JP2002058491A - New oligonucleotide, vitronectin aptamer, carcinostatic, and method for analyzing vitronectin - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a new oligonucleotide useful as the active ingredient of a carcinostatic, vitronectin aptamer and a new carcinostatic. SOLUTION: The oligonucleotide contains a base sequence among those represented by the following sequences: CAGCTCCGKS AGST, ACCTGACKSA GST or YCCVTNVYNT MSCTGCCY. The vitronectin aptamer comprises this oligonucleotide. The carcinostatic is obtained by including the oligonucleotide as an active ingredient.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a novel oligonucleotide, a vitronectin aptamer comprising the oligonucleotide, an anticancer agent comprising the oligonucleotide,
And a vitronectin analysis method. In the present specification, the "analysis" includes both "measurement" for quantitatively or semi-quantitatively determining the amount of the analyte and "detection" for determining the presence or absence of the analyte. included.

[0002]

2. Description of the Related Art As metastasis mechanisms of cancer cells, (1)
That cancer cells express a receptor (αvβ3 integrin) and infiltrate while adhering to a ligand [RGDV domain of extracellular matrix protein (eg, vitronectin)]; and (2) cancer cells are another receptor It is known that, via αvβ5 integrin, it adheres to the RGDV domain which is a ligand and migrates on vitronectin and the like which have leached out of blood into the interstitium. That is, it is known that in the metastasis of cancer cells, the binding of each of the integrins on the surface of the cancer cells to vitronectin is important.

[0003] From such a viewpoint, by adding a free RGD peptide (a peptide containing RGD) and binding it to an integrin on a cancer cell, the RG on a surface of an extracellular matrix protein (for example, vitronectin) is added.
Attempts have been made to block the binding of the D sequence to integrins on cancer cells. However, this method not only has a problem of antigenicity but also has not obtained the expected in vivo effects.

On the other hand, it specifically binds to a certain protein,
Oligonucleotides that affect its biological activity are called aptamers, and for example, a thrombin aptamer that specifically binds to thrombin is disclosed in Japanese Patent Publication No. 6-508022. But,
Aptamers that specifically bind to vitronectin (ie, vitronectin aptamers) are not known.

[0005]

SUMMARY OF THE INVENTION The present inventors aimed at obtaining an inhibitor of the growth and metastasis of cancer cells, and have a completely different mechanism of action from the above-mentioned conventional method using the RGD peptide. By searching for a compound that inhibits the binding between integrin and vitronectin by targeting the vitronectin side instead of the integrin on the side, it was found that an oligonucleotide containing a specific base sequence has an action as a vitronectin aptamer. I found it. The present invention is based on such findings.
Therefore, an object of the present invention is to provide a novel oligonucleotide useful as an active ingredient of an anticancer agent.

[0006]

Means for Solving the Problems The object is achieved by the present invention comprising an oligonucleotide containing a base sequence represented by SEQ ID NO: 1 in the sequence listing (hereinafter sometimes referred to as a first oligonucleotide). Can be solved. The present invention also relates to an oligonucleotide containing a base sequence represented by SEQ ID NO: 4 in the sequence listing (hereinafter, may be referred to as a second oligonucleotide). In addition, the present invention relates to an oligonucleotide containing a base sequence represented by SEQ ID NO: 7 in the sequence listing (hereinafter, may be referred to as a third oligonucleotide).

[0007] The present invention also relates to a vitronectin aptamer comprising the first oligonucleotide.
The present invention also relates to a vitronectin aptamer comprising the above-mentioned second oligonucleotide. The present invention also relates to a vitronectin aptamer comprising the third oligonucleotide.

[0008] The present invention also relates to an anticancer agent characterized by containing at least one of the first oligonucleotide, the second oligonucleotide, and the third oligonucleotide as an active ingredient. Furthermore, the present invention relates to a method for analyzing vitronectin, comprising using the first oligonucleotide, the second oligonucleotide, or the third oligonucleotide.

[0009]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail.
The “oligonucleotide” in the oligonucleotide of the present invention means a single-stranded oligodeoxyribonucleotide.

[0010] The first oligonucleotide according to the present invention comprises a base sequence represented by SEQ ID NO: 1 in the sequence listing. The first oligonucleotide according to the present invention is not particularly limited as long as it contains a base sequence represented by the sequence of SEQ ID NO: 1 in the sequence listing and has an activity as a vitronectin aptamer. 14mer consisting of the base sequence represented by SEQ ID NO: 1
Oligonucleotide, or SEQ ID NO: 1 in the sequence listing
5 'end and / or 3' of the base sequence represented by the sequence
Oligonucleotides comprising a base sequence having a base sequence having one or more bases added to the side terminals can be mentioned.

[0011] As an oligonucleotide comprising the base sequence represented by SEQ ID NO: 1 in the sequence listing, in the sequence of SEQ ID NO: 1 in the sequence listing, the ninth base is thymine (T) and the tenth base is Is a cytosine (C), and a 14-mer oligonucleotide consisting of a base sequence in which the 13th base is guanine (G) (that is, a base sequence represented by SEQ ID NO: 2 in the sequence listing) (described later) HVA-1 prepared in the working example
1) is preferred.

[0012] Also, an oligo-nucleotide consisting of a nucleotide sequence having one or more nucleotides added to the 5 'end and / or the 3' end of the nucleotide sequence represented by SEQ ID NO: 1 in the sequence listing. As the nucleotide, a 15-mer oligonucleotide consisting of a base sequence having a base G added to the 3 ′ end of the base sequence represented by SEQ ID NO: 1 in the sequence listing is preferable, and the ninth base is guanine. A base sequence obtained by adding a base G to the 3 ′ end of the base sequence represented by SEQ ID NO: 1 in the sequence listing in which the tenth base is guanine and the thirteenth base is cytosine (ie, A 15-mer oligonucleotide (base sequence represented by SEQ ID NO: 3 in the sequence listing) (oligonucleotide HVA-24 prepared in Examples described later)
Is more preferred.

[0013] The second oligonucleotide according to the present invention comprises a base sequence represented by SEQ ID NO: 4 in the sequence listing. The second oligonucleotide according to the present invention is not particularly limited as long as it comprises a base sequence represented by SEQ ID NO: 4 in the sequence listing and has an activity as a vitronectin aptamer. 13mer consisting of the base sequence represented by SEQ ID NO: 4
Oligonucleotide or SEQ ID NO: 4 in the sequence listing
5 'end and / or 3' of the base sequence represented by the sequence
Oligonucleotides comprising a base sequence having a base sequence having one or more bases added to the side terminals can be mentioned.

As a 13-mer oligonucleotide consisting of the base sequence represented by SEQ ID NO: 4 in the sequence listing, the eighth base is thymine and the ninth base in the sequence of SEQ ID NO: 4 in the sequence listing. The base is cytosine,
An oligonucleotide consisting of a base sequence in which the twelfth base is guanine (that is, a base sequence represented by SEQ ID NO: 6 in the sequence listing) (oligonucleotide HVA-23 prepared in Examples described later) is preferable.

[0015] Also, an oligo-nucleotide comprising a base sequence having one or more bases added to the 5 'end and / or the 3' end of the base sequence represented by SEQ ID NO: 4 in the sequence listing. As the nucleotide, a 14-mer oligonucleotide consisting of a base sequence obtained by adding a base G to the 3 'end of the base sequence represented by the sequence of SEQ ID NO: 4 in the sequence listing is preferable, and the eighth base is guanine. A base sequence obtained by adding a base G to the 3 ′ end of the base sequence represented by SEQ ID NO: 4 in the sequence listing in which the ninth base is guanine and the twelfth base is cytosine (ie, , The base sequence represented by SEQ ID NO: 5 in the sequence listing)
14-mer oligonucleotide (oligonucleotide HVA-12 prepared in Examples described later) is more preferable.

The third oligonucleotide according to the present invention comprises the base sequence represented by SEQ ID NO: 7 in the sequence listing. The third oligonucleotide according to the present invention is not particularly limited as long as it comprises a base sequence represented by SEQ ID NO: 7 in the sequence listing and has an activity as a vitronectin aptamer. 18mer consisting of the base sequence represented by SEQ ID NO: 7
Oligonucleotide or SEQ ID NO: 7 in the sequence listing
5 'end and / or 3' of the base sequence represented by the sequence
Oligonucleotides comprising a base sequence having a base sequence having one or more bases added to the side terminals can be mentioned.

As an 18-mer oligonucleotide consisting of the base sequence represented by SEQ ID NO: 7 in the sequence listing, the first base is thymine and the fourth base is The base is adenine (A), the sixth base is guanine, the seventh base is guanine, the eighth base is cytosine, the ninth base is adenine, A base sequence in which the eleventh base is adenine, the twelfth base is cytosine, and the eighteenth base is cytosine (ie, a base sequence represented by SEQ ID NO: 9 in the sequence listing)
Oligonucleotide (oligonucleotide HVA-14 prepared in Examples described later); in the sequence of SEQ ID NO: 7 in the sequence listing, the first base is cytosine, the fourth base is cytosine, The sixth base is cytosine, the seventh base is cytosine,
The eighth base is thymine, the ninth base is thymine, the eleventh base is adenine, and the twelfth base is thymine.
An oligonucleotide consisting of a base sequence in which the ninth base is guanine and the eighteenth base is thymine (ie, a base sequence represented by the sequence of SEQ ID NO: 10 in the sequence listing) (oligo prepared in Examples described later) Nucleotide HVA
-15); In the sequence of SEQ ID NO: 7 in the sequence listing, the first base is thymine, the fourth base is guanine, the sixth base is cytosine, and the seventh base is Adenine, the eighth base is cytosine, the ninth base is guanine, the eleventh base is adenine, the twelfth base is cytosine, and the eighteenth base is Oligonucleotide (oligonucleotide HVA-17 prepared in Examples described later) consisting of a nucleotide sequence that is a cytosine (that is, a nucleotide sequence represented by SEQ ID NO: 11 in the sequence listing); or SEQ ID NO: 7 in the sequence listing. In the sequence, the first base is thymine, the fourth base is cytosine, the sixth base is guanine, the seventh base is cytosine, and the eighth base is A base sequence in which the ninth base is cytosine, the ninth base is cytosine, the twelfth base is cytosine, and the twelfth base is cytosine (ie, the sequence listing (Base sequence represented by SEQ ID NO: 12) (oligonucleotide HVA-18 prepared in Examples described later) is preferable.

An oligo-nucleotide consisting of a nucleotide sequence having one or more nucleotides added to the 5 'end and / or 3' end of the nucleotide sequence represented by SEQ ID NO: 7 in the sequence listing. As the nucleotide, 23 m of a base sequence having a base sequence represented by the sequence: GTGAG added to the 5 'end of the base sequence represented by the sequence of SEQ ID NO: 7 in the sequence listing
er oligonucleotide; or a base sequence represented by SEQ ID NO: 7 in the sequence listing, a base sequence having a base sequence represented by the sequence: CCCG added to the 5 'end of the base sequence, 22 m
er oligonucleotides are preferred.

Furthermore, an oligo-nucleotide comprising a nucleotide sequence having one or more nucleotides at the 5 'end and / or the 3' end of the nucleotide sequence represented by SEQ ID NO: 7 in the sequence listing. As nucleotides, the first base is thymine, the fourth base is adenine, and the sixth base is
The ninth base is guanine, the seventh base is guanine, the eighth base is cytosine, the ninth base is adenine, the eleventh base is adenine, and the twelfth base is guanine. The base sequence represented by the sequence: GTGAG was added to the 5 ′ end of the base sequence represented by SEQ ID NO: 7 in the sequence listing in which the 18th base was cytosine and the 18th base was cytosine. A 23-mer oligonucleotide consisting of a base sequence (that is, a base sequence represented by SEQ ID NO: 8 in the sequence listing) (oligonucleotide HVA-4 prepared in Examples described later); the first base is thymine , The fourth base is adenine,
The sixth base is adenine, the seventh base is cytosine, the eighth base is cytosine,
The ninth base is adenine, the eleventh base is adenine, the twelfth base is cytosine,
The base sequence in which the base sequence represented by the sequence: CCCG is added to the 5 ′ end of the base sequence represented by SEQ ID NO: 7 in the sequence listing in which the eighth base is cytosine (ie, the sequence in the sequence listing) A 22-mer oligonucleotide consisting of the base sequence represented by the sequence of No. 13 (oligonucleotide HVA-19 prepared in Examples described later); the first base is thymine, and the fourth base is adenine. Yes, the sixth base is guanine, the seventh base is cytosine, the eighth base is cytosine,
The ninth base is guanine, the eleventh base is adenine, the twelfth base is cytosine,
SEQ ID NO: 7 in the sequence listing where the 18th base is cytosine
22mer consisting of a base sequence in which a base sequence represented by the sequence: CCCG is added to the 5 'end of the base sequence represented by the sequence of SEQ ID NO: 14 (ie, a base sequence represented by SEQ ID NO: 14 in the sequence listing) Oligonucleotide (oligonucleotide HVA-20 prepared in Examples described later); the first base is thymine, the fourth base is adenine, the sixth base is cytosine, and the seventh base is The eighth base is cytosine, the eighth base is cytosine,
The ninth base is cytosine, the eleventh base is adenine, the twelfth base is cytosine,
SEQ ID NO: 7 in the sequence listing where the 18th base is cytosine
22mer consisting of a base sequence in which a base sequence represented by the sequence: CCCG is added to the 5 'end of the base sequence represented by the sequence of SEQ ID NO: 15 (ie, a base sequence represented by the sequence of SEQ ID NO: 15 in the sequence listing) Oligonucleotide (oligonucleotide HVA-21 prepared in Examples described later);
The ninth base is thymine, the fourth base is adenine, the sixth base is thymine, the seventh base is cytosine, the eighth base is cytosine, The base represented by the sequence of SEQ ID NO: 7 in the sequence listing in which the ninth base is thymine, the eleventh base is adenine, the twelfth base is cytosine, and the eighteenth base is cytosine A 22-mer oligonucleotide consisting of a base sequence having the base sequence represented by the sequence: CCCG added to the 5 ′ end of the sequence (ie, the base sequence represented by SEQ ID NO: 16 in the sequence listing) (described later) Oligonucleotide HVA-22) prepared in Examples is more preferable, and a 22-mer oligonucleotide consisting of the base sequence represented by SEQ ID NO: 14 in the sequence listing, or
A 22-mer oligonucleotide consisting of the base sequence represented by SEQ ID NO: 16 in the sequence listing is particularly preferred.

In the oligonucleotide of the present invention, the internucleotide bond between each nucleoside can be independently a phosphodiester bond, a modified phosphodiester bond, or a peptide bond. Examples of the modified phosphodiester bond include a methylphosphonate-type bond in which one oxygen atom of two non-crosslinking oxygen atoms of a phosphodiester bond is substituted with a methyl group, and two non-crosslinking oxygen atoms of a phosphodiester bond Oxygen atom 1 of
A phosphoroamidate-type bond in which one is substituted with an amino group or a substituted amino group, a phosphorothioate-type bond in which one oxygen atom of two non-bridging oxygen atoms in a phosphodiester bond is substituted with a sulfur atom, or a phosphoric diester Two of the two non-bridging oxygen atoms in the bond are replaced with sulfur atoms 2
And the like. One or more of them can be introduced at one or more positions of the internucleoside bond.

The oligonucleotide of the present invention can be synthesized by a known method, for example, using an automatic DNA synthesizer.

The vitronectin aptamer of the present invention comprises the oligonucleotide of the present invention. That is, the first vitronectin aptamer according to the present invention comprises the first oligonucleotide according to the present invention, and similarly, the second vitronectin aptamer according to the present invention comprises the second oligonucleotide according to the present invention, and The third vitronectin aptamer consists of a third oligonucleotide according to the invention. That is, the oligonucleotide of the present invention (including the first oligonucleotide, the second oligonucleotide, and the third oligonucleotide) has an action as a vitronectin aptamer and specifically binds to vitronectin.

The anticancer agent of the present invention comprises at least one of the oligonucleotides of the present invention (including the first oligonucleotide, the second oligonucleotide, and the third oligonucleotide), alone or as desired. An animal, preferably a mammal (especially a human), together with a conventional carrier which is pharmaceutically or veterinarily acceptable.
Can be administered.

The effect of the anticancer agent of the present invention can be enhanced by using two or more of the oligonucleotides of the present invention in combination. That is, the first
At least one of the following oligonucleotides, at least one of the second oligonucleotides, and at least one of the third oligonucleotides, as an active ingredient, a combination of two or three types of oligonucleotides arbitrarily selected. The anti-cancer agent of the present invention comprises a first oligonucleotide, a second oligonucleotide, and a third oligonucleotide.
The effect is superior to the case where only one kind of the oligonucleotide is contained.

Any one selected from at least one of the first oligonucleotide of the present invention, at least one of the second oligonucleotide of the present invention, and at least one of the third oligonucleotide of the present invention. In the anticancer agent of the present invention containing a combination of two or three types of oligonucleotides as an active ingredient, the combination of each oligonucleotide of the present invention includes a combination of a first oligonucleotide and a second oligonucleotide, A combination of the first oligonucleotide and the third oligonucleotide, a combination of the second oligonucleotide and the third oligonucleotide, or a combination of the first oligonucleotide, the second oligonucleotide and the third oligonucleotide; Combinations can be mentioned, wherein the first oligonucleotide and the third oligonucleotide A combination of a nucleotide, a combination of a second oligonucleotide and a third oligonucleotide, or a combination of a first oligonucleotide, a second oligonucleotide, and a third oligonucleotide is preferable, and a first oligonucleotide is preferred. And a combination of the second oligonucleotide and the third oligonucleotide is more preferable.

Examples of parenteral administration include injection (subcutaneous, intravenous, etc.). Of these, injections are most preferably used. For example, in the preparation of an injection, in addition to the oligonucleotide of the present invention as an active ingredient, for example, a water-soluble solvent such as physiological saline or Ringer's solution, a non-water-soluble solvent such as vegetable oil or fatty acid ester, glucose or sodium chloride For example, an isotonic agent, a solubilizing agent, a stabilizing agent, a preservative, a suspending agent, an emulsifier, or the like can be used. In addition, by binding the oligonucleotide of the present invention as an active ingredient to polyethylene glycol, blood stability can be improved by intravenous injection every 12 hours (A
merchant Journal of Pathol
ogy, 154, 1, 169-179, 1999). The anticancer agent of the present invention can be used, for example, as a postoperative maintenance therapy for cancer by being added to, for example, infusions. In addition, the anticancer agent of the present invention may be administered by using a sustained-release preparation technique using a sustained-release polymer or the like. For example, the anticancer agent of the present invention can be incorporated into a pellet of ethylene vinyl acetate polymer and the pellet can be surgically implanted into the tissue to be treated or prevented.

When the anticancer agent of the present invention is used, the dosage is
It can be appropriately determined according to the type of the disease, the age, sex, body weight, degree of symptoms, administration method and the like of the patient, and can be administered parenterally.

The oligonucleotide of the present invention has an action as a vitronectin aptamer. The ability of cancer cells to proliferate is determined by integrin αvβ3 and / or integrin αvβ3, which are vitronectin receptors on the surface of cancer cells.
5 is known to be promoted by binding to vitronectin, which is an adhesion factor in serum. By adding the oligonucleotide of the present invention, integrin αv
Since the binding between β3 and / or integrin αvβ5 and vitronectin can be inhibited, the oligonucleotide of the present invention is useful as an active ingredient of an anticancer agent.

It is known that vitronectin binds to plasminogen activator inhibitor (PAI) -1 and stabilizes its activity. PAI
-1 suppresses the action of tissue plasminogen activator (tPA), which has the effect of converting plasminogen into plasmin having thrombolytic activity, and consequently is involved in the regulation of the blood coagulation system. Become. Since the oligonucleotide of the present invention suppresses the action of PAI-1 by binding to vitronectin,
The oligonucleotide of the present invention is also useful as an active ingredient of a thrombolytic agent. Currently, a thrombolytic agent having no bleeding tendency is required, but the oligonucleotide of the present invention is:
It may have a completely different effect than the conventional one.

Since the oligonucleotide of the present invention has an action as a vitronectin aptamer, it can be used for the analysis of vitronectin. For example, in the vitronectin analysis method of the present invention, a test sample which may contain vitronectin is brought into contact with the oligonucleotide of the present invention. At this time, if vitronectin is not present in the test sample, no reaction occurs with the oligonucleotide, but if vitronectin is present in the test sample, the vitronectin binds to the oligonucleotide. A conjugate of vitronectin and an oligonucleotide is produced depending on the amount of vitronectin present. This conjugate can be easily analyzed (that is, detected or measured) by a known method, so that the presence or amount of the conjugate can be used to detect the presence of vitronectin in the test sample or to determine the amount thereof. can do.

The method for analyzing the conjugate of vitronectin and the oligonucleotide is not particularly limited. For example, the oligonucleotide may be labeled in advance using various labeling substances, and the method may be used to analyze the origin of the labeling substance. A method of analyzing a signal to be analyzed can be given. More specifically, for example, after contacting a labeled oligonucleotide with a test sample that may contain vitronectin, a conjugate of the generated vitronectin and the labeled oligonucleotide, and a conjugate thereof Is separated into a portion containing no, and by analyzing a signal derived from a labeling substance contained in one of the separated portions, vitronectin can be analyzed.

Alternatively, after the test sample is fixed on a suitable carrier by a conventionally known method, the test sample can be contacted with a labeled oligonucleotide. For example, after a test sample (eg, a tissue such as a cell) is fixed on a suitable carrier (eg, a slide glass),
Alternatively, a test sample (eg, an extract of a tissue such as a cell)
Is electrophoresed and subsequently transferred to a suitable carrier (eg, a membrane filter), and then the test sample on the carrier is contacted with a labeled oligonucleotide. After removing the nucleotides
By analyzing a signal derived from a labeling substance contained in the conjugate of the generated vitronectin and the labeled oligonucleotide, vitronectin can be analyzed.

Examples of the labeling substance include a radioisotope, an enzyme, a fluorescent substance, and a luminescent substance.

As the radioisotope, for example,
¹²⁵ I, ¹³¹ I, ³ H, ¹⁴ C, ³⁵ S or the like can be used. As the enzyme, a stable enzyme having a large specific activity is preferable. For example, glycosidases (for example, β-galactosidase, β-glucosidase, β-glucuronidase, β-fructosidase, α-galactosidase, α-galactosidase,
α-glucosidase or α-mannosidase),
Carbohydrases such as amylase (eg, α-amylase, β-amylase, isoamylase, glucoamylase, or takaamylase), cellulase, or lysozyme; amidases such as urease or asparaginase; cholinesterases (eg, acetylcholinesterase); Esterases such as sulfonase or lipase; nucleases such as deoxyribonuclease or ribonuclease; iron / porphyrin enzymes such as catalase, peroxidase or cytochrome oxidase; copper enzymes such as tyrosinase or ascorbate oxidase; or alcohol dehydration. Enzyme, malate dehydrogenase, lactate dehydrogenase, or isocitrate dehydration A dehydrogenase such as an enzyme can be used. Fluorescamine as a fluorescent substance,
Alternatively, fluorescein isothiocyanate and the like, and luminescent substances include luminol, luminol derivatives, luciferin, lucigenin, and the like.

The test sample that can be used in the vitronectin analysis method of the present invention is not particularly limited as long as it may contain vitronectin, and is not limited to a biological sample such as human (particularly, human). A patient) or an extract or a specimen thereof, or blood (eg, serum or plasma), urine, or a body fluid such as cerebrospinal fluid. Also,
It is not particularly limited as long as it is a test sample in a normal clinical test or the like, and can be used.

[0036]

EXAMPLES The present invention will be described below in more detail with reference to examples, but these examples do not limit the scope of the present invention.

Example 1 << Preparation of Oligonucleotide >> In this example, the oligonucleotide of the present invention used in the evaluation examples described later was chemically synthesized by an automatic DNA synthesizer. Table 1 shows the nucleotide sequence of each oligonucleotide prepared in this example and the SEQ ID NO in the sequence listing.

Specifically, as the first oligonucleotide according to the present invention, a 14-mer oligonucleotide (hereinafter referred to as HVA-11) consisting of the base sequence represented by SEQ ID NO: 2 in the sequence listing; A 15-mer oligonucleotide (hereinafter, referred to as HVA-24) consisting of the base sequence represented by SEQ ID NO: 3 in the sequence listing was synthesized. Further, as the second oligonucleotide according to the present invention, a 14-mer oligonucleotide (hereinafter, referred to as HVA-12) consisting of the base sequence represented by SEQ ID NO: 5 in the sequence listing; and SEQ ID NO: 6 in the sequence listing
A 13-mer oligonucleotide (hereinafter, referred to as HVA-23) consisting of the base sequence represented by the following sequence was synthesized.

Further, as a third oligonucleotide according to the present invention, a 23-mer oligonucleotide consisting of a base sequence represented by the sequence of SEQ ID NO: 8 in the sequence listing (hereinafter, referred to as a third oligonucleotide)
HVA-4); an 18-mer oligonucleotide consisting of a base sequence represented by SEQ ID NO: 9 in the sequence listing (hereinafter referred to as HVA-14); a base represented by SEQ ID NO: 10 in the sequence listing An 18-mer oligonucleotide consisting of a sequence (hereinafter referred to as HVA-15); an 18-mer oligonucleotide consisting of a base sequence represented by SEQ ID NO: 11 in the sequence listing (hereinafter HVA-17)
An 18-mer oligonucleotide consisting of the base sequence represented by SEQ ID NO: 12 in the sequence listing (hereinafter referred to as HVA-18); consisting of the base sequence represented by SEQ ID NO: 13 in the sequence listing 22-mer oligonucleotide (hereinafter, referred to as HVA-19); a 22-m oligonucleotide consisting of the base sequence represented by SEQ ID NO: 14 in the sequence listing
er oligonucleotide (hereinafter referred to as HVA-20); a 22-mer oligonucleotide (hereinafter referred to as HV) consisting of the base sequence represented by SEQ ID NO: 15 in the sequence listing
A-21), and a 22-mer oligonucleotide (hereinafter, referred to as HVA-22) having a base sequence represented by SEQ ID NO: 16 in the sequence listing, respectively.

<< Table 1 >> Oligonucleotide Sequence of nucleotide sequence SEQ ID No. [First oligonucleotide according to the present invention] HVA-11 CAGCTCCGTC AGGT 2 HVA-24 CAGCTCCGGG AGCTG 3 [Second oligonucleotide according to the present invention] HVA- 12 ACCTGACGGA GCTG 5 HVA-23 ACCTGACTCA GGT 6 [third oligonucleotide according to the invention] HVA-4 GTGAG TCCATGGCAT ACCTGCCC 8 HVA-14 TCCATGGCAT ACCTGCCC 9 HVA-15 CCCCTCCTTT AGCTGCCT 10 HVA-17 TCCGTCACGT ACCTGCCC 11 HVA-18 TCCCTGCCCT CCCTGCCC 12 HVA-19 CCCG CCATACCAT ACCTGCCC 13 HVA-20 CCCG TCCATGCCGT ACCTGCCC 14 HVA-21 CCCG TCCATCCCCT ACCTGCCC 15 HVA-22 CCCG TCCATTCCTT ACCTGCCC 16

[0040]

[Evaluation Example 1] << Test of cell adhesion inhibitory activity >> The oligonucleotide of the present invention prepared in Example 1 was evaluated according to the following procedure. That is, a 48-well plate (non-tis) not treated for tissue culture was used.
sue culture treated plate
e) Each well was precoated with 100 μL of human vitronectin (concentration = 0.2 μg / well) and dried overnight in a clean bench. Phosphate buffer solution (pho
sphere-buffered saline; PB
After washing each well in S), 200 μL of 1.5% bovine serum albumin (BSA) was added to each well,
For one hour.

After replacing 1.5% BSA with 300 μL of D-MEM medium containing 0.02% BSA, NRK52E
0.25 x 10 cells (ATCC CRL 1571)
^The solution was added to each well so as to be ⁵ cells / mL or 0.5 × 10 ⁵ cells / mL. At this time, the oligonucleotide of the present invention to be evaluated (dissolved in PBS to a predetermined concentration) was also added to each well. The NRK52E cell is a cell that expresses an integrin, which is a vitronectin receptor, on its cell surface.

After incubating for 1 hour at 37 ° C. in a CO ₂ incubator, the plate was washed with PBS. MTT
[3- (4,5-dimethylthiazol-2-yl)-
2,5-diphenyltetrazolium bromide] -containing medium was added, followed by further incubation at 37 ° C for 4 hours. After the medium was removed by suction, dimethyl sulfoxide (D
(MSO) was added to lyse the cells.
The amount was measured for optical density (OD) (wavelength = 570 nm / 63)
0 nm).

With the third oligonucleotide according to the invention
Certain HVA-4, HVA-14, HVA-15, HVA
-17, HVA-18, HVA-19, or HVA-2
0 alone (additional concentration = 500 μ
M, cell concentration = 0.25 × 10 ^FiveCells / mL)
FIG. 1 shows a third oligonucleotide according to the invention.
Certain HVA-20, HVA-21, or HVA-22
When used alone (additional concentration = 450 μM, fine
Cell concentration = 0.5 × 10^Five(Cells / mL) are shown in FIG.
You. In addition, the first oligonucleotide according to the present invention
HVA-11 or HVA-24 according to the present invention
HVA-12 or H, which is an oligonucleotide of
VA-23, or a third oligonucleotide according to the invention
HVA-20 is used alone (see Appendix).
Added concentration = 450 μM, cell concentration = 0.5 × 10^Fivecell/
(mL) are shown in FIG.

In FIGS. 1 to 3, “control” refers to PBS
Instead of the oligonucleotide of the invention dissolved in
Except that BS was added, the result was the result of repeating the above-mentioned operation. “No coating” was the result of repeating the same operation as the above “Control”, except that human vitronectin was not precoated. The vertical axis in FIGS. 1 to 3 is a measured value of the optical density (OD) at a wavelength of 570 nm.

The first oligonucleotide HVA-11 according to the present invention, the second oligonucleotide HVA-12 according to the present invention, and the third oligonucleotide HVA-20 according to the present invention were simultaneously used. When used [each added concentration of HVA-11 and HVA-12 =
225.5 μM, added concentration of HVA-20 = 450 μm
M, cell concentration = 0.5 × 10 ⁵ cells / mL]
When each was used alone [additional concentration = 450μ
M, cell concentration = 0.5 × 10 ⁵ cells / mL].

In FIG. 4, "HVA-MIX" is H
These are the results when three kinds of VA-11, HVA-12 and HVA-20 were used at the same time. Further, “control” and “no coat” in FIG. 4 have the same meaning as in FIGS. 1 to 3, and the vertical axis in FIG. 4 has the same meaning as in FIGS. 1 to 3.

[0047]

The oligonucleotide according to the present invention has an action as a vitronectin aptamer. As a result, the binding between integrin αvβ3 or αvβ5 and vitronectin can be inhibited, so that cell proliferation of cancer cells can be suppressed.

[0048]

[Sequence Listing Free Text] Each oligonucleotide consisting of the nucleotide sequence represented by the sequence of SEQ ID NO: 1 to SEQ ID NO: 16 in the sequence listing is a vitronectin aptamer.

[0049]

[Sequence List] <110> Kureha Chemical Industry Co., Ltd. <120> Novel Oligonucleotide, vitronection aptamer, and Antitumor Agent <130> KRH005739P <160> 16 <210> 1 <211> 14 <212> DNA <213> Artificial Sequence <220> <223> A vitronectin aptamer. <400> 1 cagctccgks agst 14 <210> 2 <211> 14 <212> DNA <213> Artificial Sequence <220> <223> Vitronectin aptamer. <400> 2 cagctccgtc aggt 14 <210> 3 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> A vitronectin aptamer. <400> 3 cagctccggg agctg 15 <210> 4 <211> 13 <212> DNA <213> Artificial Sequence <220> <223> A vitronectin aptamer. <400> 4 acctgacksa gst 13 <210> 5 <211> 14 <212> DNA <213> Artificial Sequence <220> <223> A vitronectin aptamer. <400> 5 acctgacgga gctg 14 <210> 6 <211> 13 <212> DNA <213> Artificial Sequence <220> <223> Vitronectin aptamer. <400> 6 acctgactca ggt 13 <210> 7 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> A vitronectin aptamer. <400> 7 yccvtnvynt msctgccy 18 <210> 8 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Vitronectin aptamer. <400> 8 gtgagtccat ggcatacctg ccc 23 <210> 9 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> A vitronectin aptamer. <400> 9 tccatggcat acctgccc 18 <210> 10 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Vitronectin aptamer. <400> 10 cccctccttt agctgcct 18 <210> 11 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> A vitronectin aptamer. <400> 11 tccgtcacgt acctgccc 18 <210> 12 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Vitronectin aptamer. <400> 12 tccctgccct ccctgccc 18 <210> 13 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Vitronectin aptamer. <400> 13 cccgtccata ccatacctgc cc 22 <210> 14 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> A vitronectin aptamer. <400> 14 cccgtccatg ccgtacctgc cc 22 <210> 15 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> A vitronectin aptamer. <400> 15 cccgtccatc ccctacctgc cc 22 <210> 16 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Vitronectin aptamer. <400> 16 cccgtccatt ccttacctgc cc 22

[Brief description of the drawings]

FIG. 1 is a graph showing the results of a cell adhesion inhibitory activity test of each oligonucleotide of the present invention.

FIG. 2 is a graph showing the results of a cell adhesion inhibitory activity test of each oligonucleotide of the present invention.

FIG. 3 is a graph showing the results of a cell adhesion inhibitory activity test of each oligonucleotide of the present invention.

FIG. 4 is a graph showing the results of a cell adhesion inhibitory activity test of each oligonucleotide of the present invention.

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) // A61K 31/712 A61P 35/00 A61P 35/00 C12N 15/00 ZNAA F-term (Reference) 4B024 AA01 AA12 CA09 HA14 HA17 4B063 QA01 QA19 QQ42 QQ58 QR08 QR32 QR42 QR56 QS25 QS34 QX02 QX07 4C057 BB04 CC01 DD01 MM01 4C086 AA01 AA02 AA03 EA01 MA01 MA04 NA14 ZB26

Claims (8)

[Claims] 1. An oligonucleotide comprising a base sequence represented by SEQ ID NO: 1 in the sequence listing. 2. An oligonucleotide comprising a base sequence represented by SEQ ID NO: 4 in the sequence listing. 3. An oligonucleotide comprising a base sequence represented by SEQ ID NO: 7 in the sequence listing. 4. A vitronectin aptamer comprising an oligonucleotide having a base sequence represented by SEQ ID NO: 1 in the sequence listing. 5. A vitronectin aptamer comprising an oligonucleotide having a base sequence represented by SEQ ID NO: 4 in the sequence listing. 6. A vitronectin aptamer comprising an oligonucleotide comprising a base sequence represented by SEQ ID NO: 7 in the sequence listing. 7. An anticancer agent comprising at least one of the oligonucleotides according to claim 1 as an active ingredient. 8. A method for analyzing vitronectin, comprising using the oligonucleotide according to any one of claims 1 to 3.