RU2700584C1 - Method for assessing the affinity of an oligonucleotide - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention refers to medicine, namely to pharmacology, and can be used to assess oligonucleotide affinity to a target. That is ensured by oligonucleotide labeling with fluorochrome. Labeled oligonucleotide is then contacted with the receptor target on the cell surface. Photoregistration of the obtained sample is then carried out using a fluorescent microscope. Optical density of the coloring region of the inverted images is measured and compared with the negative control. Affinity of the oligonucleotide is then calculated for the target.

EFFECT: invention provides selection of the most highly affinity oligonucleotides to target molecules on cell surface for marking cells and tissues.

1 cl, 4 dwg, 2 ex

Description

The invention relates to medicine, biotechnology, pharmacology, and in particular, to methods for assessing the affinity of ligands for a biological target and can be used to assess the effectiveness of binding of new synthesized oligonucleotides to targets.

In modern medicine, one of the priority areas is the search for targeted drugs that act selectively on certain types of cells, without affecting cells that are not involved in the pathological process. Particularly important is the selectivity in creating antitumor drugs when it is necessary to act on malignant cells. In this case, low toxicity is desirable for the whole organism. One of the classes of substances that can recognize target molecules on the desired cells are oligonucleotide aptamers. The technology for their preparation is based on selection for binding to the target. And therefore, methods for assessing the binding of an oligonucleotide aptamer are highly sought after [Jing, M .; Bowser, M.T. Anal Chim Acta 2011, 686, 9-18].

A known method for assessing the specificity of binding of molecules to a target using a radioactive label [International application WO 00/52473 of 09/08/2000 "Set for radioactive labeling and analysis of binding"]. The subject of the invention is a set of reagents, which allows, in addition to diagnostic purposes, to evaluate the specificity of antibody binding to target cells that are positive for antigen.

However, the kit and method is intended for the analysis of antibodies and does not imply the use of oligonucleotides for the assessment of affinity. In addition, the use of radioactive tags involves the implementation of safety measures and the cost of disposing of radioactive waste, which makes the method difficult to perform in a laboratory without an isotope block.

A device is known that is used to detect binding [Patent RU 2609184 from 01/17/2013, "Device used to detect affinity"], which implements a method for evaluating affinity based on the scattering of a damped field of coherent light propagating through a planar waveguide over a surface modified by molecules - targets that bind to the test substance.

The disadvantages of the method include the fact that the target attached to the substrate can change its conformational properties and the results of the assessment of the specificity of binding will be distorted. In addition, the model is not entirely adequate for the real event of the binding of the oligonucleotide to the target receptor on the cell surface.

There is a method for evaluating affinity, based on a selector and a quantification system [Application RU 2014135576 A dated 01/30/2013, “Recognition based on a selector and a quantification system, as well as a method for the unified analysis of many analytes”]. The method uses the principle of separation of immune complexes using selective adsorption technologies on a column or cartridge.

The disadvantages of the method include the use of pre-prepared columns and cartridges necessary for separating the bound and unbound selector, which are often unavailable or require additional research to create them.

and J.R. Koke. Dynamics and Visualization of MCF7 Adenocarcinoma Cell Death by Aptamer-C1q-Mediated Membrane Attack. 2012 Nucleic Acid Ther; 22(4): 275-282] Этот способ выбран в качестве наиболее близкого к представленному изобретению. Способ осуществляют следующим образом.The most adequate model for assessing affinity in our opinion is the direct contact of the oligonucleotide with the target cell, which has a large number of target protein receptors on the outer membrane. This method for assessing the affinity of aptamers is described in the article “Dynamics and Visualization of MCF7 Adenocarcinoma Cell Death by Aptamer-C1q-Mediated Membrane Attack” [JR Stecker, AA Savage, JG Bruno, DM

and JR Koke. Dynamics and Visualization of MCF7 Adenocarcinoma Cell Death by Aptamer-C1q-Mediated Membrane Attack. 2012 Nucleic Acid Ther; 22 (4): 275-282] This method is selected as the closest to the present invention. The method is as follows.

The previously known deoxyribonucleotide aptamer MUC1-5TR-1 [Ferreira CS, Papamichael K, Guilbault G, Schwarzacher T, Gariepy J, Missailidis S. DNA aptamers against the MUC1 tumor marker: design of aptamer-antibody sandwich ELISA for the early diagnosis of epithelial tumors. Anal. Bioanal. Chem. 2008; 390: 1039-1050] was selected. Its target dissociation constant was 47.3 nM. The indicated oligonucleotide aptamer with a 5'-biotin or 5'-Alexa Fluor 546 (AF546) tag, nucleotide sequence: 5'-GGGAGACAAGAATAAACGCTCAAGAAGTGAAAATGACAGAACACAACATTCGACAGGAGGCTCACAACAGGC-3 was used for the study.

The studied aptamer was tagged along the flank of a 5'-biotin or 5'-Alexa Fluor 546 (AF546) with a label.

The target for the studied aptamer was the MUC1 marker, which is presented in large numbers on the surface of MCF7 cell lines. Cells of the MCF7 line of human breast cancer were grown on glass plates up to 80-90% coverage of the monolayer cup. The cells were washed twice with 1x binding buffer (1 × BB; 0.5 M NaCl, 10 mM Tris-HCl, and 1 mM MgCl ₂ , in nuclease-free water, pH 7.5-7.6), then 2.4 ml of fresh water was added to the cells filter-sterilized medium and 200 μl of the studied oligonucleotide (1.5 mg / ml) were incubated for 5 min at 37 ° C with 95% air mixture with the addition of 5% CO ₂ .

Cells were washed twice with 1 × BB for 5 min, and fixed in methanol at -20 ° C for 1 min. After drying, the cells were washed three times with 1 × BB, each wash for 10 min, incubated with 1:20 monoclonal mouse anti-human antibody C9 (United States Biological, Inc.) overnight at 4 ° C. Then the cells were washed three times with 1 × BB, each washing for 10 minutes, blocked with 20% milk powder for 2 hours at room temperature, after which they washed another three times with 1 × BB, each washing for 10 minutes. The cells were then incubated with an anti-mouse secondary antibody containing a Su5 tag (Invitrogen) for 2 hours at room temperature. Then the cells were washed three times again with 1 × BB, each washing for 10 min, incubated with 1: 2,000 Hoechst core dye for 20 min, washed again, the image was recorded using fluorescence microscopy.

The affinity of the aptamer of oligonucleotide nature with the target was assessed visually by looking at photographs of luminous cells.

However, the disadvantages of the method include the lack of quantitative characteristics of the visual assessment of the result. This greatly complicates the comparison of the studied aptamers by affinity. It is the solution of this problem that the invention is directed to.

The essence of the invention lies in the fact that the test oligonucleotide is used with covalently sewn fluorescent dye.

A cell culture is selected that contains a richly presented target for the oligonucleotide to be studied on the cell surface. As a control culture, a cell culture with a low representation of the target receptor on the outer surface of the cell membrane is selected.

The test oligonucleotide is incubated with positive cells containing a larger amount of the target protein receptor. At the same time, under the same conditions, the test sample of the oligonucleotide is incubated with a culture of control cells (not enriched in the target receptor).

Cell cultures are washed from unbound oligonucleotide. Fluorescence microscopy and photographing of cell samples (positive and control lines) are carried out.

The photograph of the control sample together with the image of the prototype are combined into one graphic raster file. Invert the image and measure the optical density of stained cells using a graphic file analysis program. The affinity of the oligonucleotide for the receptor is calculated by the formula:

where CO is the affinity of the oligonucleotide;

D ' _about - the optical density of the prototype minus the optical density of the background;

D ' _to - the optical density of the control sample minus the optical density of the background.

It should be noted that in analogues, the affinity of the oligonucleotide is estimated using the apparent dissociation constant (K _D ), and in the present method according to the formula [1]. Calculation of K _D is associated with the need to obtain accurate data on the current concentration of each component in the mixture. This complicates the task and does not help to achieve practical goals, namely, the assessment of the affinity of an oligonucleotide with a multicomponent living structure - a cell. Unlike the prototype, the claimed method uses a control cell culture and the proposed quantitative criterion. The conditions of exposure (incubation and washing) of the oligonucleotide to the cells were selected experimentally, under which there is a difference in the binding of control and experimental samples. The selection of incubation conditions included the use of cells immobilized on the surface of the well plates and in suspension.

The following examples disclose a specific implementation of the method described in the invention, but in no way limit the method itself.

Example 1

The TuTu22 oligonucleotide described in [D.-L. Wang, Y.-L. Songc, Z. Zhuc, X.-L. Lic, Y. Zouc, H.-T. Yanga, J.-J. Wanga, P.-S. Yaob, R.-J. Pana, C.J. Yangc, D.-Z. Kang, Selection of DNA aptamers against epidermal growth factor receptor with high affinity and specificity, BBRC, 453, 4, 681-685] to the epidermal growth factor receptor EGFR. Nucleotide sequence: 5'-FAM-TACCAGTGCGATGCTCAGTCCGTATGCTCAGTCTTACTATAACTTCTCTGCTCGTTGCTGACGCATTCGGTTGAC-3 '

This oligonucleotide was flanked with a covalently conjugated fluorescent dye fluorescein (FAM).

As an experimental cell culture, a standard A431 cell line (human kidney carcinoma cell culture) was used. The C6 line (rat cell line) was used as a control. Earlier, using real-time PCR, we showed a high level of EGFR protein expression (a 60-fold higher level of EGFR expression than in human fibroblast cells). In control C6 cells, zero expression of human EGFR was observed.

Cells were cultured under standard growing conditions, the number of cells was 3 × 10 ⁶ cells per well of a standard four-well plate. The experimental and control cell cultures were grown in the wells of the same tablet.

A solution of aptamer with a concentration of 100 nM was prepared in a mixture of 50% phosphate buffer solution and 50% serum-free cell culture medium. The culture medium for the analyzed cell lines was replaced with 200 μl of aptamer solution. The test aptamer was incubated with experimental and control cell cultures for 60 min with gentle stirring on a thermostated shaker at a temperature of 37 ° C. All manipulations were performed under darkening conditions to prevent “fading” of the aptamer fluorescent label.

The initial washing was carried out twice with a solution of phosphate buffer: the aptamer solution was previously removed and 1000 μl of buffer was added to the cells. After washing twice with buffer, the solution was poured out, 1000 μl of serum-free incubation medium was added, the cells were kept for 10 min with gentle stirring on a thermostated shaker at a temperature of 37 ° С.

After washing, the medium was removed, 200 μl of fresh incubation medium was poured onto the cells, and a four-well plate was placed on a slide of a fluorescence microscope.

In order to make sure that the staining zones coincide with the outlines of the cells, the observation zone was visualized in visible light. Photoregistration was performed using an Olympus IX fluorescence microscope.

The experimental and control cells were visualized under the same conditions of photographic registration.

Photographs of the prototype and control samples were combined into one graphic file for synchronization of image processing and analysis.

The Figure 1 shows the color (A) and black and white (B) image on the left side of which is the culture of C6, on the right - A431.

Using PhotoM 1.31 software, the obtained image of the cells of the experimental and control samples was inverted and the optical densities of the image areas corresponding to the fluorescently-stained target oligonucleotide of the target cells in the experimental and control samples were determined and the optical density of the areas outside the cells for the experimental and control image was subtracted. To increase the accuracy of the assessment, the characteristics were measured in five different zones, followed by averaging of the results.

The specified software allows you to subtract the background optical density. Figure 2 presents a screenshot (image) of the program PhotoM 1.31 demonstrating the above property for cultures C6 and A431 shown in Figure 1.

The following values were obtained:

For C6, the optical density in the selected areas:

1.1942 px (0.07%). Density 0.0137.

2.22127 px (0.08%). The density is 0.00913.

3.2081 px (0.07%). Density is 0.00508.

4.2092 px (0.08%). Density is 0.00426.

5.2331 px (0.08%). Density is 0.00794.

Property average 0.00796

For A431, the optical density in the selected areas:

1.984 px (0.04%). The density is 0.00678.

2.743 pixels (0.03%). The density is 0.00615.

3. 627 pixels (0.02%). The density is 0.0104.

4.775 pixels (0.03%). The density is 0.0104.

5.914 px (0.03%). The density is 0.00548.

The average for an object is 0.00799.

D ' _o = 0.00799;

D ' _k = 0.00796.

When substituting the obtained values of optical density in the formula [1]

got the affinity of the oligonucleotide CO = (0.00799-0.00796) / (0.00799) ⋅100% = 0.4%

Based on the result, we conclude that the tested aptamer does not have a pronounced affinity for the selected target.

Example 2

Using the inventive method, the affinity of the GREF18 oligonucleotide labeled with Cy3 for PEC-6 cells was evaluated. The control cell culture served as a line of cells A431. The method was carried out analogously to example 1.

The Figure 3 shows the photoregistration of stained cells in color (A) and black and white (B), where on the left side of the image is an A431 culture, on the right is a PEC6. Figure 4 presents a screenshot (image) of the PhotoM 1.31 program with the data on the cultures of A4631 and PEC6 shown in Figure 2 transferred to it.

The following values were obtained:

For A431, the optical density in the selected areas:

1.222426 pixels (0.80%). Density is 0.0222.

10 10714 px (0.38%). Density 0.0228.

3.14440 px (0.52%). Density 0.0288.

4. 16055 pixels (0.58%). The density is 0.0104.

5.19979 pixels (0.36%). The density is 0.0125.

The average for the facility is 0.0197.

For PEC6 optical density in the selected areas:

1.11617 px (0.42%). Density is 0.186.

2.9528 pixels (0.34%). Density 0.0701.

3. 9729 pixels (0.35%). Density 0.203.

4. 6376 pixels (0.23%). Density 0.128.

5.7629 px (0.27%). Density 0.154.

The average for an object is 0.151.

D ' _o = 0.151;

D ' _k = 0.020.

When substituting the obtained values of optical density in the formula [1]

obtained the affinity of the oligonucleotide CO = (0.151-0.020) / (0.151) ⋅100% = 87%

We conclude that the oligonucleotide under test has a strong affinity for PEC6 cell culture.

The invention allows the comparison of oligonucleotides in affinity for cells whose surface is enriched with target target molecules.

The invention introduces quantitative criteria for the selection of the highest affinity oligonucleotides for target molecules on the cell surface. On the other hand, the invention can be applied for marking cells and tissues of a certain type, for example, with the growth of a cancerous tumor.

Claims (5)

A method for assessing the affinity of an oligonucleotide to a target, including labeling the oligonucleotide with a fluorochrome, contacting a labeled oligonucleotide with a target receptor on the cell surface, photorecording the result using a fluorescence microscope, characterized in that the optical density of the stained area of the inverted images is measured, compared with a negative control, and the calculation is performed with a negative control carried out by the formula:

where CO is the affinity of the oligonucleotide; D ' _about - the optical density of the prototype minus the optical density of the background; D ' _to - the optical density of the control sample minus the optical density of the background.