RU2518368C1 - Method of selection aptamers to specified protein targets on cell surface - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to the field of biochemistry, in particular, to a method of selection of aptamers to cell receptors and surface proteins, which comprises conducting rounds of selection, each of which comprises the following stages: the positive selection followed by removal of unbound DNA; the negative selection followed by separation of the sequences bound with the negative target; amplifying of the aptamers obtained during selection with production of a PCR product. The second and all the subsequent rounds of selection are started with negative selection of aptamers, in the fourth round of selection after the stages of negative and positive selection of aptamers to aptamer-cell complexes an excess of a monoclonal antibodies is added, which are specific for the protein target on the cell surface, the aptamers passed into the solution are separated and amplified.

EFFECT: invention enables to obtain aptamers specific for the native proteins.

6 cl, 2 dwg, 1 ex

Description

The invention relates to a method for selecting aptamers for cellular receptors and surface proteins.

Today, monoclonal and polyclonal antibodies are used to diagnose diseases requiring the detection of certain receptors. Blood proteins, hormones, growth factors, cellular receptors, inflammatory and immune mediators, bacterial and viral antigens, various poisons, etc. are used to determine them. For example, monoclonal antibodies to CD-4 are used to determine immune status. CD-8, CD-20, CD-I6, etc. Using antibodies, rapid determination of blood groups is carried out, pregnancy and HIV tests are performed, cells in undifferentiated tumors are identified, they are used for diagnosis and typing of tumors [Peter Kuhn et al. Fluid biopsy in patients with metastatic prostate, pancreatic and breast cancers. Phys. Biol. 9 (2012)]. In addition, monoclonal antibodies are beginning to be successfully used for therapeutic purposes for the treatment of various pathologies.

However, the procedure for obtaining antibodies is quite time-consuming and expensive, since they require the use of animals. In addition, protein antibodies are not stable, sensitive to storage conditions and immunogenic.

Aptamers selected for given cellular receptors, due to their high affinity for target molecules, can be used as analogs of antibodies. Aptamers are short synthetic single-stranded DNAs or RNAs that can form unique tertiary structures that allow them to bind to specific targets with high specificity.

Compared to monoclonal antibodies, aptamers have several advantages: 1. Aptamers can be synthesized chemically, and unlike natural antibodies do not require the use of animals; 2. the number of selected aptamers can be easily increased by polymerase chain reaction; 3. The simple chemical structure of the aptamers makes it easy to modify them in accordance with the tasks; 4. Aptamers are more stable against temperature and chemicals; 5. With the loss of affinity of aptamers, their properties are easily restored using the denaturation procedure followed by renaturation.

In addition, the procedure for obtaining aptamers is less labor intensive and less expensive than the procedure for obtaining mono- and polyclonal antibodies.

Aptamers to a given target are obtained using in vitro selection (SELEX). SELEX technology is a technology for obtaining aptamers through the systematic evolution of ligands by exponential enrichment (Systematic Evolution of Ligands by Exponential Enrichment). [Ellington, A.D. and Szostak, J.W. (1990) In vitro selection of RNA molecules that bind specific ligands. Nature 346, 818-822]; Tuerk, C. and Gold, L. (1990) Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase. Science 249, 505-510]. The traditional SELEX method consists of several repeating rounds. Each round includes 3 main stages: 1) the library of oligonucleotides is incubated with a biological target, unbound DNA is removed; 2) the resulting aptamers are incubated with a negative target, the sequences associated with the negative target are separated; 3) the aptamers obtained during selection are amplified.

Recombinant (purified) proteins are usually used to select surface receptor aptamers. However, for analytical purposes, it is most often required to determine a protein that is not in free form, but in the composition of the cell membrane. Since the conformation of the protein is determined by the lipid bilayer of the membrane, the conformations of native and recombinant proteins may vary. Aptamers selected for recombinant proteins have a lower affinity for the same proteins that are part of the cell membrane. Therefore, the specificity of detecting whole cells whose proteins are in their native state using aptamers selected for recombinant proteins may not be quite high.

An object of the present invention is to provide aptamers specific for native surface proteins of whole cells.

The problem is solved in that in the method of selection of aptamers to given protein targets on the cell surface, including conducting rounds of selection, each of which includes the stages of: positive selection - incubation of the library of oligonucleotides with a biological target, with the removal of unbound DNA; negative selection - incubation of the obtained aptamers with a negative target, with the separation of sequences associated with a negative target; amplification of the aptamers obtained during selection to obtain the PCR product according to the invention, 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 protein target on the surface of the cell, aptamers transferred to the solution are separated and amplified.

The selection method can be carried out with a repetition of the fourth round of selection in all subsequent rounds.

The selection method can be carried out with alternating the third and fourth rounds in all subsequent rounds, after the fourth

In the third round of the selection method, after incubation of the PCR product with negative targets and separation from unbound residues, the resulting supernatant can be incubated with plasma or blood serum, after which positive selection is carried out, followed by amplification of the aptamers.

Incubation of the sample with blood plasma can be carried out through one round of selection of aptamers.

In each round of selection, the concentration of added antibodies can be increased.

The concentrations of aptamers and monoclonal antibodies during the rounds of selection can vary.

The inventive method for the selection of aptamers is based on the competitive replacement of aptamers with monoclonal and polyclonal antibodies. Candidate aptamers, being in the minority on the surface of the target receptor, are removed from it by a higher concentration of antibodies. The novelty of the invention lies in the use of the principle of competitive displacement of candidates for DNA aptamers. bound to the target receptor using monoclonal antibodies specific for these receptors.

The first few rounds of selection may not differ from the traditional ones and are aimed at enriching the pool of aptamers with sequences specific to a given cell target. Further selection to achieve the desired result requires the introduction of additional new stages of selection, namely: to the aptamer-cell complexes, an excess of monoclonal antibodies specific to the protein target on the cell surface is added, with aptamers. being in a lower concentration are displaced from the surface of the cells due to the high concentration of antibodies; the aptamers that have passed into the solution are separated and amplified.

To obtain aptamers highly specific to a given protein target on the cell surface, several rounds of selection are necessary. To ensure that the aptamer pool is enriched with precisely those sequences that are displaced by monoclonal antibodies, it is desirable to increase the concentration of added antibodies in each round. By varying the concentration of aptamers and monoclonal antibodies, aptamers of different binding strengths to the protein target can be obtained.

As a result of such selection, aptamers that specifically bind to the given receptors on the cell will be obtained.

The method is illustrated by graphs. Figure 1 shows the fluorescence intensity of aptamers bound to tumor cell cytokeratins (curve 1 - intact cells (not associated with aptamers), curve 2 - fluorescence of 50 nM aptamers pool associated with human lung cancer cells, curve 3 - fluorescence of aptamers after they competitive displacement from the cell surface by an excess of antibodies to cytokeratins). Figure 2 shows the fluorescence intensity of antibodies to cytokeratins labeled with FITC (curve 4 - intact cells (not associated with antibodies to cytokeratins), curve 5 - fluorescence of antibodies to cytokeratins associated with cells, curve 6 - fluorescence of antibodies to cytokeratins associated with cells that have displaced aptamers from the surface of cells).

The method is as follows.

The selection is carried out in a medium containing all the necessary components for the normal functioning of living cells, and in phosphate buffer in the case of molecular targets or fixed cells. A prerequisite for the selection of aptamers is the presence in the medium or incubation buffer with a library of aptamers of magnesium and calcium ions, and preferably sodium and potassium. These ions are necessary for the formation of the secondary and tertiary structure of aptamers, due to which highly specific target recognition occurs. The first three rounds of aptamer selection are practically no different from the traditional SELEX method.

In the first round of selection, the aptamer library is incubated with cells, on the surface of which there are receptors necessary for the selection of aptamers, for 10-60 minutes at 22-37 ° C (the incubation time and temperature are not fixed and may vary depending on the target, desired results and intended use). The target cells are then centrifuged at 3500 rpm for 3 min to free from unbound oligonucleotides and washed in PBS buffer containing Ca ²⁺ and Mg ²⁺ . Bound oligonucleotides precipitated after centrifugation are separated from the biological target by denaturing using a TE buffer at 95 ° C, followed by precipitation at 13,000 rpm for 15 minutes. Next, the supernatant is collected and symmetric and asymmetric polymerase chain reactions are carried out.

After the first and all subsequent rounds of selection, the PCR product is purified from the primers by DNA precipitation with ethanol or using 30 kDa filters (for example, Pall centrifugal devices). The second and all subsequent rounds begin with a negative selection of aptamers. As negative targets can be used cells that do not have on the surface of the given receptors. The PCR product obtained during the first round is incubated with negative targets for 10-40 minutes. Next, centrifugation is carried out at 3500 rpm for 3 minutes and the supernatant is collected. With the collected supernatant carry out a positive selection. Then centrifuged and washed in PBS buffer containing Ca ²⁺ and Mg ^2f ions. Bound oligonucleotides are separated from the biological target by denaturing at 95 ° C using a TE buffer, followed by precipitation at 13,000 rpm for 15 minutes. After that, symmetric and asymmetric polymerase chain reactions are carried out from the collected supernatant. In the third round, to the standard selection steps after incubation of the PCR product with negative targets and centrifugation to separate from unbound residues, the supernatant can be incubated with plasma or blood serum for 10 minutes to select nuclease-resistant aptamers. After that, a positive selection is carried out with the supernatant, followed by amplification of the aptamers. Incubation of a sample with blood plasma can be carried out on average after one round of selection of aptamers. Starting from the fourth round of selection, in order to obtain aptamers for given receptors, competitive substitution of aptamers with natural antibodies is used. Obtained in the previous round of selection, the PCR product is incubated with negative targets for 10-40 minutes. Next, centrifugation is carried out at 3500 rpm for 3 minutes and the supernatant is collected. With the collected supernatant carry out a positive selection. Then centrifuged and washed in PBS buffer. After that, antibodies specific for a given receptor are added to the precipitate and incubated for 30 minutes. After washing, antibodies are added in high concentration (0.1-7 mg / ml). You can add antibodies sequentially at different concentrations, thereby obtaining aptamer fractions with different strengths of interaction with the target. Antibodies replace aptamers that bind to receptors, as a result of which the aptamers that bind to target receptors pass into solution. Then the sample is centrifuged to separate the aptamers that have passed into solution from the precipitate. After which the supernatant is collected and symmetric and asymmetric PCR is performed. The steps described in the fourth round of selection are repeated in subsequent rounds. You can also alternate the third and fourth rounds of selection. Selection of aptamers to target receptors using competitive antibody substitution is carried out over 10-15 rounds.

The following is an example implementation of a method of selection for cytokeratins.

Example. Aptamers were selected for cytokeratins contained in lung tumor cells.

The following reagents were used to carry out the selection of aptamers for cytokeratins:

1. A randomized single-stranded DNA library with an internal variable part and nucleotides for primers to be planted on each side.

2. A mixture for symmetric PCR containing 1X PCR buffer, 2.5 mM MgCl ₂ , 0.025 U µL ⁴ KAPA2G HotStart Robust polymerase (KAPABiosystems, USA), 220 µM dNTPs, 300 nM forward primer (5′-STS STS TGA CTG TAA CCA CG-3 ′), 300 pM reverse primer (5′-GGC TTC TGG ACT ACC TAT GC-3 ′) (Integrated DNA Technologies, USA). Mix for asymmetric PCR. containing 1X PCR buffer, 2.5 mM MgCl2, 0.025 U µL-l KAPA2G HotStart Robust polymerase, 220 µM dNTPs, 1 µM Alexa-488 direct parimer (5′-A11EX488-STS STS TGA CTG TAA SSA CG-3 ′), and 50 nM reverse primer (5′-GGC TTC TGG ACT ACC TAT GC-3 ′).

3. Phosphate buffer containing Ca ²⁺ and Mg ^{2+ ions}

4. Tris EDTA buffer (TE buffer)

5. Monoclonal antibodies to cytokeratins 4, 5, 6, 8, 10, 13 and 18 labeled FITC (Sigma Aldrich, USA).

The first three rounds of aptamer selection were carried out using the standard SELEX method. Tumor cells of a human lung were used as positive targets. Lymphocytes were used as negative targets because they do not express cytokeratins.

In the first round of selection, the aptamer library was incubated with lung tumor cells for 30 minutes at room temperature. Then, the target cells were centrifuged at 3500 rpm for 3 min and freed of unbound oligonucleotides and washed in PBS buffer containing Ca ²⁺ and Mg ²⁺ . Cell-bound oligonucleotides precipitated after centrifugation were separated from the biological target by denaturing in TE buffer at 95 ° C, followed by precipitation at 13,000 rpm for 15 minutes. Next, the supernatant was collected and symmetric and asymmetric polymerase chain reactions were carried out. During symmetric PCR reactions, double-stranded DNA products were obtained; during asymmetric, single-stranded DNA aptamers. Symmetric PCR was performed using a 5 uL aptamer pool in a TE buffer with a symmetric PCR mixture. Asymmetric PCR was performed using a 5 μL symmetric PCR product mixed with a 45 uL mixture for asymmetric PCR.

Amplification was carried out according to the following program: heating for two minutes at 95 ° C, 15 cycles of 30 seconds at 95 ° C, 15 seconds at 56.3 ° C, 15 seconds at 72 ° C; further maintaining the temperature at 4 ° C.

The resulting PCR product was purified from the PCR mixture using filters using a PBS buffer containing Ca ²⁺ and Mg ^{2+ ions} .

In the second round of selection, the PCR product obtained during the first round was incubated with lymphocytes for 30 minutes. Next, centrifugation was carried out at 3500 rpm for 3 minutes and the supernatant was collected. With the collected supernatant, positive selection was carried out using lung tumor cells. Then it was centrifuged and washed in PBS buffer containing Ca ²⁺ and Mg ^{2+ ions} . Bound oligonucleotides were separated from the biological target by denaturing at 95 ° C using a TE buffer, followed by precipitation at 13,000 rpm for 15 minutes. Symmetric and asymmetric polymerase chain reactions were carried out from the collected supernatant. The resulting PCR product was purified from the examples using filters using a PBS buffer containing Ca ²⁺ and Mg ^{2+ ions} .

In the third round after negative selection, blood plasma diluted in PBS buffer containing Ca ²⁺ and Mg ²⁺ ions was added to the supernatant. And incubated for about 10 minutes. Then, with a supernatant, a positive selection was carried out, followed by amplification of the aptamers and their purification from the PCR mixture.

In the fourth round, the PCR product obtained during the previous round of selection was incubated with negative targets for 30 minutes, centrifuged at 3500 rpm for 3 minutes, non-targeting aptamers collected in the supernatant were collected and used for positive selection. During positive selection, the aptamers bind to tumor cells and are precipitated by centrifugation using a PBS buffer containing Ca ²⁺ and Mg ^{2+ ions} . After this, the pellet was resuspended and cytokeratins antibodies were added to the cell-aptamer complexes, incubation was carried out for 30 minutes at room temperature. Then, the sample was centrifuged to separate the aptamers that had passed into the solution. The supernatant with aptamers was collected and symmetric and asymmetric polymerase chain reactions were carried out, followed by purification of the product from the PCR mixture. A total of eight rounds of selection were carried out.

To confirm that aptamers specific for cytokeratins were obtained by selection using a competitive antibody displacement method, flow cytometry was used (FC-500 flow cytometer, Beckman Coulter Inc., USA).

Figure 1 shows the fluorescence intensity of aptamers associated with tumor cell cytokeratins. As a control, we used human lung tumor cells diluted in PBS buffer containing Ca ²⁺ and Mg ^{2+ ions} , Fig. 1, curve 1. Human lung tumor cells were incubated with aptamers labeled A1ex-647 at a concentration of 50 nm in 200 μl of PBS buffer containing Ca ²⁺ and Mg ^{2+ ions} . A sample incubated with aptamers showed an increase in fluorescence, which indicated the binding of cells to aptamers, Fig. 1, curve 2. After adding antibodies to cytokeratins labeled with FITC at a concentration of 2 mg / ml to the same sample, a decrease in fluorescence was observed (Fig. 1, curve 3 ), indicating the replacement of aptamers with antibodies to cytokeratins (despite the fact that antibodies to cytokeratins labeled with FITC did bind to the cells of figure 2, curve 6).

Figure 2 shows the fluorescence intensity of antibodies to cytokeratins labeled with FITC. Figure 2 curve 4 shows the intact tumor cells (not associated with antibodies to cytokeratins). After incubation of tumor cells with labeled antibodies to cytokeratins, the sample showed a significant increase in fluorescence, which indicates the binding of antibodies to cytokeratins, Fig. 2, curve 5. After labeled antibodies were added to the sample containing cells bound to aptamers, a slight decrease was observed the level of fluorescence compared with a sample containing only labeled antibodies bound to the cells, figure 2, curve 6, which may have been the result of incomplete replacement of the aptamers with antibodies ( of the strong electrostatic interactions).

Data obtained by flow cytometry showed that antibodies to cytokeratins occupied the same binding sites as aptamers obtained during competitive selection. This fact proves the specificity of the obtained aptamers to cytokeratins on the surface of cancer cells.

Thus, using the method of selection of aptamers to specific protein targets on the cell surface, based on the competitive replacement of single-stranded DNA libraries with monoclonal antibodies, it is possible to obtain aptamers specific for native proteins.

Claims (6)

1. The method of selection of aptamers to specified protein targets on the cell surface, including conducting rounds of selection, each of which includes the stages of: positive selection - incubation of the library of oligonucleotides with a biological target, with the removal of unbound DNA; negative selection - incubation of the obtained aptamers with a negative target, with the separation of sequences associated with a negative target; amplification of aptamers obtained during selection to obtain a PCR product, characterized in that 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 are added to the aptamer-cell complexes specific to the protein target on the cell surface, the aptamers that have passed into the solution are separated and amplified. 2. The selection method according to claim 1, characterized in that the fourth round of selection is repeated in all subsequent rounds. 3. The selection method according to claim 1, characterized in that it is carried out with the alternation of the third and fourth rounds in all subsequent rounds after the fourth. 4. The selection method according to claim 1, characterized in that in the third round of selection after incubation of the PCR product with negative targets and separation from unbound residues, the resulting supernatant is incubated with plasma or blood serum, after which positive selection is carried out, followed by amplification of the aptamers. 5. The selection method according to claim 4, characterized in that the incubation of the sample with blood plasma can be carried out through one round of selection of aptamers. 6. The selection method according to claim 1, characterized in that in each round of selection the concentration of added antibodies is increased.

Images