RU2734941C2 - Method of producing modified combinatorial dna libraries by solid-phase primer extension reaction on pet microparticles - Google Patents

Abstract

FIELD: organic chemistry; biochemistry; molecular biology.

SUBSTANCE: described is a method of producing combinatorial DNA libraries containing modified nucleotides for specific binding with molecular targets. Essence of invention is that combinatorial modified single-stranded DNA library is used to use a combinatorial single-strand DNA library of natural nucleotides and the complementary modified DNA is synthesized in a matrix-dependent high-specific enzymatic primer extension reaction with complete substitution of one of the natural nucleotides for the modified analogue. For primer extension reaction, Deep Vent(exo-) and KOD XL DNA polymerases and modified deoxyuridine triphosphates with C5-substituted substituents of uridine base, which are completely compatible, are proposed. In the synthesis of modified DNA one type of primers fixed on the surface of polyethylene terephthalate microparticles is used, which enables to separate and wash the carrier microparticles with the obtained fixed single-stranded modified DNA from the used DNA matrix and reaction components. Primers include groups which enable selective cleavage of the modified DNA from the carrier, without affecting its functional part by photochemical or enzymatic methods, which enables to obtain a combinatorial modified single-stranded DNA library in pure form. Besides, corresponding library is presented.

EFFECT: invention extends the methods of producing DNA libraries.

17 cl, 9 dwg, 9 ex

Description

The technical field to which the invention relates

The present invention relates to the field of organic chemistry, biochemistry and molecular biology, namely, to a method for producing combinatorial modified single-stranded DNA libraries intended for specific binding to molecular targets. The essence of the invention lies in the fact that to obtain a combinatorial modified single-stranded DNA library, a combinatorial single-stranded DNA library from natural nucleotides is used and the complementary modified DNA is synthesized during a matrix-dependent highly specific enzymatic primer extension reaction with a complete replacement of one of the natural nucleotides to a modified analogue, and subsequent elimination of the synthesized modified chains. Immobilized primers contain a special group in their structure, which is cleaved by photochemical or enzymatic methods. The proposed invention can be used to obtain modified DNA fragments, modified aptamers, functional analogs of monoclonal antibodies.

The prior art to which the invention relates

The most promising areas of application of the invention is the production of modified aptamers, which can be used for the analysis of protein, peptide and low molecular weight biologically active molecular targets, including sensors and sensor devices, as well as therapeutic agents. Modified aptamers, in contrast to antibodies, are resistant to proteases, which opens up new opportunities for their use that are unattainable for antibodies.

It is known that to obtain modified DNA fragments with functional groups on nucleic bases, a solid-phase chemical synthesis method using phosphoamidites of modified nucleosides is used. The disadvantages of this method are: the complexity of obtaining phosphoamidites of nucleosides with the necessary modifications for nucleic bases; the complexity of solid-phase chemical synthesis of DNA using phosphoamidites carrying modifying groups on nucleic bases, the conditions of chemical synthesis are quite stringent and modifying groups can be destroyed; The solid-phase phosphoamidite synthesis method has limitations on the length of the synthesized DNA molecules; with each subsequent cycle of the oligonucleotide chain extension, its yield decreases.

The authors of the patent (Phosphinoamidite carboxylates and analogs thereof in the synthesis of oligonucleotide having reduced internucleotide charge, US patent, 8846898, - 2014, Lievre Cornu LLC, USA) propose to synthesize modified DNA fragments using modified phosphoamidites of nucleosides containing various functional groups.

Known methods for obtaining single-stranded fragments of unmodified DNA using solid-phase amplification. In particular, a number of methods for conducting solid-phase PCR are known, for example, using one immobilized primer and another primer in solution (Solid-phase nucleic acid amplification method and reagent kit therefor, US patent, 6274351, - 2001, GENSET SA, USA; Single molecule nucleic acid sequence analysis processes and compositions, US patent, 9034580, - 2015, Sequenom INC, USA; Multiplex branched-chain DNA assays, US patent, 8986931, - 2015, Asymetrix INC, USA). As a result of amplification, a DNA fragment can be obtained, one chain of which is linked to a solid carrier, while the other is in solution. The method does not apply to obtaining DNA with modifications at nucleic bases.

The authors of the patent (Method of producing heterogeneous set of single-chain DNA fragments for multiplex genetic analysis, Ru patent, 2604198, - 2016, Fed Gosudarstvennoe Bjudzhetnoe Uchrezhdenie Nauki Inst Molekuljarnoj Biologii Im VA Engelgardta Ro, Russia) proposed a method for producing single-stranded DNA fragments by the method of unmodified DNA fragments solid-phase PCR on agarose microparticles followed by photochemical or enzymatic cleavage from the carrier. The method does not apply to obtaining DNA with modifications at nucleic bases.

The claimed invention differs from the above-described analogs in that to obtain modified single-stranded DNA molecules, a DNA template of natural nucleotides is used, including a degenerate (combinatorial) region, and the complementary modified DNA is synthesized during a matrix-dependent highly specific enzymatic primer extension reaction with complete replacement of one of the natural nucleotides with a modified analog. For the primer extension reaction, Deep Vent (exo-) and KOD XL DNA polymerases and modified deoxyuridine triphosphates are offered, which are fully compatible. In the synthesis of modified DNA, a primer with a fixed sequence is used, fixed on the surface of polyethylene terephthalate microparticles, which allows quantitatively separating single-stranded modified DNA, which is covalently bound to PET microparticles, from the used template DNA and reaction components. The primer contains groups that make it possible to selectively cleave the modified DNA from the carrier, without affecting its functional part, by photochemical or enzymatic methods, which makes it possible to obtain modified DNA libraries in pure form.

Disclosure of invention

When obtaining modified single-stranded DNA fragments with modified nucleotides, it is necessary to provide a full-length product and efficient purification from used reagents.

Matrix-dependent enzymatic synthesis of nucleic acids allows the synthesis of complementary DNA copies from natural nucleotides. The methodology of solid-phase synthesis of biopolymers using cleavable linkers allows you to first remove all used reagents, and then cleave off the target biopolymer and isolate biopolymers in pure form.

The aim of this invention is to combine the capabilities of matrix-dependent enzymatic and solid-phase DNA synthesis to obtain a library of single-stranded DNA fragments with a complete replacement of the natural nucleotide with a modified analogue. Incomplete replacement of a natural nucleotide with a modified one will result in nucleotide sequences containing, with a statistical probability, a natural nucleotide and a modified analogue in each position. The present invention implements a primer extension reaction, wherein the primer is covalently bonded by the 5 'end to the surface of polyethylene terephthalate (PET) microparticles.

PET, which is a linear polycondensation product of terephthalic acid and ethylene glycol, contains ester groups that can be transformed into reactive carboxyl groups as a result of alkaline hydrolysis. Carboxyl groups can be used for covalent binding to biomolecules with primary amino groups (R.A. Miftakhov et al., Obtaining active carboxyl groups on the surface of a polyethylene terephthalate film and quantitative analysis of these groups using digital luminescence microscopy, Biophysics. 2018, vol. 63 , No. 4, pp. 661-668). This publication proposes alkaline hydrolysis of the surface of PET microparticles with the formation of surface carboxyl groups. To control the specific concentration of carboxyl groups, they were activated with bis- (4-nitrophenyl) carbonate and associated with an amine-containing indodicarbocyanine dye (Fig. 1).

The primer used in the claimed invention is cleavable and consists of a functional part specific to a region of template DNA, with a free 3 'end. The sequence of the functional part of the primer is presented below (Fig. 2A-B, Olig 2):

5'-CTG TCA GCT CCA TAC TGG TAG CC-3 '

At the 5 'end of the primer there is a cleavable group, a linker oligonucleotide (Fig. 2A-B, Olig 1) with sequence (T) ₅ , and then a C6 amino linker with an amino group for covalent immobilization on a PET support (Fig. 2A-B).

The method of cleavage of the primer can be photochemical or enzymatic. For the synthesis of a photocleavable linker, it is proposed to use phosphoamidite containing a 2-nitrobenzyl group (Fig. 2A). As a cleavable sequence, it is proposed to use regions with a sequence of ribonucleotides, which are cleaved by RNAases (Fig. 2B). Areas of DNA that are recognized by restriction endonucleases can be used.

To control the specific concentration of primers immobilized on PET microparticles, they were cleaved by UV light (Fig. 3) and using RNAase (Fig. 4).

Modified 5'-triphosphates-2'-deoxyuridine linked at the C5-position of the uridine base with fragments of the amino acids phenylalanine (Fig. 5, dUTP4-6), tryptophan (Fig. 5, dUTP1-3), valine and leucine (Fig. 5 , dUTP7-8), and alanine (Fig. 5, dUTP9), obtained by chemical means. Compounds dUTP1dUTP6 and dUTP7-dUTP9 were obtained in accordance with the procedure presented in the publication (S.A. Lapa et al., Preparation of modified combinatorial DNA libraries by PCR in reverse emulsion with subsequent separation of chains. Molecular Biology, 2018, vol. 52, No. 6, pp. 984-996).

In the claimed invention, to obtain modified single-stranded DNA molecules, a synthetic universal DNA library with a flanking portion complementary to the primer and a degenerate portion (N) _{40 was used} , where N is a mixture of all four nucleotides A, T, G, C.

5 '- (N) ₄₀ -GG STA CCA GTA TGG AGC TGA CAG-3`

Complementary modified DNAs were obtained in the course of a matrix-dependent highly specific enzymatic primer extension (PEX) reaction on a solid phase with the complete replacement of one of the natural nucleotides with the corresponding modified one. For the primer extension reaction, matrix-dependent Deep Vent (exo-) and KOD XL DNA polymerases and modified deoxyuridine triphosphates are offered, which are fully compatible. When synthesizing modified DNA, a primer is used, fixed on the surface of solid PET microparticles, which allows separating single-stranded modified DNA from the used DNA template and reaction components (Fig. 6).

The primers include groups that allow selective cleavage of the modified DNA from the carrier, without affecting its functional part, by photochemical (Fig. 7) or enzymatic method (Fig. 8), which allows one to obtain single-stranded modified DNA in pure form. The specific yield of the modified single-stranded product of the solid-phase primer extension reaction was determined spectrophotometrically.

Control of the products of DNA cleavage from PET microparticles was carried out by electrophoresis in 4% agarose gel (Fig. 9). As a result, full-length modified combinatorial DNA libraries of oligonucleotides with a degenerate part were obtained.

Brief description of diagrams and figures

FIG. 1. Scheme of chemical modification of the surface of PET microparticles and binding with an amine-containing dye.

FIG. 2. Scheme of the structure of the primer photodegradable (A) and RNAase-cleavable (B).

FIG. 3. Scheme of photodegradation of primers immobilized on the surface of PET microparticles.

FIG. 4. Scheme of RNAse A cleavage of primers immobilized on the surface of PET microparticles.

FIG. 5. Diagram of the structure of 2`-deoxyuridine-5`-triphosphates modified at the C5 position.

FIG. 6. Scheme of synthesis of modified single-stranded DNA fragments by the solid-phase primer extension reaction on PET microparticles. U * -modified deoxyiridine phosphate corresponding to deoxyuridine triphosphates dUTP1-9.

FIG. 7. Scheme of photodetachment of the modified single-stranded primer extension reaction product from PET microparticles. U * -modified deoxyiridine phosphate corresponding to deoxyuridine triphosphates dUTP1-9.

FIG. 8. Scheme of rRNAase A cleavage of the modified single-stranded primer extension reaction product from PET microparticles. U * -modified deoxyiridine phosphate corresponding to deoxyuridine triphosphates dUTP1-9.

FIG. 9. Analysis of DNA cleavage products from PET microparticles in 4% agarose gel stained with ethidium bromide: 1 - dUTP1, 2 - dUTP2, 3 - dUTP3, 4 - dUTP4, 5 - dUTP5, 6 - dUTP6, 7 - natural dTTP - positive control, 8 - negative control, 9 - double-stranded DNA length marker GeneRuler 50 bp (ThermoScientific), 10 - dUTP7, 11 - dUTP8, 12 - dUTP9, 13 - natural dTTP - positive control, 14 - negative control, 15 - marker lengths of double-stranded DNA GeneRuler 50 bp (ThermoScientific).

Implementation of the invention

To carry out the invention, a commercially available polyethylene terephthalate powder was prepared in a special way. Using sieves, a fraction of PET microparticles with a diameter of 40-63 μm was seeded. Small dust-like particles were separated by sedimentation in water. The PET microparticles were washed with acetone with centrifugation and dried in a vacuum desiccator. Received fine polyethylene terephthalate with a particle size of 40-63 microns, free of fine dust particles.

Carboxylated PET microparticles were prepared by the action of an alcoholic solution of potassium hydroxide (Fig. 1). During the destruction of polyethylene terephthalate, the ester bonds of macromolecules are broken with the formation of carboxyl and hydroxyl groups on the polymer surface. The subsequent activation of carboxyl groups on the surface of PET microparticles was carried out in DMSO with bis (4-nitrophenyl) carbonate in the presence of diisopropylethylamine (DIPEA). The activation reaction was carried out for 16 h at room temperature.

To determine the concentration of 4-nitrophenyloxycarbonyl groups on the surface of PET microparticles, we used fluorescent labeling with an amine-containing indodicarbocyanine dye, which fluoresces in the near-IR region of the spectrum. The condensation reaction of the active 4-nitrophenyloxycarbonyl groups of PET microparticles and cyanine dye was carried out in DMSO in the presence of DIPEA. The amount of the dye bound to the carboxyl groups of PET microparticles was determined spectrophotometrically by determining the concentration of the solution before and after the reaction. The specific concentration of 4-nitrophenyloxycarbonyl groups was 0.60 nanomoles per 1 mg of PET microparticles.

Knowing the approximate concentration of active groups on the surface of PET microparticles, we immobilized oligonucleotide primers with a 5'-terminal amino group. The reaction was carried out in a mixture of DMSO / carbonate bicarbonate buffer solution at + 4 ° C for 16 h. Carrying out the reaction under cooling allowed the equilibrium to shift towards the formation of a peptide bond.

Determination of the concentration of oligonucleotide primers immobilized on the surface of PET microparticles was carried out according to the principle presented for determining the concentration of indodicarbocyanine dye by measuring the absorption of the solution in water at a wavelength of 260 nm. For this, it is necessary to cleave oligonucleotide primers from the surface of PET microparticles. The method of cleavage of the primer can be photochemical (UV light irradiation) (Fig. 3) or enzymatic (using RNAase) (Fig. 4), since the 5 'end of the primer contains a cleavable group (Fig. 2A-B, Olig 1) with the sequence (T) ₅ , and then a C6-amino linker with an amino group for covalent immobilization on a PET carrier (Fig. 2A-B). The specific concentration of oligonucleotides was 0.54 nanomoles per 1 mg of PET microparticles.

In the claimed invention, to obtain modified single-stranded DNA molecules, a synthetic universal DNA library with a flanking part complementary to the primer and a degenerate part (N) _{40 was used} , where N is a mixture of all four nucleotides A, T, G, C.

5 '- (N) ₄₀ -GG STA CCA GTA TGG AGC TGA CAG-3`

Complementary modified single-stranded DNA fragments were obtained during the primer extension reaction on PET microparticles with the complete replacement of one of the natural nucleotides with the corresponding modified one. To carry out the primer extension reaction, matrix-dependent Deep Vent (exo-) and KOD XL DNA polymerases and modified deoxyuridine triphosphates, which contain amino acid fragments in their structure, were used. In the synthesis of modified DNA, a primer is used, fixed on the surface of PET microparticles, which allows separating single-stranded modified DNA from the used DNA template and reaction components (Fig. 6).

The presence in the composition of the primers of groups that allow cleavage of the modified DNA from the carrier by photochemical (Fig. 7) or enzymatic (Fig. 8) methods, allows one to obtain single-stranded modified DNA in pure form. The amount of DNA is determined spectrophotometrically by measuring the absorption of a solution in water at a wavelength of 260 nm. The control of the products of DNA cleavage from PET microparticles was carried out by electrophoresis (Fig. 9), the results of which obtained full-length modified combinatorial DNA libraries of oligonucleotides with a degenerate part.

Examples of compounds and their applications

Used polyethylene terephthalate powder manufactured by Goodfellow

Cambridge Ltd, Enghland brand ES306030 p / n 264-007-76 (www.goodfellow.com). Natural color material with a density of 1.39 g / cm ³ , polydisperse with a maximum microparticle size of 300 microns.

An amine-containing indodicarbocyanine dye was synthesized according to the scheme presented in the publication (R.A. Miftakhov et al., Obtaining active carboxyl groups on the surface of a polyethylene terephthalate film and quantitative analysis of these groups using digital luminescence microscopy, Biophysics. 2018, vol. 63, No. 4, p. 661-668).

Phosphoamidite containing a 2-nitrobenzyl group (Gene Link (www.genelink.com) PC Linker (photocleavable) Cattalog No. 26-6888-10) is used for the synthesis of a photocleavable linker.

Modified 5'-triphosphates-2'-deoxyuridine, linked at the C5-position of the uridine base with fragments of the amino acids phenylalanine, tyrosine, tryptophan, valine, leucine and isoleucine, were obtained by chemical means (Fig. 5).

The primer extension (PEX) reaction was performed on a DNA Engine Tetrad 2 amplifier (Bio-Rad, United States). Temperature-time profile of the reaction: 5 min at 95 ° C, 30 sec at 60 ° C and 30 min at 72 ° C.

The amount of DNA was determined on a NanoDrop 1000 spectrophotometer (Thermo Scientific, United States).

Electrophoretic analysis of the primer extension reaction products was performed on a 4% agarose gel. The gels were stained with ethidium bromide.

We used Deep Vent polymerases (BioLabs, Great Britain) and KOD XL (Novagen, USA) with buffer solutions of the respective manufacturers; deoxynucleoside triphosphates produced by Fermentas (USA);

The rest of the reagents and solvents of the "OSCh", "KhCh" or "ChDA" brands were purchased from Aldrich, Alfa Aesar, Fluka, Himmed. Water for the preparation of solutions was obtained on a Milli-Q installation (EMD Millipore, USA).

Oligonucleotides for the PEX primer extension reaction were synthesized on an ASM-800 DNA / RNA synthesizer (Biosset, Russia).

Example 1. Preparation of PET microparticles.

With the help of sieves, a fraction of PET microparticles with a diameter of 40-63 μm is sown. Small dust-like particles are separated by sedimentation in water. The PET microparticles are washed with acetone by centrifugation and the supernatant removed. Dry in a vacuum desiccator over P ₂ O ₅ . Finely dispersed polyethylene terephthalate is obtained with a particle size of 40-63 microns, free of fine dust particles.

Example 2. Obtaining carboxylated PET microparticles.

A KOH solution in 96% ethyl alcohol (0.5 M, 1.5 ml) is added to a weighed portion of PET microparticles (100 mg) and mixed by shaking on a shaker (Vortex) for 60 min. Then washed with water, 0.05 M HCl, water. Dry and store in a vacuum desiccator over P ₂ O ₅ .

Example 3. Activation of carboxyl groups on the surface of PET microparticles.

To carboxylated PET microparticles (100 mg), 1.5 ml of a solution of bis (4-nitrophenyl) carbonate (30 nmol / μL) and diisopropylethylamine (60 nmol / μL) in DMSO are added. The mixture is stirred by shaking on a shaker (Vortex) at room temperature for 16 hours. The precipitate is separated by centrifugation and washed with anhydrous acetone (3 × 1 ml) with centrifugation. Dry and store in a vacuum desiccator over P ₂ O ₅ .

Example 4. Determination of the concentration of 4-nitrophenyloxycarbonyl groups on the surface of PET microparticles.

To a weighed portion of PET microparticles with active 4-nitrophenyloxycarbonyl groups (100 mg), add 1 ml of a solution of an amine-containing dye (0.1 mmol / μL) and diisopropylethylamine (0.25 mmol / μL) in DMSO. The mixture is stirred by shaking on a shaker (Vortex) at room temperature for 4 hours. The mixture is centrifuged, the supernatant is separated and the precipitate is washed with 50% acetonitrile in 50 mM triethylammonium bicarbonate buffer solution (TEAN) (5 × 100 μl) with stirring on a shaker ( Vortex) and centrifugation. The supernatant and washings are combined and a 50% solution of acetonitrile in 50 mM TEAN is added to a volume of 1 ml. The concentration of the dye is determined spectrophotometrically. For this, 60 μl of the solution is added to 3 ml of 50% acetonitrile in 50 mM TEAN. Dilution 51 times. The molar extinction coefficient of the amine-containing dye at a wavelength of 647 nm is 2.5 * 10 ⁵ l * M ^-1 * cm ^-1 . The optical density of the solution is measured at a wavelength of 647 nm. The measured optical density with a light path length of 1 cm is 0.196, which corresponds to a concentration of 0.196 * 51 * 1 / 2.5 * 10 ⁵ = 3.998 * 10 ^-5 M / L = 0.3998 * 10 ^-4 M / L = 0.04 mM.

When measuring the initial concentration of the dye in DMSO, a sample of 30 μl is taken and added to 3 ml of 50% acetonitrile in 50 mm TEAN. Dilution 101 times. The molar extinction coefficient of the amine-containing dye at a wavelength of 647 nm is 2.5 * 10 ⁵ l * M ^-1 * cm ^-1 . The measured optical density at a light path length of 1 cm is 0.248, which corresponds to a concentration of 0.248 * 101 * 1 / 2.5 * 10 ⁵ = 10 * 10 ^-5 M / L = 1 * 10 ^-4 M = 0.1 mM.

The amount of the dye bound to PET microparticles is calculated from the difference between the dye taken into the reaction and not bound to PET microparticles in a volume of 1 ml (0.1-0.04) mM / L * 10 ^-3 L = 6 * 10 ^-8 m = 60 nanomole. The specific concentration of 4-nitrophenyloxycarbonyl groups is 60/100 = 0.60 nanomoles per 1 mg of PET microparticles.

Example 5. Immobilization of oligonucleotide primers on activated PET microparticles.

To a weighed portion of PET microparticles (20 mg) with 4-nitrophenyloxycarbonyl groups (0.6 nmol / mg) add 25 μL of a solution containing 20 nmol of an oligonucleotide primer with a 5`-terminal amino group in 50% DMSO in 0.2 M NaHCO ₃ / Na ₂ CO ₃ (pH 9.5). The mixture is stirred on a shaker at + 4 ° С for 16 h. PET microparticles with immobilized primers are separated by centrifugation, washed with 50% DMSO in distilled water (3 × 150 μl), water (3 × 150 μl), acetone (3 × 150 μL) at 25 ° C. Dry in a vacuum desiccator over P ₂ O ₅ . Store at -18 ° C.

Example 6. Determination of the concentration of oligonucleotide primers immobilized on the surface of PET microparticles.

A weighed portion of PET microparticles with immobilized oligonucleotides (20 mg) containing a photo-cleavable linker (0.6 nanomole / mg) is suspended in 60 μl of milliQ water. The mixture was irradiated for 10 min under UV lamps (four 15W Sylvania F15T8 / 350BL lamps, 356 nm). Then add 40 μl of formamide and heat at 95 ° C for 1 min. The PET microparticles are separated by filtration and washed on the filter with 40% formamide (2 x 20 μl). Filters are combined. DNA is isolated from the combined filtrates by precipitation with a mixture (1: 2) of ethyl alcohol and 2% lithium perchlorate in acetone.

When cleavage with RNAase, a primer is used that contains two uridine nucleotides. A weighed portion of PET microparticles with immobilized oligonucleotides (20 mg) containing two uridine nucleotides (approximately 0.6 nanomole / mg) is washed with water and suspended in 30 μL of 0.1 M sodium acetate. Digestion is carried out at 60 ° C for 1 h in the presence of RNAse A. Then 20 μl of formamide is added and heated at 95 ° C for 1 min. The PET microparticles are separated by filtration and washed on the filter with 40% formamide (2 x 20 μl). Filters are combined. DNA is isolated from the combined filtrates by precipitation with a mixture (1: 2) of ethyl alcohol and 2% lithium perchlorate in acetone.

The amount of DNA is determined spectrophotometrically by measuring the absorption of a solution in water at a wavelength of 260 nm. The extinction coefficient is calculated based on the length and the ratio of nitrogenous bases. The yield is about 10.8 nanomoles, which is 90% of the capacity of PET microparticles. The specific concentration of oligonucleotides is 0.54 nanomoles per 1 mg of PET microparticles.

Example 7. Obtaining modified single-stranded DNA fragments by the method of solid-phase primer extension reaction on PET microparticles.

The primer extension (PEX) reaction is carried out using PET microparticles with an immobilized primer (20 mg, ~ 0.54 nmol / mg) and 50 μl of a mixture containing 0.2 mM each dCTP, dATP, dGTP and modified dU * TP, 12 nanomoles of template DNA, 1.5 mM MgCl ₂ , 5 units. DNA polymerase (Deep Vent (exo-) or KOD XL) and 5% DMSO in 1 × PCR buffer (pH 8.5).

The primer extension reaction (PEX) is carried out according to the following program: 5 min at 95 ° C, 30 sec at 60 ° C and 30 min at 72 ° C. Cool to room temperature. The reaction is stopped by the addition of Na-EDTA (pH 8) at a final concentration of 20 mM. Then add 35 μl of formamide and heat at 95 ° C for 1 min. PET microparticles are separated by filtration and washed on a filter with 40% formamide, water, acetone in portions of 150 μl, 3 times at a temperature of 25 ° C. Dry in a vacuum desiccator over P ₂ O ₅ . Store at -18 ° C.

Example 8. Cleavage of a modified single-stranded primer extension reaction product from PET microparticles.

For UV cleavage, DNA is used with a primer containing a photocleavable linker. A weighed portion of 20 mg PET microparticles with single-stranded primer extension reaction products (20 mg, ~ 0.54 nanomole / mg) is suspended in 60 μL of MilliQ water. The mixture was irradiated for 10 min under UV lamps (four 15W Sylvania F15T8 / 350BL lamps, 356 nm). Then add 40 μl of formamide and heat at 95 ° C for 1 min. The PET microparticles are separated by filtration and washed on the filter with 40% formamide (2 x 20 μl). Filters are combined. DNA is isolated from the combined filtrates by precipitation with a mixture (1: 2) of ethyl alcohol and 2% lithium perchlorate in acetone.

When cleavage with RNAase, DNA is used with a primer containing two uridine nucleotides. A weighed portion of 20 mg PET microparticles with single-stranded primer extension reaction products (20 mg, ~ 0.54 nanomole / mg) was washed with water and suspended in 30 μL of 0.1 M sodium acetate. Digestion is carried out at 60 ° C for 1 h in the presence of RNAse A. Then 20 μl of formamide is added and heated at 95 ° C for 1 min. The PET microparticles are separated by filtration and washed on the filter with 40% formamide (2 x 20 μl). Filters are combined. DNA is isolated from the combined filtrates by precipitation with a mixture (1: 2) of ethyl alcohol and 2% lithium perchlorate in acetone.

The amount of DNA is determined spectrophotometrically by measuring the absorption of a solution in water at a wavelength of 260 nm. The extinction coefficient is calculated based on the length and the ratio of nitrogenous bases. The length of the products and their purity (full size, individuality) are assessed by electrophoresis in a 4% agarose gel. Base-modified nucleotides slow down the speed of DNA movement in comparison with natural DNA of equal length.

Yield ~ 9.7 nanomoles. The specific yield of the modified single-stranded product of the solid-phase primer elongation reaction is 0.49 nanomolar per 1 mg of PET microparticles, which is 80% of the capacity of PET microparticles.

Example 9. Electrophoretic analysis of primer extension products.

Control of DNA cleavage products from PET microparticles is carried out by electrophoresis in 4% agarose gel. Before analysis, DNA is precipitated with 2% lithium perchlorate in acetone, the supernatant is removed, washed twice with acetone. The precipitate is dried and dissolved in a minimum volume of 10 mM phosphate buffer (PBS), pH 7.2. A mixture of a test sample (10 μl) and a special loading buffer (3 μl) containing electromotive dyes to control the passage of electrophoretic separation is applied to the wells of an agarose gel. Electrophoresis is performed at 10 V / cm for 30 minutes. The gel is stained with ethidium bromide. The results are visualized using an ultraviolet transilluminator, recorded with a gel documentation system, or with a digital camera and saved as JPEG files on electronic media.

Claims (20)

1. Libraries of modified single-stranded DNA fragments are designed to obtain aptamers, functional analogs of monoclonal antibodies, including a primer portion of natural nucleotides and a combinatorial portion in which natural deoxythymidine is completely replaced by modified deoxyuridine. 2. A method of obtaining a library of modified single-stranded DNA fragments, including carrying out alkaline hydrolysis of the surface of PET microparticles with the formation of surface carboxyl groups, then binding of surface activated carboxyl groups to a primer containing an amino group, a photodegradable or enzymatically cleavable group and a functional part, with the formation of immobilized on PET -microparticles of primers, with at least 0.5 nanomole of primers immobilized on 1 mg of dry microparticles of polyethylene terephthalate, then carrying out the primer extension (PEX) reaction with a synthetic universal DNA library with a flanking part complementary to the primer and a degenerate part, with complete replacement of natural deoxriffine phosphite on modified deoxyuridine triphosphate, with DNA polymerase, then cleavage of the synthesized library of modified single-stranded DNA fragments from PET microparticles. 3. A method according to claim 2, characterized in that PET microparticles of 40-63 microns in size, free from fine dust particles, are used. 4. The method according to claim 2, characterized in that for immobilization on PET microparticles with subsequent photocleavage of modified single-stranded DNA fragments, a primer containing an amino group, a photocleavable group and a functional part is used: 5'-NH ₂ - (CH ₂ ) ₆ -O-PO ₂ - (T) ₆ -PO ₂ -O- (CH ₂ ) ₂ -CH (2-NO ₂ -C ₆ H ₄ ) -O-PO ₂ -CGTCGTTCA TCAATGCAACT-3 ' 5. The method according to claim 2, characterized in that for immobilization on PET microparticles with subsequent enzymatic cleavage of modified single-stranded DNA fragments, a primer containing an amino group, an enzymatically cleavable group and a functional part is used: 5'-NH ₂ - (CH ₂ ) ₆ -O-PO ₂ - (T) ₆ -PO ₂ - (U) ₂ -O-PO ₂ -CGTCGTTCA TCAATGCAACT-3 ' 6. The method according to claim 2, characterized in that a synthetic universal DNA library with a flanking part of 20 nucleotides complementary to the primer and a degenerate part (N) _{40 is} used as a template in the primer extension reaction (PEX), where N is a mixture all four nucleotides A, T, G, C: 5'- AGT TGC ATT GAT GAC AGA CG - (N) ₄₀ -3 ' 7. The method according to claim 2, characterized in that as modified deoxyuridine triphosphates in the primer extension (PEX) reaction, 2'-deoxyuridine triphosphates modified at the C5 position with amino acid fragments are used, as well as a linker linking them to the C5 position pyrimidine base. 8. The method according to claim 2, characterized in that a fragment of the amino acid phenylalanine is used to modify the 2'-deoxyuridine triphosphate. 9. The method according to claim 2, characterized in that a fragment of the amino acid tryptophan is used to modify the 2-deoxyuridine triphosphate. 10. The method according to claim 2, characterized in that a fragment of the amino acid valine is used to modify the 2'-deoxyuridine triphosphate. 11. The method according to claim 2, characterized in that a fragment of the amino acid leucine is used to modify the 2'-deoxyuridine triphosphate. 12. The method according to claim 2, characterized in that the alanine amino acid fragment is used to modify the 2'-deoxyuridine triphosphate. 13. The method according to claim 2, characterized in that Deep Vent (exo-) or KOD XL DNA polymerases are used. 14. The method according to claim 2, characterized in that an enzymatic method is used to cleave the synthesized library of modified single-stranded DNA fragments from PET microparticles. 15. The method according to claim 2, characterized in that the RNAse enzyme is used for enzymatic cleavage of the synthesized library of modified single-stranded DNA fragments from PET microparticles. 16. The method according to claim. 2, characterized in that for cleavage of the synthesized library of modified single-stranded DNA fragments from PET microparticles, a photochemical method is used. 17. The method according to claim 2, characterized in that for the photochemical cleavage of the synthesized library of modified single-stranded DNA fragments from PET microparticles, UV light with a radiation wavelength with a maximum of 356 nm and a range of 350-400 nm is used.

Images