RU2723373C1 - Thermal dissociation method for selection of dna-aptamers - Google Patents

Abstract

FIELD: biology.

SUBSTANCE: invention relates to molecular biology, enzymology, biotechnology and medicine and is intended for producing DNA-aptamers. Method includes: (a) pre-enrichment of the combinatorial DNA library by forming a complex thereof with an immobilized protein target (human alpha-fetoprotein; AFP) and conducting selection rounds; (b) forming a complex of the obtained library with a native (non-immobilized) AFP solution; (c) separating DNA / AFP complexes from unbound DNA oligonucleotides by acrylamide electrophoresis; (d) extracting AFP bound DNA from acrylamide gel at 37 °C; (e) thermal dissociation of the remaining AFP-complexed DNA from gel at 60 °C; (f) amplification of DNA after thermal dissociation. After the selection rounds with the use of thermal dissociation, a rich DNA library suitable for sequencing is obtained.

EFFECT: use of thermal dissociation, due to creation of more stringent washing conditions of DNA-oligonucleotides involved with the target, makes it possible to increase efficiency of selection of the most specific aptamers to protein targets.

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Description

FIELD OF THE INVENTION

The invention relates to the field of molecular biology, enzymology, biotechnology and medicine, and provides a method for thermal dissociation of enriched combinatorial DNA libraries bound to a protein target for the selection of DNA aptamers.

Aptamers are called specially selected nucleic acid fragments (oligonucleotides) that show affinity for individual molecular targets, which allows us to consider such molecules as functional analogs of monoclonal antibodies. The set of molecular biological methods allowing the selection of highly specific aptamers to their molecular targets is called SELEX. In general, the SELEX methodology can be divided into several standard steps:

- the interaction of the DNA library, which is a combination of many individual DNA oligonucleotides with different sequences, with a molecular target;

- washing of DNA oligonucleotides not bound to the target;

- removal from the target molecule of the bound DNA oligonucleotides;

- amplification of bound DNA oligonucleotides;

- re-involvement of the bound DNA oligonucleotides in the selection rounds with a molecular target in order to enrich the combinatorial DNA library with oligonucleotides with sequences that are most specific to a given target.

In the generally accepted variant of aptamer selection, the washing principle consists in diluting the target solution and the DNA library with special buffer solutions. At the same time, both unbound and bound DNA oligonucleotides of the library, but having a low affinity for the target, are washed off.

The present invention considers a method for removing DNA molecules of a combinatorial library from a target target after the steps of washing and removing specific DNA oligonucleotides from the target, and an additional step is the thermal dissociation of DNA / target complexes. Carrying out thermal dissociation makes it possible to identify highly specific target oligonucleotides that remain in the form of a complex with the target after the stage of washing and the stage of removal of specific DNA sequences from the target. The identified set of highly specific DNA oligonucleotides obtained by thermal dissociation can be used for further selection rounds.

State of the art

Aptamers are one of the options for biodetectors, their use is based on the ability of nucleic acid fragments to interact with the substrate (target) due to the formation of three-dimensional structures that show affinity for certain spatial forms of various proteins or other macromolecules (as well as low molecular weight compounds) through the formation of hydrogen bonds, electrostatic and van der Waals interactions [1]. Thus, nucleic acid aptamers are functional analogues of protein antibodies, but are characterized by a number of advantages, such as: the possibility of using routine methods for obtaining aptamers without the use of laboratory animals; the ability to automate the process of obtaining aptamers; comparative cheapness; unique physicochemical characteristics, such as the possibility of multiple denaturation-renaturation [2].

Selection method of oligonucleotide libraries of different sequences of aptamers having affinity to a specific substrate called SELEX (S ystematic E volution of L igands by EX ponential Enrichment) and in simplified form consists of a series of oligonucleotides binding loops with the target, the selection of bound fraction and amplification of bound fractions [3, 4].

The stage of washing aptamers that did not bind to the substrate is very important, because the efficiency of enriching the library with highly specific aptamers depends on its correctness.

A description and comparison of various techniques based on the method of capillary electrophoresis for the selection of aptamers in the microfluidic format is given [5]. [6] described the use of capillary electrophoresis for the selection of aptamers for human immunoglobulin E (IgE). The separation of complexes from unbound oligonucleotides by the difference in mobility in an electric field is described.

The authors of [7] describe the nonequilibrium capillary electrophoresis of equilibrium mixtures (NECEEM) that they developed, which is applicable for the analysis of kinetic and thermodynamic parameters of a nucleic acid pool. By supplying a target-free buffer to the capillary inlet, the migrating complexes were slowly released depending on the equilibrium aptamer dissociation constant and rate constants (k _d and k _off , respectively), which were analyzed in the separation electrophoregram.

The authors of [8] proposed an electrochemical method for the selection of aptamers. The selection was carried out using a gold electrode as a solid matrix for immobilization of a protein target (DOCK8). This allowed voltammetric monitoring of bound DNA after each selection cycle. After eight rounds of selection, DNA aptamers with high affinity for DOCK8 were identified with dissociation constants in the range from 3.3 to 66 nm.

An interesting method of washing bound aptamers using the strand displacement reaction was proposed in [9]. The authors position the proposed aptamer displacement approach both for detecting target protein targets and for increasing the efficiency of cell SELEX (cell-SELEX).

A method for the rapid selection of aptamers using a magnetic field (MARAS) for screening DNA aptamers is described (US patent No. 20150119285). The described MARAS method is capable of efficiently generating aptamers with high affinity and specificity. A rotating magnetic field or an alternating magnetic field was used in combination with magnetic microparticles or nanoparticles bound to the target to select DNA aptamers with the desired degree of affinity and specificity for the target and to separate them from oligonucleotides that do not have the required affinity for the target.

The method of US Patent No. 7,745,607 provides the choice of aptamers by immobilizing each library oligonucleotide by forming a duplex with an immobilized antisense oligonucleotide, reacting the duplex molecule with the target and collecting oligonucleotide molecules that dissociate from the duplex structure due to the transition to a more energetically beneficial form. In this way, specific aptamers are washed, while aptamers that do not have sufficient affinity for the target remain in the immobilized phase.

The domestic invention (patent for the invention of the Russian Federation No. RU 2618872) describes a method for the directed depletion of oligonucleotide libraries in relation to water-soluble protein targets of glutathione-S-transferase and streptavidin. After binding of the target to the prepared pool of the source oligonucleotide library, the resulting nucleic acid / target protein mixture is separated into fractions by gel filtration chromatography. The invention allows the elimination of the maximum possible number of molecules affinity for a given target from a pool of oligonucleotides, and the repetition of rounds of such selection ensures the absence in the final pool of molecules of oligonucleotides having significant affinity for the target.

The invention (patent for the invention of the Russian Federation No. RU 2518368) relates to the field of biochemistry, in particular to a method for selecting aptamers for cell receptors and surface proteins, which includes conducting rounds of selection, each of which includes the stages of: positive selection with further removal of unbound DNA; negative selection followed by separation of sequences associated with the negative target; amplification of aptamers obtained during selection to obtain a PCR product. The second and all subsequent rounds of selection begin with a negative selection of aptamers, in the fourth round of selection after the stages of negative and positive selection of aptamers, an excess of monoclonal antibodies specific for the protein target on the cell surface are added to the aptamer-cell complexes. Aptamers that have passed into the solution are separated and amplified. The invention allows to obtain aptamers specific for native proteins.

Russian patent for the invention of the Russian Federation No. RU 2699522 describes a method for the enzymatic production of modified DNA to create reagents that specifically bind to hydrophobic regions of high molecular weight organic compounds. The invention is aimed at obtaining highly specific modified aptamers for protein targets. The described method uses a standard variant of washing unbound oligonucleotides from the target.

The claimed invention differs from the above analogues in that the developed method for the selection of target-specific aptamers consists in the use of thermal dissociation of the resulting DNA / target complexes. In this case, obviously non-specific DNA oligonucleotides are preliminarily separated from the complexes by electrophoresis in an acrylamide gel, and DNA oligonucleotides with a poor affinity for the target are removed by gel extraction before thermal dissociation.

Disclosure of the invention

Selection of DNA aptamers from combinatorial libraries is a complex task requiring an integrated approach for a successful solution. Each of the selection stages requires careful design, and currently there are many varieties of methodology aimed at optimizing the process, taking into account the selected nature of the source library (DNA or RNA), target (small molecules, macromolecules, living cells), enzymatic reactions used (PCR, primer extension reaction), available instrumentation, etc.

The aim of the present invention was to develop a method of enrichment of DNA libraries for the selection of aptamers using thermal dissociation of ligand-target complexes using DNA libraries and targets of protein nature as examples.

This goal is achieved by thermal dissociation of enriched aptamer libraries (TDA) from electrophoresis gel after conjugation with their protein target. As the target protein, human alpha-fetoprotein is used, which is a marker of pre- and neonatal diseases, as well as an oncomarker of some types of cancer. In the proposed method, five rounds of selection are initially carried out with the immobilized target protein (alpha-fetoprotein), which includes the stages: complex formation with the protein, washing, amplification of protein-specific DNA oligonucleotides. After this, the enriched library is mixed with the free target protein in micromolar concentrations and the resulting complex is subjected to electrophoretic separation in acrylamide gel. This stage allows us to exclude aptamers that do not interfere with the protein. Then elution is carried out from the corresponding gel portion of the bound aptamers into an aqueous solution of 2% lithium perchlorate while maintaining micromolar concentrations of the aptamers and their target. This allows preserving undestructed complexes of the most specific aptamers with the target protein, but removing aptamers with low affinity for the target. After this, thermal dissociation of the complexes is carried out at a temperature of 60 ° C and subsequent elution of the most highly specific aptamers. The enriched library amplifies and undergoes thermal dissociation after complexing with the target twice more.

In equilibrium, the dissociation of DNA and target complexes (T) is described as the equation of chemical equilibrium:

Where k _on and k _off are the rate constants of binding and dissociation, respectively. These two rate constants determine the equilibrium dissociation constant as K _d = k _off / k _on . With increasing temperature, the equilibrium dissociation constant of the endothermic reaction always increases, therefore, the degree of dissociation increases. We proposed to use this property in the selection of aptamers and enrichment of libraries with sequences of oligonucleotides, characterized by high affinity for the used protein target.

The result is a library enriched with highly specific aptamers, ready for sequencing to determine individual nucleotide sequences. The developed method is applicable for SELEX to protein targets, including using modified nucleotides.

Brief description of figures and tables

Fig 1. - Electrophoregram showing the separation of complexes of DNA oligonucleotides with protein from unbound DNA oligonucleotides.

1 - a complex of single-stranded DNA library No. 1 (chemically modified) with human alpha-fetoprotein (AFP);

2 - single-stranded DNA library No. 1;

3 - complex single-stranded DNA library No. 2 (natural) with AFP,

4 - single-stranded DNA library No. 2;

5 - DNA length marker “GeneRuler 50 bp”;

6 - AFP. Wells 1-5, ethidium bromide staining. Well 6 — Coomassie Blue Staining.

Fig 2. - Absorption spectrum of fluorescently-labeled DNA oligonucleotides after thermal dissociation of DNA / AFP complexes.

Fig 3. - Derived 5-aminoallyl-2'-deoxyuridine-5'-triphosphate, modified at the aminoallyl fragment of phenylpropionic acid.

Fig 4. - Electrophoregram complexes of AFP with DNA libraries.

Well No. 1 is a molecular marker of DNA lengths “GeneRuler 50bp”, Well No. 2 is an enriched DNA library obtained using the thermal dissociation method, Well No. 3 is an enriched DNA library obtained without using a thermal dissociation method. It is seen that the optical density of the DNA / AFP complex is higher in well No. 2.

The implementation of the invention

We used Taq DNA polymerases (Eurogen, Russia) and Deep Vent (BioLabs, UK) with buffer solutions of the corresponding manufacturers; deoxynucleoside triphosphates manufactured by Fermentas (USA); SYBR Green fluorescent dye (Thermo Scientific, USA); MagnaBind Streptavidin Beads magnetic particles (Thermo Scientific, USA). The remaining reagents and solvents of the “OSCh”, “ChCh” or “ChDA” brands were purchased from Aldrich, Alfa Aesar, Fluka, and Chemmed. Water for the preparation of solutions was obtained using a Milli-Q apparatus (EMD Millipore, USA).

Amplification of DNA libraries after each SELEX cycle was performed by PCR. The PCR reaction mixture contained 1.5 mM MgCl ₂ , 10 mM KCl, 10 mM Tris-HCl (pH 8.3) and 2.5 units (per reaction volume 50 μl) TaqDNA polymerase (ThermoFisher) for unmodified libraries or 0 , 5 units of Vent (exo-) polymerase (NEB) in the case of modified libraries. The mixture contained triphosphates (dATP, dCTP, dGTP and dTTP) at a concentration of 0.2 mM each. In the case of modified libraries, the mixture instead of dTTP contained modified deoxyuridine triphosphate. The working concentration of the primers was 100 nM each. One of the primers was stained with Cy5 cyanine dye to form the aptamer colored chain; the second primer carried a biotin tag. The reaction was carried out on a Rotor-Gene Q5 plex HRM DNA thermocycler according to the following program: 4 min at 95 ° C; 20 cycles of 30 sec at 95 ° C, 30 sec at 66 ° C and 40 sec at 72 ° C; complete the reaction by incubation for 3 min at 72 ° C.

The double-stranded DNA (dsDNA) obtained as a result of amplification was used for separation on magnetic streptavidin particles. The PCR product (50 μl) was mixed with an equivalent volume of 10x PCR buffer, incubated at 95 ° C for 3 min. The resulting mixture was added to magnetic particles previously suspended in 10x PCR buffer, incubated at room temperature for 30 minutes. During this time, the biotinylated DNA chain covalently binds to streptavidin. The magnetic particles were washed three times with buffer, and then incubated for 2 min with a 20% solution of ammonium hydroxide at room temperature. Then the supernatant was poured into a clean tube and reprecipitated with 5 volumes of an alcoholic solution of ammonium acetate. The precipitate was washed with 96% ethanol, dried and dissolved in deionized water (MQ-H ₂ O).

The magnetic particles were washed with ammonium hydroxide, and then incubated with it for 8 min at 60 ° C. The resulting solution was poured into a test tube and the biotinylated chain was reprecipitated with an alcoholic solution of ammonium acetate. The precipitate was washed with 96% ethanol, dried from alcohol and dissolved in MQ-H ₂ O.

The first five rounds of complex formation were carried out using an immobilized preparation of alpha-fetoprotein (AFP), a glycoprotein with a molecular weight of about 69,000 Da, consisting of one polypeptide chain including about 600 amino acids. 220 pmol libraries were incubated with the required amount of an immobilized AFP preparation. The single chain library was heated to 95 ° C for 2 minutes and slowly cooled to 37 ° C. AFP immobilized on magnetic particles with a concentration of 15 pmol / mg was washed with 1x SELEX buffer prepared from 2x SELEX buffer having a pH of 7.5 and containing 0.30 M NaCl, 60 mM HEPES, 20 mM KCl, 20 mM MgCl2, 0.1% TWEEN -twenty. The particles were diluted in 100 μl of 2x SELEX buffer. 100 μl single chain library was added. Incubated for 1 hour at 37 ° C, stirring. The supernatant was selected - unbound oligonucleotides. Microparticles containing AFPs with bound sequences were washed with 1x SELEX buffer at room temperature, dissolved in 100 μl of MQ quality H ₂ O and incubated for 10 min at 95 ° C, the supernatant was separated, its concentration was determined on a NanoDrop 1000 spectrophotometer and used in further rounds SELEX.

It is permissible to carry out five preliminary rounds of selection without the use of magnetic particles, by creating DNA / AFP complexes in solution (with native non-immobilized AFP) with their subsequent separation by electrophoresis and elution of the DNA oligonucleotides bound to the target from the gel.

The next three cycles of complex formation were carried out in a buffer with native non-immobilized AFP at 37 ° C for 60 min in a 1x SELEX buffer, then the complex was electrophoretically separated from unbound DNA oligonucleotides and subsequent thermal dissociation.

Given the molecular weight of the AFP and the length of the modified oligonucleotides that make up the library, the following gel composition was selected: 12% acrylamide / bis-acrylamide (19: 1) in 1x TBE containing 10 mM KCl and 3 mM MgCl ₂ . The presence of potassium and magnesium salts in the buffer is due to the need to stabilize noncanonical types of the secondary structure of DNA, which may be present in aptamers: G-quadruplexes and hairpin DNA structures. The analysis was carried out at a controlled temperature of 20 ° C, which prevents uncontrolled thermal dissociation of complexes during electrophoresis. For the same purpose, a relatively low voltage was maintained in the electrophoretic cell (about 120-150 volts per 20 cm of gel). Control of migration of bound and unbound DNA was carried out using a 45-mer fluorescently-labeled oligonucleotide. The path length of the oligonucleotide control was 5-7 cm. The gel was visualized by staining with SYBR Gold I. The results of electrophoresis are shown in FIG. 1.

According to the electrophoretic picture, the amount of AFP bound and unbound DNA was estimated.

The bound DNA in complex with AFP was excised from the gel and 100 μl of a solution of LiClO ₄ in H ₂ O was eluted for 60 min at 37 ° C. In the classic SELEX variant, it is precisely such bound DNA that is involved in subsequent rounds of selection. In the proposed embodiment, the resulting eluate is not used.

After elution at 37 ° C, thermal dissociation of the remaining complexes was performed. For this, 100 μl of 1x SELEX buffer was added to the gel, heated to 60 ° C and incubated for 20 min. The supernatant was separated, the DNA concentration was determined on a NanoDrop 1000 spectrophotometer (Fig. 2) and used in further (two additional) SELEX rounds followed by thermal dissociation.

It is theoretically acceptable to conduct thermal dissociation using magnetic particles and immobilized AFP.

Both a library using natural nucleotides and a modified deoxyuridine derivative, the structure of which is shown in FIG. 3. After conducting three rounds of selection using thermal dissociation, a DNA library enriched with highly specific aptamers was obtained, ready for sequencing to determine individual nucleotide sequences of aptamers.

The effectiveness of the thermal dissociation method was compared with the basic variant of aptamer selection by electrophoretic separation of DNA / AFP complexes (Fig. 4). It can be seen that the optical density is greater in the case of enrichment of the library by thermal dissociation.

Examples

Example 1. Obtaining biotinylated primer

For subsequent separation of the combinatorial DNA library chains, biotin was introduced into one of the chains formed during PCR by using a biotinylated primer. The primer was synthesized using a C6 amino modifier with a free NH ₂ group at the 5'end, followed by the introduction of a biotin label. To a solution of biotin hydroxysuccinimide ester (2.0 mg) in DMF (590 μl) was added a primer in a ratio of 10/1, respectively. The reaction took place for 30 minutes at room temperature. Next, chromatographic purification of the obtained product in an acetonitrile gradient was carried out.

Example 2. Obtaining primer labeled with cyanine dye Cy5

The primer was synthesized with a free NH ₂ group at the 5 ′ end. Dye Cy5 in the form of hydroxysuccinimide ether in DMSO was added to the primer in a ratio of 10/1. An incubation of 2 hours was carried out at room temperature. Next, chromatographic purification of the obtained product in an acetonitrile gradient was carried out.

Example 3. Polymerase chain reaction for the amplification of combinatorial (degenerate) DNA libraries

The PCR reaction mixture contained 1.5 mM MgCl ₂ , 10 mM KCl, 10 mM Tris-HCl (pH 8.3) and 2.5 units (per reaction volume 50 μl) Taq DNA polymerase (ThermoFisher) for unmodified libraries or 0.5 units of Vent (exo-) polymerase (NEB) in the case of modified libraries, triphosphates (dATP, dCTP, dGTP, as well as dTTP for unmodified libraries or a modified deoxyuridine triphosphate derivative at a concentration of 2 mM (Fig. 3) in the case of modified libraries. Primers were used: direct biotinylated and reverse fluorescently labeled, the working concentration of each primer was 10 mM. A synthetic degenerate combinatorial DNA library with the sequence was used as a template

CTGTCAGCTCCATACTGGTAGCC- (N) 40-GCGTTCGAATCTAGACGGTACGA,

where N is the combination of nucleotides A, T, G, C.

The reaction was carried out on a MiniCycler DNA Amplifier (MJResearch, Waltham, Mass.) According to the following program:

1) 4 min at 95 ° C,

2) 30 sec at 95 ° C,

3) 30 sec at 66 ° C,

4) 40 sec at 72 ° C,

5) repeat from 2 points to 4 points 19 times,

6) completed the reaction by incubation for 3 min at 72 ° C.

When using a modified derivative of deoxyuridine triphosphate as substrates, if necessary, the temperature-time profile of PCR, as well as the composition of the reaction mixture, was modified (optimized) in order to increase the yield of the target reaction product. The result of PCR was evaluated visually, electrophoretically.

Example 4. The separation of biotinylated and non-biotinylated DNA library chains after PCR using magnetic particles

To obtain single-stranded DNA with Cy5 dye at the 5'-end, chains were separated on magnetic particles of Dynabeads ™ M-270 Streptavidin (InvitrogenDynalAS, Norway) with streptavidin molecules covalently attached to their surface. The magnetic particles were washed three times with 10x buffer (pH 8.8 at 25 ° C) containing 100 mM Tris-HCl and 500 mM KCl and resuspended in a solution containing 100 μl of 5x PCR buffer. The 5 ′ end biotinylated double-stranded DNA (PCR product, 50 μl volume) was mixed with an equivalent volume of 10x PCR buffer, heated to 95 ° C, incubated for 3 min and cooled to room temperature, after which it was mixed with prepared magnetic particles. Immobilization was carried out at room temperature for 30 minutes with constant stirring. Using a magnetoseparator, magnetic particles with double-stranded DNA immobilized on them were separated from the reaction mixture and washed three times with 5X buffer containing Tris-HCl and KCl.

The non-biotinylated chain was eluted by adding 20% ammonium hydroxide solution to the magnetic particles, incubated for 2 min, then the particles were separated on a magnetic separator, and the eluate was transferred to a clean tube where 5 volumes of an alcoholic solution of ammonium acetate containing 0.125 M ammonium acetate and 70% were added to it. ethanol. Single-stranded non-biotinylated DNA was precipitated by centrifugation (16000 g, 2 min). The precipitate was washed with an alcoholic solution of ammonium acetate, then with 96% ethyl alcohol and dried in air. To the dried precipitate was added 20 μl of deionized water (MQ-H ₂ O) and the DNA concentration was determined on a NanoDrop 1000 spectrophotometer.

Biotinylated chain magnetic particles were washed with 20% ammonium hydroxide for 1 min at room temperature, then the supernatant was removed, and the particles were incubated with 50 μl of 30% ammonium hydroxide at 60 ° C with occasional manual stirring for 8 min. The eluate was transferred to a clean tube where the biotinylated chain was reprecipitated by adding 5 volumes of an alcoholic solution of ammonium acetate containing 0.125 M ammonium acetate and 70% ethanol. The precipitate was washed with an alcoholic solution of ammonium acetate, then with 96% ethyl alcohol and dried in air. To the dried precipitate was added 20 μl of deionized (MQ) H ₂ O and the DNA concentration was determined on a NanoDrop 1000 spectrophotometer. The separation efficiency of the chains was determined by electrophoretic method.

The efficiency of DNA elution was controlled by adding 40 μl of a solution containing 8 M urea and 2 M thiourea to magnetic particles, which led to the destruction of streptavidin-biotin and elution of all DNA immobilized on magnetic particles complexes. The eluate was analyzed by PAAG electrophoresis.

Example 5. The complexation of a single-stranded DNA library with alpha-fetoprotein (AFP) in the immobilized phase

The modified single chain library was heated to 95 ° C for 2 minutes and cooled to 37 ° C. AFP immobilized on magnetic particles with a concentration of 15 pmol / mg was washed with 1x SELEX buffer prepared from 2x SELEX buffer having a pH of 7.5 and containing 0.30 M NaCl, 60 mM HEPES, 20 mM KCl, 20 mM MgCl2, 0.1% TWEEN -twenty. Magnetic particles were diluted in 100 μl of 2x SELEX buffer. 100 μl of a modified single chain library was added. Incubated 1 hour with stirring, 37 ° C. The supernatant was selected - unbound oligonucleotides. Magnetic particles containing AFPs with bound sequences were washed with 1x SELEX buffer at room temperature, dissolved in 100 μl of MQ quality H ₂ O and incubated for 10 min at 95 ° C, the supernatant was separated, its concentration was determined on a NanoDrop 1000 spectrophotometer and used in further SELEX rounds.

Example 6. The formation of the complex single-stranded DNA library with alpha-fetoprotein (AFP) in solution

The modified single chain library was heated to 95 ° C for 2 min and slowly cooled to 37 ° C. To 50 μl of an aqueous solution of AFP with a concentration of 30 pmol / μl was added 50 μl of 2x SELEX buffer. 100 μl of a modified single chain library was added. Incubated for 1 hour with stirring at 37 ° C.

Example 7. Electrophoretic separation of DNA / AFP complexes and unbound oligonucleotides

An electrophoresis gel of the following composition was prepared: 12% acrylamide / bis-acrylamide (19: 1) in 1x TBE containing 10 mM KCl and 3 mM MgCl ₂ . 20 μl of the DNA / AFP complex, a free library, and controls were added to the wells. Electrophoresis was carried out at a controlled temperature of 20 ° C to prevent uncontrolled thermal dissociation of complexes. For the same purpose, a relatively low voltage was maintained in the electrophoretic cell (about 120-150 volts per 20 cm of gel). Control of migration of bound and unbound DNA was carried out using a 45-mer fluorescently-labeled oligonucleotide. Electrophoresis was carried out up to the path length of the oligonucleotide control - 5-7 cm. As additional controls were used: an AFP-unmodified modified library after the 5th round, GeneRuler 50bp DNA ladder (ThermoFisher, USA) and AFP and / or protein marker. The bands in the gel were visualized using SYBR Gold I for DNA and Coomassie for proteins. Electrophoresis results are shown in FIG. 1.

According to the electrophoretic picture, the amount of AFP bound and unbound DNA was estimated.

Example 8. Elution of DNA from the gel

The gel strips containing the DNA / AFP complex were excised with a surgical scalpel, placed in a 1.5 ml Eppendorf tube and eluted protein-bound DNA oligonucleotides were eluted with 100 μl of a solution of LiClO ₄ in H ₂ O (Milli-Q) . The mixture was incubated for 60 min at 37 ° C, the tubes were centrifuged at 2000 rpm for 2 min, the supernatant was taken and the DNA concentration was measured on a NanoDrop 1000 spectrophotometer. The DNA contained in the obtained supernatant was not used for further selection rounds. The gel remaining in the tubes was used to conduct thermal dissociation of DNA oligonucleotides with increased affinity for AFP.

Example 9. Thermal dissociation of DNA oligonucleotides with increased affinity for AFP

After elution, 100 μl of 1x SELEX buffer was added to the gel, heated to 60 ° C and incubated for 20 min. The supernatant was separated, the DNA concentration was determined on a NanoDrop 1000 spectrophotometer (Fig. 2). After thermal dissociation, the eluate was involved in the next of three rounds of selection, which includes the stage of thermal dissociation. After conducting three rounds of SELEX with thermal dissociation, an enriched DNA library was obtained that was suitable for determining the individual sequences of oligonucleotides that make up its composition.

Example 10. Electrophoresis of AFP complexes with DNA libraries obtained using and without thermal dissociation.

Electrophoretic separation was carried out similarly as described in Example 7. The efficiency of DNA binding with AFP was evaluated visually by the optical density of the band corresponding to the resulting DNA / AFP complex (Fig. 4).

LITERATURE

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Claims (12)

1. The method of thermal dissociation to obtain DNA aptamers, comprising sequential stages: (a) - preliminary enrichment of the combinatorial DNA library by forming its complex with a protein target - immobilized human alpha-fetoprotein (AFP) - and conducting rounds of selection, where the combinatorial DNA library acts as a ligand, and AFP as a substrate; (b) the formation of a complex of a pre-enriched DNA library with a solution of native (non-immobilized) AFP; (c) electrophoretic separation of DNA / AFP conjugates from unbound DNA oligonucleotides by acrylamide electrophoresis; (d) - extraction of AFP-bound DNA from acrylamide gel at 37 ° C; (e) - thermal dissociation of the DNA remaining in the form of a complex with AFP from the gel at 60 ° C; (f) - amplification of DNA after thermal dissociation; (g) the formation of a DNA complex after thermal dissociation with a solution of native (non-immobilized) AFP; (h) - a twofold repetition of points (c) - (e); (k) - establishing the primary sequence of the obtained highly specific for AFP DNA oligonucleotides (aptamers). 2. The method according to p. 1, in which unmodified (natural) DNA libraries are used as ligands. 3. The method according to p. 1, in which chemically modified DNA libraries are used as ligands, for the enzymatic preparation of which, in the PCR reaction, deoxyuridine triphosphate is completely replaced by a 5-aminoallyl-2'-deoxyuridine-5'-triphosphate derivative modified by the aminoallyl fragment phenylpropionic acid.

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