucleotide.

To determine the% immobilization of the oligonucleotide, 5'-Flu-GAAAGGTGGTGTTCTCTACGC- (CH ₂ CH (CH ₂ OH) (CH ₂ ) ₃ CH ₂ NHC (O) -CH = CH ₂ ) -3 'was obtained. Fluorescein was introduced at the 5'-end by sequential treatment with T4-polynucleotide kinase, ATPγS, 5-IAF (5-iodoacetamidofluorescein), followed by extraction with phenol / chloroform, butanol and HPLC purification. The resulting homogeneous product was introduced into the copolymerization mixture, standard for a 5% polyacrylamide gel, and polymerization was carried out under the conditions described in Example 3. Evaluation of% immobilization was carried out similarly to determination of% immobilization for phosphorus labeled at the 5'end (P ³² ): for 100% took an average fluorescence of 1 drop after completion of polymerization. After washing in water at 60 ^° C. for 45 minutes, the fluorescence intensity was re-measured and the average% immobilization was determined, which was 60 ^° C. The results are shown in FIG. 1.

 Example 5. The effect of the unsaturated group introduced into the oligonucleotide on the degree of copolymerization.

 The degree of immobilization is assessed by the binding of complementary fluorescently labeled oligonucleotides as a result of hybridization.

 It was shown that all modified oligonucleotides have a high degree of binding to a complementary oligonucleotide labeled with Texas Red, however, however, the fluorescence intensity is distributed in the studied series in this way: acrylamide = methacrylamide> 3-phenylacrylamide> 4-vinylbenzamide. Since the experiment was carried out for all of the listed compounds under the same conditions, namely, equal initial concentrations of all modified oligonucleotides in the copolymerization mixture, identical polymerization conditions and subsequent washing of the gel, equal concentration of the complementary oligonucleotide, the data obtained allow us to conclude that the listed compounds are reactive under the conditions copolymerization.

 Example 6. Hybridization on a microchip obtained by copolymerization of a modified oligonucleotide with 5% acrylamide gel.

The application was carried out using an automatic robot for applying microdrops [Froehler BC, Matteucci MD, Nucleic Acids Res. 1983, 11, P. 8031-8036, G. Yershov, V. Barsky, A. Belgovskiy, E. Kirilov, E. Kreindlin, l. lvanov, S. Parinov, D. Guschin, A. Drobishev, S. Dubiley, A. Mirzabekov. Proc.Natl. AcadSci USA 1996, V.93, P.4913-4918]. The applied droplet volume corresponded to 1 nl. The concentration of 5'-CAACTGAT- (CH ₂ CH (CH ₂ OH) - (CH ₂ ) ₃ CH ₂ NHC (O) - CH = CH ₂ ) -3 'was 1 • 10 ^-6 M. The concentration of the complementary oligonucleotide labeled with The 5'-end of Texas Red is 3 x 10 ^-6 M. The results are shown in FIG. 2.

 Example 7. Synthesis of oligonucleotides with 3'- and 5'-terminal unsaturated group.

 The synthesis of oligonucleotides with a 3'-terminal unsaturated group was carried out according to scheme 3. (Given at the end of the description).

 The formation of the target product in the acylation reaction was monitored by HPLC (Rt), and the gross composition of the obtained oligonucleotides was confirmed by the MALDI-MS method. (Table 1).

приведенной в примере 1, на синтезаторе Biosystems 394DNA/RNA synthesizer (Applied Biosystems, Foster City) в условиях стандартной фосфорамидитной химии. Образование целевого продукта контролировали по данным HPLC (Rt), а брутто состав полученных олигонуклеотидов подтверждали методом MALDI-MS. (Таблица 2).Oligonucleotides with a 5'-terminal unsaturated group were synthesized using a 0.1 M phosphoamidite solution prepared according to the procedure

described in example 1, on a Biosystems 394DNA / RNA synthesizer (Applied Biosystems, Foster City) under standard phosphoramidite chemistry. The formation of the target product was monitored according to HPLC (Rt), and the gross composition of the obtained oligonucleotides was confirmed by the MALDI-MS method. (Table 2).

 Mass spectra were recorded on a MALDI-MS spectrometer.

High performance liquid chromatography (HPLC) was performed on a Supeico LC-18 column (5μm 4.6x250 mm) using the eluent system: A) 0.05M solution of triethylammonium acetate; C) 0.05 M solution of triethylammonium acetate in 50% CH ₃ CH; gradient from 0 to 50% B) in 30 minutes.

 The efficiency of the condensation step using phosphoamidite (1) according to high performance chromatography (HPLC) was more than 92%.

вводили в олигонуклеотид с использованием 0.1М раствора фосфорамидита, полученного по методике, приведенной в примере 1, в условиях стандартной фосфорамидитной химии.Example 8. Immobilization of a modified unsaturated group of oligonucleotides in various hydrogels based on acrylamide
The oligonucleotide MAA-5'-GAACTGAG-3'-TexRed was obtained on a Biosystems 394DNA / RNA synthesizer (Applied Biosystems, Foster City). Methacrylamide group

introduced into the oligonucleotide using a 0.1 M phosphoramidite solution obtained by the method described in example 1, under standard phosphoramidite chemistry.

 The oligonucleotide in the gels for photoinduced polymerization: (A, B, C, D, E, F, G) was applied using an automatic robot, described above, on glass treated with BindSilane, UV was irradiated with λ = 312 nm and washed.

 Figure 3 presents the fluorescence picture of a microchip obtained by immobilization in hydrogels of different composition (A, B, C, D, E, F, G) of an oligonucleotide structure MAA-5'-GAACTGAG-3'-TexRed, with a 5'-terminal methacrylamide group (MAA) and fluorescent label at the 3'-end (TexRed). The efficiency of immobilization of the oligonucleotide in different gels was determined by the magnitude of the fluorescent signal. The degree of immobilization on different gels was 30-60%.

 Example 9. Hybridization of labeled oligonucleotides with modified unsaturated group of oligonucleotides immobilized in various hydrogels.

(МАА) при использовании 0.1М раствора фосфоамидита, полученного по методике, приведенной в примере 1, на синтезаторе Biosystems 394DNA/RNA synthesizer (Applied Biosystems, Foster City) в условиях стандартной фосфорамидитной химии. Полученные олигонуклеотиды добавляли в составе гелей для фотоиндуцируемой полимеризации наносили с помощью автоматического робота, охарактеризованного на стр.6, на стекло, обработанное BindSilane, облучали УФ с λ = 312 нм, отмывали и использовали для проведения гибридизационного анализа. Использовали гидрогели следующего состава: А{акриламид:N,N'-метиленбис-акриламид-Т10%, С5%, глицерин-64.5%} ; В {акриламид:N,N'-метиленбисакриламид-Т5%, С5%, глицерин-64.5%}, С {(акриламид+N- [трис(гидроксиметил)метил]акриламид(1:3)}
(N, N'-метиленбисакриламид+N, N'-(1,2-дигидроксиэтил-акриламид))(3: 7), Т5%, С25%; глицерин-64.5%}.For the manufacture of a microchip, the oligonucleotides MAA-GAACTGAG and MAA-CAACTGAC with a 5'-terminal methacrylamide group were synthesized

(MAA) using a 0.1 M phosphoamidite solution obtained according to the procedure described in Example 1 on a Biosystems 394DNA / RNA synthesizer (Applied Biosystems, Foster City) under standard phosphoramidite chemistry. The obtained oligonucleotides were added as part of the gels for photoinduced polymerization, applied using an automatic robot, described on page 6, to the glass treated with BindSilane, UV with λ = 312 nm was irradiated, washed and used for hybridization analysis. Hydrogels of the following composition were used: A {acrylamide: N, N'-methylenebis-acrylamide-T10%, C5%, glycerol-64.5%}; B {acrylamide: N, N'-methylenebisacrylamide-T5%, C5%, glycerin-64.5%}, C {(acrylamide + N- [tris (hydroxymethyl) methyl] acrylamide (1: 3)}
(N, N'-methylenebisacrylamide + N, N '- (1,2-dihydroxyethyl-acrylamide)) (3: 7), T5%, C25%; glycerin-64.5%}.

 In FIG. 4 shows the results of hybridization on an oligonucleotide microchip using immobilized oligonucleotides of the GAACTGAG and CAACTGAC structure and a hybridizable oligonucleotide of the CTCAGTTC structure. The resulting hybridization pattern corresponds to theoretical concepts. A larger fluorescent signal is observed on the immobilized GAACTGAG oligonucleotide, a fully complementary hybridizable probe, regardless of the type of gel and linker connecting the methacrylamide group to the oligonucleotide. A smaller signal is observed on cells with immobilized CAACTGAC oligonucleotide containing terminal substitutions.

 Example 10. Immobilization of a modified unsaturated group of oligonucleotides in various hydrogels.

 The oligonucleotide in the gels for photoinduced polymerization (A, B) was applied using an automatic robot, described above, onto a glass treated with BindSilane, UV irradiated with λ = 312 nm and washed. The efficiency of immobilization of the oligonucleotide was determined by the magnitude of the fluorescent signal on the rank before and after washing the chip. In FIG. Figure 5 shows the fluorescence picture of a microchip obtained by immobilizing an oligonucleotide of different composition (A, B) in hydrogels: MAA-5'-GAACTGAG-3'-TexRed, with a 5'-end methacrylamide group (MAA) and a fluorescent label at the 3'-end (TexRed). The percentage of immobilized oligonucleotide in the gel was A-75-80%, in B-68-76%.

 This example shows that oligonucleotides with a terminal unsaturated group can be effectively immobilized not only in hydrogels based on acrylamide and N, N'-methylene bisacrylamide, but also on the basis of any other water-soluble monomers capable of copolymerizing with the terminal unsaturated group of the oligonucleotide.

Claims (18)

1. A method of immobilizing oligonucleotides in polymer hydrogels, comprising copolymerizing modified oligonucleotides with unsaturated monomers to form polymer gels, in which a high degree of immobilization is achieved by using oligonucleotides modified with unsaturated groups of the general formula I

where R ¹ , R ² , R ³ represent H, alkyl C ₁ -C ₆ Ph, PhCH ₂ -;
Z represents (CH ₂ ) _n CH (CH ₂ OH) CH ₂ OX, where n = 1-6; or (CH ₂ ) _n —OX, where n = 2-6, and X is a phosphodiester group linking the unsaturated fragment to the 5 ′ and / or 3 ′ end of the oligonucleotide;
R ⁴ represents H, (CH ₂ ) _n OH, where n = 2-6;
Y represents (pC ₆ H ₄ ) _n , where n = 0-2.  2. The method according to claim 1, in which the polymer hydrogel is a polyacrylamide gel obtained by copolymerization of unsaturated monomers of acrylamide and bis-acrylamide, or any other monomers based on them.  3. The method according to claim 1, in which the oligonucleotides modify unsaturated groups in automatic mode. 4. The method according to claim 3, in which the oligonucleotides are modified with unsaturated groups using phosphoamidite General formula II

where R ¹ , R ² represent H, alkyl C ₁ -C ₆ , Ph, PhCH ₂ -;
R ³ is C ₁ -C ₆ alkyl;
R ⁴ represents H, (CH ₂ ) _n -ODMT, where n = 2-6;
Y represents (pC ₆ H ₄ ) _n , where n = 0-2,
n is 2-6;
with a type of bond of phosphorus to the unsaturated group P-O.  5. The method according to claim 1, in which the oligonucleotides are modified with unsaturated groups in a post-automatic mode.  6. The method according to claim 5, in which the oligonucleotides are modified with unsaturated groups by acylating an oligonucleotide containing aminoline with an activated unsaturated ester.  7. The method of claim 6, wherein the activated unsaturated ester is nitrophenyl ether.  8. The method according to claim 3 or 5, in which the oligonucleotides are modified with unsaturated groups at the 5'-end of the oligonucleotide.  9. The method according to claim 3 or 5, in which the oligonucleotides are modified with unsaturated groups at the 3'-end of the oligonucleotide.  10. The method according to claim 1, in which the oligonucleotides are immobilized by photopolymerization under the influence of UV radiation.  11. The method according to p. 10, in which the photopolymerization is carried out by the drip method by applying drops to pre-modified glass.  12. The method according to claim 11, in which the drops are applied using a microsyringe.  13. The method of claim 12, wherein the droplet volume is about 0.2 μl.  14. The method according to claim 11, in which the drops are applied using an automatic robot for applying microdrops.  15. The method according to 14, in which the volume of drops is about 1 nl. 16. Oligonucleotide modified by unsaturated groups of the general formula I

where R ¹ , R ² , R ³ represent H, alkyl C ₁ -C ₆ Ph, PhCH ₂ -;
Z represents (CH ₂ ) _n CH (CH ₂ OH) CH ₂ OX, where n = 1-6; or (CH ₂ ) _n —OX, where n = 2-6, and X represents a phosphodiester group linking the unsaturated fragment to the 5 ′ and / or 3 ′ end of the oligonucleotide,
R ⁴ represents H, (CH ₂ ) _n OH, where n = 2-6;
Y represents (pC ₆ H ₄ ) _n , where n = 0-2.  17. The oligonucleotide according to claim 16, modified by a unsaturated group at the 5'-end.  18. The oligonucleotide according to claim 16, modified by a unsaturated group at the 3'-end.

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