WO2019093503A1

WO2019093503A1 - Aptamer and use thereof

Info

Publication number: WO2019093503A1
Application number: PCT/JP2018/041759
Authority: WO
Inventors: 信介山東; 亮介植木; 彩香植木; 早紀熱田
Original assignee: 国立大学法人東京大学
Priority date: 2017-11-11
Filing date: 2018-11-09
Publication date: 2019-05-16
Also published as: JPWO2019093503A1; JP6970458B2

Abstract

Provided is an aptamer comprising a polynucleotide that includes the base sequence described in SEQ ID NO: 1, and that exhibits activity for binding to the fibroblast growth factor receptor 1 (FGFR1).

Description

Aptamers and uses thereof

The present invention relates to aptamers and their use. More specifically, the present invention relates to an aptamer, a preventive or therapeutic agent for a Fibroblast Growth Factor Receptor 1 (FGFR1) signaling related disease, a composition for cell culture, a cell culture method, and a combination of Fibroblast Growth Factor 2 (bFGF) and FGFR1. The present invention relates to a method of inhibition, a method of suppressing cell growth, a method of activating FGFR1, a method of promoting cell growth, and a method of maintaining pluripotency of pluripotent stem cells. The present application claims priority based on US Patent No. 62 / 584,755 filed on Nov. 11, 2017 provisionally filed in the United States, the contents of which are incorporated herein by reference.

In receptor signaling, when a cell receives a ligand at a receptor on a cell membrane, it is known that a signal is transmitted into the cell to control cell differentiation, proliferation and the like. By receiving signal molecules, cells can detect the surrounding environment and conditions.

Fibroblast growth factors (FGFs) are one of the growth factors that activate tyrosine kinase type receptors and play an important role in cell proliferation and cell differentiation (for example, non-patent literature) See 1). Among them, bFGF is one of the important factors added to the culture medium to culture pluripotent stem cells such as induced pluripotent stem cells (iPS cells) in the state where their pluripotency and self-replication ability are maintained. It is.

However, production of bFGF requires the use of an expression system using animal cells or microorganisms. For this reason, bFGF is expensive. Moreover, the quality control of the recombinant protein is complicated. Therefore, there is a need for the development of synthetic substitutes that can mimic the function of bFGF. Under such background, the present invention aims to provide a technique for mimicking the activity of bFGF with an aptamer.

The present invention includes the following aspects.
[1] An aptamer comprising a polynucleotide comprising the nucleotide sequence set forth in SEQ ID NO: 1 and having an activity of binding to Fibroblast Growth Factor Receptor 1 (FGFR1).
[2] The aptamer according to [1], which has a loop structure in which a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 1 forms at least a part.
[3] The aptamer according to [2], wherein the loop structure is composed of a polynucleotide strand having 28 to 40 bases.
[4] The aptamer according to [2] or [3], which has a stem structure composed of a double stranded polynucleotide linked to the loop structure.
[5] The aptamer according to any one of [1] to [4], which forms a guanine quadruplex structure.
[6] An aptamer having an activity of binding to FGFR1 and activating FGFR1 to which two or more of the aptamers described in any of [1] to [5] are linked.
[7] The aptamer according to [6], wherein the aptamer according to any one of [1] to [5] is linked by a linker, and the length of the linker is 80 bases or less in length in terms of polynucleotide.
[8] The nucleotide sequence set forth in SEQ ID NO: 11 or the nucleotide sequence set forth in SEQ ID NO: 11 consisting of a nucleotide sequence in which one or several bases have been deleted, substituted or added and binding to FGFR1 to activate FGFR1 The aptamer according to [6] or [7], which has an activity of
[9] A preventive or therapeutic agent for an FGFR1 signaling related disease, which comprises the aptamer according to any one of [1] to [8] as an active ingredient.
[10] A composition for cell culture, comprising the aptamer according to any one of [1] to [8] as an active ingredient.
[11] A cell culture method comprising culturing an FGFR1 positive cell in a medium containing the aptamer according to any one of [1] to [8].
[12] A method for inhibiting the binding of bFGF to the FGFR1 comprising contacting the aptamer according to any one of [1] to [5] with an FGFR1 positive cell.
[13] A method for suppressing the growth of cells, which comprises bringing the aptamer according to any one of [1] to [5] into contact with FGFR1 positive cells.
[14] The method for activating FGFR1, which comprises contacting the aptamer according to any one of [6] to [8] with an FGFR1 positive cell.
[15] A method for promoting the growth of cells, comprising contacting the aptamer according to any one of [6] to [8] with FGFR1 positive cells.
[16] A method for maintaining pluripotency of the pluripotent stem cells, comprising contacting the aptamer according to any one of [6] to [8] with an FGFR1 positive pluripotent stem cell.

According to the present invention, it is possible to provide a technique for mimicking the activity of bFGF with an aptamer.

It is a schematic diagram which shows the example of an aptamer. It is a schematic diagram which shows the example of a multi-structure aptamer. It is a schematic diagram which shows the example of a multi-structure aptamer. It is a schematic diagram which shows the example of a multi-structure aptamer. It is a schematic diagram which shows the example of a multi-structure aptamer. (A) and (b) is a schematic diagram which shows an example of the aptamer whose linker is double stranded DNA. It is a schematic diagram which shows an example of the multi-structure aptamer containing several linker. (A) and (b) are schematics showing an example of an aptamer fixed carrier. In Experimental example 2, it is a schematic diagram which shows the result of having analyzed the three-dimensional structure of the aptamer. In Experimental Example 2, it is a graph which shows the result of having analyzed binding with an aptamer and FGFR1-Fc bead. It is a photograph which shows the result of example 3 of an experiment. (A) is a graph which shows the result of the flow cytometry analysis in example 4 of an experiment. (B) is a graph which shows the result of SPR analysis in example 4 of an experiment. 15 is a graph showing the results of flow cytometry in Experimental Example 5. (A) And (b) is a graph which shows the result of CD spectrum measurement in example 6 of an experiment. It is a graph which shows the result of CD spectrum measurement in example 7 of an experiment. (A) And (b) is a graph which shows the result of the flow cytometry analysis in Experimental example 8. It is a graph which shows the result of CD spectrum measurement in example 9 of an experiment. (A) is a figure which shows the result of having predicted the structure of an aptamer in Experimental example 10. (B) and (c) is a photograph which shows the result of having analyzed the activation of FGFR1 in example 10 of an experiment. FIG. 16 is a photograph showing the results of analysis of FGFR1 activation in Experimental Example 11. FIG. (A) is a graph showing the results of quantitative RT-PCR (RT-qPCR) analysis of Oct-4 and Nanog expression levels in Experimental Example 12. (B) is a graph which shows the result of having measured cell proliferation in Experimental example 12. It is a photograph which shows the result of the fluorescence immunostaining in Experimental example 13. (A) and (b) is a schematic diagram which shows the structure of the aptamer produced in Experimental example 14. FIG. (A) is a graph which shows the result of having measured phosphorylation of FGFR1 in example 15 of an experiment. (B) is a photograph which shows the result of having measured phosphorylation of Erk1 / 2 in example 15 of an experiment. (A) is a graph which shows the result of having analyzed expression of SSEA-4 protein in example 16 of an experiment. (B) is the graph which quantified the result of (a).

[Aptamer]
In one embodiment, the present invention provides an aptamer comprising a nucleotide sequence set forth in SEQ ID NO: 1 and consisting of a polynucleotide having an activity of binding to FGFR1.

The aptamer is a molecule capable of binding to a target molecule, and an aptamer comprising a nucleic acid or a peptide is known. The aptamer of the present embodiment is a nucleic acid aptamer that binds to FGFR1. The aptamer of the present embodiment preferably binds to FGFR1 to inhibit the binding of bFGF to FGFR1.

The human FGFR1 protein has multiple isoforms, and the NCBI accession numbers are NP_056934.2, NP_001167534.1, NP_001341297.1, NP_001167537.1, NP_001341299.1 and the like. As FGFR1 to which the aptamer of this embodiment binds, FGFR1 whose NCBI accession number is NP_056934.2 is preferable among them.

In addition, multiple isoforms also exist for human bFGF protein, and the NCBI accession numbers are, for example, NP — 00134854.1, NP — 0019937.5, and the like. Among the bFGFs for which the aptamer of the present embodiment inhibits the binding to FGFR1, bFGF in which the NCBI accession number is NP — 010034854.1 is particularly preferable.

In the present specification, the nucleic acid may be a natural nucleic acid such as DNA or RNA, or may be an artificial nucleic acid such as LNA (locked nucleic acid) or BNA (bridged nucleic acid), and has a function equivalent to a nucleic acid A nucleic acid analog represented by a peptide nucleic acid such as PNA (Peptide Nucleic Acid) may be used as long as it has the The nucleic acid which comprises an aptamer can combine multiple types of nucleic acids, such as a combination of DNA and LNA.

The FGF receptor is a protein capable of binding to FGF, and one of the receptor tyrosine kinases FGFR1 to 4 is known as an FGF receptor. Receptor-type tyrosine kinases are known as transmembrane proteins, and have a ligand binding domain on the cell outer side and an intracellular domain on the cytoplasmic side. The ligand binding domain is capable of binding a ligand. The intracellular domain has a kinase activity.

Receptor-type tyrosine kinases are known to form dimers upon ligand binding. Once the dimer is formed, the tyrosine residues of the intracellular domain of the dimer are phosphorylated on one another. The FGFR1 to which the aptamer of the present embodiment binds may be a monomer or a complex of dimer or more.

FIG. 1 shows a schematic view of an aptamer according to one embodiment. The aptamer 1 is a polynucleotide 10 consisting of a polynucleotide and containing the base sequence shown in SEQ ID NO: 1.

As described later in the Examples, the inventors screened for an aptamer having the ability to bind to FGFR1. As a result, it was found that a polynucleotide containing the nucleotide sequence set forth in SEQ ID NO: 1 has an ability to bind to FGFR1. The nucleotide sequence set forth in SEQ ID NO: 1 is a consensus sequence of several tens of aptamers capable of binding to FGFR1 obtained by screening.

In the nucleotide sequence set forth in SEQ ID NO: 1 (5′-kywtgghwkdggatggyrkggkyt-3 ′), k represents g (guanine) or t (thymine), y represents c (cytosine) or t (thymine), w is a (Adenine) or t (thymine) is represented, d is a (adenine), g (guanine) or t (thymine), h is a (adenine), c (cytosine) or t (thymine). In addition, the base sequence shown in SEQ ID NO: 1 may be one in which one or more bases at any position are inserted or deleted. Here, one or several may be one to three, one or two, or one.

More specifically, for example, the second "y" from the 5 'side may be deleted. Alternatively, the eighth "w" from the 5 'side may be deleted. Alternatively, there may be an insertion of one base between the 17th "y" and the 18th "r" from the 5 'side.

More specifically, the base sequence set forth in SEQ ID NO: 1 may be the base sequence set forth in any of SEQ ID NOs: 24 to 35 below. As described later in the Examples, the nucleotides containing the nucleotide sequences set forth in SEQ ID NOS: 24 to 35 are nucleotides having the consensus sequence shown in SEQ ID NO: 1, and those confirmed to have an activity of binding to FGFR1. is there.

5'-ttatggctggggatggtgtgggtt-3 '(SEQ ID NO: 24)
5'-ttatggcaggggatggtgtgggtt-3 '(SEQ ID NO: 25)
5'-gtttggtgtggatggcaggggct-3 '(SEQ ID NO: 26)
5'-ttatggtgtggatggctatgggct-3 '(SEQ ID NO: 27)
5'-ttatggtgtggatggctggggttt-3 '(SEQ ID NO: 28)
5'-tcatggtgtggatggcaggggct-3 '(SEQ ID NO: 29)
5'-ttttggtgtggatggcaggggcg-3 '(SEQ ID NO: 30)
5'-ttatgtgtgtggatggcaggggct-3 '(SEQ ID NO: 31)
5 '-tatggtgtggatggcaggggct-3' (SEQ ID NO: 32)
5'-tcatggatgtgatggcaggggtt-3 '(SEQ ID NO: 33)
5'-tcatggtgtggatggcaggggtt-3 '(SEQ ID NO: 34)
5'-ttatggttaggatggtgtggttt-3 '(SEQ ID NO: 35)

The number of bases of the aptamer of this embodiment may be, for example, 28 to 48 bases, 33 to 43 bases, or 38 bases.

The aptamer of the present embodiment preferably has a loop structure in which a polynucleotide consisting of the base sequence set forth in SEQ ID NO: 1 forms at least a part. As described later in the Examples, an aptamer having a loop structure in which a polynucleotide consisting of the base sequence set forth in SEQ ID NO: 1 forms at least a part tends to have a high ability to bind to FGFR1.

In the present specification, a loop structure refers to a cyclic structure formed by binding one or more places of a chain compound to each other. For example, the loop structure may be a cyclic structure in which one or more pairs of complementary bases form a base pair in a single-stranded nucleic acid. The loop structure may be formed in part by a polynucleotide consisting of the base sequence shown in SEQ ID NO: 1, or may be formed only from a polynucleotide consisting of the base sequence shown in SEQ ID NO: 1. The means for binding to form a loop structure is not limited to the formation of base pairs, and may be, for example, the ligation of the 5 'end and the 3' end of nucleic acid, or any other cross-linked structure It is also good.

When a polynucleotide consisting of the base sequence shown in SEQ ID NO: 1 forms a part of a loop structure, the remainder of the loop structure has any base sequence as long as the aptamer has an activity of binding to FGFR1. It is also good.

The number of bases in the loop structure may be, for example, 28 to 40 bases, 28 to 35 bases, or 28 to 30 bases.

As shown in FIG. 1, the aptamer 2 is composed of a polynucleotide, and has a loop structure 20 in which the polynucleotide 10 including the base sequence set forth in SEQ ID NO: 1 forms at least a part.

The aptamer of this embodiment preferably has a stem structure composed of a double stranded polynucleotide linked to the loop structure. As described later in the Examples, an aptamer having a stem structure consisting of a double-stranded polynucleotide linked to the above-described loop structure tends to have a higher ability to bind to FGFR1.

In the present specification, a stem structure refers to a chain structure formed by connecting two or more portions of a chain compound to each other. For example, in a single-stranded nucleic acid, the stem structure may be a chain-like structure formed by base pairing one or more pairs of complementary bases. The means for binding to form a stem structure is not limited to the formation of base pairs, and may be any other cross-linked structure.

The form in which the stem structure is linked to the loop structure is not particularly limited, and the form in which the loop structure and the stem structure are formed from a series of polynucleotides can be exemplified. Such a form of nucleic acid is a structure generally called a stem-loop structure, and is found in tRNA and the like. The stem structure is represented by a double-stranded polynucleotide formed by pairing complementary bases to each other.

As shown in FIG. 1, the aptamer 3 is composed of a polynucleotide, and a loop structure 20 formed of at least a part of a polynucleotide 10 including the nucleotide sequence of SEQ ID NO: 1 and a double strand linked to the loop structure 20 And a stem structure 30 consisting of a polynucleotide.

By having a stem structure composed of a double stranded polynucleotide, the aptamer can easily form a loop structure, and the ability to bind to an FGF receptor is improved.

The stem structure may be composed of only double stranded polynucleotide. In that case, one single-stranded polynucleotide forming a double-stranded polynucleotide may be a polynucleotide strand having 2 to 50 bases, or may be a polynucleotide strand having 4 to 30 bases, It may be several 5 to 15 polynucleotide strands.

The aptamer of this embodiment preferably forms a guanine quadruplex structure. As described later in the Examples, aptamers that form a guanine quadruple chain structure tend to have higher binding ability to FGFR1.

The guanine quadruplex structure may be identified in a nucleic acid capable of binding to a target. As the name guanine quadruple chain, guanine quadruple chain structure is a specific three-dimensional structure formed by 4 sets of G sequences (Nonaka Y., et al., Screening and improvement of an anti-VEGF DNA aptamer , Molecules, 15 (1), 215-225, 2010.).

Guanine quadruplexes can be classified into "parallel" and "antiparallel" from their topology. Here, the guanine quadruplex structure formed by the aptamer of the present embodiment may be parallel or antiparallel.

Whether or not the polynucleotide forms a guanine quadruplex structure can be confirmed by known methods. The presence of the parallel guanine quadruplex structure can be confirmed, for example, by detection of a negative peak around 245 nm and a positive peak around 265 nm by CD spectral measurement. Moreover, since the formation of guanine quadruplex structures that require the K ⁺ ions, for example, a CD spectrum measurements of the polynucleotide in a solution containing K ⁺ ions, the polynucleotide in solution without K ⁺ ion CD The presence of the guanine quadruplex structure can be more reliably confirmed by comparing the spectrum measurement results.

The aptamer of the present embodiment preferably includes four or more sets of two or more consecutive G sequences from the viewpoint of forming a guanine quadruplex structure. When the aptamer of the present embodiment has a loop structure, the polynucleotide forming the loop structure preferably contains four or more sets of two or more consecutive G sequences.

The number of bases of the polynucleotide of the aptamer of this embodiment may be 500 bases or less, 250 bases or less, 150 bases or less, or 80 bases or less. It may be 70 bases or less, 60 bases or less, or 55 bases or less.

Moreover, the number of bases of the polynucleotide of the aptamer of the present embodiment may be 28 bases or more, 35 bases or more, 40 bases or more, or 45 bases or more. Good.

Further, the number of bases of the polynucleotide of the aptamer of the present embodiment may be, for example, 28 to 500 bases, 35 to 250 bases, or 40 to 150 bases, and may be 40 to It may be 150 bases, 40 to 80 bases, 40 to 70 bases, 40 to 60 bases, or 45 to 55 bases.

The aptamer of the present embodiment may be a polynucleotide having a base sequence shown in SEQ ID NO: 1 with a polynucleotide having any base sequence added thereto, as long as it has an activity of binding to FGFR1. Further, the aptamer of the present embodiment may be, for example, various modifications for enhancing the stability in vivo, or a label such as a dye.

The aptamer of the embodiment can be produced by a known method. Methods of nucleic acid synthesis and methods of their modification are widely used in the life sciences field.

[Multi-structure aptamer]
In one embodiment, the present invention provides an aptamer to which two or more of the aforementioned aptamers are linked, and which has an activity of binding to FGFR1 and activating FGFR1.

The aptamer of this embodiment has a multi-structure in which two or more units of the aptamer are linked, with the above-described aptamer as one unit. In the present specification, an aptamer in which two or more units of aptamers are linked may be referred to as a "multistructural aptamer". As a multi-structural aptamer, for example, a dimer in which one unit of aptamer is linked can be exemplified. The linked aptamers may be the same as or different from each other.

The multistructural aptamer preferably has a stem structure consisting of a double stranded polynucleotide linked to a loop structure. In addition, the loop structure may be composed of a polynucleotide strand having 28 to 40 bases. In addition, it is preferable that the multi-structure aptamer form a guanine quadruplex structure. Therefore, it is preferable that the multi-structure aptamer contains four or more sets of consecutive two or more G base sequences.

FIG. 2 is a schematic view showing an example of a multistructural aptamer. Multi-structure aptamer 4 is composed of a polynucleotide, and has a multi-structure in which two or more polynucleotides 10 including the base sequence described in SEQ ID NO: 1 are linked. Multi-structure aptamer 4 is one in which two units of aptamer 1 are linked. The polynucleotides 10 may be polynucleotides having the same base sequence. Alternatively, the polynucleotides 10 may be polynucleotides having different base sequences.

Multi-structural aptamers have the activity of binding to FGFR1 and activating FGFR1. The activation state of FGFR1 can be confirmed by a known detection method. Activation of FGFR1 can be detected, for example, by detecting phosphorylation of FGFR1. Phosphorylation of FGFR1 can be detected using known antibodies capable of specifically detecting phosphorylation of FGFR1.

Whether or not the aptamer has an FGF receptor activation effect can be confirmed by a known detection method. For example, a sample solution C containing cells expressing an FGF receptor and not containing an aptamer, and a sample solution D containing cells expressing an FGF receptor and an aptamer are prepared. After culturing the cells in each sample solution, the values indicating the phosphorylation of the FGF receptor may be compared between the sample solution C treatment and the sample solution D treatment. Comparison experiments shall be conducted under the same conditions that can be compared. When the value indicating the phosphorylation of the FGF receptor in the sample solution D is higher than the value indicating the phosphorylation of the FGF receptor in the sample solution C, the aptamer has an FGF receptor activating action. It can be judged. In addition, the method of judging that an aptamer has FGFR1 activation effect is not restricted to said method.

As shown in FIG. 2, the multi-structure aptamer 5 has a multi-loop structure in which two or more loop structures 20 in which a polynucleotide 10 including the nucleotide sequence set forth in SEQ ID NO: 1 forms at least a part are linked. Thus, the multi-structure aptamer 5 is one in which two units of one aptamer 2 having a loop structure 20 are linked.

The multistructural aptamer preferably has a stem structure consisting of a double stranded polynucleotide linked to a loop structure.

As shown in FIG. 2, the multi-structure aptamer 6 is composed of a loop structure 20 in which the polynucleotide 10 containing the nucleotide sequence of SEQ ID NO: 1 at least partially forms, and a double-stranded polynucleotide linked to the loop structure 20 A structure having a stem structure 30 has a multi-loop structure in which two or more are connected.

Thus, the multi-structural aptamer 6 is one in which two aptamers 3 each having a loop structure 20 and a stem structure 30 are linked. When a plurality of stem structures are included in a molecule, the base sequences forming each stem structure may be different from each other. When there is a base sequence that forms two or more identical stem structures in a molecule, it is possible that the opposite nucleotide strand forming the complementary strand may be replaced. In this regard, by making the base sequences forming each stem structure different from each other, it is possible to prevent the replacement of the nucleotide strand of the partner forming the complementary strand. In addition, even if it is the multi structure aptamer which made the completely same stem structure and the completely same aptamer sequence continue, an FGFR1 activation effect can be exhibited.

An example of the multi-structure aptamer is schematically shown in FIGS. The multi-structure aptamer 7 shown in FIG. 3 is one in which one unit of aptamer 3 is linked in three or more in parallel. Further, in the multi-structure aptamers 8a and 8b shown in FIG. 4, three or more one units of aptamers 3 are radially linked.

The polynucleotide forming the multistructural aptamer may be DNA. Further, the nucleotides constituting the multi-structure aptamer may contain DNA at least in part, or may consist of only DNA.

In the multi-structural aptamer, the above-mentioned aptamer may be linked by a linker, and the length of the linker may be 80 bases or less in terms of polynucleotide.

In FIG. 5, the multi-structure aptamer 6 is taken as an example to describe the linker of the multi-structure aptamer. The linker 40 is a region between the ends of the loop structure in which the polynucleotide 10 consisting of the base sequence set forth in SEQ ID NO: 1 forms at least a part. When the aptamer has a stem structure 30, the stem structure is also included in part of the linker. That is, in the present specification, a structure connecting the loop structure and the loop structure constituting the multistructural aptamer is used as a linker.

A part of the linker may be composed of single stranded DNA. Alternatively, all of the linkers may be composed of double stranded DNA. In addition, when the multistructural aptamer contains a plurality of linkers, some of the linkers may include single-stranded DNA portions, and some of the linkers may be composed of double-stranded DNA.

FIGS. 6 (a) and 6 (b) are schematic diagrams showing an example of an aptamer in which all the linkers are double-stranded DNA. As shown in FIG. 6 (a), the multi-structure aptamer 9a has two loop structures 20 in which a polynucleotide 10 consisting of the base sequence described in SEQ ID NO: 1 forms at least a part. The first loop structure 20 and the second loop structure 20 are linked by a linker 40 consisting of double-stranded DNA.

Further, as shown in FIG. 6 (b), the multi-structure aptamer 9 b has two loop structures 20 in which the polynucleotide 10 consisting of the base sequence described in SEQ ID NO: 1 forms at least a part. The first loop structure 20 and the second loop structure 20 are linked by a linker 40 consisting of double-stranded DNA.

FIG. 7 is a schematic view showing an example of a multi-structure aptamer comprising a plurality of linkers. As shown in FIG. 7, the multistructural aptamer 10 has four loop structures 20 in which a polynucleotide 10 consisting of the base sequence set forth in SEQ ID NO: 1 forms at least a part. And multi-structure aptamer 10 contains a plurality of

linkers

40a, 40b, and 40c. The linker 40a and the linker 40c are linkers including a single-stranded DNA portion, and the linker 40b is composed only of double-stranded DNA. Also, the linker 40a and the linker 40b share part of the linker. Also, the linker 40b and the linker 40c share part of the linker.

In the multistructural aptamer, the length per linker is preferably 80 or less in length in terms of polynucleotide. The linker may be formed of a material other than a polynucleotide. For example, materials other than polynucleotides include, for example, polyethylene glycol (PEG). If the linker is formed of a material other than a polynucleotide, the length of the linker shall be calculated based on the length of the polynucleotide. The length of the reference polynucleotide is calculated based on the distance between base pairs of 3.4 angstroms in the double stranded DNA. One angstrom is 0.1 nm.

The length of the linker may be 0 to 80 bases in length, 5 to 70 bases in length, 10 to 60 bases in length, or 15 to 50 bases in terms of polynucleotide. It may be 15 to 30 bases long or 16 to 20 bases long.

In the multi-structure aptamer, the number of bases of the polynucleotide may be 1000 bases or less, 700 bases or less, 500 bases or less, 250 bases or less, or 150 bases. It may be less than 100 bases, may be up to 80 bases, may be up to 60 bases.

The number of bases of the polynucleotide possessed by the multistructural aptamer may be 56 bases or more, 60 bases or more, 80 bases or more, 100 bases or more, 200 bases It may be more than.

The number of bases of the polynucleotide possessed by the multi-structural aptamer may be, for example, 56 to 1000 bases, 60 to 700 bases, 80 to 500 bases, or 90 to 250 bases. It may be 90 to 150 bases or 90 to 100 bases.

Multi-structure aptamers can be produced by known methods. Methods of nucleic acid synthesis and methods of their modification are widely used in the life sciences field.

The multi-structure aptamer may consist of the nucleotide sequence set forth in SEQ ID NO: 11. As described later in the Examples, it has been confirmed that an aptamer consisting of the nucleotide sequence set forth in SEQ ID NO: 11 has the same activity as bFGF.

The multi-structure aptamer may have a mutation with respect to the base sequence set forth in SEQ ID NO: 11. Specifically, the multi-structure aptamer consists of a nucleotide sequence in which one or several bases are deleted, substituted or added in the nucleotide sequence set forth in SEQ ID NO: 11 and has an activity of binding to FGFR1 and activating FGFR1. It may have the

In the present specification, one or several may be one to ten, may be one to five, may be one to four, or may be one to three. It may be one or two.

[Aptamer fixed carrier]
In one embodiment, the present invention provides an aptamer-immobilized carrier, wherein the above-mentioned aptamer or multi-structure aptamer is immobilized on the surface of a solid phase carrier.

The shape of the solid phase carrier is not particularly limited, and examples thereof include sheet, plate, particle, and membrane. Further, the material of the solid phase carrier is not particularly limited as long as it is a material capable of immobilizing the aptamer, and resin, silica, glass, metal and the like can be exemplified.

FIGS. 8A and 8B schematically show an example of the form of the aptamer-immobilized carrier. The aptamer immobilization carrier 100 is one in which the aptamer 3 is immobilized on the surface of a sheet-like solid phase carrier 110. The aptamer-immobilizing carrier 101 is one in which the aptamer 3 is immobilized on the surface of a columnar solid-phase carrier 120.

[Prophylaxis or Therapeutic Agent for FGFR1 Signaling Related Disease]
In one embodiment, the present invention provides a preventive or therapeutic agent for an FGFR1 signaling related disease, which comprises the above-described aptamer or multi-structure aptamer as an active ingredient. Here, an aptamer means a monomer aptamer (1 unit of aptamer), and a multi-structure aptamer means an aptamer in which 2 units or more of aptamers are linked.

The monomeric aptamer has an activity of binding to FGFR1 and inhibiting the binding of bFGF to FGFR1. On the other hand, multi-structure aptamers have an activity of binding to FGFR1 and activating FGFR1.

FGFR1 signaling can be controlled by administering the aptamer of this embodiment to a living body. As a result, FGFR1 signaling related diseases can be prevented or treated. The FGFR1 signaling related diseases include cancer, diabetes and the like. Further, in the present specification, FGFR1 signaling related diseases also include wounds such as abrasions and cuts.

By administering a monomeric aptamer to a patient, FGFR1 signaling can be suppressed. As a result, cell proliferation can be suppressed and, for example, cancer can be treated.

In addition, by applying a multi-structure aptamer to a wound, FGFR1 can be activated to promote cell proliferation. As a result, the wound can be treated promptly. Moreover, the prophylactic or therapeutic agent of this embodiment can also be used for cosmetic purposes.

In one embodiment, the present invention provides a method for the prophylaxis or treatment of a disease associated with FGFR1 signaling, comprising the step of administering an effective amount of a monomeric aptamer or a multistructural aptamer to a patient in need of treatment.

In one embodiment, the present invention provides a monomeric or multi-structural aptamer for the prevention or treatment of a FGFR1 signaling related disease.

In one embodiment, the present invention provides the use of a monomeric aptamer or a multi-structural aptamer for producing a preventive or therapeutic agent for a FGFR1 signaling-related disease.

The preventive or therapeutic agent for an FGFR1 signaling related disease of the present embodiment may be in the form of a pure monomeric aptamer or a multi-structural aptamer, or a pharmaceutical composition mixed with an appropriate pharmacologically acceptable additive. It may be in the form of a product or a cosmetic composition. The pharmaceutical composition or cosmetic composition may be orally administered in the form of tablets, capsules, granules and the like, or in the form of ointments, creams, lotions, injections and the like. It may be orally administered.

These preparations can be manufactured by known methods using additives such as excipients, binders, disintegrants, lubricants, emulsifiers, stabilizers, diluents, solvents for injections and the like.

Examples of the excipient include organic excipients, inorganic excipients and the like. Organic excipients include hydrocarbons such as white petrolatum and liquid paraffin; sugar derivatives such as lactose and sucrose; starch derivatives such as corn starch and potato starch; cellulose derivatives such as crystalline cellulose; gum arabic etc. . Examples of inorganic excipients include sulfates such as calcium sulfate.

As the binder, the above-mentioned excipients, gelatin, polyvinyl pyrrolidone, polyethylene glycol and the like can be mentioned.

As the disintegrant, the above-mentioned excipients; starch or derivatives of cellulose such as croscarmellose sodium, sodium carboxymethyl starch etc .; crosslinked polyvinyl pyrrolidone etc. may be mentioned.

Lubricants include talc, stearic acid, colloidal silica, waxes such as beeswax and geewol, sulfates such as sodium sulfate, lauryl sulfates such as sodium lauryl sulfate, starch derivatives in the above-mentioned excipients, etc. Be

Emulsifying agents include colloidal clays such as bentonite and veegum; anionic surfactants such as sodium lauryl sulfate; cationic surfactants such as benzalkonium chloride; nonionic surfactants such as polyoxyethylene alkyl ether etc. It can be mentioned.

As the stabilizer, parahydroxybenzoic acid esters such as methylparaben and propylparaben; alcohols such as chlorobutanol; and phenols such as phenol and cresol.

Examples of diluents include water, ethanol, propylene glycol and the like. Examples of solvents for injection include water, ethanol, glycerin and the like.

The dose of the prophylactic or therapeutic agent of the present embodiment varies depending on the condition of the patient etc., but in the case of oral administration, it is generally 0.1 to 100 mg per day for adults (60 kg body weight) in general. Administration of a certain amount of active ingredient (aptamer or multi-structure aptamer) can be mentioned.

When administered parenterally, the dose varies depending on the administration subject, target organ, symptoms, administration method, for example, in the case of an ointment, for example, about 0.01 to 30 mg once a day Application to the affected area several times may be mentioned.

The prophylactic or therapeutic agent of the present embodiment may be in the form of a vector capable of expressing a monomeric aptamer or a multistructural aptamer.

[Composition for cell culture]
In one embodiment, the present invention provides a composition for cell culture, which comprises the above-described monomeric aptamer or multi-structured aptamer as an active ingredient. The composition for cell culture of the present embodiment may be a culture medium, or may be a culture medium additive.

By culturing cells in the presence of the above-described monomer aptamer or multi-structure aptamer, it is possible to control various cell states involved in FGFR1 signaling, such as cell differentiation and proliferation.

The composition for cell culture may contain a basic medium, serum or the like in addition to the monomeric aptamer or the multistructural aptamer.

The concentration of the aptamer contained in the composition for cell culture can be appropriately adjusted depending on the purpose of use, and may be, for example, 0.01 nM to 10 μM, or 0.1 nM to 5 μM. It may be 1 nM to 5 μM, 50 nM to 3 μM, or 100 nM to 2 μM. In addition, "M" represents "mol / L".

[Cell culture method]
In one embodiment, the present invention provides a cell culture method comprising culturing an FGFR1 positive cell in a medium containing the above-mentioned monomer aptamer or multi-structure aptamer. When the FGFR1 positive cells are cultured in the presence of the monomeric aptamer, the monomeric aptamer binds to FGFR1 and the binding of bFGF to FGFR1 is inhibited. As a result, FGFR1 signaling in cells can be suppressed. In addition, when FGFR1-positive cells are cultured in the presence of a multi-structural aptamer, the multi-structural aptamer can bind to FGFR1 and activate FGFR1 signaling in the cells.

[Method for inhibiting the binding of bFGF to the aforementioned FGFR1]
In one embodiment, the present invention provides a method for inhibiting the binding of bFGF to FGFR1, which comprises contacting the above-mentioned monomeric aptamer with an FGFR1 positive cell. When the monomeric aptamer is brought into contact with FGFR1 positive cells, the monomeric aptamer binds to FGFR1 and the binding of bFGF to FGFR1 is inhibited. As a result, FGFR1 signaling in cells can be suppressed.

In the present specification, the method for contacting a monomeric aptamer or multi-structure aptamer with an FGFR1 positive cell is not particularly limited, and various methods can be used. For example, by culturing cells in a culture medium containing a monomer aptamer or a multi-structure aptamer, the monomer aptamer or the multi-structure aptamer may be brought into contact with the cells, or the monomer aptamer or A monomer aptamer or a multi-structure aptamer may be brought into contact with the cell by containing the multi-structure aptamer and the cell, or a single amount is dropped by dropping the composition containing the monomer aptamer or the multi-structure aptamer into the cell. Body aptamers or multi-structure aptamers may be contacted with cells. Also, the contact of the monomeric aptamer or multi-structural aptamer with the FGFR1 positive cells may be performed in vivo or in vitro.

[Method for suppressing the growth of FGFR1 positive cells]
In one embodiment, the present invention provides a method for suppressing the growth of cells, which comprises contacting the above-mentioned monomeric aptamer with an FGFR1 positive cell. When the monomeric aptamer is brought into contact with FGFR1 positive cells, the monomeric aptamer binds to FGFR1 and the binding of bFGF to FGFR1 is inhibited. As a result, cell proliferation can be suppressed.

Whether the aptamer suppresses the growth of the cells is, for example, comparing the cells in contact with the aptamer with the cells not in contact with the aptamer, and if the cells in contact with the aptamer have a smaller degree of cell growth, the aptamer Can be judged to be suppressing cell proliferation.

[Method of activating FGFR1]
In one embodiment, the present invention provides the aforementioned FGFR1 activation method, which comprises contacting the multi-structure aptamer described above with an FGFR1 positive cell. When the multistructural aptamer is contacted with FGFR1 positive cells, the multistructural aptamer can bind to FGFR1 and activate FGFR1 signaling in the cell.

For example, whether the multi-structure aptamer activated FGFR1 or not is compared with a cell in contact with the multi-structure aptamer with a cell in non-contact with the multi-structure aptamer, and the cells in contact with the multi-structure aptamer have FGFR1 When the degree of phosphorylation is large, it can be judged that the multi-structure aptamer activated FGFR1.

[Method for promoting the growth of FGFR1 positive cells]
In one embodiment, the present invention provides a method for promoting the growth of cells, which comprises contacting the multi-structure aptamer described above with an FGFR1 positive cell. When the multistructural aptamer is contacted with FGFR1 positive cells, the multistructural aptamer can bind to FGFR1 and activate FGFR1 signaling in the cell. As a result, cell proliferation can be promoted.

Whether or not the multi-structure aptamer promotes cell growth can be compared, for example, with cells in contact with the multi-structure aptamer with cells not in contact with the multi-structure aptamer, and cells in contact with the multi-structure aptamer are cells. When the degree of proliferation is large, it can be judged that the multi-structure aptamer promoted cell proliferation.

[Method for maintaining pluripotency of pluripotent stem cells]
In one embodiment, the present invention provides the method for maintaining pluripotency of pluripotent stem cells, comprising contacting the multi-structure aptamer described above with FGFR1 positive pluripotent stem cells. Examples of FGFR1 positive pluripotent stem cells include pluripotent stem cells such as iPS cells and Embryonic stem cells (ES cells).

Whether or not the multistructural aptamer maintains the pluripotency of the pluripotent stem cells, for example, compares the cell contacted with the multistructural aptamer with the cell not contacted with the multistructural aptamer, and the multistructural aptamer contacts When the expression of pluripotent stem cell markers is higher in these cells, it can be determined that the multistructural aptamer maintained pluripotency of pluripotent stem cells. Examples of pluripotent stem cell marker genes include Oct-4, Nanog, SSEA-4, SSEA-1, SOX2 and the like.

[Detection method]
In one embodiment, the present invention provides a method of detecting FGFR1 comprising binding the above-described monomeric aptamer or multistructural aptamer to FGFR1. Here, it is preferable that the monomer aptamer or the multi-structure aptamer is labeled with a labeling substance. As a result, FGFR1 can be detected by detecting the labeling substance. Examples of the labeling substance include dyes, fluorescent dyes, radioisotopes, antibodies, antigens, enzymes and the like.

Hereinafter, the present invention will be described by way of examples, but the present invention is not limited to the following examples.

[Materials and Methods]
(material)
The compounds were purchased from common suppliers and used without further purification. All DNA samples were purchased from Fasmac. Annealing of the DNA (cooling at 95 ° C. for 5 minutes, 0.1 ° C./minute to 25 ° C. or room temperature) was performed by a thermal cycler. Dulbecco's phosphate buffered saline (DPBS) was purchased from Fujifilm Wako Pure Chemical Industries, Ltd. Recombinant basic FGF (# 100-18B) and recombinant human TGF-β (# 100-21) were purchased from Peprotech. Recombinant human FGFR1-Fc chimeric protein (# 661-FR) and recombinant human Met-Fc chimeric protein (# 358-MT) were purchased from R & D Systems. Recombinant FGFR1 extracellular domain (# 7421-20) was purchased from Biovision. The fluorescence image was acquired by an inverted microscope (IX-81, Olympus Co., Ltd.) equipped with a sCMOS camera (Zyla 4.2, Andor Technology Co., Ltd.). Absorbance and fluorescence intensity were measured by Infinite M200 pro (Tecan).

(FGFR1 binding DNA aptamer)
FGFR1-Fc was incubated with Dynabeads Protein G (# 10004D, Thermo Fisher Scientific) for 30 minutes in PBS-T buffer (DPBS with 0.02% Tween 20 (wt / vol)). A slurry of beads (12.6 μL; binding capacity 20 pmol) was used to immobilize 20 pmol of protein. After incubation, the beads were washed 3 times with PBS-T buffer and used for selection. Human Met-Fc fixed beads were prepared by the same procedure and used for negative selection. The amount of recombinant protein used for each selection is summarized in Table 1.

(Separation of FGFR1 binding sequence)
A mixture of single-stranded DNA (single-stranded DNA pool) represented by SEQ ID NO: 2 was prepared. In SEQ ID NO: 2, N is any of a (adenine), t (thymine), g (guanine) and c (cytosine). The single stranded DNA pool was dissolved in DPBS, dissociated for 5 minutes at 95 ° C and slowly cooled to 25 ° C at 0.1 ° C / min. After refolding, equal volumes of DPBS containing 0.04% Tween 20 (wt / vol) were added to the single stranded DNA pool. Single-stranded DNA pools were incubated with Dynabeads Protein G for 30 minutes at room temperature to remove bead-bound sequences. Negative selection was also performed using human Met-Fc fixed beads during the 5 to 6 rounds of selection. The supernatant was incubated with FGFR1 fixed beads for 10-30 minutes at room temperature. After incubation, the beads were washed with PBS-T buffer. The beads and elution buffer (100 mM sodium acetate containing 7 M urea, 3 mM EDTA) were incubated at 95 ° C. for 3 minutes to obtain a FGFR1 binding sequence. The eluted DNA was extracted with phenol / chloroform / isoamyl alcohol solution and ultrafiltered with Amicon Ultra (#UFC 501096). The conditions for each selection are summarized in Table 1.

(Single-stranded DNA pool)
The eluted DNA was amplified by PCR using KOD DNA polymerase according to the manual. The primers used were a primer consisting of the nucleotide sequence of SEQ ID NO: 3 and a primer consisting of the nucleotide sequence of SEQ ID NO: 4. After PCR, the reaction mixture and streptavidin magnetic beads (# 21344, Thermo Fisher Scientific) were incubated for 10 minutes in buffer (10 mM Tris-HCl, 1 M NaCl, 1 mM EDTA). The beads were incubated for 5 minutes in a solution containing 150 mM NaOH to obtain single-stranded DNA. The supernatant was neutralized with 150 mM HCl and desalted on a G-25 spin column. Single stranded DNA was eluted from the column with nuclease free water and used for the next selection.

(Sequencing)
After six rounds of selection, the recovered DNA was amplified with unmodified primers. The PCR product was inserted into a cloning vector using TArget Clonetm-Plus kit (sha-puTAK-201, Toyobo Co., Ltd.). Aptamer candidate sequences were identified by DNA sequencing according to standard E. coli transformation and cloning methods.

(Flow cytometry)
The fluorescence intensity of each bead and cells was measured using flow cytometry (Guava easyCyte, Merck).

(Aptamer bound to FGFR1 immobilized beads)
Human FGFR1-Fc (1 pmol for each sample) and Dynabeads Protein G were incubated in PBS-T for 30 minutes. After incubation, the beads were washed 3 times with PBS-T. 20 pmol of 5'-FITC-modified DNA and FGFR1 fixed beads were incubated in 50 μL of PBS-T for 30 minutes at 37 ° C. After incubation, the beads were washed with PBS-T and analyzed by flow cytometry. The increase in fluorescence intensity of the beads incubated with the aptamer relative to the fluorescence intensity of the untreated beads is shown as the average fluorescence intensity.

(Expression of SSEA-4)
409B2 cells were detached from the dishes and washed with DPBS. Cells and Alexa 488-labeled anti-SSEA-4 antibody (1: 100 dilution, # 53-8843, Thermo Fisher Scientific) were incubated on ice for 30 minutes in FCM buffer (DPBS with 1% BSA). After incubation, cells were washed twice with FCM buffer and analyzed by flow cytometry. The increase in fluorescence intensity of antibody-stained cells relative to the fluorescence intensity when untreated was shown as the average fluorescence intensity.

(SPR analysis)
SPR analysis was performed by Biacore T100 (GE Healthcare) using Series S Sensor Chip SA (# 29104992, GE Healthcare). The 5'-biotinylated DNA was immobilized on a sensor chip. Various concentrations of FGFR1 protein (2.5, 10, 25, 50, 100 nM) were injected onto the surface of fixed DNA at a flow rate of 30 μL / min (flow buffer at 25 ° C .: 0.2% Nonidet-P40) DPBS containing, Regeneration solution: 1 M NaCl solution containing 50 mM NaOH, contact time: 180 seconds, dissociation time: 600 seconds).

(Cell culture)
Human rhabdomyosarcoma cell line A204 cells were cultured in DMEM containing 10% fetal bovine serum (FBS) and 1% anti-mycoplasma antibiotic (# 15240, Thermo Fisher Scientific) . 409B2 cells were cultured in StemFit® AK02N (# AK02N, Ajinomoto Co., Ltd.) containing 1% penicillin-streptomycin mixed solution (# 26253-84, Nacalai Tesque, Inc.). iMatrix-511 (0.25 μg / cm ² , # 892011, Nippi) was used as a culture substrate. All cells were cultured in an incubator at 37 ° C. in a humidified 5% CO ₂ atmosphere.

(ELISA analysis)
The analysis was performed according to a manual protocol using PathScan (registered trademark) Phospho-FGF Receptor 1 (panTyr) Sandwich ELISA Kit (# 12909C, Cell Signaling Technology). Three independent experiments were performed and the mean values are shown with error bars (SD).

(Immunoblot analysis)
The cell lysate was separated by SDS-PAGE and transferred to a PVDF membrane. The PVDF membrane was reacted with the primary antibody overnight at 4 ° C. and then with the appropriate secondary antibody at room temperature for 1 hour. As primary antibodies, phospho-ERK1 / 2 (# 4370), ERK1 / 2 (# 4695), beta-actin (# 4967) were purchased from Cell Signaling Technologies. The secondary antibody (Anti-Rabbit Immunoglobulins / HRP, P0488) was purchased from Dako. The membrane was detected by ImmunoStar LD (# 296-69901, Fujifilm Wako Pure Chemical Industries, Ltd.).

Maintenance of pluripotency of hiPSC cells in the presence of FGFR agonist
In order to evaluate the potential effects of FGFR agonists on the maintenance of pluripotency of iPS cells, "ABT medium" was prepared and used in experiments as a basal medium. ABT medium is composed of StemFit® AK02N solution A (400 mL) and solution B (100 mL), recombinant TGF-β (final concentration 2 ng / mL), complete StemFit® AK02N is solution A ( It consists of 400 mL), solution B (100 mL), solution C (2 mL). It was confirmed that the pluripotency of 409B2 iPS cells can not be maintained in ABT medium in the absence of recombinant bFGF. 409B2 cells (2 × 10 ³ cells / cm ² ) were seeded on culture dishes containing ABT medium containing the appropriate FGFR agonist, Y-27632 (10 μM), iMatrix-511 (0.3 μg / cm ² ) did. The next day, the medium was replaced with ABT medium containing the appropriate FGFR agonist. The medium was changed every other day to maintain the cells.

(Immuno staining imaging)
409B2 cells were washed twice with DPBS and fixed with 4% paraformaldehyde phosphate buffer for 30 minutes at room temperature. After fixation, cells were washed three times with DPBS and incubated in permeation buffer (DPBS with 0.5% Triton X-100) for 15 minutes at room temperature. After permeabilization, cells were washed 3 times with DPBS and incubated in blocking buffer (DPBS with 3% BSA) for 15 minutes at room temperature. The cells were then reacted with Alexa 488 labeled anti-SSEA-4 antibody (1: 100 dilution, # 53-8843, Thermo Scientific Pierce) overnight at 4 ° C. The cells were washed three times with DPBS and stained with Hoechst 33258 (1: 1000, # H341, Dojin Chemical Laboratories) prior to fluorescence imaging observation.

(RT-qPCR analysis)
Total RNA was purified by FastGenetm RNA Premium Kit (# FG-81050, Japan Genetics). The purified RNA was subjected to reverse transcription using an iScript cDNA Synthesis Kit (# 1708890, Bio-Rad) according to the manual protocol. KOD SYBR qPCR Mix (# QKD-201, Toyobo Co., Ltd.) was used to quantify the amount of gene expression. For quantification of the expression level of OCT4, the primer represented by SEQ ID NO: 20 and the primer represented by SEQ ID NO: 21 were used. For quantification of the expression level of RPLP0, the primer represented by SEQ ID NO: 22 and the primer represented by SEQ ID NO: 23 were used.

(Cell proliferation analysis)
409B2 cells were washed twice with DPBS and detached from petri dishes with DPBS containing 0.5 mM EDTA. The cells were stained with 0.4% trypan blue solution (# 207-17081, Fujifilm Wako Pure Chemical Industries, Ltd.) and counted with a cell counter.

[Experimental Example 1]
(Screening of DNA aptamers that bind to FGFR1)
Among single-stranded DNA pools having random sequences, single-stranded DNA binding to FGFR1 was concentrated and cloned by PCR and affinity purification.

First, a pool of single-stranded DNA containing a 40 base random sequence shown in SEQ ID NO: 2 was prepared. The pool was brought into contact with beads immobilized with FGFR1 to select DNA that binds to FGFR1. Subsequently, the selected DNA was amplified by PCR. In PCR, a primer in which the 5 'end of the DNA represented by SEQ ID NO: 3 was labeled with FITC and a primer in which the 5' end of the DNA represented by SEQ ID NO: 4 was labeled with biotin were used. These steps were repeated six times to concentrate DNA binding to FGFR1.

Next, the concentrated DNA pool was brought into contact with FGFR1 positive cells to select DNA that binds to FGFR1. Subsequently, the selected DNA was amplified by PCR. These steps were repeated seven times to concentrate DNA binding to FGFR1.

The concentrated DNA was subsequently cloned into a vector and the sequence of the inserted DNA was analyzed by sequencing. As a result, several dozen kinds of aptamers were obtained, including an aptamer consisting of the nucleotide sequences of SEQ ID NOs: 36 to 50.

As a result of aligning the aptamers consisting of the nucleotide sequences set forth in SEQ ID NOS: 36 to 50, it became clear that these aptamers contain the nucleotide sequences set forth in SEQ ID NOs: 24 to 35 as partial sequences. Furthermore, the nucleotide sequences set forth in SEQ ID NOs: 24 to 35 were specified to use the nucleotide sequence set forth in SEQ ID NO: 1 as a consensus sequence.

Among the aptamers obtained as a result of the screening, the following examination was conducted focusing on the aptamer showing a nucleotide sequence in SEQ ID NO: 5. The nucleotide sequence set forth in SEQ ID NO: 5 is obtained by binding the nucleotide sequence of the primer for amplification of aptamer (SEQ ID NOs: 3 and 4) to the 5 'side and 3' side of the nucleotide sequence set forth in SEQ ID NO: 38 is there.

[Experimental Example 2]
(Site of DNA aptamer that binds to FGFR1)
From the full-length 84-base DNA aptamer (SEQ ID NO: 5) obtained in Experimental Example 1, a site that binds to FGFR1 was identified.

Analysis of the three-dimensional structure of a full-length 84-base DNA aptamer having a nucleotide sequence shown in SEQ ID NO: 5 indicated that two loop structures were linked. This structure is shown in FIG. For analysis of the three-dimensional structure, Mfold software was used (see Zuker M., Mfold web server for nucleic acid folding and hybridization prediction, Nucleic Acids Res., 31 (13), 3406-3415, 2003).

As a result, a loop structure and a stem structure connected to the loop structure were found. The sequences other than the stem structure and the loop structure are the sequences of the primers used in Experimental Example 1. The loop is named loop30 (L30), and the structure consisting of stem and loop is named stem-loop 38 (SL38). The nucleotide sequence of L30 is represented by SEQ ID NO: 6, and the nucleotide sequence of SL38 is represented by SEQ ID NO: 7.

The 5 'end of FITC-labeled DNA was synthesized from SL38 Rev, which is the reverse sequence of full-length 84 bases SL38, L30, and SL38. The sequence of SL38 Rev is the sequence represented by SEQ ID NO: 8. The binding of these sequences to FGFR1-Fc was analyzed by flow cytometry. The results are shown in FIG.

FIG. 10 is a graph showing the results of analysis of the binding of each DNA to FGFR1-Fc beads by flow cytometry. In FIG. 10, the vertical axis of the graph indicates the average value of the fluorescence intensity. Also, "Full 84" indicates that the result is a full-length DNA of 84 bases, "SL 38" indicates that it is the result of SL 38, "L 30" indicates that it is the result of L 30, and "SL 38 Rev" indicates that it is SL 38 Rev Show that it is the result of

As a result, it was revealed that the full-length 84 bases SL38 and L30 bind to FGFR1. Of the three DNAs, SL38 bound most strongly to FGFR1.

[Experimental Example 3]
(Stem structure and stability)
The role of stem sequences in the stability of the aptamer was analyzed. SL38 was predicted to have a 4-base stem structure consisting of A and T linked to the loop structure. In order to enhance the stability of SL38, SL38.2 was synthesized in which the base sequence of the stem structure was modified to C or G. The nucleotide sequence of SL38.2 is shown in SEQ ID NO: 9.

Subsequently, SL38 and SL38.2 were incubated in 10% fetal bovine serum and analyzed for resistance to nucleases. The sample after incubation was developed by polyacrylamide gel electrophoresis.

FIG. 11 is a photograph showing the results of analysis of DNA after 1 hour to 4 hours by adding SL38 or SL38.2 to serum. As a result, it was revealed that SL38 decomposed but SL38.2 did not decompose after 4 hours. From these results, it was revealed that SL38.2 had higher resistance to nucleases than SL38.

[Experimental Example 4]
(Binding of SL38.2 with FGFR1)
The avidity of SL38.2 and FGFR1 was analyzed by flow cytometry and surface plasmon resonance (SPR).

SL38 and SL38.2 in which the 5 'end was labeled with FITC were prepared. In addition, FGFR1-Fc was immobilized on beads. The avidity of labeled SL38 or SL38.2 and FGFR1-Fc was analyzed by flow cytometry. The results are shown in FIG. 12 (a). In FIG. 12 (a), "Vehicle" is the result of a negative control.

As a result, it was revealed that SL38.2 has the same binding ability as that of SL38 for FGFR1. Given the stability to nucleases, SL38.2 was considered to be a better aptamer than SL38.

Next, a solution containing the extracellular domain of FGFR1 was flowed to the sensor chip on which SL38.2 was immobilized, and the SPR signal was analyzed. FIG. 12 (b) is a graph showing the analysis results of the binding strength of SL38.2 and FGFR1 extracellular domain (FGFR1_ECD).

As a result, the dissociation constant of SL38.2 and FGFR1 extracellular domain was 13 nM. The analysis results of the binding strength of SL38.2 and bFGF are shown in Table 2. The data of bFGF are from Ibrahimi O. A., Kinetic model for FGF, FGFR, and proteoglycan signal transduction complex assembly, Biochemistry, 43 (16), 4724-4730, 2004. As a result, it was revealed that SL38.2 has an avidity equivalent to bFGF for the FGFR1 extracellular domain.

[Experimental Example 5]
(Binding of SL38.2 and FGFR1 to 4)
The binding specificity of SL38.2 and FGFR1-4 was analyzed by flow cytometry. Specifically, first, SL38.2 labeled with FITC at the 5 'end was prepared. In addition, FGFR1-Fc, FGFR2-Fc, FGFR3-Fc, and FGFR4-Fc were fixed to beads, respectively. Subsequently, the binding force of the labeled SL38.2 and each FGFR-Fc fixed bead was analyzed by flow cytometry.

FIG. 13 is a graph showing the results of analysis of the binding strength between the labeled SL38.2 and each FGFR-fixed bead. As a result, it was revealed that SL38.2 specifically binds to FGFR1 among FGFR1 to 4.

[Experimental Example 6]
Role of stem structure in formation of guanine quadruplex structure
The guanine quadruplex structure of SL38 and SL38.2 was analyzed by CD spectroscopy. Guanine quadruplexes can be classified into "parallel" and "antiparallel" from the topology. The presence of the parallel guanine quadruple chain structure can be confirmed by CD spectral measurement, which detects a negative peak around 245 nm and a positive peak around 265 nm. Further, since the formation of guanine quadruplex structure requires the K ⁺ ions, and the polynucleotide in a solution containing K ⁺ ions, to the polynucleotide in a solution containing no K ⁺ ions, the CD spectrum measurements By comparison, the guanine quadruplex structure can be confirmed more reliably.

First, the guanine quadruplex structure of SL38 was analyzed by CD spectroscopy. 14 (a) is a ^{K +} ion absence were measured in the presence ^{K +} ions, a CD spectrum of SL38.

As a result, it was revealed that SL38 changed its spectrum by the addition of K ⁺ ions. From these results, it was revealed that SL38 has an antiparallel guanine quadruplex structure.

The guanine quadruplex structures of SL38 and SL38.2 were compared by CD spectroscopy. The results are shown in FIG. 14 (b). As a result, it was revealed that SL38 and SL38.2 changed their spectra upon addition of K ⁺ ions. This result indicates that SL38 and SL38.2 both have antiparallel guanine quadruplex structure.

[Experimental Example 7]
(Role of G-rich loop in formation of guanine quadruplex structure)
The base sequence of the G-rich loop was modified to analyze the role of the G-rich loop in the formation of guanine quadruplex structure.

SL38 has guanine at positions 14 to 15, 18 to 21, 24 to 25, and 29 to 31 from the 5 'end. These four locations are G-rich loops, which have high guanine content.

"G14T" was generated by mutating the 14th guanine from the 5 'end of SL38 to thymine. In addition, "G24T" was prepared by mutating the 24th guanine from the 5 'end of SL38 to thymine. The CD spectrum was analyzed about these. The results are shown in FIG.

Figure 15 is a ^{K +} ion absence was measured under ^{K +} ions present, a CD spectrum of G14T and G24T. As a result, it became clear that G14T and G24T do not have guanine quadruple chain structure. From these results, it was revealed that the 14th and 24th guanines from the 5 'end of SL38 are required for the formation of the guanine quadruplex structure.

[Experimental Example 8]
(Role of guanine quadruplex structure in the binding of SL38 and FGFR1)
We analyzed the binding strength of FGFR1 with G14T or G24T, which does not form guanine quadruplex structure, and examined the role of guanine quadruplex structure in the binding of SL38 and FGFR1.

A DNA in which the 5 'end of SL38 Rev, G14T, G24T, which is the reverse sequence of SL38, SL38, was labeled with FITC was prepared. In addition, FGFR1-Fc was immobilized on beads. Subsequently, the binding power of the labeled DNA and the FGFR1-Fc fixed beads was analyzed by flow cytometry. The results are shown in FIGS. 16 (a) and (b).

FIG. 16 (a) is a graph showing the results of analysis of the binding of each DNA to FGFR1-Fc beads by flow cytometry. "Vehicle" shows that it is a result of a negative control in FIG. 16 (a). FIG. 16 (b) is a graph showing the average value of fluorescence intensity. As a result, it was revealed that G14T and G24T do not bind to FGFR1.

This result revealed that the 14th and 24th guanines of SL38 are important in binding to FGFR1. In addition, it was revealed that the presence or absence of binding to FGFR1 was correlated with the presence or absence of guanine quadruplex structure.

[Experimental Example 9]
Role of stem structure in thermal stability
The melting temperatures of SL38 and SL38.2 were calculated, and the role of stem structure in thermal stability was analyzed. Specifically, the temperature was changed from 10 ° C. to 70 ° C. for SL38 and SL38.2, and the CD spectrum at 295 nm was measured. The results are shown in FIG. FIG. 17 is a graph showing the results of measurement of CD spectra at 295 nm of SL38 and SL38.2 under each temperature.

As a result, it was revealed that the melting temperature of SL38 was 39 ° C., and the melting temperature of SL38.2 was 47 ° C. From this result, it was revealed that the thermostability of the aptamer structure is enhanced by mutating the stem structure and replacing the binding between adenine and thymine with the stronger binding between guanine and cytosine.

[Experimental Example 10]
(Aptamer bound to tandem)
The activation of FGFR1 was analyzed by the aptamer bound in tandem. FGFR1 is known to dimerize and activate upon binding to bFGF. It was examined whether aptamers linked in tandem dimerize and activate FGFR1. The predicted structure of SL38.2_dimer in which SL38.2 is linked in tandem is shown in FIG.

We synthesized SL38 and SL38.2, which are aptamers having a stem-loop structure. The sequence of SL38 is the sequence represented by SEQ ID NO: 7. In addition, SL38_dimer in which SL38 was coupled in tandem, and SL38.2_dimer in which SL38.2 were coupled in tandem were prepared. The sequence of SL38_dimer is the sequence shown in SEQ ID NO: 10, and the sequence of SL38.2_dimer is the sequence shown in SEQ ID NO: 11.

After contacting 2 nM bFGF, 200 nM SL38, 200 nM SL38.2, 50 nM or 100 nM SL38_dimer, 50 nM or 100 nM SL38.2_dimer respectively with A204 cells, the lysate of A204 cells was developed by SDS-PAGE. Subsequently, immunoblot analysis was performed using an anti-FGFR1 antibody and an anti-phosphorylated FGFR1 antibody. The results are shown in FIG. 18 (b).

As a result, SL38_dimer or SL38.2_dimer phosphorylated FGFR1, but SL38 or SL38.2 did not phosphorylate FGFR1. From this result, it was revealed that the aptamer bound in tandem strongly activated FGFR1.

Subsequently, Rev-SL38.2_dimer having a reverse base sequence of SL38.2_dimer was produced. The nucleotide sequence of Rev-SL38.2_dimer is shown in SEQ ID NO: 12. Subsequently, after contacting 2 nM bFGF, 10, 50, 100 and 500 nM SL38.2_dimer and 500 nM Rev-SL38.2_dimer respectively with A204 cells, the disrupted A204 cells were developed by SDS-PAGE.

Subsequently, immunoblot analysis was performed using an anti-FGFR1 antibody, an anti-phosphorylated FGFR1 antibody, an anti-phosphorylated Akt antibody, and an anti-phosphorylated Erk1 / 2 antibody. FIG. 18 (c) shows the results of analysis of phosphorylation of FGFR, Akt and Erk1 / 2 by bFGF, SL38.2_dimer and Rev-SL38.2_dimer.

As a result, it was revealed that 10 nM SL38.2_dimer phosphorylates FGFR1, Akt and Erk1 / 2 to the same extent as 2 nM bFGF.

From the above results, it has become clear that monomeric SL38 and SL38.2 bind to FGFR1 but can not activate FGFR1. On the other hand, it was revealed that SL38_dimer and SL38.2_dimer which dimerized SL38 and SL38.2 activate FGFR1.

[Experimental Example 11]
(Activation of FGFR1 pathway by aptamer bound to tandem)
Activation of FGFR, downstream PI3K / Akt pathway and MAPK pathway was analyzed by SL38.2_dimer.

The cells were recovered after 1, 5, 10, 15, 30 minutes by contacting A204 cells with 2 nM bFGF or 50 nM SL38.2_dimer. Subsequently, the disrupted cells were analyzed by SDS-PAGE and immunoblot.

FIG. 19 shows the results obtained by contacting A204 cells with bFGF or SL38.2_dimer, and analyzing phosphorylation of FGFR1, Akt, and Erk1 / 2 after each time. In FIG. 19, "Vehicle" is the result of a negative control. As a result, bFGF was confirmed to phosphorylate FGFR continuously for 1 minute to 30 minutes after stimulation. On the other hand, when stimulated with SL38.2_dimer, it was revealed that the phosphorylation of FGFR1 was attenuated 5 to 30 minutes after stimulation, compared with 1 minute after stimulation.

[Experimental Example 12]
Maintenance of pluripotency of human iPS cells by aptamers linked in tandem
The expression level of pluripotency markers was analyzed in SL38.2 dimer-stimulated human iPS cells. As pluripotent markers, Oct-4, Nanog and SSEA-4 were examined.

Human iPS cells (409B2 cells) were stimulated by 3 nM bFGF, 500 nM SL38.2_dimer, 500 nM Rev-SL38.2_dimer, and the expression levels of Oct-4 and Nanog were analyzed by RT-qPCR.

FIG. 20 (a) is a graph showing the results of analysis of the expression levels of Oct-4 and Nanog after stimulation by RT-qPCR and normalization with the housekeeping gene RPLP0. "Vehicle" shows the result of a negative control in FIG. 20 (a). As a result, like bFGF, SL38.2_dimer was found to have the effect of maintaining the expression of pluripotency marker gene.

Subsequently, the effects of stimulation by bFGF, SL38.2_dimer and Rev-SL38.2_dimer on cell proliferation were analyzed. Specifically, human iPS cells (409B2 cells) were stimulated with 3 nM bFGF, 500 nM SL38.2_dimer, 500 nM Rev-SL38.2_dimer, and then the number of cells was counted.

FIG. 20 (b) is a graph showing the results of measuring the number of cells. As a result, it was revealed that stimulation by SL38.2_dimer promotes proliferation of iPS cells to the same extent as bFGF.

Experimental Example 13
Human iPS cells were stimulated with SL38.2_dimer to analyze changes in the expression level of the pluripotency marker SSEA-4 protein.

Human iPS cells (409B2 cells) were stimulated with 3 nM bFGF, 500 nM SL38.2_dimer, 500 nM Rev-SL38.2_dimer, and the expression level of SSEA-4 protein was analyzed by fluorescence immunostaining.

FIG. 21 is a photograph showing the results of fluorescent immunostaining. In FIG. 21, "PH image" shows that it is a result of a phase-contrast photomicrograph, "Nuclei (Hoechst)" shows that it is a result of staining a nucleus with Hoechst 33342, and "SSEA-4" shows fluorescence immunostaining. It shows that it is the result of detecting the expression of SSEA-4 protein by. Moreover, "Vehicle" shows that it is a result of a negative control.

As a result, it was revealed that stimulation with SL38.2_dimer promotes the expression of SSEA-4 protein to the same extent as bFGF. This result further supports that SL38.2_dimer can be used as a substitute for bFGF.

[Experimental Example 14]
(Synthesis of dimeric DNA aptamer)
Dimeric DNA aptamers of various binding modes were synthesized. Specifically, an aptamer in which the linker for linking the aptamers is a single-stranded DNA, and an aptamer in which the linker for linking the aptamers is a double-stranded DNA were synthesized.

FIG. 22 (a) is a schematic view showing the structures of DP20 and DP0, which are aptamers in which the linker for linking the dimers is double-stranded DNA. DP20 is a dimer DNA aptamer in which DP_A represented by SEQ ID NO: 13 and DP_B represented by SEQ ID NO: 14 are hybridized and the linker for linking the aptamers is a double-stranded DNA. DP_A has a base sequence in which the linker sequence represented by SEQ ID NO: 15 is linked to the 3 'end of the base sequence of SL38.2. DP_B has a base sequence in which the linker sequence represented by SEQ ID NO: 16 is linked to the 3 'end of the base sequence of SL38.2.

Further, DP0 is a dimer DNA aptamer in which DP_A represented by SEQ ID NO: 13 and DP_C represented by SEQ ID NO: 17 are hybridized, and the linker for linking the aptamers is a double-stranded DNA. is there. DP_C has a base sequence in which the linker sequence represented by SEQ ID NO: 18 is linked to the 5 'end of the base sequence of SL38.2. In addition, the stem sequence in DP_C was replaced with a nucleotide sequence different from DP_A in order to suppress hybridization with the stem sequence of DP_A.

FIG. 22 (b) is a schematic view showing the structures of SL38.2_dimer and SL38.2_dimer 20, which are aptamers in which the linker for linking each other is single-stranded DNA.

The nucleotide sequence of SL38.2_dimer 20 is a sequence represented by SEQ ID NO: 19. SL38.2_dimer 20 has a base sequence in which two SL38.2s are linked by 20 t (thymine) consecutive linkers.

The two loop structures of SL38.2_dimer are close, but the two loop structures of SL38.2_dimer20 are more distant than SL38.2_dimer. Also, while the two loop structures of DP0 are close, the two loop structures of DP20 are further apart than DP0.

[Experimental Example 15]
(Activation of FGFR1 pathway by dimeric DNA aptamer)
The activation of the FGFR1 pathway by dimeric DNA aptamers was analyzed. Specifically, SL38.2_dimer, Rev-L38.2_dimer, SL38.2_dimer20, DP20, DP0, and 2 nM bFGF were respectively contacted with A204 cells at 500 nM, respectively.

Subsequently, after 5 minutes, each cell was disrupted to obtain a disrupted cell solution. Phosphorylation of FGFR1 was analyzed by PathScan® phospho-FGF receptor 1 (panTyr) sandwich ELISA kit.

FIG. 23 (a) is a graph showing the results of measuring the phosphorylation of FGFR1. In FIG. 23 (a), "vehicle" is a negative control.

As a result, it was revealed that SL38.2_dimer, SL38.2_dimer20, DP20 and DP0, like bFGF, promote the phosphorylation of FGFR1. From these results, it was revealed that SL38.2_dimer, SL38.2_dimer20, DP20, and DP0 activate FGFR1.

Next, SL38.2_dimer, Rev-L38.2_dimer, SL38.2_dimer20, DP20, DP0, and 2 nM bFGF of 500 nM, respectively, were contacted with A204 cells and analyzed for phosphorylation of Erk1 / 2.

FIG.23 (b) is a photograph which shows an analysis result. As a result, it was revealed that SL38.2_dimer, SL38.2_dimer20, DP20 and DP0 promote Erk1 / 2 phosphorylation similarly to bFGF. From these results, it was revealed that SL38.2_dimer, SL38.2_dimer20, DP20 and DP0 activate Erk1 / 2.

[Experimental Example 16]
(Maintaining pluripotency of iPS cells by stimulation of dimeric DNA aptamer)
The expression of pluripotency markers in iPS cells by stimulation of dimeric DNA aptamer was analyzed.

The iPS cells (409B2 cells) were cultured for 7 days in a medium containing 500 nM of SL38.2_dimer, Rev-L38.2_dimer, SL38.2_dimer20, DP20, DP0, and 3 nM bFGF, respectively. The culture medium was changed at 1, 3 and 5 days after the start of culture. Subsequently, iPS cells were stained with Alexa 488-labeled anti-SSEA-4 antibody, and the expression of SSEA-4 protein was analyzed by flow cytometry.

FIG. 24 (a) is a graph showing the results of analysis of expression of SSEA-4 protein. In FIG. 24 (a), the horizontal axis shows fluorescence intensity, and the vertical axis shows the number of cells. Further, FIG. 24 (b) is a graph showing an average value of fluorescence intensity in each cell of FIG. 24 (a). In FIG. 24 (a) and (b), "Vehicle" shows the result of a negative control.

As a result, when SL38.2_dimer was added to the medium, it became clear that SSEA-4 protein was expressed to the same extent as when bFGF was added to the medium. On the other hand, when Rev-L38.2_dimer, SL38.2_dimer20, DP20, and DP0 were added to the medium, the expression level of SSEA-4 protein was equivalent to that in the control group.

As a result, SL38.2_dimer, SL38.2_dimer20, DP20, DP0, like bFGF, can activate the FGFR1 pathway, but the activity to maintain pluripotency of iPS cells is highest at SL38.2_dimer Indicates that.

1, 2, 3 ... aptamers, 4, 5, 6, 7, 8a, 8b, 9a, 9b, 10 ... multistructural aptamers, 10 ... polynucleotides, 20 ... loop structures, 30 ... stem structures, 40, 40a, 40b , 40c ... linker, 100, 101 ... aptamer fixed carrier, 110, 120 ... solid phase carrier.

Claims

An aptamer comprising a nucleotide sequence set forth in SEQ ID NO: 1 and comprising a polynucleotide having an activity of binding to Fibroblast Growth Factor Receptor 1 (FGFR1).
The aptamer according to claim 1, which has a loop structure in which a polynucleotide consisting of the base sequence set forth in SEQ ID NO: 1 forms at least a part.
The aptamer according to claim 2, wherein the loop structure consists of a polynucleotide strand having 28 to 40 bases.
The aptamer of Claim 2 or 3 which has a stem structure which consists of a double stranded polynucleotide linked to the said loop structure.
The aptamer according to any one of claims 1 to 4, which forms a guanine quadruplex structure.
An aptamer having an activity of binding to FGFR1 and activating FGFR1 in which two or more of the aptamers according to any one of claims 1 to 5 are linked.
The aptamer according to claim 6, wherein the aptamer according to any one of claims 1 to 5 is linked by a linker, and the length of the linker is 80 bases or less in length in terms of polynucleotide.
The nucleotide sequence set forth in SEQ ID NO: 11 or the nucleotide sequence set forth in SEQ ID NO: 11 consisting of a nucleotide sequence in which one or several bases have been deleted, substituted or added, and binding to FGFR1 to activate FGFR1 The aptamer of Claim 6 or 7 which has these.
An agent for the prophylaxis or treatment of a disease associated with FGFR1 signaling, which comprises the aptamer according to any one of claims 1 to 8 as an active ingredient.
A composition for cell culture, comprising the aptamer according to any one of claims 1 to 8 as an active ingredient.
A cell culture method comprising culturing an FGFR1 positive cell in a culture medium containing the aptamer according to any one of claims 1 to 8.
A method for inhibiting the binding of bFGF to the FGFR1 comprising contacting the aptamer according to any one of claims 1 to 5 with an FGFR1 positive cell.
A method for suppressing the growth of cells, which comprises bringing the aptamer according to any one of claims 1 to 5 into contact with FGFR1 positive cells.
The method for activating FGFR1, which comprises contacting the aptamer according to any one of claims 6 to 8 with FGFR1 positive cells.
A method for promoting the growth of cells, comprising contacting the aptamer according to any one of claims 6 to 8 with FGFR1 positive cells.
A method for maintaining pluripotency of the pluripotent stem cells, comprising contacting the aptamer according to any one of claims 6 to 8 with FGFR1 positive pluripotent stem cells.