WO2016053015A1 - Method for preparing aptamers targeting new cells - Google Patents

Abstract

The present invention relates to a method for preparing aptamers binding to new cells and, specifically, to a method for preparing aptamers, comprising a step of eluting aptamers bound to cells by culturing cells in a chip comprising a single or a plurality of chambers, and treating the same with an elution buffer. Cell SELEX-on-a-chip has been developed by applying a microfluid for eluting specific aptamers without damaging cells. It is shown that SELEX can be effectively carried out in a SELEX-on-a-chip, compared with a chip SELEX and a conventional SELEX. It is shown that the method, which has excellent performance, can be applied to aptamers specific to various cancer cells.

Description

New cell target aptamer production method

The present invention relates to a method for producing aptamer binding to a novel cell, specifically comprising culturing cells in a chip including a single or a plurality of chambers, and eluting the aptamer bound to the cells by treatment with an elution buffer. It relates to an aptamer manufacturing method.

Cell SELEX-on-a-chip was developed by applying microfluid to elute specific aptamer without damaging cells. Comparing Chip SELEX and conventional SELEX shows that SELEX can be efficiently performed on SELEX on SELEX-on-a-chip. It can be used to apply various cancer cell specific aptamers with excellent performance.

With the development of drugs, many diseases are treatable, but there are still many diseases to be treated. Above all, cancer is a representative disease that is difficult to treat completely. Most treatments require a combination of surgery and chemotherapy, but because it affects cells throughout the body, it is difficult to say that there is an appropriate treatment.

Recently, aptamers are in the spotlight in the treatment and diagnosis of cancer. Aptamers are single stranded oligonucleotides that form specific three dimensional formation. Allows aptamers to have high affinity and specificity for the target molecule. Aptamers have similar properties to antibodies but have a lower molecular weight, making them easier to synthesize in vitro and more stable at higher temperatures. In addition, the toxicity and antigenicity is lower. Aptamers can be isolated by in vitro evolution processes such as SELEX (systematic evolution of ligands by exponential enrichment). Target cells from 10 up ^{to 14 -} 10 ¹⁵ random incubated with the ssDNA or RNA library comprising a nucleotide, then by washing the unbound sequence collect only the oligonucleotide bound to the cells.

A disadvantage of the conventional cell SELEX method is that the cells may be damaged in the elution step, which is caused by the fact that by-products from the cells can be obtained with bound aptamers because the cells are peeled off the plate.

Under this technical background, the inventors of the present application have developed a novel cell SELEX method capable of eluting aptamers without damaging the cells and completed the present invention.

The above information described in this Background section is only for improving the understanding of the background of the present invention, and therefore does not include information that forms a prior art known to those of ordinary skill in the art. You may not.

Summary of the Invention

A method is characterized by separating aptamers bound to cells without damaging the cells. Chip SELEX is economical, effective and can perform faster. Since the chip has a small volume, it is possible to culture many different cells without contamination.

The present invention provides a method for producing aptamer that binds to a cell comprising the following steps: culturing the cell in a chip comprising a single or a plurality of chambers; Binding aptamer specific to the cell; Washing the cells to which the aptamer is bound; And eluting the aptamer bound to the cells by treatment with an elution buffer.

1 shows the shape of a chip according to the invention.

Figure 2 shows a schematic diagram of a conventional SELEX and a chip SELEX according to the present invention.

FIG. 3 shows the incubation of Cy3 labeled aptamers with cells, (A): BNL 1ME A. 7R. Optical image of 1 cell, (B) BNL 1ME A. 7R after incubation with aptamer. Fluorescence image of 1 cell. Bound aptamers are eluted with the elution buffer and (C): BNL 1ME A. 7R. Optical image of 1 cell, (D) BNL 1ME A. 7R after incubation with elution buffer. An optical image of 1 cell is shown.

4 is a graph showing normalized data of elution amount in a conventional SELEX and a chip SELEX according to the present invention.

Detailed description of the invention and preferred Embodiment

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In general, the nomenclature used herein is well known and commonly used in the art.

In one aspect, the present invention relates to a method for producing aptamer that binds to a cell comprising the following steps: culturing the cell in a chip comprising a single or a plurality of chambers; Binding aptamer specific to the cell; Washing the cells to which the aptamer is bound; And eluting the aptamer bound to the cells by treatment with an elution buffer. The method can separate aptamers bound to cells without damaging the cells.

Biomarkers, such as antibodies, aptamers or peptides, can be used to diagnose a disease or to detect certain metabolites. As used herein, "aptamer" refers to a small single-stranded oligonucleotide that can specifically recognize a target with high affinity. Aptamers can be obtained by a specific method called SELEX (Systematic Evolution of Ligands by Exponential enrichment). In contrast to the target purified protein, Cell SELEX is used to select aptamers that target cell specific cells. Cell SELEX may be a better method because it can screen for aptamers that bind in vivo. However, the conventionally used Cell SELEX has a large background problem, and in particular, by removing the whole cell instead of eluting the aptamer, a long round of SELEX was caused to obtain the aptamer. Thus, Cell SELEX-on-a-chip was developed by applying a microfluid to elute specific aptamers without interfering with the cells. Comparing Chip SELEX and conventional SELEX shows that SELEX can be efficiently performed on SELEX on SELEX-on-a-chip. It can be used to apply various cancer cell specific aptamers with excellent performance.

Many diseases are being treated, but cancer and HIV disease still remain in need of treatment. While considerable research is underway on initial diagnosis, identification of treatment targets and identification of biomarkers for drug design, conventional research methods are inefficient and time consuming. The present invention has developed a method for efficiently improving conventional cell SELEX and cell SELEX-on-a-chip. Compared to conventional methods requiring large amounts of reagents and instruments, Ibidi® chips can run with much smaller volumes of reagents and cells. Moreover, through solution-based elution, contamination of DNA with cell debris prepared from the scraping method can be avoided. In terms of the efficiency of the new system, it is more than six times more efficient than the conventional Cell-SELEX method and can be used more efficiently to generate aptamers for markers such as cancer biomarkers.

Unlike the SELEX method for purified protein, “Cell SELEX” herein refers to a method for selecting aptamers having the highest binding ability to proteins or structures on the cell surface by performing SELEX on all living cells. Currently, the development of aptamers for most cell surface receptors is accomplished using proteins purified by overexpression in E. coli. The use of SELEX for live cells is because membrane proteins play an indispensable role in life, especially for cell signaling, growth and proliferation, and cell-cell interactions. In cancer cells, cancerous mutations are caused by variations in the function of cancer receptors that affect expression levels or cancer progression. Purified proteins (recombinant proteins) contain the same genetic information as the membrane proteins on the cell surface, but are modified or physiologically masked. Under these circumstances, aptamers may not be able to recognize unmodified prototype proteins on the cell surface.

However, in order to use aptamer as a therapeutic agent or as an in vivo diagnostic marker, it is necessary to be able to recognize the prototype of the protein in vivo that has undergone post-translational modification. However, when simply performing cell-SELEX, the types of receptors and surface proteins located on the cell surface are so complex that it is difficult to select aptamers that bind to the desired target receptor proteins. The most difficult thing in Cell-SELEX is aptamer selection, which only binds to target positive cell lines. Since the cell surface has one or more receptors, the diversity of membrane proteins makes it difficult to screen for aptamers that target cancer proteins, and the selection takes about 10 rounds or more. Positive and negative cell lines share a specific group of membrane proteins, which means that there is a high likelihood of choosing aptamers that can bind to both cell lines. In order to screen for aptamers with high affinity and specificity for positive cell lines, cell concentration and number of passages must be carefully controlled.

Indeed, recently, there are examples of using cell-SELEX as a biodiagnosis by incorporating fluorescent materials for diagnosis or treatment of tumors or using nanoparticles for drug delivery. In the past, cancer diagnosis was judged by cell shape or tissue change, but there was an example of using aptamer, which selectively binds only Burkitt's lymphoma cells, with Cell-SELEX to diagnose cancer. In addition, they combined aptamers, nanoparticles, and control-release polymer systems to provide a platform for the selective treatment of cancer cells.

In the present invention, a novel cellular SELEX method has been developed that can elute aptamers without cell damage, and higher amounts of aptamers can be obtained. For efficiency evaluation, the murine liver cancer cells BNL 1ME A.7R.1 cell line was used, which was obtained by transforming BNL CL.2 with methylcholanthrene epoxide, in which the TLS11 aptamer was converted to BNL 1ME A. Targeting R7 cells is known. Using these cell lines and aptamers, experiments were carried out on conventional and novel methods using 100 mm and chips. The chip contains a smaller growth area than the 100 mm plate used in the prior art, so a smaller volume of reagent is sufficient. Instead of scraping the cells, an elution buffer was used to obtain bound aptamers (FIG. 2). This makes it possible to separate bound aptamers from cells without cell damage and separation. This method is expected to produce improved results in various aspects.

Definitions of main terms used in the detailed description of the present invention are as follows.

As used herein, "nucleic acid" refers to single- or double-stranded DNA, RNA and chemical modifications thereof, which modifications do not interfere with the amplification of selected nucleic acids, but are not limited to, but are not limited to, backbone. Modification, methylation, biphasic combination, substitution of 5-bromo-uracil, and the like.

As used herein, “microfluidic device” refers to a device capable of controlling or manipulating the flow of microscopic microscopic solutions, such as μl, nL, pL, fL, and the like.

In one aspect, the present invention provides an aptamer binding to a cell using a multiplex microfluidic device for nucleic acid aptamer selection that can be applied to an improved SELEX (Systematic Evolution of Ligands by Exponential Enrichment) process as shown in FIG. 1. It is about how to.

The multiplex microfluidic device used in the present invention has a great advantage over macro-scale cultures such as flasks, dishes and well-plates. First, the flexibility in the design of the microfluidic device is great, which allows individual cell types to be tailored for the purpose and co-culture on the same chip. Moreover, under these microfluidic cell culture conditions, it is possible to mimic the natural microenvironment of the cells. This allows for continuous perfusion cultures and through direct connection to the construction and automation of chemical gradients, parallelization, on-chip analysis or downstream analytical chemical platforms. It allows to study a small number of cells or single cells at high lateral and / or spatial resolution. At the same time, microfluidic cell culture allows for reduced consumption of reagents, reduced risk of contamination and efficient high throughput experimentation. Overall, the advantages of flexible design, single cell analysis, real-time on-chip analysis and low reagent consumption through time-lapse microscopy make microfluidic cell cultures more attractive for cell-SELEX applications. Make it suitable.

The chip herein may be a single or a plurality of chambers, for example μ-slide (ibidi®), the shape of Figure 1 having a 0.6-2.5 cm ² per channel growth area and 30-100ul channel volume May be a chip. However, the single chamber may have a problem that the cells fall off a lot in the washing step, a plurality of chambers is preferable.

In one embodiment, the chamber may comprise 1 × 10 ⁵ ˜9 × 10 ⁵ cells. As confirmed in the Example, the existing SELEX 5 × 10 ⁶ cells were used, in the Chip SELEX 3 × 10 ⁵ cells were used. The chamber of the chip is small in volume, so the amount of cells required is small. About 10 ⁵ cells per chamber is sufficient.

In addition, the cell culture may be performed at 37 ℃. As confirmed in the Example, when the cell culture at 37 ℃ small number of cells to be isolated was suitable. For example, the elution buffer may be at least one selected from the group consisting of MgCl ₂ , NaCl, NaOH, and HCl, and preferably MgCl ₂ . In this case, the concentration of the buffer may be, for example, 5 mM to 5M, preferably 100 mM to 3M, more preferably 500 mM. In addition, the elution buffer may be treated for 10 to 30 minutes, as confirmed in the embodiment, 30 minutes is the highest aptamer obtained concentration is preferred.

Hereinafter, the present invention will be described in more detail by the following examples. These examples are only for illustrating the present invention in more detail, it will be apparent to those skilled in the art that the scope of the present invention is not limited to these examples.

Example 1

1-1. Cell culture

BNL.1.ME.7R.1 cells were purchased from the American Tissue Culture Collection (ATCC TIB-73), which is a murine hepatocellular carcinoma cell and transforms BNL CL.2 with methylcholanthrene epoxide. Induced.

Medium was used DMEM containing 10% serum and incubated in 37 ℃ 5% CO ₂ incubator. To find out if the amount of seeding in the chip is suitable for the experiment, we looked for a point where the amount of cells doubled. As a result, it was observed that 4 times the amount of existing cells after 1 day and 4 times after 2 days. At this time, the seeding number was 1 × 10 ⁵ , 2 × 10 ⁵ , 4 × 10 ⁴ / 100mm.

1-2. Setup for cell SELEX on-a-chip

To set up the optimal experimental conditions for the new cell SELEX method using μ-slide, the following tests were run:

First, a temperature test was performed to determine the conditions suitable for binding. In general, the cell SELEX binding is 4 ℃, if too many cells are separated at 4 ℃ is carried out at 37 ℃. Both conditions were tested. About 30-40% of the cells were separated from the chip surface at 4 ° C and only about 20% of the cells were separated at 37 ° C.

Second, four DNA elution buffers were tested at different concentrations based on HCl, NaOH, MgCl ₂ or NaCl (HCl: 5 mM, 10 mM, 25 mM, 75 mM, 100 mM, 200 mM, 300 mM, 400). NaOH: 5 mM, 10 mM, 25 mM, 75 mM, 100 mM; NaCl: 50 mM, 100 mM, 500 mM, 1 M, 2 M, 3 M, 4 M; MgCl ₂ : 100 mM, 500 mM , 1 M, 2 M, 3 M, 4 M, 5 M).

After checking the performance of the other buffers to elute DNA in the cells, the best candidates were obtained: HCl: 200 mM, 300 mM, 400 mM; NaCl: 2 M, 3 M; MgCl ₂ : 500 mM, 1 M. Finally, 500 mM MgCl ₂ was selected as the elution buffer. HCl has a problem that the acid is too strong in the cell, NaCl concentration is too high than the concentration of MgCl ₂ and showed similar performance for DNA elution.

Third, the time required to elute DNA without cell damage was determined. After binding, cells were incubated with the elution buffer for 10, 20, or 30 minutes. Thereafter, the eluted DNA was quantified by RT-PCR. 3.34 × 10 ⁶ , 9 × 10 ⁷ , and 2 × 10 ⁸ cells were obtained for 10, 20 and 30 minute elution times, respectively. Through this, it was confirmed that 30 minutes is the most appropriate elution time.

In summary, in the elution buffer used for Chip SELEX, MgCl ₂ , NaOH, NaCl, HCl, etc. were tested by concentration, but 500 mM MgCl ₂ was selected because it can be used in experiments without damaging the cells. .

In addition, when testing the cell SELEX binding temperature conditions, 37 ℃ was the most suitable.

In order to find the optimal aptamer binding and elution time, 30 minutes was the most suitable when quantified using Q-PCR with different elution times after binding.

1-3. Cell selex

400 pmole of SELEX 5'-Cy3-labeled TLS11 aptamer (5'-cy3-ACGCTCGGATGCCACTACAGGCATCCCCATGTGAACAATCGCATTGTGATTGTTACGGTTTCCGCCTCATGGACGTGCTGGTGAC, Bioneer: SEQ ID NO: 1) was prepared. It has already been demonstrated that TLS11 aptamers target BNL 1ME A.7R.1 cells. Boil 5 minutes at 95 ℃ and cooled on ice for 3 minutes to form a secondary structure. 5 × 10 ⁶ cells were washed twice with 5 ml of wash buffer [5 mM MgCl ₂ , 4.5 g glucose / L of DPBS], and then 5 ml binding buffer [4.5 g / L glucose, 5 mM MgCl containing 400 pmole of TLS11 aptamer] ₂ , 0.1 mg / ml yeast tRNA, and 1 mg / mL bovine serum albumin (BSA) in DPBS] were added to the plate and bound for 30 min in a 37 ° C, 5% CO ₂ incubator. After 30 minutes, the supernatant was removed and washed twice with 5 ml of wash buffer. The cells of the plate were then collected by scraping with wash buffer and centrifuged at 1000 rpm for 3 minutes. After that, the supernatant was removed and the pellet was freed with wash buffer.

Heat was applied at 95 ° C. for 5 minutes to remove the aptamer from the cells. Then return the sentry for 5 minutes at 13200 rpm. At this time, only the supernatant was obtained and PCI phenol: chloroform: isoamyl alchohol (PCI, sigma) was subjected to the process and ethanol.

1-4. Chip cell The SELEX μ-slide chip contains six chambers. Since 3 pmole of aptamer (cy3-labeled TLS11 aptamer) is required for each chamber, a total of 18 pmole of aptamer was prepared. Boil 5 minutes at 95 ℃ and cooled on ice for 3 minutes to form a secondary structure. Each chamber contains 5 × 10 ⁴ cells and was pre-washed twice with 30 μl wash buffer. Then, binding was performed by incubating at 37 ° C. for 30 min with a binding buffer in which aptamer (cy3-labeled TLS11 aptamer) was dissolved. At the end of the incubation, unbound aptamers were washed twice with 30 μl wash buffer and the supernatant collected in another tube. At this time, it was checked whether the aptamer is bound to each cell by fluorescence microscope.

30 μl of the elution buffer [500 mM MgCl ₂ ] was added to remove the aptamer from the cells and allowed to stand at room temperature for 30 minutes. The cells were then washed twice with 30 μl of elution buffer, and the chip was confirmed by fluorescence microscopy to determine whether the aptamer was well separated from the cells. The aptamer obtained through the elution process was obtained by PCI phenol: chloroform: isoamyl alchohol (PCI, sigma) and prepared with ethanol.

1-5. Realtime-PCR for quantification.

To proceed with real-time PCR (RT-PCR), 20 μl of the mixture was made using Power SYBR Green PCR Mastermix (Applied Biosystems). 7 μL HPLC water, 10 μL 2x SYBR, 1 μL forward primer (ACG CTC GGA TGC CAC TAC AG, Bioneer: SEQ ID NO: 2) and reverse primer (GTC ACC AGC ACG TCC ATG AG, Bioneer: SEQ ID NO: 3), 1 μL Template aptamer.

The heating cycling protocol is as follows. Denaturation at 95 ℃ for 10 minutes, 95 ℃ 30 seconds → 52 ℃ 1 minutes → 72 ℃ 1 minutes This process was repeated 40 cycle. After amplification, the PCR product was liver inactivated at 95 ° C. for 15 seconds. The melting curve was analyzed from 52 ° C. to 95 ° C. with a temperature gradient of 0.3 ° C./s. Proceed to confirm that only a specific product will be amplified.

Results and analysis

Aptamers were successfully elution without the Chip detaching from the surface (FIG. 3). 3A and 3C, it was confirmed by optical microscopy that the majority of cells were attached to the surface of the chip after elution.

In addition, the cells were observed by fluorescence microscopy after binding, and the fluorescence signal was detected, thereby demonstrating that the aptamer labeled with cy-3 was attached to the surface of the cells. However, after elution the fluorescence signal was greatly reduced, thus confirming that the aptamer labeled cy-3 was successfully desorbed from the surface of the cells (FIGS. 3B, 3D).

The difference in efficiency between the chip cell SELEX and the existing cell SELEX was measured by quantitative analysis using RT-PCR. As shown in Table 1, 18 pmole (1.08 × 10 ¹³ ) aptamer was used for the Chip SELEX, and 1.11 × 10 ¹¹ aptamer was eluted. As a conventional SELEX process, 2.4 × 10 ¹⁴ aptamers were used to elute 4.07 × 10 ¹¹ aptamers. Therefore, it was confirmed that the chip selex according to the present invention used an aptamer about 22 times less. Regarding the aptamer extraction rate, the chip SELEX according to the present invention was proved more efficient than the conventional SELEX method.

Conventional cell SELEX and Chip Cell SELEX used different amounts of aptamer and normalized for more accurate calculation (FIG. 4). After normalization, it was confirmed that the Chip Cell SELEX according to the present invention obtained about 6.07 times more elute than the existing Cell SELEX method, which proved that the Chip Cell SELEX method according to the present invention is more efficient than the existing method.

In experiments demonstrating the effectiveness of the new SELEX using Chip, 5 × 10 ⁶ cells were used, 40 pmol of aptamer was used, and a total of 30.0 ml of buffer was used. Chip SELEX, on the other hand, used 3 × 10 ⁵ cells, 10 pmol of aptamer, and 1.44 ml of buffer. Comparing the materials used in the experiments, Chip SELEX required 16 times fewer cells, 22 times less aptamers, and 21 times less buffer than conventional methods.

Traditional methods require flasks or plates, which require a very large amount of cells, aptamers, and buffers. In addition, cells must be scraped off the plate, causing damage and bursting. However, with the Chip SELEX technology according to the present invention, the aptamer can be obtained without using the elution buffer in the elution step without affecting the cells and by-products from the dead cells.

As a result, the Chip SELEX according to the present invention is economical, effective, and can be performed faster. Since the chip has a small volume, it is possible to culture various different cells without contamination. It may be preferred to culture hard to obtain cells or small amounts of cells.

In addition, the Chip SELEX according to the present invention can be used for multiplex selection. Since the chip contains six chambers, it is possible to culture other cell lines without contamination. The chamber of the chip is small in volume, so the amount of cells required is small. About 10 ⁵ cells per chamber is sufficient.

The specific parts of the present invention have been described in detail above, and it is apparent to those skilled in the art that such specific descriptions are merely preferred embodiments, and thus the scope of the present invention is not limited thereto. something to do. Thus, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

According to the present invention, instead of scraping and separating the cells, only the bound aptamer is separated using the elution buffer, so that the aptamer bound to the cells can be separated without damaging the cells.

Electronic file attached.

Claims (7)

Method for producing aptamer binding to a cell comprising the following steps: Culturing the cells in a chip comprising a single or a plurality of chambers; Binding aptamer specific to the cell; Washing the cells to which the aptamer is bound; And Eluting the aptamer bound to the cells by treatment with an elution buffer. The method of claim 1, The chamber is an aptamer manufacturing method comprising 1 × 10 ⁵ ~ 9 × 10 ⁵ cells. The method of claim 1, The cell culture is aptamer production method characterized in that carried out at 37 ℃. The method of claim 1, The elution buffer is aptamer manufacturing method characterized in that it comprises one or more selected from the group consisting of MgCl ₂ , NaCl, NaOH and HCl. The method of claim 1, The elution buffer is aptamer production method characterized in that the MgCl ₂ . The method of claim 5, The elution buffer is aptamer manufacturing method characterized in that the concentration of 5mM-5M. The method of claim 1, The elution buffer is aptamer manufacturing method characterized in that the treatment for 10 to 30 minutes.

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