KR20230131326A - Insulin receptor aptamer and uses thereof - Google Patents

Abstract

The present invention relates to a DNA aptamer that specifically binds to an insulin receptor (IR), a pharmaceutical composition for preventing or treating insulin-related diseases, and a diagnostic composition containing the same.
The aptamer of the present invention can be applied to the prevention, treatment, and diagnosis of insulin-related diseases such as diabetes, diabetic complications, metabolic syndrome, obesity, and cardiovascular disease.

Description

Insulin receptor specific aptamer and uses thereof {Insulin receptor aptamer and uses thereof}

The present invention relates to an aptamer that specifically binds to an insulin receptor (IR), a pharmaceutical composition for preventing or treating insulin-related diseases, and a diagnostic composition containing the same.

Insulin is a blood sugar control hormone secreted by the human pancreas. It transports excess glucose in the blood to cells, supplies energy to the cells, and maintains blood sugar at a normal level. However, in the case of diabetic patients, insulin does not perform its normal function due to insufficient insulin or insulin resistance and loss of beta cell function. As a result, diabetic patients are unable to use glucose in the blood as an energy source and exhibit symptoms of hyperglycemia with high blood glucose levels, ultimately leading to sugar being excreted through urine, which causes various complications. Therefore, patients who have abnormalities in insulin production (Type 1, Type I) or who develop diabetes due to insulin resistance (Type 2, Type II) can control their blood sugar to a normal level by administering insulin.

Today, many types of insulin derivatives have been developed and used to normally control blood sugar in diabetic patients. However, insulin induces cell division in addition to glucose uptake, and changes in the amino acid sequence introduced into some insulin derivatives increase binding to and activation of the insulin-like growth factor 1 (IGF-1) receptor. Long-term insulin administration for the treatment of diabetes can cause atherosclerosis, and it has been reported that there is a significant correlation between continued insulin administration and an increase in the incidence of cancer. Concerns about the side effects caused by insulin have been continuously raised. .

Therefore, the development of a biased agonist for the insulin receptor that only increases glucose uptake without inducing cell division could be a good alternative.

Meanwhile, an aptamer is a single-stranded nucleotide such as DNA or RNA that has a stable tertiary structure and the ability to bind to target molecules with high affinity and specificity. Since 1990, many aptamers that can bind to various target molecules, including low-molecular-weight organic substances, peptides, and membrane proteins, have been discovered. Aptamers have the property of being able to selectively bind to target molecules with high binding force and specificity, so they can be used to identify specific substances.

Currently, most efforts to discover functional aptamers using the high binding affinity and specificity of aptamers are focused on the aptamer's ability to inhibit the target. In particular, for clinical applications, various types of inhibitory aptamers that interfere with the activity of target molecules have been developed to treat diseases (e.g. Macugen, Fovista). However, considering that interactions between molecules are inevitably accompanied by structural changes, it is believed that activation of protein function will be possible if aptamer-protein binding can induce appropriate structural changes in the protein. Therefore, in theory, aptamers have the potential to act as functional agents by mimicking specific protein-protein bonds.

However, currently the development of agonist aptamers that activate the function of the target remains a difficult problem.

Under this background, the present inventors attempted to develop a treatment for insulin-related diseases with reduced side effects, and as a result, developed an aptamer that induces selective phosphorylation of the insulin receptor and activates only signaling related to glucose uptake, and developed an insulin-related aptamer containing the same. The present invention was completed by developing pharmaceutical compositions for disease prevention or treatment and diagnostic compositions.

1. Mary E Herman, et al., Prog Cardiovasc Dis. Nov-Dec 2017;60(3):422-434.

One object of the present invention is to provide an insulin receptor-specific aptamer.

Another object of the present invention is to provide an insulin receptor agonist comprising the insulin receptor-specific aptamer.

Another object of the present invention is to provide a pharmaceutical composition for preventing or treating insulin-related diseases, comprising the insulin receptor-specific aptamer as an active ingredient.

Another object of the present invention is to provide a method for preventing or treating insulin-related diseases, comprising administering the pharmaceutical composition to an individual.

Another object of the present invention is to provide a composition for diagnosing diabetes or diabetic complications, including the insulin receptor-specific aptamer.

This is explained in detail as follows. Meanwhile, each description and embodiment disclosed in the present invention may also be applied to each other description and embodiment. That is, all combinations of the various elements disclosed in the present invention fall within the scope of the present application. Additionally, the scope of the present invention cannot be considered limited by the specific description described below.

One aspect of the present invention provides an insulin receptor specific aptamer.

In the present invention, the term "aptamer" refers to a "Chemical Antibody", which is a short-length (20 to 80 bases) single strand that has the property of binding to various types of target ligands, from specific compounds to proteins, with high specificity and affinity. refers to a nucleic acid molecule. Aptamers can be manufactured in vitro through SELEX (Systematic evolution of ligands by exponential enrichment).

Aptamers are considered to be oligonucleotide molecules with similar properties to antibodies in that they have high binding affinity and selectivity at nanomolar (nM) to femtomolar (fM) levels for target proteins. Meanwhile, compared to antibodies, aptamers have several advantages: (1) aptamers manufactured through chemical synthesis are easier to achieve the desired chemical mutation than protein-based materials such as antibodies, and (2) the SELEX process selectivity and affinity can be maximized through chemical synthesis, (3) the purity is high because it is made through chemical synthesis, (4) the created material can be identified through instrumental analysis, and (5) it is heat stable and can be stored for a long time at room temperature. do.

In the present invention, the term "SELEX (Systematic evolution of ligands by exponential enrichment)" refers to a method of selecting aptamers that bind to a target substance. In a generally known method, a target protein is reacted with an oligonucleotide library (DNA or RNA) having a random base sequence at a certain temperature, and then the DNA/RNA library that does not bind to the target is removed. After separating the nucleotide bound to the target, it is amplified using a gene amplification method and the above process is repeated several times to select an aptamer with high binding affinity to the target.

In the case of the present invention, an aptamer was prepared by modifying the general SELEX method as described above, and the specific method is described below.

The aptamer selection process generally requires the process of securing a single-stranded nucleic acid pool from a library having about 10 ¹⁴ to 10 ¹⁵ different sequences, that is, diversity. There are several methods used for this, but generally, asymmetric PCR is used to amplify only one strand, and biotin is attached to the end of one strand of a double-stranded nucleic acid and then a bead is wrapped with streptavidin. The most widely used method is to selectively separate only one strand using a bead.

Afterwards, a selection process is performed to select aptamers with high binding affinity by binding the secured library to the target molecule. When the target molecule is a protein, the biotin labeled on the protein is usually pulled down using streptavidin beads. After inducing binding of the target protein and the modified nucleic acid library, the modified nucleic acid library that does not bind to the target protein is removed by washing with buffer.

Similarly, in the case of plates, the binding between the nucleic acid library and the target protein is induced and then washed with buffer to remove nucleic acids that do not bind to the target protein. Using these methods, aptamers with affinity for the ligand can be obtained. Usually, an aptamer with high affinity can be obtained by repeating the selection-amplification process 5-15 times. When the selection process is completed, the amplified nucleic acid is cloned, the sequence is confirmed through sequence analysis from each clone, and an aptamer is synthesized to measure affinity and binding force with the target molecule.

In the present invention, the term “target molecule” refers to a substance that can be detected with the aptamer of the present invention. Specifically, the target molecule is present in the separated sample and may be a protein, peptide, carbohydrate, polysaccharide, glycoprotein, hormone, receptor, antigen, antibody, virus, cofactor, drug, or protein to which the capture aptamer can bind. It may be one or more selected from the group consisting of dyes, growth factors, and controlled substances, but is not limited thereto. For the purposes of the present invention, the target molecule may be, but is not limited to, an insulin receptor.

In the present invention, the term “insulin receptor aptamer” refers to an aptamer that can bind to insulin with specific affinity. The insulin receptor (IR) may be derived from a human insulin receptor, but is not limited thereto. That is, the aptamer of the present invention may be characterized by specifically binding to the insulin receptor.

The aptamer of the present invention may consist of 10 to 90 nucleotides, specifically 15 to 80 nucleotides, and more specifically 20 to 50 nucleotides, but is not limited thereto.

The polynucleotide sequence constituting the aptamer of the present invention may include a chemically or physically modified form of one or more of the nucleotides constituting the polynucleotide sequence.

The chemical or physical modification may include, for example, the nucleotides constituting the base sequence, wherein at least one of the nucleotides is phosphorothioate nucleic acid, phosphorodithioate nucleic acid, phosphoroamidate nucleic acid, amide-linked nucleic acid, MMI -It may contain one or more modifications selected from the group consisting of linked nucleic acids, 2'-O-methyl RNA, alpha-nucleic acids and methylphosphonate nucleic acids, sugar-modified nucleotides and nucleotides with base modifications, but Not limited.

The sugar modified nucleotides include 2'-O-methyl RNA, 2'-fluoro RNA, 2'-amino RNA, 2'-O-alkyl nucleic acid, 2'-O-allyl nucleic acid, 2'-O-alkynyl It may be one or more selected from the group consisting of nucleic acid, hexose nucleic acid, pyranosyl RNA, and anhydrohexitol nucleic acid, but is not limited thereto.

Nucleotides having the base modification include pyrimidine with a substituent at C-5 (5' carbon, 5th carbon), thymine with a substituent at C-5, dUTP (deoxyuracil) with a substituent at C-5, and C-7. It may be one or more selected from the group consisting of 7-deazapurine, inosine, and diaminopurine having a substituent, but is not limited thereto.

Specifically, in pyrimidine having a substituent at C-5, which is one of the nucleotides having the base modification, the substituent is a naphthyl group, fluoro-, bromo-, chloro-, iodo-, methyl-, ethyl-, It may be vinyl-, formyl-, ethyl-, propynyl-, alkynyl-, thiazoryl-, imidazoryl-, and pyridyl-, etc., but is not limited thereto.

In dUTP, which has a substituent at C-5, which is one of the nucleotides with the base modification, the substituent may be a hydrophobic substituent, and the hydrophobic substituent is, for example, a benzyl group, pyrrolebenzyl group, phenylethyl group, and phenylpropyl group. , thiophenylmethyl group, fluorobenzyl group, naphthyl group, 2-naphthyl group, naphthyl-2-ethyl group, 2-naphthyl-2-ethyl group, tryptophan group, 3-benzothiophenyl-2-ethyl group, 3-benzofura Nyl-2-ethyl group, benzimidazole-2-ethyl group, tyrosine group, pyridinylmethyl group, methylenedioxybenzyl group, 3-methylenedioxyphenyl-2-ethyl group, 3-methoxybenzyl group, 4-methoxybenzyl group, 3,4-dimethoxybenzyl group, R-tetrahydrofuran group, S-tetrahydrofuran group, morpholino-2-ethyl group, threonine group, tryptophan group, and isobutyl group, but is not limited thereto.

For example, substitution with the benzyl group includes substitution with 5-(N-benzylcarboxyamide)-2'-deoxyuridine (5'-(N-benzylcarboxyamide)-2'-deoxyuridine, BndU), and the phenylethyl group. Substitution with 5-(N-2-phenylethylcarboxyamide)-2'-deoxyuridine (5-(N-2-phenylethylcarboxyamide)-2'-deoxyuridine, PedU), substitution with the phenylpropyl group Substitution with 5-(N-3-phenylpropylcarboxyamide)-2'-deoxyuridine (5-(N-3-phenylpropylcarboxyamide)-2'-deoxyuridine, PpdU), with the thiophenylmethyl group Substitution is with 5-(N-2-thiophenylmethylcarboxyamide)-2'-deoxyuridine (5-(N-2-thiophenylmethylcarboxyamide)-2'-deoxyuridine, ThdU), with the fluorobenzyl group. Substitution with 5-(N-4-fluorobenzylcarboxyamide)-2'-deoxyuridine (5-(N-4-fluorobenzylcarboxyamide)-2'-deoxyuridine, FBndU), with the naphthyl group Substitution with 5-(N-1-naphthylmethylcarboxyamide)-2'-deoxyuridine (5-(N-1-naphthylmethylcarboxyamide)-2'-deoxyuridine, NapdU), the 2-naph Substitution with a thyl group is 5-(N-2-naphthylmethylcarboxyamide)-2'-deoxyuridine (5-(N-2-naphthylmethylcarboxyamide)-2'-deoxyuridine, 2NapdU), the naphthyl-2 -Substitution with an ethyl group is 5-(N-1-naphthyl-2-ethylcarboxyamide)-2'-deoxyuridine (5-(N-1-naphthyl-2-ethylcarboxyamide)-2'-deoxyuridine, Substitution with NedU), substitution with the 2-naphthyl-2-ethyl group is 5-(N-2-naphthyl-2-ethylcarboxyamide)-2'-deoxyuridine (5-(N-2 -naphthyl-2-ethylcarboxyamide)-2'-deoxyuridine, 2NedU), substitution with the tryptophan group is 5-(N-3-indole-2-ethylcarboxyamide)-2'-deoxyuridine (5 -(N-3-indole-2-ethylcarboxyamide)-2'-deoxyuridine, TrpdU), substitution with the 3-benzothiophenyl-2-ethyl group is 5-(N-3-benzothiophenyl-2 -Ethylcarboxyamide)-2'-deoxyuridine (5-(N-3-benzothiophenyl-2-ethylcarboxyamide)-2'-deoxyuridine, BtdU), substituted with the 3-benzofuranyl-2-ethyl group Substitution of 5-(N-3-benzofuranyl-2-ethylcarboxyamide)-2'-deoxyuridine (5-(N-3-benzofuranyl-2-ethylcarboxyamide)-2'-deoxyuridine, BfdU) Substitution with, substitution with the benzimidazole-2-ethyl group is 5-(N-1-benzimidazole-2-ethylcarboxyamide)-2'-deoxyuridine (5-(N-1-benzimidazol -2-ethylcarboxyamide)-2'-deoxyuridine, BidU), substitution with the tyrosine group is 5-(N-4-hydrophenyl-2-ethylcarboxyamide)-2'-deoxyuridine (5- Substitution with (N-1-hydroxyphenyl-2-ethylcarboxyamide)-2'-deoxyuridine, TyrdU), and substitution with the pyridinylmethyl group is 5-(N-4-pyridinylmethylcarboxyamide)-2'-deoxy Substitution with uridine (5-(N-4-pyridinylmethylcarboxyamide)-2'-deoxyuridine, PyrdU), and substitution with the methylenedioxybenzyl group is 5-(N-3,4-methylenedioxybenzylcarboxyamide)- Substitution with 2'-deoxyuridine (5-(N-3,4,-methylenedioxybenzylcarboxyamide)-2'-deoxyuridine, MBndU), substitution with 3-methylenedioxyphenyl-2-ethyl group is 5-(N -3,4-methylenedioxyphenyl-2ethylcarboxyamide)-2'-deoxyuridine (5-(N-3,4-methylenedioxyphenyl-2ethylcarboxyamide)-2'-deoxyuridine, MPedU), above Substitution with 3-methoxybenzyl group is 5-(N-3-methoxybenzylcarboxyamide)-2'-deoxyurinde (5-(N-3-methoxybenzylcarboxyamide)-2'-deoxyurinde, 3MBndU) Substitution, substitution with the 4-methoxybenzyl group is 5-(N-4-methoxybenzylcarboxyamide)-2'-deoxyuridine (5-(N-4-methoxybenzylcarboxyamide)-2'-deoxyuridine, 4MBndU ), substitution with the 3,4-dimethoxybenzyl group is 5-(N-3,4-dimethoxybenzylcarboxyamide)-2'-deoxyuridine (5-(N-3,4- substitution with dimethoxybenzylcarboxyamide)-2'-deoxyuridine, 3,4MBndU), and substitution with the R-tetrahydrofuran group is 5-(N-R-tetrahydrofuranylmethylcarboxyamide)-2'-deoxyuridine (5- Substitution with (N-R-tetrahydrofuranylmethylcarboxyamide)-2'-deoxyuridine, RTHFdU), , Substitution with the S-tetrahydrofuran group is 5-(N-S-tetrahydrofuranylmethylcarboxyamide)-2'-deoxyuridine ( Substitution with 5-(N-S-tetrahydrofuranylmethylcarboxyamide)-2'-deoxyurinde, STHFdU), substitution with the morpholino-2-ethyl group is 5-(N-morpholino-2-ethylcarboxyamide)-2'-deoxy Substitution with uridine (5-(N-morpholino-2-ethylcarboxyamide)-2'-deoxyuridine, MoedU), and substitution with the threonine group is 5-(N-R-2-hydropropylcarboxyamide)-2'-deoxy Uridine (5-(N-R-2-hydroxypropylcarboxyamide)-2'-deoxyuridine, ThrdU) and substitution with the isobutyl group is 5-(N-isobutylcarboxyamide)-2'-deoxyuridine (5-( It may be substituted with N-iso-butylcarboxyamide)-2'-deoxyuridine, iBudU), but is not limited thereto.

Specifically, the aptamer of the present invention may include a polynucleotide sequence of General Formula 1 below.

[General Formula 1]

5'- CABBACGCAB GAGBCBAGAB CCGBCAG- 3' (SEQ ID NO: 4);

Here, B in General Formula 1 is deoxyribose uracil (dUTP) in which the 5th carbon position of the nucleotide is substituted with a hydrophobic functional group,

C in General Formula 1 may be a modified or unmodified nucleotide, and the modification may include a linker (C3 linker) binding to the nucleotide; Introduction of a methoxy group (2-O-Me, 2-O-Methoxy-DNA) at the 2nd carbon position of the nucleotide; and introduction of fluorine (2-F, 2-Fluorine-DNA) at the second carbon position of the nucleotide; and one or more modifications selected from the group consisting of,

G in General Formula 1 may be a modified or unmodified nucleotide, and the modification may include a linker bond to the nucleotide; introduction of a methoxy group at the second carbon position of the nucleotide; and introduction of fluorine at the second carbon position of the nucleotide; and one or more modifications selected from the group consisting of,

A of General Formula 1 may be a modified or unmodified nucleotide, and the modification may include a linker bond to the nucleotide; introduction of a methoxy group at the second carbon position of the nucleotide; and introduction of fluorine at the second carbon position of the nucleotide.

In the present invention, the hydrophobic functional group is specifically benzyl group, pyrrolebenzyl group, phenylethyl group, phenylpropyl group, thiophenylmethyl group, fluorobenzyl group, naphthyl group, 2-naphthyl group, naphthyl-2-ethyl group, and 2-naphthyl group. -2-ethyl group, tryptophan group, 3-benzothiophenyl-2-ethyl group, 3-benzofuranyl-2-ethyl group, benzimidazole-2-ethyl group, tyrosine group, pyridinylmethyl group, methylenedioxybenzyl group, 3 -Methylenedioxyphenyl-2-ethyl group, 3-methoxybenzyl group, 4-methoxybenzyl group, 3,4-dimethoxybenzyl group, R-tetrahydrofuran group, S-tetrahydrofuran group, morpholino- It may be any one or more selected from the group consisting of 2-ethyl group, threonine group, tryptophan group, and isobutyl group, and more specifically, any one selected from the group consisting of naphthyl group, benzyl group, pyrrolebenzyl group, isobutyl group, and tryptophan. It may be more than one, and more specifically, it may be one or more selected from a naphthyl group or a benzyl group.

Deoxyribose uracil modified by substitution with the naphthyl group is named naphthyl-uracil nucleotide (Naphthyl-dU) and is represented by W in the base sequence described in the present invention. In addition, uracil modified by substitution with the benzyl group is named benzyl-uracil nucleotide (Benzyl-dU) and is indicated as Y in the base sequence described in the present invention. That is, B in General Formula 1 is naphthyl-uracil nucleotide (5-(N-1-naphthylmethylcarboxyamide)-2'-deoxyuridine, Naphthyl-dU, NapdU) or benzyl-uracil nucleotide (5 -(N-Benzylcarboxyamide)-2'-deoxyuridine, Benzyl-dU, BndU) It may be any one or more selected from among.

The naphthyl group is shown in Formula 1 below, and the benzyl group is shown in Formula 2 below.

[Formula 1]

[Formula 2]

In the present invention, the linker is a nucleotide substituted with a linker, and the base of the nucleotide or the nucleoside containing the base and pentose sugar is removed, but the phosphate backbone remains and the empty space is maintained. It may mean that the nucleotides on both sides of the empty space are connected while maintaining a certain original distance, but it is not limited to this.

The linker of the present invention can be used interchangeably with a 'spacer'. The linker or spacer may be composed of three carbons, but is not limited thereto, as long as it can maintain a certain distance between nucleotides. Specifically, the linker or spacer of the present invention may be a linker or spacer consisting of three carbons, and may be used interchangeably with C3 linker, C3 spacer, or L. The C3 linker may be bound to nucleotides at the 5' end, 3' end, middle, or both ends of the aptamer of the present invention. The structure in which the C3 linker is linked to the 5'-end (Formula 3), 3'-end (Formula 4), and middle (Formula 5) nucleotides of the nucleotide sequence is shown in the formula below.

[Formula 3]

[Formula 4]

[Formula 5]

The aptamer may be one in which 1 to 3 nucleotides are excluded from either the 3' end or the 5' end of the sequence of General Formula 1.

Specifically, the aptamer may include the following polynucleotide sequence.

5'- CABBACGCAB GAGBCBAGAB CCGBC- 3' (SEQ ID NO: 82);

Here, B of the general formula 1 is deoxyribose uracil in which the 5th carbon position of the nucleotide is substituted with a hydrophobic functional group, and a naphthyl-uracil nucleotide in which the 5th carbon position of the dUTP nucleotide is substituted with a naphthyl group; or a benzyl-uracil nucleotide in which the 5th carbon of the dUTP nucleotide is substituted with a benzyl group; One or more of the following,

C in General Formula 1 may be a modified or unmodified nucleotide, and the modification may include a linker bond to the nucleotide; introduction of a methoxy group at the second carbon position of the nucleotide; and introduction of fluorine at the second carbon position of the nucleotide; and one or more modifications selected from the group consisting of,

G in General Formula 1 may be a modified or unmodified nucleotide, and the modification may include a linker bond to the nucleotide; introduction of a methoxy group at the second carbon position of the nucleotide; and introduction of fluorine at the second carbon position of the nucleotide; and one or more modifications selected from the group consisting of,

A of General Formula 1 may be a modified or unmodified nucleotide, and the modification may include a linker bond to the nucleotide; introduction of a methoxy group at the second carbon position of the nucleotide; and introduction of fluorine at the second carbon position of the nucleotide. The description of the nucleotides included in the present sequence is as described above.

The aptamer of the present invention may include one or more polynucleotide sequences selected from the group consisting of polynucleotide sequences of SEQ ID NO: 5 to SEQ ID NO: 81.

The aptamer of the present invention is one or more polynucleotide sequences selected from the group comprising the polynucleotide sequences of SEQ ID NO: 5 to SEQ ID NO: 81, or has at least 70%, at least 80%, at least 85%, specifically at least 90% homology thereto. It may contain more than one polynucleotide sequence.

In the present invention, the term 'homology' or 'identity' refers to the degree to which two given amino acid sequences or base sequences are related and can be expressed as a percentage. The terms homology and identity can often be used interchangeably.

Sequence homology or identity of a conserved polynucleotide or polypeptide is determined by standard alignment algorithms, and may be used with a default gap penalty established by the program used. Substantially homologous or identical sequences are generally defined under medium or high stringency conditions along the entire sequence or at least about 50%, 60%, 70%, 80% or 90% of the full-length. It can hybridize under stringent conditions. Hybridization is also contemplated for polynucleotides that contain degenerate codons instead of codons in the polynucleotide.

Whether any two polynucleotide or polypeptide sequences have homology, similarity or identity can be determined, for example, by Pearson et al (1988) [Proc. Natl. Acad. Sci. USA 85]: It can be determined using a known computer algorithm such as the "FASTA" program using default parameters as in 2444. Or, as performed in the Needleman program in the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277) (version 5.0.0 or later), It can be determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) (GCG program package (Devereux, J., et al, Nucleic Acids Research 12: 387 (1984)), BLASTP, BLASTN, FASTA (Atschul, [S.] [F.,] [ET AL, J MOLEC BIOL 215]: 403 (1990); Guide to Huge Computers, Martin J. Bishop , [ed.,] Academic Press, San Diego, 1994, and [CARILLO ETA/.] (1988) SIAM J Applied Math 48: 1073. For example, BLAST from the National Center for Biotechnology Information Database, or ClustalW can be used to determine homology, similarity, or identity.

Homology, similarity or identity of polynucleotides or polypeptides is defined in, for example, Smith and Waterman, Adv. Appl. Math (1981) 2:482, see, for example, Needleman et al. (1970), J Mol Biol. This can be determined by comparing sequence information using a GAP computer program such as 48:443. In summary, the GAP program defines the number of similarly aligned symbols (i.e., nucleotides or amino acids) divided by the total number of symbols in the shorter of the two sequences. The default parameters for the GAP program are (1) a binary comparison matrix (containing values 1 for identity and 0 for non-identity) and Schwartz and Dayhoff, eds., Atlas Of Protein Sequence And Structure, National Biomedical Research Foundation , pp. 353-358 (1979), Gribskov et al (1986) Nucl. Acids Res. 14: Weighted comparison matrix of 6745 (or EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix); (2) a penalty of 3.0 for each gap and an additional 0.10 penalty for each symbol in each gap (or a gap opening penalty of 10 and a gap extension penalty of 0.5); and (3) no penalty for end gaps.

In addition, whether any two polynucleotide or polypeptide sequences have homology, similarity, or identity can be confirmed by comparing the sequences by Southern hybridization experiments under defined stringent conditions, and the appropriate hybridization conditions defined are within the scope of the relevant technology. , methods well known to those skilled in the art (e.g., J. Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory press, Cold Spring Harbor, New York, 1989; F.M. Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., New York).

In addition, the aptamer has PEG (polyethylene glycol), idT (inverted deoxythymidine), LNA (Locked Nucleic Acid), amine linker, and thiol linker at one or more selected from its 3' or 5' end. It may be a combination of any one or more selected from the group consisting of linker) and cholesterol.

Specifically, when PEG is bound to one or more selected from the 3' end or the 5' end of the aptamer, the half-life of the aptamer may increase, and the insulin receptor and Akt phosphorylation activities may increase. In particular, 3 The aptamer with PEG bound to the 'end can exhibit stronger IR and Akt phosphorylation activities at low concentrations compared to the aptamer with PEG bound to the 5' end (FIG. 28).

The aptamer of the present invention can bind to the insulin receptor competitively or non-competitively with insulin.

In one embodiment of the present invention, as a result of confirming the binding ability of the produced single nucleic acid substitution aptamer (SEQ ID NO: 5 to 71) to the insulin receptor, it was confirmed that some aptamers have a Kd value for the insulin receptor that is superior to that of the existing A62, , this was selected as the final variant through sequence optimization (Figures 11 and 12).

The aptamer of the present invention can phosphorylate Y1150 of the insulin receptor.

In one embodiment of the present invention, to confirm IR-selective phosphorylation of the modified aptamer (A62m) combi 7 (A62ms) selected as the insulin receptor-specific aptamer of the present invention, six tyrosines in the intracellular region of the insulin receptor As a result of analyzing the phosphorylation activity of each region (Y960, Y1146, Y1150, Y1151, Y1316, and Y1322), insulin has phosphorylation activity in all regions of Y960, Y1146, Y1150, Y1151, Y1316, and Y1322, but A62ms only has phosphorylation activity at Y1150. It was confirmed that specific phosphorylation occurred only at , and phosphorylation at other tyrosine positions was very low (Figure 19).

The aptamer of the present invention can phosphorylate AKT.

The aptamer of the present invention can sensitize the phosphorylation of ERK.

In one embodiment of the present invention, as a result of confirming the effect of selective Y1150 phosphorylation of A62m combi 7 (A62ms), an insulin receptor-specific aptamer of the present invention, on insulin downstream signaling, phosphorylation of Y1150 was not observed. It was confirmed that phosphorylation of downstream factors was well induced when treated with low concentration of insulin alone, and that treatment with A62m combi 7 alone did not affect phosphorylation. On the other hand, it was confirmed that when A62m combi 7 and insulin were treated together, the downstream signaling of insulin was increased (FIG. 21).

The aptamer of the present invention can increase cellular glucose uptake.

In one embodiment of the present invention, in order to confirm whether A62m combi 7 (A62ms), an insulin receptor-specific aptamer of the present invention, has glucose uptake activity in the normal state or insulin resistance state as an IR aptamer, fully differentiated 3T3- As a result of performing an in vitro glucose uptake experiment using L1 adipocytes, the EC ₅₀ of glucose uptake in the case of A62ms is about 197 nM, which is about 4 times higher than that of insulin, which has an EC ₅₀ of 49 nM, but the maximum glucose uptake is lower than that of insulin. It was confirmed that a value of over 100% was reached (Figure 22). This shows that the aptamer of the present invention has similar efficacy in terms of insulin and glucose absorption effects.

The aptamer of the present invention may not promote cell division.

In one embodiment of the present invention, in order to determine whether A62m combi 7 (A62ms), an insulin receptor-specific aptamer of the present invention, exhibits the side effect of promoting division of cancer cells like insulin, MCF-7 cells, a breast cancer cell line, were used. As a result of checking whether cell division was performed, it was confirmed that insulin promoted division of MCF-7, but A62ms did not promote cell division (FIG. 24). This shows that the aptamer of the present invention can be safely administered without the side effect of promoting cancer cell division.

The aptamer of the present invention may exhibit blood sugar lowering activity.

In one embodiment of the present invention, A62m combi 7 (A62ms), an insulin receptor-specific aptamer of the present invention, was tested to determine whether A62m combi 7 (A62ms) exhibits a blood sugar lowering effect in vivo on diet and energy homeostasis using an animal model of type II diabetes. As a result of confirmation using db/db mice that have a mutation in the gene that expresses the leptin receptor, an important hormone, A62ms showed a blood sugar lowering effect similar to that of Lantus, a long-acting insulin preparation. It was confirmed that the duration of blood sugar lowering was longer than that of Lantus (Figure 25).

The aptamer of the present invention may exhibit weight gain inhibitory activity.

In one embodiment of the present invention, as a result of confirming whether A62m combi 7 (A62ms), an insulin receptor-specific aptamer of the present invention, exhibits an in vivo weight gain inhibitory effect using a type 2 diabetes animal model, A62ms It was confirmed that it exhibited a significant weight gain inhibition effect compared to Lantus (FIG. 26).

In addition, the lipolysis inhibitory activity of A62ms was confirmed in vitro using fully differentiated 3T3-L1 adipocytes, and the EC ₅₀ of lipolysis by insulin was 7.3 nM, which is about 6 times lower than that of glucose uptake. However, the EC ₅₀ of lipolysis by aptamer was 81 nM, which was 1.5 times lower than that of glucose absorption (Figure 23). As a result of comparing the efficacy of inhibiting lipolysis compared to the same amount of glucose absorption, A62ms showed a significantly lower degree of inhibition of lipolysis compared to insulin (Figure 23). This shows that the aptamer of the present invention has a relatively low efficacy of inhibiting lipolysis compared to the amount of glucose absorption, indicating that the same It can be seen that in situations where positive glucose absorption is induced, the effect of inhibiting lipolysis is lower than that of insulin.

In another embodiment of the present invention, A62ms showed a significant weight gain inhibition effect compared to Lantus, a positive control (FIG. 26).

The aptamer of the present invention may exhibit a bioavailability of 40% or more.

The aptamer of the present invention may exhibit a blood half-life of 5 hours or more.

In one embodiment of the present invention, after administering a single dose of 10 mg/kg to ICR mice by intravenous injection (IV) or subcutaneous injection (SC), blood samples were collected at 8 points for 24 hours and pressure was measured. As a result of measuring the tamer, when A62m combi 7 (A62ms), the insulin receptor-specific aptamer of the present invention, was injected subcutaneously, A62ms had a half-life of 4.2 hours when injected intravenously, bioavailability of more than 40% and bioavailability of 5.2% when injected subcutaneously. It was confirmed that the blood half-life of time appeared (Figure 27). The maximum concentration in blood (C _max ) was 125321.0 ng/mL for intravenous injection and 6400.4 ng/mL for subcutaneous injection, and differences were observed depending on the route of administration.

The present invention is based on the insulin receptor-specific aptamer (IR-A62) disclosed in Korean Patent Publication No. 10-2017-0013178, which is a prior patent of the present inventors, and is based on the binding affinity to the insulin receptor, phosphorylation activity of the insulin receptor, and serum stability. wanted to improve.

Accordingly, in order to perform optimization of the nucleotide sequence and optimization using chemical modification, as described above, nucleotides at specific sites in the nucleotide sequence of the present invention may be replaced with other types of nucleotides; Binding a nucleotide at a specific site to a linker; Modified aptamers were prepared by additional chemical modification of nucleotides.

In addition, the modified aptamer has superior IR phosphorylation activity and stability in serum compared to the existing unmodified aptamer (A62), has superior IR phosphorylation activity and glucose uptake activity compared to insulin, has low lipolysis inhibitory activity, and has a lower lipolysis inhibitory activity than the existing aptamer (A62). It exhibits a blood sugar lowering effect comparable to Lantus, a long-acting insulin preparation, has a longer duration of blood sugar lowering than Lantus, exhibits an excellent weight gain inhibition effect, has excellent bioavailability and half-life in the blood, and does not have the side effect of promoting cancer cell division. It is significant in confirming that it can be administered safely and can be provided for the prevention, treatment, and diagnosis of insulin-related diseases such as diabetes, diabetic complications, metabolic syndrome, obesity, and cardiovascular disease.

Another aspect of the present invention provides an insulin receptor agonist comprising the insulin receptor-specific aptamer of the present invention.

The terms used here are the same as described above.

As used herein, the term “insulin receptor agonist” refers to a pharmaceutically acceptable agent that selectively binds to the insulin receptor. Insulin receptor agonists generally refer to a new type of treatment for insulin-related diseases that was developed to effectively control blood sugar. The insulin receptor agonist of the present invention not only specifically binds to the insulin receptor but also has the characteristic of having no side effects such as cancer. Therefore, the agonist of the insulin receptor can be used for diagnosis or treatment of various diseases related to insulin.

Another aspect of the present invention provides a pharmaceutical composition for preventing or treating insulin-related diseases, comprising the insulin receptor-specific aptamer of the present invention as an active ingredient.

The terms used here are the same as described above.

In the present invention, the term "insulin-related disease" refers to a disease related to abnormal secretion of insulin, abnormal binding between insulin and the insulin receptor, or abnormal activity of the insulin receptor and downstream signaling proteins of insulin. For example, the disease is diabetes. , diabetes complications, metabolic syndrome, obesity, cardiovascular disease, etc., but is not limited thereto.

In the present invention, the term “prevention” refers to all actions to suppress or delay insulin-related diseases by administering the composition of the present invention to an individual, and “treatment” refers to all actions to prevent or delay insulin-related diseases by administering the composition of the present invention to an individual. It refers to any action that improves symptoms or makes it beneficial.

In addition to containing the insulin receptor-specific aptamer as an active ingredient, the pharmaceutical composition of the present invention may further include a pharmaceutically acceptable carrier. At this time, the content of the active ingredient included in the pharmaceutical composition is not particularly limited, but may include 0.0001% by weight to 10% by weight, specifically 0.001% by weight to 1% by weight, based on the total weight of the composition.

In the present invention, “pharmaceutically acceptable” means that the compound is commonly used in the pharmaceutical field and does not irritate the organism upon administration and does not inhibit the biological activity and properties of the administered compound.

In the present invention, the type of the carrier is not particularly limited, and any carrier commonly used in the art can be used. Non-limiting examples of the carrier include saline solution, sterile water, Ringer's solution, buffered saline solution, albumin injection solution, lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, maltodextrin, glycerol, ethanol, etc. You can. These may be used alone or in combination of two or more types.

In addition, the pharmaceutical composition of the present invention can be used by adding other pharmaceutically acceptable additives, such as excipients, diluents, antioxidants, buffers or bacteriostatic agents, if necessary, as well as fillers, extenders, wetting agents, disintegrants, dispersants, and surfactants. , the binder can be used by additionally adding a lubricant, etc.

In addition, the pharmaceutical composition is selected from the group consisting of tablets, pills, powders, granules, capsules, suspensions, oral solutions, emulsions, syrups, sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations and suppositories. It may have any one dosage form, and may be various oral or parenteral dosage forms. When formulated, it is prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc. These solid preparations contain one or more compounds and at least one excipient, such as starch, calcium carbonate, sucrose, or lactose ( It is prepared by mixing lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral administration include suspensions, oral solutions, emulsions, and syrups. In addition to the commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included. there is. Preparations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable esters such as ethyl oleate. As a base for suppositories, witepsol, macrogol, tween 61, cacao, laurel, glycerogelatin, etc. can be used.

The pharmaceutical composition can be formulated with the carrier as described above and used as food, medicine, feed additive, drinking water additive, etc.

The pharmaceutical composition may be formulated and used in various dosage forms suitable for oral administration or parenteral administration.

Non-limiting examples of the preparation for oral administration include troches, lozenges, tablets, aqueous suspensions, oily suspensions, prepared powders, granules, emulsions, hard capsules, soft capsules, syrups or elixirs, etc. can be mentioned.

In order to formulate the pharmaceutical composition of the present invention for oral administration, binders such as lactose, saccharose, sorbitol, mannitol, starch, amylopectin, cellulose or gelatin; excipients such as dicalcium phosphate; disintegrants such as corn starch or sweet potato starch; Lubricants such as magnesium stearate, calcium stearate, sodium stearyl fumarate, or polyethylene glycol wax can be used, and sweeteners, fragrances, syrups, etc. can also be used.

Furthermore, in the case of capsules, in addition to the above-mentioned substances, a liquid carrier such as fatty oil can be additionally used.

Non-limiting examples of the parenteral preparations include injection solutions, suppositories, powders for respiratory inhalation, aerosol preparations for sprays, ointments, powders for application, oils, creams, etc.

To prepare the pharmaceutical composition of the present invention for parenteral administration, sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, topical preparations, etc. can be used. The non-aqueous solutions and suspensions include propylene glycol, Polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, etc. may be used.

In addition, more specifically, when the pharmaceutical composition of the present invention is formulated as an injection solution, the composition of the present invention is mixed in water with a stabilizer or buffer to prepare a solution or suspension, which is then placed in an ampoule or vial. It can be formulated for unit administration. Additionally, when the pharmaceutical composition of the present invention is formulated as an aerosol, a propellant or the like may be mixed with additives to disperse the water-dispersed concentrate or wet powder.

The insulin receptor-specific aptamer of the present invention has a blood sugar lowering effect in vivo compared to Lantus, a long-acting insulin preparation (Example 3-4), a glucose absorption effect in vitro compared to insulin (Example 3-1), and lipolysis. As it was confirmed that the inhibition reduction effect (Example 3-2) was superior, the pharmaceutical composition containing the aptamer as an active ingredient can be used for the prevention or treatment of insulin-related diseases.

The composition of the present invention can be administered in a pharmaceutically effective amount.

In the present invention, the term "pharmaceutically effective amount" refers to an amount sufficient to treat a disease with a reasonable benefit/risk ratio applicable to medical treatment, and the effective dose level is determined by the type and severity of the individual, age, gender, and severity of the disease. It can be determined based on factors including the type, activity of the drug, sensitivity to the drug, time of administration, route of administration and excretion rate, duration of treatment, drugs used simultaneously, and other factors well known in the medical field. The composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with conventional therapeutic agents. And it can be administered single or multiple times. Considering all of the above factors, it is important to administer an amount that can achieve maximum effect with the minimum amount without side effects, and can be easily determined by a person skilled in the art. The preferred dosage of the composition of the present invention varies depending on the patient's condition and weight, degree of disease, drug form, administration route and period, but for desirable effects, the composition of the present invention is administered in an amount of 0.0001 mg/kg to 100 mg/kg per day. , more preferably 0.001 mg/kg to 10 mg/kg. Administration may be administered once a day, or may be administered several times. The composition can be administered to various mammals such as rats, livestock, and humans by various routes, and the method of administration includes without limitation any method conventional in the art, for example, orally, rectally, intravenously, intramuscularly, or intravenously. It may be administered by subcutaneous, intraperitoneal, intrathecal, or intracerebrovascular injection. Specifically, it may be oral administration, but is not limited thereto.

In addition, the pharmaceutical composition of the present invention can be used not only in the form of a medicine for humans, but also in the form of an animal medicine. Here, animals are a concept that includes livestock and companion animals.

Another aspect of the present invention provides a method for preventing or treating insulin-related diseases, comprising administering the pharmaceutical composition of the present invention to a subject.

The terms used here are the same as described above.

In the present invention, the term “individual” refers to all animals, including mammals, including rats, livestock, etc., that have developed or may develop insulin-related diseases. By administering the pharmaceutical composition of the present invention to a subject suspected of having an insulin-related disease, the subject can be treated efficiently.

In the present invention, the term "administration" means introducing the pharmaceutical composition of the present invention into a subject suspected of having cachexia by any appropriate method, and the route of administration is oral or parenteral, as long as it can reach the target tissue. It can be.

The pharmaceutical composition of the present invention can be administered in a pharmaceutically effective amount, as described above.

The pharmaceutical composition of the present invention is not particularly limited and can be applied to any entity as long as it is intended to prevent or treat cachexia. For example, non-human animals such as monkeys, dogs, cats, rabbits, guinea pigs, rats, mice, cows, sheep, pigs, goats, birds, and fish can be used, and the pharmaceutical composition can be administered parenterally, subcutaneously, or intraperitoneally. It can be administered intranasally, intrapulmonaryly and intranasally, and for topical treatment, if necessary, by any suitable method, including intralesional administration. The preferred dosage of the pharmaceutical composition of the present invention varies depending on the individual's condition and weight, degree of disease, drug form, administration route and period, but can be appropriately selected by a person skilled in the art. For example, it may be administered orally, rectally, or by intravenous, intramuscular, subcutaneous, intrauterine intrathecal or intracerebrovascular injection, but is not limited thereto.

The pharmaceutical composition of the present invention can be administered alone or in combination with methods using surgery, hormone therapy, drug therapy, and biological response regulators to prevent, improve, or treat insulin-related diseases.

Another aspect of the present invention provides a composition for diagnosing diabetes or diabetic complications, comprising the aptamer of the present invention.

The terms used here are the same as described above.

In the present invention, the term “diagnosis” means confirming the existence or characteristics of a pathological condition, and may specifically mean determining whether diabetes or diabetes complications have developed.

Additionally, the present invention provides a method of providing information on diagnosing diabetes or diabetic complications using the insulin receptor-specific aptamer.

The method of providing information for diagnosing diabetes or diabetic complications includes preparing a separated biological sample; reacting the biological sample with an insulin receptor-specific aptamer according to the present invention; And a step of measuring the binding degree of the insulin receptor-specific aptamer in the biological sample, and if the binding degree of the insulin receptor-specific aptamer in the biological sample is higher than that of the normal sample, diabetes is diagnosed. It may be done by.

The step of measuring the binding degree of the insulin receptor-specific aptamer in the biological sample can be performed using a DNA aptamer binding measurement technique commonly used in the related art, for example, at the end of the insulin receptor-specific aptamer. Methods such as labeling a fluorescent or radioactive material to measure the fluorescence or radioactivity intensity or imaging and observing the method may be used, but are not limited thereto.

The aptamer of the present invention has excellent insulin receptor (IR) phosphorylation activity and stability in serum compared to existing aptamers, has excellent IR phosphorylation activity and glucose uptake activity compared to insulin, has low lipolysis inhibitory activity, and has long-term stability. It exhibits a blood sugar lowering effect comparable to Lantus, a type insulin preparation, has a longer duration of blood sugar lowering than Lantus, exhibits an excellent weight gain inhibition effect, has excellent bioavailability and half-life in the blood, and has the side effect of promoting cancer cell division. Since it can be safely administered without any medication, it can be applied to the prevention, treatment, and diagnosis of insulin-related diseases such as diabetes, diabetic complications, metabolic syndrome, obesity, and cardiovascular disease.

Figure 1 is a diagram confirming the insulin receptor (IR) phosphorylation activity upon treatment with 10 nM of an aptamer library modified by linking linkers at each nucleotide position.
Figure 2 is a diagram confirming the IR phosphorylation activity upon treatment with 5 nM of the aptamer library modified by linking the linker at each nucleotide position.
Figure 3 shows the IR phosphorylation activity following treatment with 10 nM or 5 nM of the aptamer library modified by introducing a methoxy group (2-O-Me, 2-O-Methoxy-DNA) at the second carbon position for each C nucleotide position. It is also a degree.
Figure 4 is a diagram confirming the IR phosphorylation activity according to treatment with 10 nM or 5 nM of an aptamer library modified by introducing fluorine (2-F, 2-Fluorine-DNA) at the second carbon position for each C nucleotide position.
Figure 5 is a diagram confirming the IR phosphorylation activity upon treatment with 10 nM or 5 nM of an aptamer library modified by introducing fluorine at the second carbon position for each A nucleotide position.
Figure 6 is a diagram confirming the IR phosphorylation activity upon treatment with 10 nM or 5 nM of an aptamer library modified by introducing a methoxy group at the second carbon position for each A nucleotide position.
Figure 7 is a diagram confirming the IR phosphorylation activity according to treatment with 10 nM or 5 nM of an aptamer library modified by introducing fluorine at the second carbon position for each G nucleotide position.
Figure 8 is a diagram confirming the IR phosphorylation activity upon treatment with 10 nM or 5 nM of an aptamer library modified by introducing a methoxy group at the second carbon position for each G nucleotide position.
Figure 9 is a diagram confirming the IR phosphorylation activity upon treatment with 10 nM or 5 nM of an aptamer library modified with benzyl-uracil nucleotide (Benzyl-dU, Y) for each naphthyl-uracil nucleotide (Naphthyl-dU, W) position.
Figure 10 is a diagram showing the chemical optimization of a single sequence for each nucleotide position, creating a MAP for IR phosphorylation and protein binding results, and showing the results of substitution scanning.
Figure 11 shows the results of confirming Kd and Bmax for IR of each aptamer library.
Figure 12 shows the results of confirming Kd and Bmax for IR of each aptamer library, following Figure 11.
Figure 13 shows the results of confirming the IR phosphorylation activity of A62m combi 1 to combi 4.
Figure 14 shows the results of confirming the IR phosphorylation activity of A62m combi 5 to combi 10.
Figure 15 shows the results of confirming the IR phosphorylation activity of A62m combi 5 to combi 10 according to treatment concentration.
Figure 16 shows the results of confirming the IR phosphorylation activity of A62m combi 6 to combi 9 according to treatment concentration.
Figure 17 shows the results of confirming the IR phosphorylation activity of A62m combi 7 to combi 9 according to treatment concentration.
Figure 18 shows the results of confirming the serum stability of A62m combi 7 to combi 9.
Figure 19 shows the results of confirming the IR phosphorylation site of A62m combi 7 (A62ms).
Figure 20 shows the results of confirming the degree of phosphorylation at the Y1150 position for each treatment time and concentration of A62m combi 7 (A62ms).
Figure 21 shows the results of treating cells with A62m combi 7 (A62ms), insulin, or each of A62m combi 7 and insulin to confirm the effect on insulin's downstream signaling under each condition.
Figure 22 shows the results of confirming the in vitro glucose uptake activity of A62m combi 7 (A62ms).
Figure 23 shows the results of confirming the in vitro lipolysis inhibitory activity of A62m combi 7 (A62ms).
Figure 24 shows the results of confirming whether A62m combi 7 (A62ms) promotes cancer cell division in vitro .
Figure 25 shows the results confirming the in vivo blood sugar lowering activity of A62m combi 7 (A62ms).
Figure 26 shows the results confirming the weight gain inhibitory activity of A62m combi 7 (A62ms).
Figure 27 shows the results of confirming the bioavailability and blood half-life of A62m combi 7 (A62ms) according to the administration method.
Figure 28 shows that after chemically binding polyethylene glycol (PEG) to the end of A62m combi 7 (A62ms) to increase the pharmacokinetics (PK) half-life, IR and Akt phosphorylation activity were checked to determine whether the activity changed. It is a result.

Hereinafter, the present invention will be described in more detail through examples and experimental examples. However, these examples and experimental examples are for illustrative purposes only and the scope of the present invention is not limited to these examples and experimental examples.

Example 1: Preparation and selection of a modified insulin receptor (IR) aptamer library through chemical optimization of A62

In order to improve the effect of the IR-A62 aptamer (hereinafter referred to as A62) (Korean Patent Publication No. 10-2017-0013178), known as the existing insulin receptor-specific aptamer, part of the polynucleotide sequence of A62 (22nd to 48th positions) Sequence optimization was performed to modify the polynucleotide sequence corresponding to the position, SEQ ID NO: 1). The A62 variant produced through the above sequence optimization had improved binding ability to IR compared to the existing A62, thereby producing an aptamer more effective than A62, the existing IR aptamer.

Example 1-1: Aptamer library production

To optimize the sequence by modifying some sequences in A62, it was confirmed which nucleotides were modified to improve the effect. For this purpose, the nucleotides in the sequence of A62 (A62 Ori, SEQ ID NO: 1) are linked to the remaining nucleotides except for the naphthyl-uracil nucleotide (Naphthyl-dU, W) in which the 5th carbon of the dUTP (deoxyuracil) nucleotide is substituted with a naphthyl group. (Linker, C3-L, L) is attached, or a methoxy group (2'-Me, 2-O-Methoxy-DNA, N _me ) or fluorine (2'-F, 2-Fluorine-DNA) is attached to the 2nd carbon position. , N _f ) were modified with introduced nucleotides to produce a total of 60 aptamer libraries (SEQ ID NOs: 5 to 64). In addition, seven additional aptamer libraries (SEQ ID NOs: 65 to 71) were created by modifying W in the sequence to a benzyl-uracil nucleotide (Benzyl-dU, Y) in which the 5th carbon of the dUTP nucleotide is substituted with a benzyl group.

The linker may be composed of three carbons and may be bound to nucleotides at the 5' end, 3' end, middle, or both ends of the aptamer. The structure in which the C3 linker is linked to the 5'-end (Formula 3), 3'-end (Formula 4), and middle (Formula 5) nucleotides of the nucleotide sequence is shown in the following formula.

[Formula 3]

[Formula 4]

[Formula 5]

To prepare a library required for aptamer SELEX (Systematic evolution of ligands by exponential enrichment), an antisense library with biotin bound to the 5' was synthesized. To synthesize the antisense library, 1 mM dNTPs (dATP, dGTP, dCTP, Nap-dU), 0.25 U/ul KOD XL, 10X extension buffer (1.2 M Tris-HCl pH 7.8, 100 mM KCl, 60 mM ( Double-stranded DNA was prepared by reacting with 50 uM of the forward primer (SEQ ID NO: 2) on NH ₄ ) ₂ SO ₄ , 70 mM MgSO ₄ , 1% TritonX100, 1 mg/ml BSA) at 70°C for 2 hours. Here, single-stranded modified DNA was eluted using 20 mM NaOH and then neutralized with HCl solution. The prepared library was quantified using a 10% urea gel. The sequences of primers used in Example 1 are shown in Table 1 below.

sequence number sequence name Sequence (5' -> 3') 2 forward_F GAGTGACCGTCCGCCTG 3 reverse_R GGCTGGTGTGTGGCTG

Example 1-2: Evaluation of IR phosphorylation activity

The IR phosphorylation activity according to chemical optimization of the aptamer library (SEQ ID NOs: 5 to 71) prepared in Example 1-1 was confirmed through the following experiment.

Specifically, 105 Rat-1/hIR cells, ^which are mouse embryonic fibroblasts that stably express the human insulin receptor (hIR) under the control of the SV40 early promoter, were plated in each well of a 12-well plate. Then, the cells were cultured overnight in high glucose Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS) at 37°C and humidified conditions containing 5% CO ₂ . Before processing the aptamer sample, cells were cultured in DMEM without FBS for 1 hour. Cultured cells were washed three _times with ₁ and 1.3 mM KH ₂ PO ₄ ] for 1 hour. The aptamer sample was prepared by heating at 95°C for 5 minutes, cooling slowly at room temperature to reconstitute the tertiary structure of the aptamer, and dissolving in Krebs-Ringer HEPES buffer for the experiment.

Krebs-Ringer HEPES buffer containing 10 nM or 5 nM of the aptamer sample was cultured for 1 hour after treatment in each well where Rat-1/hIR cells were cultured.

After washing three times with 1xPBS, cells were removed from the plate and lysed with lysis buffer. The cell lysate was centrifuged at 14,000 rpm for 15 minutes at 4°C to separate proteins. The separated sample was heated at 100°C for 5 minutes with 5X sample buffer and then slowly cooled at room temperature to prepare a Western blot sample. The prepared sample was subjected to electrophoresis on 4% to 12% SDS-PAGE for 1 hour and 30 minutes, and then transferred to a nitrocellulose membrane for 1 hour and 30 minutes. Afterwards, the membrane was blocked in a blocking buffer solution (1xPBST, 3% skim milk powder) at room temperature for 40 minutes, and the primary antibody [anti-phospho-IR (10C3), Santa Cruz Biotechnology was added to the blocking buffer solution. or anti-β-actin (D6A8), Cell Signaling Technology] was diluted 1:500 or 1:1500 and incubated at room temperature for 1 hour. Afterwards, the membrane was washed three times with 1xPBS, and the secondary antibody bound to the blocking buffer solution was diluted 1:20,000, incubated at room temperature for 1 hour, and then washed three times with 1xPBST. After spraying the enhanced chemiluminescence (ECL) solution on the membrane and incubating it at room temperature for 1 minute, the membrane was exposed to an x-ray film. The intensity of the band formed on the membrane was measured, quantified, and compared with A62.

As a result, the degree of insulin receptor phosphorylation was different for each nucleotide position and type of chemical modification, and the degree of phosphorylation is shown in Figures 1 to 10 based on the phosphorylation activity of A62 that has not been chemically modified.

Example 1-3: Clonal binding affinity assay

A clonal binding affinity assay was performed to confirm whether the binding affinity of the aptamer library (SEQ ID NOs: 5 to 71) prepared in Example 1-1 to the insulin receptor was superior to that of the A62 aptamer.

Specifically, 10 fmole of _each aptamer sample was added to 1 . IR was sequentially diluted 4.64 times to 7 points at 100 nM using 1X SB18 buffer, and then 30 ul of aptamer was added and reacted at 37°C for 30 minutes. Afterwards, 5.5 ul of Dynabead TALON was added to the formed aptamer and protein conjugate and reacted at room temperature for 5 minutes. Then, it was placed in a Durapore filter plate previously moistened with 30ul of 1X SB18 buffer and placed under vacuum. Then, 100ul of 1X SB18 buffer was added, vacuumed again, and washed. A 2mM NaOH solution was added and reacted at room temperature for 5 minutes to elute and then neutralized with an 8mM HCl solution.

The aptamer and protein polymer above were amplified using QPCR (quantitative PCR). Specifically, the forward primer (SEQ ID NO: 2) and reverse primer (SEQ ID NO: 3) used in library preparation were each containing 0.2 uM dNTP (dATP, dGTP, dCTP, dTTP), 5 mM MgCl ₂ , 0.025 U/ul KOD XL, and Mix the DNA protein conjugate to a total volume of 20 ul, once at 96°C for 15 seconds, 55°C for 10 seconds, and 70°C for 30 minutes, and then at 96°C for 15 seconds, 55°C for 10 seconds, and 70°C for 1 minute. Amplification was repeated 40 times. After normalizing based on the Ct value, Kd and Bmax were calculated using SigmaPlot.

As a result, as shown in Figures 11 and 12, a sequence was identified that showed a Kd value superior to that of the existing A62 when a single sequence was replaced. In subsequent examples, the optimized sequence was derived by applying it together with the IR phosphorylation result map.

In the case of the C3 linker variant, Kd and Bmax were not measured because polymerase could not be applied.

Example 1-4: Selection of variant aptamer (A62m) according to chemical modification combination

The results of confirming the degree of IR phosphorylation activation according to chemical modification of each nucleotide position of the aptamer library (SEQ ID NOs: 5 to 71) produced in Example 1-1 were as shown in FIG. 10.

Based on this, numerous combination experiments were performed according to chemical modification at each position, and 10 candidate sequences that appeared to be chemically optimized were selected. Candidate sequences were selected in consideration of the degree of phosphorylation activity and the stability of the aptamer itself, and with a focus on improving stability, candidate sequences were selected in a direction that would improve stability unless there was a severe decrease in phosphorylation activity.

Additionally, when methoxylating thymine, methoxylation originates from RNA, so U methoxy is more common than T methoxy. Therefore, thymine was transformed into uracil, which exists in RNA, and then methoxylated.

The candidate sequences were named A62m combi 1 to combi 10 (Co 1 to Co 10, respectively) (SEQ ID NOs. 72 to 81), and each sequence is shown in Table 2 below.

sequence number sequence name Sequence (5' -> 3') size 72 A62m combination 1 CA _F YYA C _F GC _F AW G _Me A _Me G _Me YC WAGA _F WC _Me C _F GYC _Me A _Me G _Me 27mer 73 A62m combination 2 CA _F YYA C _F GC _F AW G _Me A _Me G _Me YC WAGA _F WC _Me C _F GYC _Me A _Me 26mer 74 A62m combination 3 CA _F YYA C _F GC _F AW G _Me A _Me G _Me YC WAGA _F WC _Me C _F GYC _Me 25mer 75 A62m combination 4 CA _F YYA C _F GC _F AW G _Me A _Me G _Me YC WAGA _F WC _Me C _F GY 24mer 76 A62m combination 5 CA _F YYA C _F GC _F AW G _Me A _F G _Me YC WAGA _F WC _Me C _F GYC _Me A _Me G _Me 27mer 77 A62m combination 6 CA _F YYA C _F GC _F AW G _Me A _F G _Me YC WAGA _F WC _Me C _F GYC _Me A _Me K 27mer 78 A62m combination 7 CA _F YYA C _F GC _F AW G _Me A _F G _Me YC WAGA _F WC _Me C _F GYC _Me 25mer 79 A62m combination 8 CA _F YYA C _F GC _F AW G _Me A _F G _Me YC WAGA _F WC _Me C _F GYC _Me A _F G _Me 27mer 80 A62m combination 9 CA _F YYA C _F GC _F AW G _F A _Me G _F YC WAGA _F WC _Me C _F GYC _Me A _F G _Me 27mer 81 A62m combination 10 CA _F YYA C _F GC _F AW G _Me A _F G _Me YC WAGA _F WC _Me C _F GYK A _Me K 27mer

W is a naphthyl-uracil nucleotide (Naphthyl-dU). Y is a benzyl-uracil nucleotide (Benzyl-dU).

The K is a nucleotide to which a linker (C3 linker) is attached.

The N _Me is a nucleotide in which a methoxy group (2-O-Me, 2-O-Methoxy-DNA) is introduced at the second carbon position.

The _NF is a nucleotide in which fluorine (2-F, 2-Fluorine-DNA) is introduced at the second carbon position.

The aptamer had idT (inverted deoxythymidine) attached to the 3' or 5' end of each sequence.

Example 2: Activity evaluation of chemically optimized A62m

Example 2-1: Evaluation of IR phosphorylation activity

As a result of confirming the degree of IR phosphorylation of A62m combi 1 to combi 10 (SEQ ID NOs: 72 to 81) in the same manner as in Example 1-2, many candidates with improved EC ₅₀ compared to A62 Ori were identified, among which A62m combi The IR phosphorylation EC ₅₀ of 7 was confirmed to be 7.37 nM, which was the lowest compared to A62Ori. The measured EC ₅₀ of Combi 7, 8, and 9 were confirmed to be 7.37, 10.43, and 9.831 nM, respectively (Figures 13 to 17).

Example 2-2: Confirmation of serum stability of aptamer

In Example 2-1, the serum stability of A62m combi 7 to combi 9 (SEQ ID NOs: 72 to 81), which had an excellent degree of IR phosphorylation, was confirmed. The serum stability determines how long an aptamer can exist in the body when administered as a therapeutic agent. The higher the serum stability, the longer the aptamer can exist in the body and maintain its effect for a long time. Therefore, the dosing interval is longer. It has the advantage of being wider.

Specifically, 10 μM of 10 uM aptamer was added to 45 μl of 100% serum and cultured at 37°C for 0, 4, 24, 48, and 72 hours, respectively. After incubation, 5 ul of 10 uM control aptamer was added, and then diluted with 165 ul of distilled water. The same volume of phenol:chloroform:isoamyl alcohol (25:24:1) as the solution was added to the sample, mixed with a vortex mixer for 20 seconds, and centrifuged at 16,000 g for 10 minutes. After centrifugation, the supernatant was transferred to a new tube, 5X sample buffer was added, and heated at 95°C for 5 minutes. The sample was electrophoresed in urea gel at 220V for 25 minutes, then the gel was stained with SYBR gold and confirmed using the Gel Doc™ EZ system infrared imaging system.

As a result, A62m combi 7 to combi 9 all showed superior serum stability than A62, showing that they are stably active in vivo as a treatment for diabetes (FIG. 18).

A62m combi 7 (SEQ ID NO: 78), which showed the best effect in IR phosphorylation in Example 2-1 and serum stability in Example 2-2, was named A62ms, and its specific activity was further confirmed in the following examples.

Example 2-3: Confirmation of IR selective phosphorylation

In order to confirm the IR selective phosphorylation of A62m combi 7 (A62ms), which showed the best effect on IR phosphorylation in Example 2-1 and serum stability in Example 2-2, six tyrosines in the intracellular region of the insulin receptor ( The phosphorylation activity of each region (Y960, Y1146, Y1150, Y1151, Y1316, and Y1322) was analyzed by Western blotting using various types of insulin receptor phosphorylation antibodies. For reference, the phosphorylation antibody for Y953 has not been developed, so the experiment was performed excluding this region.

Specifically, anti-phospho-IR antibody (Y1150/Y1151) (10C3, Santa Cruz Biotechnology), anti-phospho-IR antibody (Y960) (Invitrogen), anti-phospho-IR antibody (Y1146) (Invitrogen), anti-phospho-IR antibody (Y1316) (Invitrogen), anti-phospho-IR antibody (Y1322) (Invitrogen), anti-phospho-IR antibody (Py) (4G10, Millipore), anti- IR-antibody (Santa Cruz, CA, USA) and anti-β-actin (D6A8) (Cell Signaling Technology) were used as primary antibodies, using Western blotting in the same manner as in Example 2-1 above. Phosphorylation activity was confirmed, but before processing the aptamer sample, cells were cultured in DMEM without FBS for 3 hours.

As a result, insulin has phosphorylation activity in all regions of Y960, Y1146, Y1150, Y1151, Y1316, and Y1322, but A62m combi 7 (A62ms) caused specific phosphorylation only at the Y1150 position, and phosphorylation at other tyrosine positions was not observed. It was confirmed that it was very low (Figure 19).

Next, A62m combi 7 (A62ms) by treatment time (0, 1 min, 5 min, 10 min, 30 min, 1 hr, 2 hr, 4 hr, 8 hr) and by treatment concentration (0, 0.98, 195, . The degree of phosphorylation at the Y1150 position was compared with that upon insulin treatment (91, 7.81, 15.63, 31.25, 62.5, 125, 250, 500, 1000 nM).

As a result, in the case of insulin, phosphorylation occurs 1 min to 10 min after treatment and saturates quickly within a short period of time, whereas in the case of A62m combi 7, phosphorylation begins to be observed gradually after treatment and reaches 10 min to 4 hr. It was confirmed that phosphorylation occurred.

In addition, insulin and A62m combi 7 showed similar behavior by concentration, but the EC ₅₀ of A62 combi 7 was confirmed to be 18.4 nM, which is about 4.7 times lower than that of insulin, and faster saturation was observed compared to insulin, confirming that the phosphorylation intensity was higher. (Figure 20).

Example 2-4: Phosphorylation of insulin signaling protein

In Example 2-3, it was confirmed that A62m combi 7 (A62ms) induces phosphorylation of only Y1150 among several tyrosines present in insulin, and it was confirmed how the selective phosphorylation affects downstream signaling of insulin. .

Specifically, fully differentiated 3T3-L1 cells were heated at 95°C for 5 minutes and cooled at room temperature before being treated with 200 nM of A62m combi 7, 50 nM of insulin, or 200 nM of A62m combi 7 and 50 nM of insulin, respectively (insulin 5 min for aptamers, 1 hr for aptamers), and sampled to produce anti-phospho-IR antibodies (Y1150/Y1151) and anti-phospho-IR substrate 1 (phospho-Insulin receptor substrate 1, pIRS-1). Antibodies, anti-phospho-Akt antibody (S473), anti-phospho-Akt antibody (T308), and anti-phospho-ERK (1/2) antibody (T202/Y204) were used as primary antibodies; Phosphorylation of insulin signaling protein was confirmed through Western blot.

As a result, it was confirmed that the phosphorylation of downstream factors was well induced when treated with insulin alone at a low concentration such that phosphorylation of Y1150 was not observed, and that treatment with A62m combi 7 alone did not affect phosphorylation. On the other hand, it was confirmed that when A62m combi 7 and insulin were treated together, the downstream signaling of insulin was increased (FIG. 21).

Example 3: Confirmation of diabetes improvement effect of A62ms

Example 3-1: A62ms in vitro Check glucose uptake activity

In order to confirm whether A62m combi 7 (A62ms) selected in Example 2 has glucose uptake activity as an IR aptamer in a normal state or an insulin resistance state, it was tested in vitro for unit time using fully differentiated 3T3-L1 adipocytes. The glucose absorption rate during the period was confirmed.

Specifically, fully differentiated 3T3-L1 adipocytes without any treatment were determined to be in a normal state, and fully differentiated 3T3-L1 adipocytes were treated with 10 ng/ml of TNF (Tumor necrosis factor)-α to achieve an insulin resistance state. The experiment was conducted by inducing .

Fully differentiated 3T3-L1 adipocytes were cultured in DMEM without FBS for 3 hours and then treated with Krebs-Ringer HEPES buffer for 1 hour. Afterwards, the cells were treated with insulin or A62ms at concentrations of 1.6 nM, 8 nM, 40 nM, 200 nM, and 1000 nM for the corresponding time, and then treated with 2-deoxy[ ¹⁴ C]glucose for 10 minutes. The cells were washed three times with PBS containing 20 mM glucose, and the cells were lysed with a solution containing 0.5 N NaOH and 1% SDS. The amount of 2-Deoxy-D-glucose absorbed into cells was observed using a liquid scintillation counter.

As a result, in the case of A62ms, the EC ₅₀ of glucose absorption was about 197 nM, which is about 4 times higher than that of insulin with an EC ₅₀ of 49 nM, but it was confirmed that the maximum glucose absorption reached a value of more than 100% compared to insulin (Figure 22 ). Accordingly, it can be seen that the aptamer of the present invention has similar efficacy in terms of insulin and glucose absorption effects.

Example 3-2: A62ms in vitro Confirmation of lipolysis inhibition activity

To confirm the effect of the selected A62m combi 7 (A62ms) on inhibiting lipolysis as an IR aptamer, the amount of glycerol secreted during lipolysis was measured using fully differentiated 3T3-L1 adipocytes. .

Specifically, fully differentiated 3T3-L1 adipocytes were treated with DMEM without FBS for 3 hours to starve serum, and then treated with Krebs-Ringer HEPES buffer for 1 hour to induce lipolysis by glucose starvation. . After treating the cells with insulin or A62ms at concentrations of 0.93nM, 2.59nM, 7.26nM, 20.34nM, 56.94nM, 159.44nM, 446.43nM, 1250nM, and 3500nM for 4 hours, the cells were collected. 20ul of the collected buffer was added to each 96 well plate, 200ul of free glycerol reagent (Sigma F6428) was added, and the mixture was reacted at room temperature for 30 minutes. Afterwards, color development due to the concentration of glycerol secreted from the cells was measured through absorbance at 540 nm.

As a result, the EC ₅₀ of lipolysis by insulin was 7.3 nM, which was about 6 times lower than that of glucose uptake, but the EC ₅₀ of lipolysis by aptamer was 81 nM, which was 1.5 times lower than that of glucose uptake (Figure 23 ). As a result of comparing the efficacy of inhibiting lipolysis compared to the same amount of glucose absorption, A62ms showed a significantly lower degree of inhibition of lipolysis compared to insulin (Figure 23). This shows that the aptamer of the present invention has a relatively low efficacy of inhibiting lipolysis compared to the amount of glucose absorption, indicating that the same It can be seen that in situations where positive glucose absorption is induced, the effect of inhibiting lipolysis is lower than that of insulin.

Example 3-3: A62ms in vitro Check whether cancer cell division is promoted

To confirm whether the selected A62m combi 7 (A62ms) showed a side effect that promotes the division of cancer cells like insulin, the amount of divided cells was analyzed using MCF-7 cells, a breast cancer cell line.

Specifically, MCF-7 cells were cultured in a 24-well plate and then treated with insulin or A62ms at different concentrations for 3 days under 2% FBS conditions. Afterwards, intracellular DNA was stained with PBS containing 1 μM SYTO60 fluorescent dye, and fluorescence was measured using a LI-COR Odyssey scanner to quantify the amount of divided cells.

As a result, it was confirmed that insulin promoted the division of MCF-7, but A62ms did not promote cell division (FIG. 24). This shows that the aptamer of the present invention can be safely administered without the side effect of promoting cancer cell division.

Example 3-4: A62ms in vivo Confirmed activity in lowering blood sugar and inhibiting weight gain

To confirm whether the selected A62m combi 7 (A62ms), as an IR aptamer, has the effect of lowering blood sugar and suppressing weight gain in vivo , it was used as an animal model of type II diabetes, which plays an important role in diet and energy homeostasis. An insulin tolerance test (ITT) assay was performed using db/db mice with a mutation in the gene expressing the leptin receptor, a hormone.

Specifically, 8-week-old db/db mice were fasted for 12 hours, and then A62ms was dissolved in PBS and administered via intraperitoneal injection (IP) at doses of 5, 10, and 20 mg/kg. At 2, 4, 6, and 8 hours after administration, blood was collected from the tail and changes in blood sugar were observed using a blood glucose meter (Accu-Check Active; Roche Diagnostics). As a positive control, 3IU/kg of Lantus, a long-acting insulin preparation, was administered to db/db mice.

As a result, A62ms showed a similar level of blood sugar lowering effect as the positive control Lantus, and the duration of blood sugar lowering was confirmed to be longer than that of Lantus (FIG. 25).

Next, A62ms dissolved in PBS was administered to the db/db mice (n=8) daily via intraperitoneal injection at doses of 10, 20, and 30 mg/kg for 28 days, and body weight changes were observed. As a positive control, 6IU/kg of Lantus, a long-acting insulin preparation, was administered to db/db mice.

As a result, it was confirmed that A62ms showed a significant weight gain inhibition effect compared to Lantus, a positive control (FIG. 26).

Example 4: Pharmacokinetic analysis of A62ms

To measure the blood half-life, a single intravenous injection (IV) or subcutaneous injection (SC) of 10 mg/kg of A62ms was administered to ICR mice, and 8 points (0.083, 0.25, 0.5, 1, 2, 6, 12, and 24 hours) Blood was collected to obtain serum. 24 hours after injection, liver, kidney, and thigh muscle tissue was collected from the individual, pulverized using Precellys® Lysing Kits (France), and sampled by centrifugation at 6000 RPM for 15 seconds. Aptamers were measured from serum and tissue samples prepared through ELOHA (enzyme-linked oligonucleotide hybridization assay). The measured values were analyzed using Phoenix® WinNonlin® (Ver. 8.2, Certara).

As a result, it was confirmed that A62ms had a half-life of 4.2 hours when injected intravenously, and bioavailability of more than 40% and a blood half-life of 5.2 hours when injected subcutaneously (FIG. 27). The maximum concentration in blood (C _max ) was 125321.0 ng/mL for intravenous injection and 6400.4 ng/mL for subcutaneous injection, and differences were observed depending on the route of administration.

Next, to improve the half-life of A62ms in vivo , 40kD polyethylene glycol (PEG) was chemically conjugated to the 5' or 3' end of A62ms, and then IR and Akt phosphorylation of A62ms depending on PEG binding. It was confirmed whether there was any effect on the effect. Rat-1 cells overexpressing IR were treated with 100 and 1000 nM of each aptamer (A62ms, A62ms-PEG, PEG-A62ms) and reacted for 1 hour. Insulin was treated at 20 nM. After 1 hour, cells were recovered, ptotein was extracted using lysis buffer, and changes in p-1150 and p-AKT were observed using Western blot.

As a result, both A62ms (PEG-A62ms) with PEG bound to the 5' end and A62ms (A62ms-PEG) with PEG bound to the 3' end showed IR and Akt phosphorylation activities, but A62ms with PEG bound to the 3' end showed IR and Akt phosphorylation activities. It was confirmed that A62ms showed stronger IR and Akt phosphorylation activity at a low concentration (100 nM) compared to A62ms with PEG bound to the 5' end (FIG. 28). Insulin was treated for a long time, so the expression of p-IR(1150) was observed to be low.

From the results of the above examples, the A62ms aptamer, which is a chemically optimized modification of A62, an existing insulin receptor-specific aptamer according to the present invention, has superior IR phosphorylation activity and stability in serum compared to the A62 aptamer, and has superior IR phosphorylation activity and stability in serum compared to insulin. It has excellent phosphorylation activity and glucose uptake activity, low lipolysis inhibitory activity, shows a blood sugar lowering effect comparable to that of Lantus, a long-acting insulin preparation, has a longer duration of blood sugar lowering than Lantus, and has an excellent weight gain suppressing effect. , has excellent bioavailability and half-life in the blood, can be administered safely without the side effect of promoting cancer cell division, and can be applied to the prevention, treatment, and diagnosis of insulin-related diseases such as diabetes, diabetic complications, metabolic syndrome, obesity, and cardiovascular disease. there is.

From the above description, those skilled in the art to which the present invention pertains will understand that the present invention can be implemented in other specific forms without changing its technical idea or essential features. In this regard, the embodiments described above should be understood in all respects as illustrative and not restrictive. The scope of the present invention should be construed as including the meaning and scope of the patent claims described below rather than the detailed description above, and all changes or modified forms derived from the equivalent concept thereof are included in the scope of the present invention.

<110> Aptamer Sciences Inc. POSTECH Research and Business Development Foundation <120> Insulin receptor aptamer and uses its <130> KPA220172-KR <160> 82 <170> KoPatentIn 3.0 <210> 1 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> IR-A62F(22-48) <400> 1 cannacgcan gagncnagan ccgncag 27 <210> 2 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> forward_F <400> 2 gagtgaccgt ccgcctg 17 <210> 3 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> reverse_R <400> 3 ggctggtggt gtggctg 17 <210> 4 <211> 27 <212> DNA <213> Artificial Sequence <220 > <223> General formula 1 <220> <221> misc_feature <222> (3) <223> B is dUTP in which the 5th carbon position of the nucleotide is substituted with a hydrophobic functional group. <220> <221> misc_feature <222> (4) <223> B is dUTP in which the 5th carbon position of the nucleotide is substituted with a hydrophobic functional group. <220> <221> misc_feature <222> (10) <223> B is dUTP in which the 5th carbon position of the nucleotide is substituted with a hydrophobic functional group. <220> <221> misc_feature <222> (14) <223> B is dUTP in which the 5th carbon position of the nucleotide is substituted with a hydrophobic functional group. <220> <221> misc_feature <222> (16) <223> B is dUTP in which the 5th carbon position of the nucleotide is substituted with a hydrophobic functional group. <220> <221> misc_feature <222> (20) <223> B is dUTP in which the 5th carbon position of the nucleotide is substituted with a hydrophobic functional group. <220> <221> misc_feature <222> (24) <223> B is dUTP in which the 5th carbon position of the nucleotide is substituted with a hydrophobic functional group. <400> 4 cabbacgcab gagbcbagab ccgbcag 27 <210> 5 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> A62 L 1 <220> <221> misc_feature <222> (1) <223> K is a nucleotide to which a C3 linker is attached. <220> <221> misc_feature <222> (3) <223> W is NapdU. <220> <221> misc_feature <222> (4) <223> W is NapdU. <220> <221> misc_feature <222> (10) <223> W is NapdU. <220> <221> misc_feature <222> (14) <223> W is NapdU. <220> <221> misc_feature <222> (16) <223> W is NapdU. <220> <221> misc_feature <222> (20) <223> W is NapdU. <220> <221> misc_feature <222> (24) <223> W is NapdU. <400> 5 kawwacgcaw gagwcwagaw ccgwcag 27 <210> 6 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> A62 Me 2 <220> <221> misc_feature <222> (1) <223> C is a nucleotide in which a methoxy group is introduced at the second carbon position. <220> <221> misc_feature <222> (3) <223> W is NapdU. <220> <221> misc_feature <222> (4) <223> W is NapdU. <220> <221> misc_feature <222> (10) <223> W is NapdU. <220> <221> misc_feature <222> (14) <223> W is NapdU. <220> <221> misc_feature <222> (16) <223> W is NapdU. <220> <221> misc_feature <222> (20) <223> W is NapdU. <220> <221> misc_feature <222> (24) <223> W is NapdU. <400> 6 cawwacgcaw gagwcwagaw ccgwcag 27 <210> 7 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> A62 F 3 <220> <221> misc_feature <222> (1) <223> C is a nucleotide in which a fluorine group is introduced at the second carbon position. <220> <221> misc_feature <222> (3) <223> W is NapdU. <220> <221> misc_feature <222> (4) <223> W is NapdU. <220> <221> misc_feature <222> (10) <223> W is NapdU. <220> <221> misc_feature <222> (14) <223> W is NapdU. <220> <221> misc_feature <222> (16) <223> W is NapdU. <220> <221> misc_feature <222> (20) <223> W is NapdU. <220> <221> misc_feature <222> (24) <223> W is NapdU. <400> 7 cawwacgcaw gagwcwagaw ccgwcag 27 <210> 8 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> A62 L 4 <220> <221> misc_feature <222> (2) <223> K is a nucleotide to which a C3 linker is attached. <220> <221> misc_feature <222> (3) <223> W is NapdU. <220> <221> misc_feature <222> (4) <223> W is NapdU. <220> <221> misc_feature <222> (10) <223> W is NapdU. <220> <221> misc_feature <222> (14) <223> W is NapdU. <220> <221> misc_feature <222> (16) <223> W is NapdU. <220> <221> misc_feature <222> (20) <223> W is NapdU. <220> <221> misc_feature <222> (24) <223> W is NapdU. <400> 8 ckwwacgcaw gagwcwagaw ccgwcag 27 <210> 9 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> A62 Me 5 <220> <221> misc_feature <222> (2) <223> A is a nucleotide in which a methoxy group is introduced at the second carbon position. <220> <221> misc_feature <222> (3) <223> W is NapdU. <220> <221> misc_feature <222> (4) <223> W is NapdU. <220> <221> misc_feature <222> (10) <223> W is NapdU. <220> <221> misc_feature <222> (14) <223> W is NapdU. <220> <221> misc_feature <222> (16) <223> W is NapdU. <220> <221> misc_feature <222> (20) <223> W is NapdU. <220> <221> misc_feature <222> (24) <223> W is NapdU. <400> 9 cawwacgcaw gagwcwagaw ccgwcag 27 <210> 10 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> A62 F 6 <220> <221> misc_feature <222> (2) <223> A is a nucleotide in which a fluorine group is introduced at the second carbon position. <220> <221> misc_feature <222> (3) <223> W is NapdU. <220> <221> misc_feature <222> (4) <223> W is NapdU. <220> <221> misc_feature <222> (10) <223> W is NapdU. <220> <221> misc_feature <222> (14) <223> W is NapdU. <220> <221> misc_feature <222> (16) <223> W is NapdU. <220> <221> misc_feature <222> (20) <223> W is NapdU. <220> <221> misc_feature <222> (24) <223> W is NapdU. <400> 10 cawwacgcaw gagwcwagaw ccgwcag 27 <210> 11 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> A62 L 7 <220> <221> misc_feature <222> (3) <223> W isNapdU. <220> <221> misc_feature <222> (4) <223> W is NapdU. <220> <221> misc_feature <222> (5) <223> K is a nucleotide to which a C3 linker is attached. <220> <221> misc_feature <222> (10) <223> W is NapdU. <220> <221> misc_feature <222> (14) <223> W is NapdU. <220> <221> misc_feature <222> (16) <223> W is NapdU. <220> <221> misc_feature <222> (20) <223> W is NapdU. <220> <221> misc_feature <222> (24) <223> W is NapdU. <400> 11 cawwkcgcaw gagwcwagaw ccgwcag 27 <210> 12 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> A62 Me 8 <220> <221> misc_feature <222> (3) <223> W isNapdU. <220> <221> misc_feature <222> (4) <223> W is NapdU. <220> <221> misc_feature <222> (5) <223> A is a nucleotide in which a methoxy group is introduced at the second carbon position. <220> <221> misc_feature <222> (10) <223> W is NapdU. <220> <221> misc_feature <222> (14) <223> W is NapdU. <220> <221> misc_feature <222> (16) <223> W is NapdU. <220> <221> misc_feature <222> (20) <223> W is NapdU. <220> <221> misc_feature <222> (24) <223> W is NapdU. <400> 12 cawwacgcaw gagwcwagaw ccgwcag 27 <210> 13 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> A62 F 9 <220> <221> misc_feature <222> (3) <223> W isNapdU. <220> <221> misc_feature <222> (4) <223> W is NapdU. <220> <221> misc_feature <222> (5) <223> A is a nucleotide in which a fluorine group is introduced at the second carbon position. <220> <221> misc_feature <222> (10) <223> W is NapdU. <220> <221> misc_feature <222> (14) <223> W is NapdU. <220> <221> misc_feature <222> (16) <223> W is NapdU. <220> <221> misc_feature <222> (20) <223> W is NapdU. <220> <221> misc_feature <222> (24) <223> W is NapdU. <400> 13 cawwacgcaw gagwcwagaw ccgwcag 27 <210> 14 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> A62 L 10 <220> <221> misc_feature <222> (3) <223> W isNapdU. <220> <221> misc_feature <222> (4) <223> W is NapdU. <220> <221> misc_feature <222> (6) <223> K is a nucleotide to which a C3 linker is attached. <220> <221> misc_feature <222> (10) <223> W is NapdU. <220> <221> misc_feature <222> (14) <223> W is NapdU. <220> <221> misc_feature <222> (16) <223> W is NapdU. <220> <221> misc_feature <222> (20) <223> W is NapdU. <220> <221> misc_feature <222> (24) <223> W is NapdU. <400> 14 cawwakgcaw gagwcwagaw ccgwcag 27 <210> 15 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> A62 Me 11 <220> <221> misc_feature <222> (3) <223> W isNapdU. <220> <221> misc_feature <222> (4) <223> W is NapdU. <220> <221> misc_feature <222> (6) <223> C is a nucleotide in which a methoxy group is introduced at the second carbon position. <220> <221> misc_feature <222> (10) <223> W is NapdU. <220> <221> misc_feature <222> (14) <223> W is NapdU. <220> <221> misc_feature <222> (16) <223> W is NapdU. <220> <221> misc_feature <222> (20) <223> W is NapdU. <220> <221> misc_feature <222> (24) <223> W is NapdU. <400> 15 cawwacgcaw gagwcwagaw ccgwcag 27 <210> 16 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> A62 F 12 <220> <221> misc_feature <222> (3) <223> W isNapdU. <220> <221> misc_feature <222> (4) <223> W is NapdU. <220> <221> misc_feature <222> (6) <223> C is a nucleotide in which a fluorine group is introduced at the second carbon position. <220> <221> misc_feature <222> (10) <223> W is NapdU. <220> <221> misc_feature <222> (14) <223> W is NapdU. <220> <221> misc_feature <222> (16) <223> W is NapdU. <220> <221> misc_feature <222> (20) <223> W is NapdU. <220> <221> misc_feature <222> (24) <223> W is NapdU. <400> 16 cawwacgcaw gagwcwagaw ccgwcag 27 <210> 17 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> A62 L 13 <220> <221> misc_feature <222> (3) <223> W isNapdU. <220> <221> misc_feature <222> (4) <223> W is NapdU. <220> <221> misc_feature <222> (7) <223> K is a nucleotide to which a C3 linker is attached. <220> <221> misc_feature <222> (10) <223> W is NapdU. <220> <221> misc_feature <222> (14) <223> W is NapdU. <220> <221> misc_feature <222> (16) <223> W is NapdU. <220> <221> misc_feature <222> (20) <223> W is NapdU. <220> <221> misc_feature <222> (24) <223> W is NapdU. <400> 17 cawwackcaw gagwcwagaw ccgwcag 27 <210> 18 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> A62 Me 14 <220> <221> misc_feature <222> (3) <223> W isNapdU. <220> <221> misc_feature <222> (4) <223> W is NapdU. <220> <221> misc_feature <222> (7) <223> G is a nucleotide in which a methoxy group is introduced at the second carbon position. <220> <221> misc_feature <222> (10) <223> W is NapdU. <220> <221> misc_feature <222> (14) <223> W is NapdU. <220> <221> misc_feature <222> (16) <223> W is NapdU. <220> <221> misc_feature <222> (20) <223> W is NapdU. <220> <221> misc_feature <222> (24) <223> W is NapdU. <400> 18 cawwacgcaw gagwcwagaw ccgwcag 27 <210> 19 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> A62 F 15 <220> <221> misc_feature <222> (3) <223> W isNapdU. <220> <221> misc_feature <222> (4) <223> W is NapdU. <220> <221> misc_feature <222> (7) <223> G is a nucleotide in which a fluorine group is introduced at the second carbon position. <220> <221> misc_feature <222> (10) <223> W is NapdU. <220> <221> misc_feature <222> (14) <223> W is NapdU. <220> <221> misc_feature <222> (16) <223> W is NapdU. <220> <221> misc_feature <222> (20) <223> W is NapdU. <220> <221> misc_feature <222> (24) <223> W is NapdU. <400> 19 cawwacgcaw gagwcwagaw ccgwcag 27 <210> 20 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> A62 L 16 <220> <221> misc_feature <222> (3) <223> W isNapdU. <220> <221> misc_feature <222> (4) <223> W is NapdU. <220> <221> misc_feature <222> (8) <223> K is a nucleotide to which a C3 linker is attached. <220> <221> misc_feature <222> (10) <223> W is NapdU. <220> <221> misc_feature <222> (14) <223> W is NapdU. <220> <221> misc_feature <222> (16) <223> W is NapdU. <220> <221> misc_feature <222> (20) <223> W is NapdU. <220> <221> misc_feature <222> (24) <223> W is NapdU. <400> 20 cawwacgkaw gagwcwagaw ccgwcag 27 <210> 21 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> A62 Me 17 <220> <221> misc_feature <222> (3) <223> W isNapdU. <220> <221> misc_feature <222> (4) <223> W is NapdU. <220> <221> misc_feature <222> (8) <223> C is a nucleotide in which a methoxy group is introduced at the second carbon position. <220> <221> misc_feature <222> (10) <223> W is NapdU. <220> <221> misc_feature <222> (14) <223> W is NapdU. <220> <221> misc_feature <222> (16) <223> W is NapdU. <220> <221> misc_feature <222> (20) <223> W is NapdU. <220> <221> misc_feature <222> (24) <223> W is NapdU. <400> 21 cawwacgcaw gagwcwagaw ccgwcag 27 <210> 22 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> A62 F 18 <220> <221> misc_feature <222> (3) <223> W isNapdU. <220> <221> misc_feature <222> (4) <223> W is NapdU. <220> <221> misc_feature <222> (8) <223> C is a nucleotide in which a fluorine group is introduced at the second carbon position. <220> <221> misc_feature <222> (10) <223> W is NapdU. <220> <221> misc_feature <222> (14) <223> W is NapdU. <220> <221> misc_feature <222> (16) <223> W is NapdU. <220> <221> misc_feature <222> (20) <223> W is NapdU. <220> <221> misc_feature <222> (24) <223> W is NapdU. <400> 22 cawwacgcaw gagwcwagaw ccgwcag 27 <210> 23 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> A62 L 19 <220> <221> misc_feature <222> (3) <223> W isNapdU. <220> <221> misc_feature <222> (4) <223> W is NapdU. <220> <221> misc_feature <222> (9) <223> K is a nucleotide to which a C3 linker is attached. <220> <221> misc_feature <222> (10) <223> W is NapdU. <220> <221> misc_feature <222> (14) <223> W is NapdU. <220> <221> misc_feature <222> (16) <223> W is NapdU. <220> <221> misc_feature <222> (20) <223> W is NapdU. <220> <221> misc_feature <222> (24) <223> W is NapdU. <400> 23 cawwacgckw gagwcwagaw ccgwcag 27 <210> 24 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> A62 Me 20 <220> <221> misc_feature <222> (3) <223> W isNapdU. <220> <221> misc_feature <222> (4) <223> W is NapdU. <220> <221> misc_feature <222> (9) <223> A is a nucleotide in which a methoxy group is introduced at the second carbon position. <220> <221> misc_feature <222> (10) <223> W is NapdU. <220> <221> misc_feature <222> (14) <223> W is NapdU. <220> <221> misc_feature <222> (16) <223> W is NapdU. <220> <221> misc_feature <222> (20) <223> W is NapdU. <220> <221> misc_feature <222> (24) <223> W is NapdU. <400> 24 cawwacgcaw gagwcwagaw ccgwcag 27 <210> 25 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> A62 F 21 <220> <221> misc_feature <222> (3) <223> W isNapdU. <220> <221> misc_feature <222> (4) <223> W is NapdU. <220> <221> misc_feature <222> (9) <223> A is a nucleotide in which a fluorine group is introduced at the second carbon position. <220> <221> misc_feature <222> (10) <223> W is NapdU. <220> <221> misc_feature <222> (14) <223> W is NapdU. <220> <221> misc_feature <222> (16) <223> W is NapdU. <220> <221> misc_feature <222> (20) <223> W is NapdU. <220> <221> misc_feature <222> (24) <223> W is NapdU. <400> 25 cawwacgcaw gagwcwagaw ccgwcag 27 <210> 26 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> A62 L 22 <220> <221> misc_feature <222> (3) <223> W isNapdU. <220> <221> misc_feature <222> (4) <223> W is NapdU. <220> <221> misc_feature <222> (10) <223> W is NapdU. <220> <221> misc_feature <222> (11) <223> K is a nucleotide to which a C3 linker is attached. <220> <221> misc_feature <222> (14) <223> W is NapdU. <220> <221> misc_feature <222> (16) <223> W is NapdU. <220> <221> misc_feature <222> (20) <223> W is NapdU. <220> <221> misc_feature <222> (24) <223> W is NapdU. <400> 26 cawwacgcaw kagwcwagaw ccgwcag 27 <210> 27 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> A62 Me 23 <220> <221> misc_feature <222> (3) <223> W isNapdU. <220> <221> misc_feature <222> (4) <223> W is NapdU. <220> <221> misc_feature <222> (10) <223> W is NapdU. <220> <221> misc_feature <222> (11) <223> G is a nucleotide in which a methoxy group is introduced at the second carbon position. <220> <221> misc_feature <222> (14) <223> W is NapdU. <220> <221> misc_feature <222> (16) <223> W is NapdU. <220> <221> misc_feature <222> (20) <223> W is NapdU. <220> <221> misc_feature <222> (24) <223> W is NapdU. <400> 27 cawwacgcaw gagwcwagaw ccgwcag 27 <210> 28 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> A62 F 24 <220> <221> misc_feature <222> (3) <223> W isNapdU. <220> <221> misc_feature <222> (4) <223> W is NapdU. <220> <221> misc_feature <222> (10) <223> W is NapdU. <220> <221> misc_feature <222> (11) <223> G is a nucleotide in which a fluorine group is introduced at the second carbon position. <220> <221> misc_feature <222> (14) <223> W is NapdU. <220> <221> misc_feature <222> (16) <223> W is NapdU. <220> <221> misc_feature <222> (20) <223> W is NapdU. <220> <221> misc_feature <222> (24) <223> W is NapdU. <400> 28 cawwacgcaw gagwcwagaw ccgwcag 27 <210> 29 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> A62 L 25 <220> <221> misc_feature <222> (3) <223> W isNapdU. <220> <221> misc_feature <222> (4) <223> W is NapdU. <220> <221> misc_feature <222> (10) <223> W is NapdU. <220> <221> misc_feature <222> (12) <223> K is a nucleotide to which a C3 linker is attached. <220> <221> misc_feature <222> (14) <223> W is NapdU. <220> <221> misc_feature <222> (16) <223> W is NapdU. <220> <221> misc_feature <222> (20) <223> W is NapdU. <220> <221> misc_feature <222> (24) <223> W is NapdU. <400> 29 cawwacgcaw gkgwcwagaw ccgwcag 27 <210> 30 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> A62 Me 26 <220> <221> misc_feature <222> (3) <223> W isNapdU. <220> <221> misc_feature <222> (4) <223> W is NapdU. <220> <221> misc_feature <222> (10) <223> W is NapdU. <220> <221> misc_feature <222> (12) <223> A is a nucleotide in which a methoxy group is introduced at the second carbon position. <220> <221> misc_feature <222> (14) <223> W is NapdU. <220> <221> misc_feature <222> (16) <223> W is NapdU. <220> <221> misc_feature <222> (20) <223> W is NapdU. <220> <221> misc_feature <222> (24) <223> W is NapdU. <400> 30 cawwacgcaw gagwcwagaw ccgwcag 27 <210> 31 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> A62 F 27 <220> <221> misc_feature <222> (3) <223> W isNapdU. <220> <221> misc_feature <222> (4) <223> W is NapdU. <220> <221> misc_feature <222> (10) <223> W is NapdU. <220> <221> misc_feature <222> (12) <223> A is a nucleotide in which a fluorine group is introduced at the second carbon position. <220> <221> misc_feature <222> (14) <223> W is NapdU. <220> <221> misc_feature <222> (16) <223> W is NapdU. <220> <221> misc_feature <222> (20) <223> W is NapdU. <220> <221> misc_feature <222> (24) <223> W is NapdU. <400> 31 cawwacgcaw gagwcwagaw ccgwcag 27 <210> 32 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> A62 L 28 <220> <221> misc_feature <222> (3) <223> W isNapdU. <220> <221> misc_feature <222> (4) <223> W is NapdU. <220> <221> misc_feature <222> (10) <223> W is NapdU. <220> <221> misc_feature <222> (13) <223> K is a nucleotide to which a C3 linker is attached. <220> <221> misc_feature <222> (14) <223> W is NapdU. <220> <221> misc_feature <222> (16) <223> W is NapdU. <220> <221> misc_feature <222> (20) <223> W is NapdU. <220> <221> misc_feature <222> (24) <223> W is NapdU. <400> 32 cawwacgcaw gakwcwagaw ccgwcag 27 <210> 33 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> A62 Me 29 <220> <221> misc_feature <222> (3) <223> W isNapdU. <220> <221> misc_feature <222> (4) <223> W is NapdU. <220> <221> misc_feature <222> (10) <223> W is NapdU. <220> <221> misc_feature <222> (13) <223> G is a nucleotide in which a methoxy group is introduced at the second carbon position. <220> <221> misc_feature <222> (14) <223> W is NapdU. <220> <221> misc_feature <222> (16) <223> W is NapdU. <220> <221> misc_feature <222> (20) <223> W is NapdU. <220> <221> misc_feature <222> (24) <223> W is NapdU. <400> 33 cawwacgcaw gagwcwagaw ccgwcag 27 <210> 34 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> A62 F 30 <220> <221> misc_feature <222> (3) <223> W isNapdU. <220> <221> misc_feature <222> (4) <223> W is NapdU. <220> <221> misc_feature <222> (10) <223> W is NapdU. <220> <221> misc_feature <222> (13) <223> G is a nucleotide in which a fluorine group is introduced at the second carbon position. <220> <221> misc_feature <222> (14) <223> W is NapdU. <220> <221> misc_feature <222> (16) <223> W is NapdU. <220> <221> misc_feature <222> (20) <223> W is NapdU. <220> <221> misc_feature <222> (24) <223> W is NapdU. <400> 34 cawwacgcaw gagwcwagaw ccgwcag 27 <210> 35 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> A62 L 31 <220> <221> misc_feature <222> (3) <223> W isNapdU. <220> <221> misc_feature <222> (4) <223> W is NapdU. <220> <221> misc_feature <222> (10) <223> W is NapdU. <220> <221> misc_feature <222> (14) <223> W is NapdU. <220> <221> misc_feature <222> (15) <223> K is a nucleotide to which a C3 linker is attached. <220> <221> misc_feature <222> (16) <223> W is NapdU. <220> <221> misc_feature <222> (20) <223> W is NapdU. <220> <221> misc_feature <222> (24) <223> W is NapdU. <400> 35 cawwacgcaw gagwkwagaw ccgwcag 27 <210> 36 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> A62 Me 32 <220> <221> misc_feature <222> (3) <223> W isNapdU. <220> <221> misc_feature <222> (4) <223> W is NapdU. <220> <221> misc_feature <222> (10) <223> W is NapdU. <220> <221> misc_feature <222> (14) <223> W is NapdU. <220> <221> misc_feature <222> (15) <223> C is a nucleotide in which a methoxy group is introduced at the second carbon position. <220> <221> misc_feature <222> (16) <223> W is NapdU. <220> <221> misc_feature <222> (20) <223> W is NapdU. <220> <221> misc_feature <222> (24) <223> W is NapdU. <400> 36 cawwacgcaw gagwcwagaw ccgwcag 27 <210> 37 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> A62 F 33 <220> <221> misc_feature <222> (3) <223> W isNapdU. <220> <221> misc_feature <222> (4) <223> W is NapdU. <220> <221> misc_feature <222> (10) <223> W is NapdU. <220> <221> misc_feature <222> (14) <223> W is NapdU. <220> <221> misc_feature <222> (15) <223> C is a nucleotide in which a fluorine group is introduced at the second carbon position. <220> <221> misc_feature <222> (16) <223> W is NapdU. <220> <221> misc_feature <222> (20) <223> W is NapdU. <220> <221> misc_feature <222> (24) <223> W is NapdU. <400> 37 cawwacgcaw gagwcwagaw ccgwcag 27 <210> 38 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> A62 L 34 <220> <221> misc_feature <222> (3) <223> W isNapdU. <220> <221> misc_feature <222> (4) <223> W is NapdU. <220> <221> misc_feature <222> (10) <223> W is NapdU. <220> <221> misc_feature <222> (14) <223> W is NapdU. <220> <221> misc_feature <222> (16) <223> W is NapdU. <220> <221> misc_feature <222> (17) <223> K is a nucleotide to which a C3 linker is attached. <220> <221> misc_feature <222> (20) <223> W is NapdU. <220> <221> misc_feature <222> (24) <223> W is NapdU. <400> 38 cawwacgcaw gagwcwkgaw ccgwcag 27 <210> 39 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> A62 Me 35 <220> <221> misc_feature <222> (3) <223> W isNapdU. <220> <221> misc_feature <222> (4) <223> W is NapdU. <220> <221> misc_feature <222> (10) <223> W is NapdU. <220> <221> misc_feature <222> (14) <223> W is NapdU. <220> <221> misc_feature <222> (16) <223> W is NapdU. <220> <221> misc_feature <222> (17) <223> A is a nucleotide in which a methoxy group is introduced at the second carbon position. <220> <221> misc_feature <222> (20) <223> W is NapdU. <220> <221> misc_feature <222> (24) <223> W is NapdU. <400> 39 cawwacgcaw gagwcwagaw ccgwcag 27 <210> 40 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> A62 F 36 <220> <221> misc_feature <222> (3) <223> W isNapdU. <220> <221> misc_feature <222> (4) <223> W is NapdU. <220> <221> misc_feature <222> (10) <223> W is NapdU. <220> <221> misc_feature <222> (14) <223> W is NapdU. <220> <221> misc_feature <222> (16) <223> W is NapdU. <220> <221> misc_feature <222> (17) <223> A is a nucleotide in which a fluorine group is introduced at the second carbon position. <220> <221> misc_feature <222> (20) <223> W is NapdU. <220> <221> misc_feature <222> (24) <223> W is NapdU. <400> 40 cawwacgcaw gagwcwagaw ccgwcag 27 <210> 41 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> A62 L 37 <220> <221> misc_feature <222> (3) <223> W isNapdU. <220> <221> misc_feature <222> (4) <223> W is NapdU. <220> <221> misc_feature <222> (10) <223> W is NapdU. <220> <221> misc_feature <222> (14) <223> W is NapdU. <220> <221> misc_feature <222> (16) <223> W is NapdU. <220> <221> misc_feature <222> (18) <223> K is a nucleotide to which a C3 linker is attached. <220> <221> misc_feature <222> (20) <223> W is NapdU. <220> <221> misc_feature <222> (24) <223> W is NapdU. <400> 41 cawwacgcaw gagwcwakaw ccgwcag 27 <210> 42 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> A62 Me 38 <220> <221> misc_feature <222> (3) <223> W isNapdU. <220> <221> misc_feature <222> (4) <223> W is NapdU. <220> <221> misc_feature <222> (10) <223> W is NapdU. <220> <221> misc_feature <222> (14) <223> W is NapdU. <220> <221> misc_feature <222> (16) <223> W is NapdU. <220> <221> misc_feature <222> (18) <223> G is a nucleotide in which a methoxy group is introduced at the second carbon position. <220> <221> misc_feature <222> (20) <223> W is NapdU. <220> <221> misc_feature <222> (24) <223> W is NapdU. <400> 42 cawwacgcaw gagwcwagaw ccgwcag 27 <210> 43 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> A62 F 39 <220> <221> misc_feature <222> (3) <223> W isNapdU. <220> <221> misc_feature <222> (4) <223> W is NapdU. <220> <221> misc_feature <222> (10) <223> W is NapdU. <220> <221> misc_feature <222> (14) <223> W is NapdU. <220> <221> misc_feature <222> (16) <223> W is NapdU. <220> <221> misc_feature <222> (18) <223> G is a nucleotide in which a fluorine group is introduced at the second carbon position. <220> <221> misc_feature <222> (20) <223> W is NapdU. <220> <221> misc_feature <222> (24) <223> W is NapdU. <400> 43 cawwacgcaw gagwcwagaw ccgwcag 27 <210> 44 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> A62 L 40 <220> <221> misc_feature <222> (3) <223> W isNapdU. <220> <221> misc_feature <222> (4) <223> W is NapdU. <220> <221> misc_feature <222> (10) <223> W is NapdU. <220> <221> misc_feature <222> (14) <223> W is NapdU. <220> <221> misc_feature <222> (16) <223> W is NapdU. <220> <221> misc_feature <222> (19) <223> K is a nucleotide to which a C3 linker is attached. <220> <221> misc_feature <222> (20) <223> W is NapdU. <220> <221> misc_feature <222> (24) <223> W is NapdU. <400> 44 cawwacgcaw gagwcwagkw ccgwcag 27 <210> 45 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> A62 Me 41 <220> <221> misc_feature <222> (3) <223> W isNapdU. <220> <221> misc_feature <222> (4) <223> W is NapdU. <220> <221> misc_feature <222> (10) <223> W is NapdU. <220> <221> misc_feature <222> (14) <223> W is NapdU. <220> <221> misc_feature <222> (16) <223> W is NapdU. <220> <221> misc_feature <222> (19) <223> A is a nucleotide in which a methoxy group is introduced at the second carbon position. <220> <221> misc_feature <222> (20) <223> W is NapdU. <220> <221> misc_feature <222> (24) <223> W is NapdU. <400> 45 cawwacgcaw gagwcwagaw ccgwcag 27 <210> 46 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> A62 F 42 <220> <221> misc_feature <222> (3) <223> W isNapdU. <220> <221> misc_feature <222> (4) <223> W is NapdU. <220> <221> misc_feature <222> (10) <223> W is NapdU. <220> <221> misc_feature <222> (14) <223> W is NapdU. <220> <221> misc_feature <222> (16) <223> W is NapdU. <220> <221> misc_feature <222> (19) <223> A is a nucleotide in which a fluorine group is introduced at the second carbon position. <220> <221> misc_feature <222> (20) <223> W is NapdU. <220> <221> misc_feature <222> (24) <223> W is NapdU. <400> 46 cawwacgcaw gagwcwagaw ccgwcag 27 <210> 47 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> A62 L 43 <220> <221> misc_feature <222> (3) <223> W isNapdU. <220> <221> misc_feature <222> (4) <223> W is NapdU. <220> <221> misc_feature <222> (10) <223> W is NapdU. <220> <221> misc_feature <222> (14) <223> W is NapdU. <220> <221> misc_feature <222> (16) <223> W is NapdU. <220> <221> misc_feature <222> (20) <223> W is NapdU. <220> <221> misc_feature <222> (21) <223> K is a nucleotide to which a C3 linker is attached. <220> <221> misc_feature <222> (24) <223> W is NapdU. <400> 47 cawwacgcaw gagwcwagaw kcgwcag 27 <210> 48 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> A62 Me 44 <220> <221> misc_feature <222> (3) <223> W isNapdU. <220> <221> misc_feature <222> (4) <223> W is NapdU. <220> <221> misc_feature <222> (10) <223> W is NapdU. <220> <221> misc_feature <222> (14) <223> W is NapdU. <220> <221> misc_feature <222> (16) <223> W is NapdU. <220> <221> misc_feature <222> (20) <223> W is NapdU. <220> <221> misc_feature <222> (21) <223> C is a nucleotide in which a methoxy group is introduced at the second carbon position. <220> <221> misc_feature <222> (24) <223> W is NapdU. <400> 48 cawwacgcaw gagwcwagaw ccgwcag 27 <210> 49 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> A62 F 45 <220> <221> misc_feature <222> (3) <223> W isNapdU. <220> <221> misc_feature <222> (4) <223> W is NapdU. <220> <221> misc_feature <222> (10) <223> W is NapdU. <220> <221> misc_feature <222> (14) <223> W is NapdU. <220> <221> misc_feature <222> (16) <223> W is NapdU. <220> <221> misc_feature <222> (20) <223> W is NapdU. <220> <221> misc_feature <222> (21) <223> C is a nucleotide in which a fluorine group is introduced at the second carbon position. <220> <221> misc_feature <222> (24) <223> W is NapdU. <400> 49 cawwacgcaw gagwcwagaw ccgwcag 27 <210> 50 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> A62 L 46 <220> <221> misc_feature <222> (3) <223> W isNapdU. <220> <221> misc_feature <222> (4) <223> W is NapdU. <220> <221> misc_feature <222> (10) <223> W is NapdU. <220> <221> misc_feature <222> (14) <223> W is NapdU. <220> <221> misc_feature <222> (16) <223> W is NapdU. <220> <221> misc_feature <222> (20) <223> W is NapdU. <220> <221> misc_feature <222> (22) <223> K is a nucleotide to which a C3 linker is attached. <220> <221> misc_feature <222> (24) <223> W is NapdU. <400> 50 cawwacgcaw gagwcwagaw ckgwcag 27 <210> 51 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> A62 Me 47 <220> <221> misc_feature <222> (3) <223> W isNapdU. <220> <221> misc_feature <222> (4) <223> W is NapdU. <220> <221> misc_feature <222> (10) <223> W is NapdU. <220> <221> misc_feature <222> (14) <223> W is NapdU. <220> <221> misc_feature <222> (16) <223> W is NapdU. <220> <221> misc_feature <222> (20) <223> W is NapdU. <220> <221> misc_feature <222> (22) <223> C is a nucleotide in which a methoxy group is introduced at the second carbon position. <220> <221> misc_feature <222> (24) <223> W is NapdU. <400> 51 cawwacgcaw gagwcwagaw ccgwcag 27 <210> 52 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> A62 F 48 <220> <221> misc_feature <222> (3) <223> W isNapdU. <220> <221> misc_feature <222> (4) <223> W is NapdU. <220> <221> misc_feature <222> (10) <223> W is NapdU. <220> <221> misc_feature <222> (14) <223> W is NapdU. <220> <221> misc_feature <222> (16) <223> W is NapdU. <220> <221> misc_feature <222> (20) <223> W is NapdU. <220> <221> misc_feature <222> (22) <223> C is a nucleotide in which a fluorine group is introduced at the second carbon position. <220> <221> misc_feature <222> (24) <223> W is NapdU. <400> 52 cawwacgcaw gagwcwagaw ccgwcag 27 <210> 53 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> A62 L 49 <220> <221> misc_feature <222> (3) <223> W isNapdU. <220> <221> misc_feature <222> (4) <223> W is NapdU. <220> <221> misc_feature <222> (10) <223> W is NapdU. <220> <221> misc_feature <222> (14) <223> W is NapdU. <220> <221> misc_feature <222> (16) <223> W is NapdU. <220> <221> misc_feature <222> (20) <223> W is NapdU. <220> <221> misc_feature <222> (23) <223> K is a nucleotide to which a C3 linker is attached. <220> <221> misc_feature <222> (24) <223> W is NapdU. <400> 53 cawwacgcaw gagwcwagaw cckwcag 27 <210> 54 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> A62 Me 50 <220> <221> misc_feature <222> (3) <223> W isNapdU. <220> <221> misc_feature <222> (4) <223> W is NapdU. <220> <221> misc_feature <222> (10) <223> W is NapdU. <220> <221> misc_feature <222> (14) <223> W is NapdU. <220> <221> misc_feature <222> (16) <223> W is NapdU. <220> <221> misc_feature <222> (20) <223> W is NapdU. <220> <221> misc_feature <222> (23) <223> G is a nucleotide in which a methoxy group is introduced at the second carbon position. <220> <221> misc_feature <222> (24) <223> W is NapdU. <400> 54 cawwacgcaw gagwcwagaw ccgwcag 27 <210> 55 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> A62 F 51 <220> <221> misc_feature <222> (3) <223> W isNapdU. <220> <221> misc_feature <222> (4) <223> W is NapdU. <220> <221> misc_feature <222> (10) <223> W is NapdU. <220> <221> misc_feature <222> (14) <223> W is NapdU. <220> <221> misc_feature <222> (16) <223> W is NapdU. <220> <221> misc_feature <222> (20) <223> W is NapdU. <220> <221> misc_feature <222> (23) <223> G is a nucleotide in which a fluorine group is introduced at the second carbon position. <220> <221> misc_feature <222> (24) <223> W is NapdU. <400> 55 cawwacgcaw gagwcwagaw ccgwcag 27 <210> 56 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> A62 L 52 <220> <221> misc_feature <222> (3) <223> W isNapdU. <220> <221> misc_feature <222> (4) <223> W is NapdU. <220> <221> misc_feature <222> (10) <223> W is NapdU. <220> <221> misc_feature <222> (14) <223> W is NapdU. <220> <221> misc_feature <222> (16) <223> W is NapdU. <220> <221> misc_feature <222> (20) <223> W is NapdU. <220> <221> misc_feature <222> (24) <223> W is NapdU. <220> <221> misc_feature <222> (25) <223> K is a nucleotide to which a C3 linker is attached. <400> 56 cawwacgcaw gagwcwagaw ccgwkag 27 <210> 57 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> A62 Me 53 <220> <221> misc_feature <222> (3) <223> W isNapdU. <220> <221> misc_feature <222> (4) <223> W is NapdU. <220> <221> misc_feature <222> (10) <223> W is NapdU. <220> <221> misc_feature <222> (14) <223> W is NapdU. <220> <221> misc_feature <222> (16) <223> W is NapdU. <220> <221> misc_feature <222> (20) <223> W is NapdU. <220> <221> misc_feature <222> (24) <223> W is NapdU. <220> <221> misc_feature <222> (25) <223> C is a nucleotide in which a methoxy group is introduced at the second carbon position. <400> 57 cawwacgcaw gagwcwagaw ccgwcag 27 <210> 58 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> A62 F 54 <220> <221> misc_feature <222> (3) <223> W isNapdU. <220> <221> misc_feature <222> (4) <223> W is NapdU. <220> <221> misc_feature <222> (10) <223> W is NapdU. <220> <221> misc_feature <222> (14) <223> W is NapdU. <220> <221> misc_feature <222> (16) <223> W is NapdU. <220> <221> misc_feature <222> (20) <223> W is NapdU. <220> <221> misc_feature <222> (24) <223> W is NapdU. <220> <221> misc_feature <222> (25) <223> C is a nucleotide in which a fluorine group is introduced at the second carbon position. <400> 58 cawwacgcaw gagwcwagaw ccgwcag 27 <210> 59 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> A62 L 55 <220> <221> misc_feature <222> (3) <223> W isNapdU. <220> <221> misc_feature <222> (4) <223> W is NapdU. <220> <221> misc_feature <222> (10) <223> W is NapdU. <220> <221> misc_feature <222> (14) <223> W is NapdU. <220> <221> misc_feature <222> (16) <223> W is NapdU. <220> <221> misc_feature <222> (20) <223> W is NapdU. <220> <221> misc_feature <222> (24) <223> W is NapdU. <220> <221> misc_feature <222> (26) <223> K is a nucleotide to which a C3 linker is attached. <400> 59 cawwacgcaw gagwcwagaw ccgwckg 27 <210> 60 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> A62 Me 56 <220> <221> misc_feature <222> (3) <223> W isNapdU. <220> <221> misc_feature <222> (4) <223> W is NapdU. <220> <221> misc_feature <222> (10) <223> W is NapdU. <220> <221> misc_feature <222> (14) <223> W is NapdU. <220> <221> misc_feature <222> (16) <223> W is NapdU. <220> <221> misc_feature <222> (20) <223> W is NapdU. <220> <221> misc_feature <222> (24) <223> W is NapdU. <220> <221> misc_feature <222> (26) <223> A is a nucleotide in which a methoxy group is introduced at the second carbon position. <400> 60 cawwacgcaw gagwcwagaw ccgwcag 27 <210> 61 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> A62 F 57 <220> <221> misc_feature <222> (3) <223> W isNapdU. <220> <221> misc_feature <222> (4) <223> W is NapdU. <220> <221> misc_feature <222> (10) <223> W is NapdU. <220> <221> misc_feature <222> (14) <223> W is NapdU. <220> <221> misc_feature <222> (16) <223> W is NapdU. <220> <221> misc_feature <222> (20) <223> W is NapdU. <220> <221> misc_feature <222> (24) <223> W is NapdU. <220> <221> misc_feature <222> (26) <223> A is a nucleotide in which a fluorine group is introduced at the second carbon position. <400> 61 cawwacgcaw gagwcwagaw ccgwcag 27 <210> 62 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> A62 L 58 <220> <221> misc_feature <222> (3) <223> W isNapdU. <220> <221> misc_feature <222> (4) <223> W is NapdU. <220> <221> misc_feature <222> (10) <223> W is NapdU. <220> <221> misc_feature <222> (14) <223> W is NapdU. <220> <221> misc_feature <222> (16) <223> W is NapdU. <220> <221> misc_feature <222> (20) <223> W is NapdU. <220> <221> misc_feature <222> (24) <223> W is NapdU. <220> <221> misc_feature <222> (27) <223> K is a nucleotide to which a C3 linker is attached. <400> 62 cawwacgcaw gagwcwagaw ccgwcak 27 <210> 63 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> A62 Me <220> <221> misc_feature <222> (3) <223> W isNapdU. <220> <221> misc_feature <222> (4) <223> W is NapdU. <220> <221> misc_feature <222> (10) <223> W is NapdU. <220> <221> misc_feature <222> (14) <223> W is NapdU. <220> <221> misc_feature <222> (16) <223> W is NapdU. <220> <221> misc_feature <222> (20) <223> W is NapdU. <220> <221> misc_feature <222> (24) <223> W is NapdU. <220> <221> misc_feature <222> (27) <223> G is a nucleotide in which a methoxy group is introduced at the second carbon position. <400> 63 cawwacgcaw gagwcwagaw ccgwcag 27 <210> 64 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> A62 F <220> <221> misc_feature <222> (3) <223> W isNapdU. <220> <221> misc_feature <222> (4) <223> W is NapdU. <220> <221> misc_feature <222> (10) <223> W is NapdU. <220> <221> misc_feature <222> (14) <223> W is NapdU. <220> <221> misc_feature <222> (16) <223> W is NapdU. <220> <221> misc_feature <222> (20) <223> W is NapdU. <220> <221> misc_feature <222> (24) <223> W is NapdU. <220> <221> misc_feature <222> (27) <223> G is a nucleotide in which a fluorine group is introduced at the second carbon position. <400> 64 cawwacgcaw gagwcwagaw ccgwcag 27 <210> 65 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> A62 b 1 <220> <221> misc_feature <222> (3) <223> Y is BndU. <220> <221> misc_feature <222> (4) <223> W is NapdU. <220> <221> misc_feature <222> (10) <223> W is NapdU. <220> <221> misc_feature <222> (14) <223> W is NapdU. <220> <221> misc_feature <222> (16) <223> W is NapdU. <220> <221> misc_feature <222> (20) <223> W is NapdU. <220> <221> misc_feature <222> (24) <223> W is NapdU. <400> 65 caywacgcaw gagwcwagaw ccgwcag 27 <210> 66 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> A62 b 2 <220> <221> misc_feature <222> (3) <223> W isNapdU. <220> <221> misc_feature <222> (4) <223> Y is BndU. <220> <221> misc_feature <222> (10) <223> W is NapdU. <220> <221> misc_feature <222> (14) <223> W is NapdU. <220> <221> misc_feature <222> (16) <223> W is NapdU. <220> <221> misc_feature <222> (20) <223> W is NapdU. <220> <221> misc_feature <222> (24) <223> W is NapdU. <400> 66 cawyacgcaw gagwcwagaw ccgwcag 27 <210> 67 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> A62 b 3 <220> <221> misc_feature <222> (3) <223> W isNapdU. <220> <221> misc_feature <222> (4) <223> W is NapdU. <220> <221> misc_feature <222> (10) <223> Y is BndU. <220> <221> misc_feature <222> (14) <223> W is NapdU. <220> <221> misc_feature <222> (16) <223> W is NapdU. <220> <221> misc_feature <222> (20) <223> W is NapdU. <220> <221> misc_feature <222> (24) <223> W is NapdU. <400> 67 cawwacgcay gagwcwagaw ccgwcag 27 <210> 68 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> A62 b 4 <220> <221> misc_feature <222> (3) <223> W isNapdU. <220> <221> misc_feature <222> (4) <223> W is NapdU. <220> <221> misc_feature <222> (10) <223> W is NapdU. <220> <221> misc_feature <222> (14) <223> Y is BndU. <220> <221> misc_feature <222> (16) <223> W is NapdU. <220> <221> misc_feature <222> (20) <223> W is NapdU. <220> <221> misc_feature <222> (24) <223> W is NapdU. <400> 68 cawwacgcaw gagycwagaw ccgwcag 27 <210> 69 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> A62 b 5 <220> <221> misc_feature <222> (3) <223> W isNapdU. <220> <221> misc_feature <222> (4) <223> W is NapdU. <220> <221> misc_feature <222> (10) <223> W is NapdU. <220> <221> misc_feature <222> (14) <223> W is NapdU. <220> <221> misc_feature <222> (16) <223> Y is BndU. <220> <221> misc_feature <222> (20) <223> W is NapdU. <220> <221> misc_feature <222> (24) <223> W is NapdU. <400> 69 cawwacgcaw gagwcyagaw ccgwcag 27 <210> 70 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> A62 b 6 <220> <221> misc_feature <222> (3) <223> W isNapdU. <220> <221> misc_feature <222> (4) <223> W is NapdU. <220> <221> misc_feature <222> (10) <223> W is NapdU. <220> <221> misc_feature <222> (14) <223> W is NapdU. <220> <221> misc_feature <222> (16) <223> W is NapdU. <220> <221> misc_feature <222> (20) <223> Y is BndU. <220> <221> misc_feature <222> (24) <223> W is NapdU. <400> 70 cawwacgcaw gagwcwagay ccgwcag 27 <210> 71 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> A62 b 7 <220> <221> misc_feature <222> (3) <223> W isNapdU. <220> <221> misc_feature <222> (4) <223> W is NapdU. <220> <221> misc_feature <222> (10) <223> W is NapdU. <220> <221> misc_feature <222> (14) <223> W is NapdU. <220> <221> misc_feature <222> (16) <223> W is NapdU. <220> <221> misc_feature <222> (20) <223> W is NapdU. <220> <221> misc_feature <222> (24) <223> Y is BndU. <400> 71 cawwacgcaw gagwcwagaw ccgycag 27 <210> 72 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> A62m combi 1 <220> <221> misc_feature <222> (2) <223> A is a nucleotide in which a fluorine group is introduced at the second carbon position. <220> <221> misc_feature <222> (3) <223> Y is BndU. <220> <221> misc_feature <222> (4) <223> Y is BndU. <220> <221> misc_feature <222> (6) <223> C is a nucleotide in which a fluorine group is introduced at the second carbon position. <220> <221> misc_feature <222> (8) <223> C is a nucleotide in which a fluorine group is introduced at the second carbon position. <220> <221> misc_feature <222> (10) <223> W is NapdU. <220> <221> misc_feature <222> (11) <223> G is a nucleotide in which a methoxy group is introduced at the second carbon position. <220> <221> misc_feature <222> (12) <223> A is a nucleotide in which a methoxy group is introduced at the second carbon position. <220> <221> misc_feature <222> (13) <223> G is a nucleotide in which a methoxy group is introduced at the second carbon position. <220> <221> misc_feature <222> (14) <223> Y is BndU. <220> <221> misc_feature <222> (16) <223> W is NapdU. <220> <221> misc_feature <222> (19) <223> A is a nucleotide in which a fluorine group is introduced at the second carbon position. <220> <221> misc_feature <222> (20) <223> W is NapdU. <220> <221> misc_feature <222> (21) <223> C is a nucleotide in which a methoxy group is introduced at the second carbon position. <220> <221> misc_feature <222> (22) <223> C is a nucleotide in which a fluorine group is introduced at the second carbon position. <220> <221> misc_feature <222> (24) <223> Y is BndU. <220> <221> misc_feature <222> (25) <223> C is a nucleotide in which a methoxy group is introduced at the second carbon position. <220> <221> misc_feature <222> (26) <223> A is a nucleotide in which a methoxy group is introduced at the second carbon position. <220> <221> misc_feature <222> (27) <223> G is a nucleotide in which a methoxy group is introduced at the second carbon position. <400> 72 cayyacgcaw gagycwagaw ccgycag 27 <210> 73 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> A62m combi 2 <220> <221> misc_feature <222> (2) <223> A is a nucleotide in which a fluorine group is introduced at the second carbon position. <220> <221> misc_feature <222> (3) <223> Y is BndU. <220> <221> misc_feature <222> (4) <223> Y is BndU. <220> <221> misc_feature <222> (6) <223> C is a nucleotide in which a fluorine group is introduced at the second carbon position. <220> <221> misc_feature <222> (8) <223> C is a nucleotide in which a fluorine group is introduced at the second carbon position. <220> <221> misc_feature <222> (10) <223> W is NapdU. <220> <221> misc_feature <222> (11) <223> G is a nucleotide in which a methoxy group is introduced at the second carbon position. <220> <221> misc_feature <222> (12) <223> A is a nucleotide in which a methoxy group is introduced at the second carbon position. <220> <221> misc_feature <222> (13) <223> G is a nucleotide in which a methoxy group is introduced at the second carbon position. <220> <221> misc_feature <222> (14) <223> Y is BndU. <220> <221> misc_feature <222> (16) <223> W is NapdU. <220> <221> misc_feature <222> (19) <223> A is a nucleotide in which a fluorine group is introduced at the second carbon position. <220> <221> misc_feature <222> (20) <223> W is NapdU. <220> <221> misc_feature <222> (21) <223> C is a nucleotide in which a methoxy group is introduced at the second carbon position. <220> <221> misc_feature <222> (22) <223> C is a nucleotide in which a fluorine group is introduced at the second carbon position. <220> <221> misc_feature <222> (24) <223> Y is BndU. <220> <221> misc_feature <222> (25) <223> C is a nucleotide in which a methoxy group is introduced at the second carbon position. <220> <221> misc_feature <222> (26) <223> A is a nucleotide in which a methoxy group is introduced at the second carbon position. <400> 73 cayyacgcaw gagycwagaw ccgyca 26 <210> 74 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> A62m combi 3 <220> <221> misc_feature <222> (2) <223> A is a nucleotide in which a fluorine group is introduced at the second carbon position. <220> <221> misc_feature <222> (3) <223> Y is BndU. <220> <221> misc_feature <222> (4) <223> Y is BndU. <220> <221> misc_feature <222> (6) <223> C is a nucleotide in which a fluorine group is introduced at the second carbon position. <220> <221> misc_feature <222> (8) <223> C is a nucleotide in which a fluorine group is introduced at the second carbon position. <220> <221> misc_feature <222> (10) <223> W is NapdU. <220> <221> misc_feature <222> (11) <223> G is a nucleotide in which a methoxy group is introduced at the second carbon position. <220> <221> misc_feature <222> (12) <223> A is a nucleotide in which a methoxy group is introduced at the second carbon position. <220> <221> misc_feature <222> (13) <223> G is a nucleotide in which a methoxy group is introduced at the second carbon position. <220> <221> misc_feature <222> (14) <223> Y is BndU. <220> <221> misc_feature <222> (16) <223> W is NapdU. <220> <221> misc_feature <222> (19) <223> A is a nucleotide in which a fluorine group is introduced at the second carbon position. <220> <221> misc_feature <222> (20) <223> W is NapdU. <220> <221> misc_feature <222> (21) <223> C is a nucleotide in which a methoxy group is introduced at the second carbon position. <220> <221> misc_feature <222> (22) <223> C is a nucleotide in which a fluorine group is introduced at the second carbon position. <220> <221> misc_feature <222> (24) <223> Y is BndU. <220> <221> misc_feature <222> (25) <223> C is a nucleotide in which a methoxy group is introduced at the second carbon position. <400> 74 cayyacgcaw gagycwagaw ccgyc 25 <210> 75 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> A62m combi 4 <220> <221> misc_feature <222> (2) <223> A is a nucleotide in which a fluorine group is introduced at the second carbon position. <220> <221> misc_feature <222> (3) <223> Y is BndU. <220> <221> misc_feature <222> (4) <223> Y is BndU. <220> <221> misc_feature <222> (6) <223> C is a nucleotide in which a fluorine group is introduced at the second carbon position. <220> <221> misc_feature <222> (8) <223> C is a nucleotide in which a fluorine group is introduced at the second carbon position. <220> <221> misc_feature <222> (10) <223> W is NapdU. <220> <221> misc_feature <222> (11) <223> G is a nucleotide in which a methoxy group is introduced at the second carbon position. <220> <221> misc_feature <222> (12) <223> A is a nucleotide in which a methoxy group is introduced at the second carbon position. <220> <221> misc_feature <222> (13) <223> G is a nucleotide in which a methoxy group is introduced at the second carbon position. <220> <221> misc_feature <222> (14) <223> Y is BndU. <220> <221> misc_feature <222> (16) <223> W is NapdU. <220> <221> misc_feature <222> (19) <223> A is a nucleotide in which a fluorine group is introduced at the second carbon position. <220> <221> misc_feature <222> (20) <223> W is NapdU. <220> <221> misc_feature <222> (21) <223> C is a nucleotide in which a methoxy group is introduced at the second carbon position. <220> <221> misc_feature <222> (22) <223> C is a nucleotide in which a fluorine group is introduced at the second carbon position. <220> <221> misc_feature <222> (24) <223> Y is BndU. <400> 75 cayyacgcaw gagycwagaw ccgy 24 <210> 76 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> A62m combi 5 <220> <221> misc_feature <222> (2) <223> A is a nucleotide in which a fluorine group is introduced at the second carbon position. <220> <221> misc_feature <222> (3) <223> Y is BndU. <220> <221> misc_feature <222> (4) <223> Y is BndU. <220> <221> misc_feature <222> (6) <223> C is a nucleotide in which a fluorine group is introduced at the second carbon position. <220> <221> misc_feature <222> (8) <223> C is a nucleotide in which a fluorine group is introduced at the second carbon position. <220> <221> misc_feature <222> (10) <223> W is NapdU. <220> <221> misc_feature <222> (11) <223> G is a nucleotide in which a methoxy group is introduced at the second carbon position. <220> <221> misc_feature <222> (13) <223> G is a nucleotide in which a methoxy group is introduced at the second carbon position. <220> <221> misc_feature <222> (14) <223> Y is BndU. <220> <221> misc_feature <222> (16) <223> W is NapdU. <220> <221> misc_feature <222> (19) <223> A is a nucleotide in which a fluorine group is introduced at the second carbon position. <220> <221> misc_feature <222> (20) <223> W is NapdU. <220> <221> misc_feature <222> (21) <223> C is a nucleotide in which a methoxy group is introduced at the second carbon position. <220> <221> misc_feature <222> (22) <223> C is a nucleotide in which a fluorine group is introduced at the second carbon position. <220> <221> misc_feature <222> (24) <223> Y is BndU. <220> <221> misc_feature <222> (25) <223> C is a nucleotide in which a methoxy group is introduced at the second carbon position. <220> <221> misc_feature <222> (26) <223> A is a nucleotide in which a methoxy group is introduced at the second carbon position. <220> <221> misc_feature <222> (27) <223> G is a nucleotide in which a methoxy group is introduced at the second carbon position. <220> <221> misc_feature <222> (12) <223> A is a nucleotide in which a fluorine group is introduced at the second carbon position. <400> 76 cayyacgcaw gagycwagaw ccgycag 27 <210> 77 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> A62m combi 6 <220> <221> misc_feature <222> (2) <223> A is a nucleotide in which a fluorine group is introduced at the second carbon position. <220> <221> misc_feature <222> (3) <223> Y is BndU. <220> <221> misc_feature <222> (4) <223> Y is BndU. <220> <221> misc_feature <222> (6) <223> C is a nucleotide in which a fluorine group is introduced at the second carbon position. <220> <221> misc_feature <222> (8) <223> C is a nucleotide in which a fluorine group is introduced at the second carbon position. <220> <221> misc_feature <222> (10) <223> W is NapdU. <220> <221> misc_feature <222> (11) <223> G is a nucleotide in which a methoxy group is introduced at the second carbon position. <220> <221> misc_feature <222> (12) <223> A is a nucleotide in which a fluorine group is introduced at the second carbon position. <220> <221> misc_feature <222> (13) <223> G is a nucleotide in which a methoxy group is introduced at the second carbon position. <220> <221> misc_feature <222> (14) <223> Y is BndU. <220> <221> misc_feature <222> (16) <223> W is NapdU. <220> <221> misc_feature <222> (19) <223> A is a nucleotide in which a fluorine group is introduced at the second carbon position. <220> <221> misc_feature <222> (20) <223> W is NapdU. <220> <221> misc_feature <222> (21) <223> C is a nucleotide in which a methoxy group is introduced at the second carbon position. <220> <221> misc_feature <222> (22) <223> C is a nucleotide in which a fluorine group is introduced at the second carbon position. <220> <221> misc_feature <222> (24) <223> Y is BndU. <220> <221> misc_feature <222> (25) <223> C is a nucleotide in which a methoxy group is introduced at the second carbon position. <220> <221> misc_feature <222> (26) <223> A is a nucleotide in which a methoxy group is introduced at the second carbon position. <220> <221> misc_feature <222> (27) <223> K is a nucleotide to which a C3 linker is attached. <400> 77 cayyacgcaw gagycwagaw ccgycak 27 <210> 78 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> A62m combi 7(A62ms) <220> <221> misc_feature <222> (2) <223> A is a nucleotide in which a fluorine group is introduced at the second carbon position. <220> <221> misc_feature <222> (3) <223> Y is BndU. <220> <221> misc_feature <222> (4) <223> Y is BndU. <220> <221> misc_feature <222> (6) <223> C is a nucleotide in which a fluorine group is introduced at the second carbon position. <220> <221> misc_feature <222> (8) <223> C is a nucleotide in which a fluorine group is introduced at the second carbon position. <220> <221> misc_feature <222> (10) <223> W is NapdU. <220> <221> misc_feature <222> (11) <223> G is a nucleotide in which a methoxy group is introduced at the second carbon position. <220> <221> misc_feature <222> (12) <223> A is a nucleotide in which a fluorine group is introduced at the second carbon position. <220> <221> misc_feature <222> (13) <223> G is a nucleotide in which a methoxy group is introduced at the second carbon position. <220> <221> misc_feature <222> (14) <223> Y is BndU. <220> <221> misc_feature <222> (16) <223> W is NapdU. <220> <221> misc_feature <222> (19) <223> A is a nucleotide in which a fluorine group is introduced at the second carbon position. <220> <221> misc_feature <222> (20) <223> W is NapdU. <220> <221> misc_feature <222> (21) <223> C is a nucleotide in which a methoxy group is introduced at the second carbon position. <220> <221> misc_feature <222> (22) <223> C is a nucleotide in which a fluorine group is introduced at the second carbon position. <220> <221> misc_feature <222> (24) <223> Y is BndU. <220> <221> misc_feature <222> (25) <223> C is a nucleotide in which a methoxy group is introduced at the second carbon position. <400> 78 cayyacgcaw gagycwagaw ccgyc 25 <210> 79 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> A62m combi 8 <220> <221> misc_feature <222> (2) <223> A is a nucleotide in which a fluorine group is introduced at the second carbon position. <220> <221> misc_feature <222> (3) <223> Y is BndU. <220> <221> misc_feature <222> (4) <223> Y is BndU. <220> <221> misc_feature <222> (6) <223> C is a nucleotide in which a fluorine group is introduced at the second carbon position. <220> <221> misc_feature <222> (8) <223> C is a nucleotide in which a fluorine group is introduced at the second carbon position. <220> <221> misc_feature <222> (10) <223> W is NapdU. <220> <221> misc_feature <222> (11) <223> G is a nucleotide in which a methoxy group is introduced at the second carbon position. <220> <221> misc_feature <222> (12) <223> A is a nucleotide in which a fluorine group is introduced at the second carbon position. <220> <221> misc_feature <222> (13) <223> G is a nucleotide in which a methoxy group is introduced at the second carbon position. <220> <221> misc_feature <222> (14) <223> Y is BndU. <220> <221> misc_feature <222> (16) <223> W is NapdU. <220> <221> misc_feature <222> (19) <223> A is a nucleotide in which a fluorine group is introduced at the second carbon position. <220> <221> misc_feature <222> (20) <223> W is NapdU. <220> <221> misc_feature <222> (21) <223> C is a nucleotide in which a methoxy group is introduced at the second carbon position. <220> <221> misc_feature <222> (22) <223> C is a nucleotide in which a fluorine group is introduced at the second carbon position. <220> <221> misc_feature <222> (24) <223> Y is BndU. <220> <221> misc_feature <222> (25) <223> C is a nucleotide in which a methoxy group is introduced at the second carbon position. <220> <221> misc_feature <222> (26) <223> A is a nucleotide in which a fluorine group is introduced at the second carbon position. <220> <221> misc_feature <222> (27) <223> G is a nucleotide in which a methoxy group is introduced at the second carbon position. <400> 79 cayyacgcaw gagycwagaw ccgycag 27 <210> 80 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> A62m combi 9 <220> <221> misc_feature <222> (2) <223> A is a nucleotide in which a fluorine group is introduced at the second carbon position. <220> <221> misc_feature <222> (3) <223> Y is BndU. <220> <221> misc_feature <222> (4) <223> Y is BndU. <220> <221> misc_feature <222> (6) <223> C is a nucleotide in which a fluorine group is introduced at the second carbon position. <220> <221> misc_feature <222> (8) <223> C is a nucleotide in which a fluorine group is introduced at the second carbon position. <220> <221> misc_feature <222> (10) <223> W is NapdU. <220> <221> misc_feature <222> (11) <223> G is a nucleotide in which a fluorine group is introduced at the second carbon position. <220> <221> misc_feature <222> (12) <223> A is a nucleotide in which a methoxy group is introduced at the second carbon position. <220> <221> misc_feature <222> (13) <223> G is a nucleotide in which a fluorine group is introduced at the second carbon position. <220> <221> misc_feature <222> (14) <223> Y is BndU. <220> <221> misc_feature <222> (16) <223> W is NapdU. <220> <221> misc_feature <222> (19) <223> A is a nucleotide in which a fluorine group is introduced at the second carbon position. <220> <221> misc_feature <222> (20) <223> W is NapdU. <220> <221> misc_feature <222> (21) <223> C is a nucleotide in which a methoxy group is introduced at the second carbon position. <220> <221> misc_feature <222> (22) <223> C is a nucleotide in which a fluorine group is introduced at the second carbon position. <220> <221> misc_feature <222> (24) <223> Y is BndU. <220> <221> misc_feature <222> (25) <223> C is a nucleotide in which a methoxy group is introduced at the second carbon position. <220> <221> misc_feature <222> (26) <223> A is a nucleotide in which a fluorine group is introduced at the second carbon position. <220> <221> misc_feature <222> (27) <223> G is a nucleotide in which a methoxy group is introduced at the second carbon position. <400> 80 cayyacgcaw gagycwagaw ccgycag 27 <210> 81 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> A62m combi 10 <220> <221> misc_feature <222> (2) <223> A is a nucleotide in which a fluorine group is introduced at the second carbon position. <220> <221> misc_feature <222> (3) <223> Y is BndU. <220> <221> misc_feature <222> (4) <223> Y is BndU. <220> <221> misc_feature <222> (6) <223> C is a nucleotide in which a fluorine group is introduced at the second carbon position. <220> <221> misc_feature <222> (8) <223> C is a nucleotide in which a fluorine group is introduced at the second carbon position. <220> <221> misc_feature <222> (10) <223> W is NapdU. <220> <221> misc_feature <222> (11) <223> G is a nucleotide in which a methoxy group is introduced at the second carbon position. <220> <221> misc_feature <222> (12) <223> A is a nucleotide in which a fluorine group is introduced at the second carbon position. <220> <221> misc_feature <222> (13) <223> G is a nucleotide in which a methoxy group is introduced at the second carbon position. <220> <221> misc_feature <222> (14) <223> Y is BndU. <220> <221> misc_feature <222> (16) <223> W is NapdU. <220> <221> misc_feature <222> (19) <223> A is a nucleotide in which a fluorine group is introduced at the second carbon position. <220> <221> misc_feature <222> (20) <223> W is NapdU. <220> <221> misc_feature <222> (21) <223> C is a nucleotide in which a methoxy group is introduced at the second carbon position. <220> <221> misc_feature <222> (22) <223> C is a nucleotide in which a fluorine group is introduced at the second carbon position. <220> <221> misc_feature <222> (24) <223> Y is BndU. <220> <221> misc_feature <222> (25) <223> K is a nucleotide to which a C3 linker is attached. <220> <221> misc_feature <222> (26) <223> A is a nucleotide in which a methoxy group is introduced at the second carbon position. <220> <221> misc_feature <222> (27) <223> K is a nucleotide to which a C3 linker is attached. <400> 81 cayyacgcaw gagycwagaw ccgykak 27 <210> 82 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> General formula 1' <220> <221> misc_feature <222> (3) <223 > B is NapdU or BndU. <220> <221> misc_feature <222> (4) <223> B is NapdU or BndU. <220> <221> misc_feature <222> (10) <223> B is NapdU or BndU. <220> <221> misc_feature <222> (14) <223> B is NapdU or BndU. <220> <221> misc_feature <222> (16) <223> B is NapdU or BndU. <220> <221> misc_feature <222> (20) <223> B is NapdU or BndU. <220> <221> misc_feature <222> (24) <223> B is NapdU or BndU.<400> 82 cabbacgcab gagbcbagab ccgbc 25

Claims (20)

An insulin receptor-specific aptamer comprising a polynucleotide sequence of General Formula 1 below:
[General Formula 1]
5'- CABBACGCAB GAGBCBAGAB CCGBCAG- 3' (SEQ ID NO: 4);
Here, B in General Formula 1 is deoxyribose uracil in which the 5th carbon position of the nucleotide is substituted with a hydrophobic functional group,
C in General Formula 1 may be a modified or unmodified nucleotide, and the modification may include a linker (C3 linker) binding to the nucleotide; Introduction of a methoxy group (2-O-Me, 2-O-Methoxy-DNA) at the 2nd carbon position of the nucleotide; and introduction of fluorine (2-F, 2-Fluorine-DNA) at the second carbon position of the nucleotide; and one or more modifications selected from the group consisting of,
G in General Formula 1 may be a modified or unmodified nucleotide, and the modification may include a linker bond to the nucleotide; introduction of a methoxy group at the second carbon position of the nucleotide; and introduction of fluorine at the second carbon position of the nucleotide; and one or more modifications selected from the group consisting of,
A of General Formula 1 may be a modified or unmodified nucleotide, and the modification may include a linker bond to the nucleotide; introduction of a methoxy group at the second carbon position of the nucleotide; and introducing fluorine at the second carbon position of the nucleotide.
The insulin receptor-specific aptamer according to claim 1, wherein the hydrophobic functional group is at least one selected from the group consisting of a naphthyl group, benzyl group, pyrrolebenzyl group, isobutyl group, and tryptophan.
The insulin receptor-specific aptamer according to claim 1, wherein 1 to 3 nucleotides are excluded from either the 3' end or the 5' end of the sequence of General Formula 1.
The insulin receptor-specific aptamer of claim 3, wherein the aptamer comprises the following polynucleotide sequence:
5'- CABBACGCAB GAGBCBAGAB CCGBC- 3' (SEQ ID NO: 82);
Here, B in General Formula 1 is a naphthyl-uracil nucleotide in which the 5th carbon of the dUTP nucleotide is substituted with a naphthyl group; or a benzyl-uracil nucleotide in which the 5th carbon of the dUTP nucleotide is substituted with a benzyl group; One or more of the following,
C in General Formula 1 may be a modified or unmodified nucleotide, and the modification may include a linker bond to the nucleotide; introduction of a methoxy group at the second carbon position of the nucleotide; and introduction of fluorine at the second carbon position of the nucleotide; and one or more modifications selected from the group consisting of,
G in General Formula 1 may be a modified or unmodified nucleotide, and the modification may include a linker bond to the nucleotide; introduction of a methoxy group at the second carbon position of the nucleotide; and introduction of fluorine at the second carbon position of the nucleotide; and one or more modifications selected from the group consisting of,
A of General Formula 1 may be a modified or unmodified nucleotide, and the modification may include a linker bond to the nucleotide; introduction of a methoxy group at the second carbon position of the nucleotide; and introducing fluorine at the second carbon position of the nucleotide.
The insulin receptor-specific aptamer according to claim 1, wherein the aptamer comprises one or more polynucleotide sequences selected from the group consisting of polynucleotide sequences of SEQ ID NO: 5 to SEQ ID NO: 81.
The method of claim 1, wherein the aptamer has PEG (polyethylene glycol), idT (inverted deoxythymidine), LNA (Locked Nucleic Acid), amine linker, An insulin receptor-specific aptamer, wherein at least one selected from the group consisting of a thiol linker and cholesterol is combined.
The insulin receptor-specific aptamer of claim 1, wherein the aptamer binds to the insulin receptor competitively or non-competitively with insulin.
The insulin receptor-specific aptamer according to claim 1, wherein the aptamer phosphorylates Y1150 of the insulin receptor.
The insulin receptor-specific aptamer according to claim 1, wherein the aptamer phosphorylates AKT.
The insulin receptor-specific aptamer according to claim 1, wherein the aptamer sensitizes phosphorylation of ERK.
The insulin receptor-specific aptamer according to claim 1, wherein the aptamer increases cellular glucose uptake.
The insulin receptor-specific aptamer according to claim 1, wherein the aptamer does not promote cell division.
The insulin receptor-specific aptamer according to claim 1, wherein the aptamer exhibits blood sugar lowering activity.
The insulin receptor-specific aptamer according to claim 1, wherein the aptamer exhibits weight gain inhibitory activity.
The insulin receptor-specific aptamer according to claim 1, wherein the aptamer exhibits a bioavailability of 40% or more.
The insulin receptor-specific aptamer according to claim 1, wherein the aptamer exhibits a blood half-life of 5 hours or more.
An insulin receptor agonist comprising the aptamer of any one of claims 1 to 16.
A pharmaceutical composition for preventing or treating insulin-related diseases, comprising the aptamer of any one of claims 1 to 16 as an active ingredient.
The pharmaceutical composition according to claim 18, wherein the insulin-related disease is at least one selected from the group consisting of diabetes, diabetic complications, metabolic syndrome, obesity, and cardiovascular disease.
A composition for diagnosing diabetes or diabetic complications, comprising the aptamer of any one of claims 1 to 16.