WO2007094218A1 - Modified oligonucleotide - Google Patents

Abstract

A modified oligonucleotide comprising an oligonucleotide containing at its 3’end a modified nucleoside as a structural component, wherein the modified nucleoside is one having a carboxylic acid or steroid skeleton containing compound linked via a linker to the 3’end of nucleoside. The modified oligonucleotide can be produced by solid-phase synthesis using any of the unit compounds for solid-phase synthesis represented by the formula (XIA) or the unit compounds for solid-phase synthesis represented by the formula (XIS) as a starting material. (XIA) (XIS) wherein R1, R2, n, m, RA and RS are as defined in the description.

Description

 Modified oligonucleotide

 Technical field

 [0001] The present invention relates to a modified oligonucleotide.

 Background art

 [0002] In recent years, a methodology for capturing genetic information itself as a treatment target has been spreading. One of the methodologies is an antisense method using oligonucleotides. The anti-sense method involves adding chemically synthesized oligonucleotides (for example, single-stranded DNA consisting of 15 to 20 base pairs) to cells to form ZmRNA double-stranded nucleic acid such as target messenger RNA (mRNA) and DNA. Thus, the expression of the target gene is specifically suppressed in the base sequence, and the translation process into mRNA protein is inhibited. In the antisense method, if the base sequence of the pathogenic virus or gene is known, it is possible to theoretically design an antisense molecule and synthesize it. It is expected as one of the effective treatment methods for diseases caused by various viruses and genetic diseases.

Recently, attention has been focused on a method using RNAi (RNA interference) as a gene expression suppression method using oligonucleotides. RNAi refers to a phenomenon in which RNA derived from a cell chromosome having the same base sequence is degraded and cleaved by introducing double-stranded RNA into a cell. The mechanism of RNAi is currently considered as follows. First, a long double-stranded RNA is called a double-stranded RNA (siRNA (short interfering RNA)) with a length of about 21 bases with a 3-UU-type dangling end structure by an enzyme (called Dicer). ). The siRNA forms an RNAZ mRNA double-stranded nucleic acid with the target mRNA and recognizes this double-stranded nucleic acid (called RNA-induced silencing complex (RISC)), and the double-stranded nucleic acid Are bound, and this conjugate cleaves the target mRNA. In many cases, this RNAi-based method achieves the same effect using RNA at a concentration of about 1Z100 compared to the antisense method. Therefore, the method using RNAi is Expectations are growing as an effective treatment method for diseases and genetic diseases caused by various viruses that have been considered difficult to cure.

 [0004] In methods using oligonucleotides, such as antisense methods and RNAi-based methods, it is necessary to make oligonucleotides introduced into cells stable. ), And the introduced oligonucleotides, in particular, the natural oligonucleotides, were easily degraded.

 [0005] Various oligonucleotide analogs have also been developed for the purpose of improving the ability to form a double strand with a natural oligonucleotide and improving the stability of the formed double strand. Examples of such oligonucleotide analogues include oligonucleotide analogues in which the 2 ′ hydroxyl group of ribose on the nucleoside of a natural oligonucleotide is replaced with an alkoxy group (eg, methoxy group) or a halogen atom (eg, fluorine atom) (eg, And non-patent document 1). These oligonucleotide analogues have been confirmed to improve the ability to form double strands with natural oligonucleotides and to improve the stability of the double strands formed. However, these oligonucleotide analogues were similar in structure to natural oligonucleotides, and thus improved nuclease resistance was not achieved.

 [0006] On the other hand, it has been confirmed that the modified oligonucleotide has a modified siRNA ability in which cholesterol is bound to the third end of the sense strand and is useful in the treatment of hyperlipidemia in an animal model (for example, Non-patent document 2).

 [0007] Thus, it is difficult to satisfy both the excellent nuclease resistance and the properties, the excellent ability to form a duplex and the excellent stability of the formed duplex. There was a problem! However, when oligonucleotide analogues are used for DNA chips, gene diagnostics, etc., it has been desired to provide modified oligonucleotides with excellent properties in order to obtain stable diagnostic results.

Non-Special Reference 1: Andrew M. Kawasaki, Martin D. Casper, busan M. Freier, Elena A. Lesnik, Maryann C. Zounes, Lendell L. Cummins, Carolyn Gonzalez and P. Dan Cook, "Uniformly modified 2 ' -deoxy-2 and fluoro phophorothioate oligonucleotides as nucl ease-resistant antisense compounds with highly affinity and specificity for RNA targe ts ", Journal of Medicinal Chemistry, 1993, 36, p.831. Non-Patent Document 2: Jurgen Sontschek et al., Nature, 2004, No. 432, p.173 Disclosure of the Invention

 Problems to be solved by the invention

[0008] Therefore, an object of the present invention is to provide an oligonucleotide having a modified 3 'end that is excellent in two properties of nuclease resistance and duplex forming ability.

 Means for solving the problem

[0009] The present invention is a modified oligonucleotide containing a modified nucleoside as a constituent component at the 3, terminus of the oligonucleotide. The modified nucleoside force nucleoside has the following formula (末端) via a linker at the 3, terminus: A modified oligonucleotide which is a modified nucleoside bound with a carboxylic acid represented by the formula:

R ^A -COOH (III)

In the above formula, R ^A means a saturated aliphatic hydrocarbon group, an unsaturated aliphatic hydrocarbon group, an aryl group, or a heterocyclic group.

 [0010] The present invention is a double-stranded modified oligonucleotide,

 A sense strand having the same base sequence as a part of the target mRNA, and an antisense strand against the sense strand,

 Among the antisense strand and the sense strand, at least the antisense strand is an oligonucleotide analogue containing a modified nucleoside as a constituent component at the 3, end of the oligonucleotide, and at the 3, end of the modified nucleoside force nucleoside. And a double-stranded modified oligonucleotide which is a modified nucleoside to which a compound having a steroid skeleton represented by the following formula (Π) is bound via a linker.

R ^S -OH (II)

In the above formula, R ^s means a steroid skeleton optionally having one or more substituents, wherein one or more single bonds in the skeleton are optionally replaced with double bonds.

 The invention's effect

[0011] In the present invention, a compound having a carboxylic acid or a steroid skeleton is bound to the 3, terminus of a nucleoside contained as a constituent component at the 3, terminus of an oligonucleotide via a linker. To improve the nuclease resistance and the ability to form double strands of the modified oligonucleotide.

 Brief Description of Drawings

 [0012] [FIG. 1 (a)] FIG. 1 (a) shows an example of a modified double-stranded oligonucleotide and an example of an unmodified double-stranded oligonucleotide in which palmitic acid is bound to the 3 ′ end via a linker. 2 is a graph showing the expression concentration of RNaseL. Lane 1 represents a control, lane 2 represents a 50 nM modified double-stranded oligonucleotide, lane 3 represents a ΙΟΟηΜ modified double-stranded oligonucleotide, and lane 4 represents a 200 nM modified double-stranded oligonucleotide.

 [Fig. 1 (b)] Fig. 1 (b) shows the RNaseL expression levels of examples of modified DNA sense strands with oleic acid bound to the end via a linker and examples of unmodified double-stranded oligonucleotides. It is a graph which shows. Lane 1 represents a control, lane 2 represents a 50 nM modified double-stranded oligonucleotide, lane 3 represents a ΙΟΟηΜ modified double-stranded oligonucleotide, and lane 4 represents a 200 ηΜ modified double-stranded oligonucleotide.

 [Fig. 1 (c)] Fig. 1 (c) shows the RNaseL expression levels of a modified DN Α sense strand and a non-modified double-stranded oligonucleotide example in which cholesterol is bound to the 3 'end via a linker. It is a graph to show. Lane 1 represents a control, lane 2 represents a 50 nM modified double-stranded oligonucleotide, lane 3 represents a ΙΟΟηΜ modified double-stranded oligonucleotide, and lane 4 represents a 20 OnM modified double-stranded oligonucleotide.

 BEST MODE FOR CARRYING OUT THE INVENTION

[0013] The present invention, as described above, is a modified oligonucleotide containing a modified nucleoside as a constituent component at the 3 'end of the oligonucleotide, and the modified nucleoside force nucleoside via a linker at the 3' end, A modified oligonucleotide which is a modified nucleoside to which a carboxylic acid represented by the following formula (III) is bound.

R ^A -COOH (III)

In the above formula, R ^A means a saturated aliphatic hydrocarbon group, an unsaturated aliphatic hydrocarbon group, an aryl group, or a heterocyclic group.

[0014] The modified oligonucleotide to which the carboxylic acid represented by the formula (ΠΙ) is bound is preferably a modified oligonucleotide represented by the following formula (IA). [0015] [Chemical 4]

 O H

R ¹ — OPOL— — ^ -R ^A (IA)

 ό- O In the above formula,

 R, 1 OH means oligonucleotide,

 The hydroxyl group in R′—OH means the hydroxyl group at the 3 ′ end of the modified nucleoside contained as a constituent component at the 3 ′ end of the oligonucleotide,

 L means a divalent group,

R ^A represents a saturated aliphatic hydrocarbon group, an unsaturated aliphatic hydrocarbon group, an aryl group, or a heterocyclic group.

 [0016] In the formula (IA), the saturated aliphatic hydrocarbon group is preferably a saturated aliphatic hydrocarbon group having 1 to 25 carbon atoms. In the formula (IA), the unsaturated aliphatic hydrocarbon group is preferably an unsaturated aliphatic hydrocarbon group having 3 to 25 carbon atoms.

[0017] In the formula (IA), R ^A represents CH-(CH) — or CH-(CH) — CH = CH—

 3 2 14 3 2 7

 More preferably, (CH 2) —.

 2 7

 [0018] In the formula (IA), the divalent group is preferably a divalent group represented by the following formula (IVA).

 -X-M-Y- (IVA)

 In the above formula,

 —X— is — (CH) or (CH) —CH (CH OH) —,

 2 m 2 m 2

 —Y— is one (CH) or one (CH) -CH (CH OH) one;

 2 n 2 n 2

 —M is one 0—C (= 0) NH, one O—, one (= 0) —? << 1-or-? << 1-ji (= O)-

 m and n are each independently an integer of 1 to 20.

In the formula (IA), the divalent group is preferably a divalent group represented by the following formula (IVA-1).

— CH— CH (CH OH) -0-C (= 0) NH- (CH) One (IVA—1) In the above formula, n is an integer of 1-20.

 [0020] The modified oligonucleotide to which the carboxylic acid represented by the formula (ΠΙ) is bound may be a single-stranded oligonucleotide. Preferably, the modified oligonucleotide force is antisense DNA or antisense RNA, and has a sequence complementary to at least a part of mRNA encoding the modified oligonucleotide force endonuclease.

 [0021] The modified oligonucleotide to which the carboxylic acid represented by the above formula (ΠΙ) is bound may be a double-stranded oligonucleotide.

 [0022] The present invention is also a double-stranded modified oligonucleotide as described above,

 A sense strand having the same base sequence as a part of the target mRNA, and an antisense strand against the sense strand,

 Among the antisense strand and the sense strand, at least the antisense strand is an oligonucleotide analogue containing a modified nucleoside as a constituent component at the 3, end of the oligonucleotide, and at the 3, end of the modified nucleoside force nucleoside. And a double-stranded modified oligonucleotide which is a modified nucleoside to which a compound having a steroid skeleton represented by the following formula (Π) is bound via a linker.

R ^S -OH (II)

In the above formula, R ^S means a steroid skeleton optionally having one or more substituents, wherein one or more single bonds in the skeleton are optionally replaced with double bonds.

[0023] The oligonucleotide analogue is preferably represented by the formula (IS).

[0024] [Chemical 5]

 0

 † H

R'-OPOL— ^ OR ^s (IS)

 O-O in the above formula,

 R, 1 OH means oligonucleotide,

 The hydroxyl group in R′—OH means the hydroxyl group at the 3 ′ end of the modified nucleoside contained as a constituent component at the 3 ′ end of the oligonucleotide,

L means a divalent group, R ^s means a steroid skeleton that optionally has one or more substituents, and one or more single bonds in the skeleton are optionally replaced with double bonds.

[0025] In the formula (IS), R ^s is

[0026] [Chemical 6]

 It is preferable that the group power to be power is selected.

 In the formula (IS), the divalent group is preferably a divalent group represented by the following formula (IVS).

 -X-M-Y- (IVS)

 In the above formula,

 —X— is — (CH) or (CH) —CH (CHOH) —

 2 m 2 m 2

 —Y— is one (CH) or one (CH) -CH (CH OH) one;

 2 n 2 n 2

 —M is one 0—C (= 0) NH, one O—, one (= 0) —? << 1-or-? << 1-ji (= O)-

 m and n are each independently an integer of 1 to 20.

 [0028] In the formula (IS), the divalent group is preferably a divalent group represented by the following formula (IVS-1).

 — CH— CH (CH 0H) -0-C (= 0) NH- (CH) One (IVS— 1)

 2 2 2 n

In the above formula, n is an integer of 1-20. [0029] The modified oligonucleotide is an oligonucleotide that causes RNAi (RNA interference), and preferably has the same base sequence as a part of mRNA encoding the modified oligonucleotide force endonuclease! /.

[0030] The modified oligonucleotide is siRNA (short interfering RNA),

 A portion of the mRNA is

 It is a 19-base sequence following the first AA sequence, more than 75 bases upstream from the start codon, and is a partial force of the mRNA that is specific for the mRNA,

 The modified oligonucleotide force is more preferably a combination of a sense strand and an antisense strand of the 19-base sequence. At least one force of the sense strand and the antisense strand further includes two base sequences at the 3 ′ end. More preferably it has nucleotides.

 [0031] The modified oligonucleotide of the present invention is preferably resistant to the oligonucleotide force nuclease.

 [0032] The present invention is also a gene expression inhibitor comprising an oligonucleotide, wherein the oligonucleotide is a modified oligonucleotide of the present invention.

 [0033] The present invention is also a pharmaceutical composition for treating a disease associated with gene expression, wherein the pharmaceutical composition comprises the gene expression inhibitor of the present invention. The pharmaceutical composition preferably further contains a cell introduction excipient. It is preferable that the cell introduction excipient is a transfection reagent.

 [0034] The present invention is also an RNAi kit, wherein the kit comprises the modified oligonucleotide of the present invention.

 [0035] The present invention is also a reagent for RNAi research, wherein the reagent contains the modified oligonucleotide of the present invention.

[0036] The present invention is also a method for suppressing gene expression using an oligonucleotide, wherein the oligonucleotide force is a modified oligonucleotide of the present invention.

[0037] The present invention is also a method of causing RNAi using an oligonucleotide, wherein the oligonucleotide is a modified oligonucleotide of the present invention.

[0038] The present invention also provides a solid phase synthesis compound represented by the following formula (XIA) as a starting material. A method for producing a modified oligonucleotide represented by the following formula (IA), wherein R ^{1 of the} unit compound for solid phase synthesis represented by the formula (XIA) is removed and converted to a free hydroxyl group, In this production method, the modified oligonucleotide is obtained by extending the oligonucleotide to a free hydroxyl group and then cutting out the solid phase carrier force.

[0039] [Chemical 7]

R'-0-

In the above formula,

 R, —OH means oligonucleotide,

 The hydroxyl group in R′—OH means the hydroxyl group at the 3 ′ end of the modified nucleoside contained as a constituent component at the 3 ′ end of the oligonucleotide,

R ^A means a saturated aliphatic hydrocarbon group, an unsaturated aliphatic hydrocarbon group, an aryl group, or a heterocyclic group;

R ¹ is a hydroxyl protecting group,

R ² is a solid support,

 L is a group represented by the formula (IVA).

 -X-M-Y- (IVA)

 In the above formula,

 —X— is — (CH) or (CH) —CH (CH OH) —,

 2 m 2 m 2

 —Y— is one (CH) or one (CH) -CH (CH OH) one;

 2 n 2 n 2

 —M is one 0—C (= 0) NH, one O—, one (= 0) —? << 1-or-? << 1-ji (= O)-

 m and n are each independently an integer of 1 to 20.

[0040] The present invention also provides the following formula for producing a modified oligonucleotide represented by the following formula (IA): It is a unity compound for solid phase synthesis represented by (XIA).

[0041] [Chemical 8]

R'-0-

 In the above formula,

 R, —OH means oligonucleotide,

 The hydroxyl group in R′—OH means the hydroxyl group at the 3 ′ end of the modified nucleoside contained as a constituent component at the 3 ′ end of the oligonucleotide,

R ^A means a saturated aliphatic hydrocarbon group, an unsaturated aliphatic hydrocarbon group, an aryl group, or a heterocyclic group;

R ¹ is a hydroxyl protecting group,

R ² is a solid support,

 L is a group represented by the formula (IVA).

 -X-M-Y- (IVA)

 In the above formula,

 —X— is — (CH) or (CH) —CH (CH OH) —,

 2 m 2 m 2

 —Y— is one (CH) or one (CH) -CH (CH OH) one;

 2 n 2 n 2

 —M is one 0—C (= 0) NH, one O—, one (= 0) —? << 1-or-? << 1-ji (= O)-

 m and n are each independently an integer of 1 to 20.

[0042] The present invention is also a method for producing a modified oligonucleotide represented by the following formula (IS) using a compound for solid phase synthesis represented by the following formula (XIS) as a starting material,

R ^{1 of the} unit compound for solid phase synthesis represented by the formula (XIS) is removed and converted to a free hydroxyl group, the oligonucleotide is extended to the free hydroxyl group, and then the solid phase carrier is cleaved. This is a production method for obtaining the modified oligonucleotide.

 [Chemical 9]

In the above formula,

 R, —OH means oligonucleotide,

 The hydroxyl group in R′—OH means the hydroxyl group at the 3, terminal end of the modified nucleoside contained as a constituent component at the 3 ′ end of the oligonucleotide.

R ^S means a steroid skeleton optionally having one or more substituents, wherein one or more single bonds in the skeleton are arbitrarily replaced with double bonds,

R ¹ is a hydroxyl protecting group,

R ² is a solid support,

 L is a group represented by the formula (IVS),

 -X-M-Y- (IVS)

In the above formula,

 X— is one (CH) or (CH) — CH (CH OH) —,

 2 m 2 m 2

 Y— is one (CH) or one (CH) CH (CH OH) one;

 2 n 2 n 2

 —M is one 0—C (= 0) NH, one O, one C (= 0) —NH or one NH—C (= O) —,

m and n are each independently an integer of 1 to 20.

The present invention is also a unit compound for solid phase synthesis represented by the following formula (XIS) for producing a modified oligonucleotide represented by the following formula (IS). [0045] [Chemical 10]

 In the above formula,

 R, —OH means oligonucleotide,

 The hydroxyl group in R′—OH means the hydroxyl group at the 3 ′ end of the modified nucleoside contained as a constituent component at the 3 ′ end of the oligonucleotide,

R ^S means a steroid skeleton optionally having one or more substituents, wherein one or more single bonds in the skeleton are arbitrarily replaced with double bonds,

R ¹ is a hydroxyl protecting group,

R ² is a solid support,

 L is a group represented by the formula (IVS),

 -X-M-Y- (IVS)

 In the above formula,

 —X— is — (CH) or (CH) —CH (CH OH) —,

 2 m 2 m 2

 —Y— is one (CH) or one (CH) -CH (CH OH) one;

 2 n 2 n 2

 —M is one 0—C (= 0) NH, one O—, one (= 0) —? << 1-or-? << 1-ji (= O)-

 m and n are each independently an integer of 1 to 20.

 Next, the present invention will be specifically described.

[0047] In the present invention, "oligonucleotide" refers to, for example, a polymer of nucleoside subunits, and the number of subunits is not particularly limited. In particular, when the modified oligonucleotide of the present invention is DNA, the number of subunits is 2 to: LOO strength S, preferably 4 to 30 is more preferred RNA. 2 to 50 is preferred. 4 to 30 is more preferred. The “oligonucleotide” in the present invention is not particularly limited except that the 3′-hydroxy group of the 3 ′ terminal nucleoside is modified. For example, for a nucleoside that is a subunit of an oligonucleotide, the sugar moiety (eg 2′-substitution) and base may be modifications known to those skilled in the art.

 [0048] As described above, the present invention includes a modified nucleoside as a constituent component at the 3 'end of the oligonucleotide, and the modified nucleoside force nucleoside via the linker at the 3, terminus of the modified nucleoside. For the sake of convenience, this is referred to as an acid-modified oligonucleotide.

 [0049] The oligonucleotide includes a modified nucleoside as a constituent component at the 3 'end, and a carboxylic acid represented by the following formula (III) is bonded to the modified nucleoside force nucleoside at the 3' end via a linker. The acid-modified oligonucleotide is preferably a modified oligonucleotide represented by the following formula (IA).

 [0050] [Chemical 11]

0

 γ Η

R'-OPOL— N— f R ^A (IA)

 O O In the above formula,

 R, 1 OH means oligonucleotide,

 The hydroxyl group in R′—OH means the hydroxyl group at the 3 ′ end of the modified nucleoside contained as a constituent component at the 3 ′ end of the oligonucleotide,

 L means a divalent group,

R ^A represents a saturated aliphatic hydrocarbon group, an unsaturated aliphatic hydrocarbon group, an aryl group, or a heterocyclic group.

 [0051] The modified oligonucleotide represented by the formula (IA) can be represented, for example, as follows.

[0052] [Chemical 12]

In the above formula, W ¹ and W ² are hydrogen or a hydroxyl group, Basel and Base 2 are a group represented by the following formula (1), a group represented by the following formula (2), and a formula (3) Independently selected from the group represented by the group represented by the following formula (4) and the group represented by the following formula (5).

[Chemical 13]

(1) (2) (3) (4) (5) The saturated aliphatic hydrocarbon group means a saturated hydrocarbon substituted with a carboxy group, for example, having 1 to 25 carbon atoms, preferably 10 ˜25, more preferably 15-20 saturated aliphatic hydrocarbon groups. Examples of saturated aliphatic hydrocarbon groups include methanic acid (formic acid), ethanoic acid (acetic acid), propanoic acid (propionic acid), butanoic acid (butyric acid), 2-methylbutanoic acid (isobutyric acid), pentanoic acid ( Valeric acid), 3-methylbutanoic acid (isovaleric acid), 2,2-dimethylpropanoic acid (pivalic acid), hexanoic acid, heptanoic acid, octanoic acid, nonanoic acid, decanoic acid, dodecanoic acid (lauric acid), Examples include tridecanoic acid, tetradecanoic acid (myristic acid), pentadecanoic acid, hexadecanoic acid (palmitic acid), heptadecanoic acid, octadecanoic acid (stearic acid), nonadecanoic acid, and icosanoic acid. Of these, hexadecanoic acid (palmitic acid) is preferred. [0054] The unsaturated aliphatic hydrocarbon group means an unsaturated hydrocarbon such as an alkene, alkyne and the like, in which a carboxy group is substituted, for example, having 3 to 25 carbon atoms, preferably 10 to 25 carbon atoms. Preferably they are 15-20 saturated aliphatic hydrocarbon groups. Examples of unsaturated aliphatic hydrocarbon groups include propenoic acid (acrylic acid), propionic acid (propiolic acid), 2-methylpropenoic acid (methacrylic acid), trans butter-2-enoic acid (crotonic acid), cis butter Examples include 2-enoic acid (isocrotonic acid), cis-octadeca-9-enoic acid (oleic acid), trans-otatadecaenoic acid (raidic acid), and the like. Among them, cis-octadeca 9 enoic acid (oleic acid) is preferable.

 [0055] The aryl group has a total of 5 to 50 ring atoms, at least one ring is aromatic, and each ring has 5 to 8 ring atoms, monocyclic, bicyclic, Refers to tricyclic and tetracyclic carbon cyclic groups. Examples of the aryl group include phenyl, indul, naphthyl, fluorenyl and the like.

 [0056] The heterocyclic group has a total of 5 to 50 ring atoms, at least one ring has a heteroatom, and each ring has 5 to 8 ring atoms, It means bicyclic, tricyclic and tetracyclic carbocyclic groups. Examples of the heterocyclic group include thiophenyl, fuller, viral, xanthur, pyrrolyl, imidazolyl, pyridyl and the like.

 [0057] Examples of the divalent group in the formula (IA) include, but are not limited to, a divalent group represented by the following formula (IVA).

 —X— M—Y— (IVA)

 In the above formula,

 —X— is — (CH) or (CH) —CH (CH OH) —,

 2 m 2 m 2

 —Y— is one (CH) or one (CH) —CH (CH OH) one;

 2 n 2 n 2

 —M is one 0—C (= 0) NH, one O—, one (= 0) —? << 1-or-? << 1-ji (= O)-

 m and n are each independently an integer of 1 to 20.

[0058] The acid-modified oligonucleotide of the present invention may be a single-stranded oligonucleotide, a double-stranded oligonucleotide, or the like. The acid-modified oligonucleotide of the present invention is preferable for use as antisense, siRNA and the like because of its excellent ability to form double strands. Acid-modified oligonucleotide When the oxide is double-stranded, it is preferably a single-stranded oligonucleotide force-modified oligonucleotide of one or both of the double-stranded oligonucleotides. When both single-stranded oligonucleotides of the double-stranded oligonucleotide are acid-modified oligonucleotides, the two acid-modified oligonucleotides may be the same or different.

 [0059] When the acid-modified oligonucleotide is single-stranded, the modified oligonucleotide is antisense DNA or antisense RNA, and the modified oligonucleotide is a part of mRNA encoding endonuclease or the like It is preferable to have a sequence complementary to the whole. Modified oligonucleotide force This is a force capable of forming double strands with such mRNA and suppressing the expression of endonucleases and the like.

 [0060] Further, as described above, the present invention is a double-stranded modified oligonucleotide, and includes a sense strand having the same base sequence as a part of the modified oligonucleotide-force target mRNA, and an antisense to the sense strand. A strand of the antisense strand and the sense strand

At least the antisense strand is an oligonucleotide analogue containing a modified nucleoside as a constituent component at the 3 ′ end of the oligonucleotide, and the modified nucleoside force nucleoside via the linker at the 3, end of the modified nucleoside A force that is a modified nucleoside to which a compound having a steroid skeleton represented by (ii) is bound. Hereinafter, this is referred to as a steroid-modified oligonucleotide for convenience.

 [0061] The oligonucleotide analogue is preferably a modified oligonucleotide represented by the following formula (IS).

[0062] [Chemical 14]

 0

 H

R'-OPOL— N- ^ pOR ^s (IS)

 O O In the above formula,

 R, 1 OH means oligonucleotide,

 The hydroxyl group in R′—OH means the hydroxyl group at the 3 ′ end of the modified nucleoside contained as a constituent component at the 3 ′ end of the oligonucleotide,

L means a divalent group, R ^s means a steroid skeleton that optionally has one or more substituents, and one or more single bonds in the skeleton are optionally replaced with double bonds.

[0063] The oligonucleotide analog represented by the formula (IS) can be expressed, for example, as follows.

[0064] [Chemical 15]

In the above formula, W ¹ and W ² are hydrogen or a hydroxyl group, Basel and Base 2 are a group represented by the following formula (1), a group represented by the following formula (2), and a formula (3) Independently selected from the group represented by the group represented by the following formula (4) and the group represented by the following formula (5).

 [Chemical 16]

(1) (2) (3) (4) (5) The steroid skeleton means a cyclopentahyofuronananthrene derivative and has a structure in which three six-membered rings and one five-membered ring are condensed. Have. In the R ^S , one or more single bonds in the sterol skeleton may be arbitrarily replaced with a double bond. Examples of the substituent in the above include a lower alkyl group (for example, methyl, ethyl, propyl, butyl, 1,5-dimethylhexyl, etc.), a hydroxyl group, and the like. The substituent has 0 to: LO It may be. Examples of R ^s include those represented by the following formulas _c

 [Chemical 17]

Examples of the divalent group in the formula (IS) include, but are not limited to, a divalent group represented by the following formula (IVS).

 -X-M-Y- (IVS)

 In the above formula,

 —X— is — (CH) or (CH) —CH (CH OH) —,

 -Y— is one (CH) or — (H) —

M is O— C (= 0) NH—

—NH— or NH— C (: O) —

 m and n are each independently an integer of 1 to 20.

 [0067] When the modified oligonucleotide (acid-modified oligonucleotide and steroid-modified oligonucleotide) is double-stranded, it is an oligonucleotide that causes RNAi (RNA interference), and encodes the modified oligonucleotide force endonuclease, etc. Preferred are modified oligonucleotides having the same base sequence as part of the mRNA. Such modified oligonucleotides are also useful for RNAi research and the like. Examples of the endonuclease include RNaseL.

[0068] When the modified oligonucleotide is double-stranded, it is the modified oligonucleotide force siRNA (short interfering RNA), and a part of the mRNA is 75% from the start codon. A 19-base sequence following the first AA sequence, upstream of the base, and having a partial force of the mRNA, specific to the mRNA, and the modified oligonucleotide force The sense strand and the antisense strand of the 19-base sequence And a combination thereof. Such a sense chain can be obtained, for example, as follows. First, using a known gene database such as NCBI (National Center for Biotechnology Information), EMBL-EBI (European Molecular Biology Laboratory-European Bioinformatics Institute), for example, an exonuclease mRNA sequence is obtained. In the gene sequence, find the first AA sequence that is more than 75 bases downstream from the start codon. A 19-base sequence following the AA sequence, a total of 21 bases. Again, using a known gene database, confirm that it is specific for the target gene. It is also confirmed that this 19-base sequence has a GC content of around 50%. By confirming these two, the obtained 19-base sequence ability is the sense strand as described above.

[0069] In the case of a modified oligonucleotide-strength RNA as described above, it is preferable that one or both of the sense strand and the antisense strand further have two base sequences at the 3 'end. Having such a sequence at the 3 ′ end also tends to cause RNAi. Such two base sequences are not limited to, for example, forces such as TT and cocoon.

 [0070] The modified oligonucleotide of the present invention is preferably nuclease resistant. The modified oligonucleotide of the present invention can be prevented from being degraded by a nuclease when incorporated into cells, and as a result, the modified oligonucleotide can maintain its activity in cells. is there.

 [0071] The gene expression inhibitor of the present invention includes the modified oligonucleotide of the present invention. Such a gene expression inhibitor can modify the ability of modified oligonucleotide, for example, cleave the mRNA of the target gene or form a double strand with the mRNA of the target gene, thereby suppressing the gene expression.

[0072] The pharmaceutical composition of the present invention is for treating a disease associated with gene expression, and includes the gene expression inhibitor. When a disease associated with gene expression, for example, a disease is caused by the expression of a protein, the pharmaceutical composition It can be used to suppress expression and treat diseases associated with its gene expression.

 [0073] Such a pharmaceutical composition preferably further contains an excipient for cell introduction. This is because it becomes possible to facilitate introduction into cells when treating diseases associated with gene expression. Examples of the cell introduction excipient include a transfection reagent and the like. The transfection reagent includes, for example, a DNA molecule (the pharmaceutical composition) encapsulated in artificial lipid vesicles (ribosomes) composed of phospholipids, and the artificial lipid vesicles in a cell suspension. In addition, it is a reagent that forms artificial lipid vesicles that are used in the lipofection method to attach DNA molecules in artificial lipid vesicles by attaching them to the cell surface and fusing with cell membranes. Examples of the transfer reagent include, for example, Lipofectamine (Invitrogen), Lipofectamine 2000 (Invitrogen), Oligofectamin (Invitrogen), Transmessenger (Kigen (Kigen)) QIAGEN))), siRNA Transfection 'kit. Jet SI (Ambion), Gene Slicer SiRNA Transfection Reagent (Gene Therapy Systems), etc. It is done.

 [0074] In addition to including the cell introduction excipient as described above, the pharmaceutical composition of the present invention can also be introduced into cells by using an electroporation method, a particle gun method, or the like. . The electopore position method is, for example, a method in which an electric pulse is applied to a cell to make a hole in the cell wall, and the pharmaceutical composition of the present invention is introduced into the cell through the hole. In the particle gun method, molecules such as DNA molecules (the pharmaceutical composition) are attached to gold fine particles, and using a particle gun (particle gun), the compressed fine helium gas is used to shoot the gold fine particles into the cell membrane. In this method, the DNA molecule (the pharmaceutical composition) is introduced into cells.

[0075] The RNAi kit of the present invention includes a modified oligonucleotide that is an siRNA. Other examples of such kits include a plate on which a well and a modified oligonucleotide are fixed, a fixed carrier such as a fiber and a nanochip, and the like. Such a kit may contain, in addition to the modified oligonucleotide, for example, a drug, a coloring reagent that reacts to develop color, a detection reagent that facilitates detection, and the like. [0076] The RNAi research reagent of the present invention contains a modified oligonucleotide that is siRNA. During RNAi research, when dsRNA of 30bp or more is introduced into a cell, the cell-specific defense reaction is activated, and a reaction that randomly degrades mRNA occurs, which indicates whether RNAi is generated in the cell. It may not be possible to judge. This random degradation of mRNA is considered to occur by the following mechanism. First, dsRNA activates 2-5 oligoadreic acid synthase (2-5AS) and activates 2-5A RNAaseL produced thereby. The RNAaseL degrades mRNA at random. Since the modified oligonucleotide, which is siRNA, can suppress the expression of RNAaseL, random degradation of mRNA can be suppressed. As a result, for example, it is easy to determine whether RNAi is occurring in the cell.

 [0077] The gene expression suppression method of the present invention is a method of suppressing gene expression using a modified oligonucleotide. In this method, the modified oligonucleotide can, for example, cleave the mRNA of the target gene or form a double strand with the mRNA of the target gene, thereby suppressing gene expression.

[0078] The method for causing RNAi of the present invention is a method for causing RNAi using a modified oligonucleotide that is siRNA. This method can cause RNAi since it is a modified oligonucleotide force RNA.

[0079] Next, a production method for producing the modified oligonucleotide of the present invention will be described by way of examples. Such a production method made it possible to produce the modified oligonucleotide of the present invention that could not be produced conventionally.

[0080] First, an example of producing an acid-modified oligonucleotide will be described.

[0081] For example, R ¹ of the unit compound for solid-phase synthesis represented by the following formula (XIA) is removed and converted to a free hydroxyl group,

 The modified oligonucleotide can be obtained by extending the oligonucleotide to the free hydroxyl group and then cutting out the solid phase carrier force.

[0082] [Chemical 18]

In the above formula, R ^A is a saturated aliphatic hydrocarbon group, an unsaturated aliphatic hydrocarbon group, an aryl group.

Or a heterocyclic group,

R ¹ is a hydroxyl protecting group,

R ² is a solid support,

 m is an integer from 1 to 20,

 n is an integer of 1-20.

[0083] In the formula (XIA), the saturated aliphatic hydrocarbon group, the unsaturated aliphatic hydrocarbon group, the aryl group, and the heterocyclic group, the saturated aliphatic hydrocarbon group in the formula (IA), As defined for unsaturated aliphatic hydrocarbon groups, aryl groups, and heterocyclic groups.

 [0084] Next, production examples of steroid-modified oligonucleotides will be described.

[0085] For example, R ¹ of the unit compound for solid phase synthesis represented by the following formula (XIS) is removed and converted to a free hydroxyl group, and the oligonucleotide is extended to the free hydroxyl group. The modified oligonucleotide can be obtained by cutting out the phase carrier force.

[0086] [Chemical 19]

In the above formula, R 1 optionally has one or more substituents, and one or more single bonds in the skeleton are optional. The steroid skeleton is replaced by a double bond,

R ¹ is a hydroxyl protecting group,

R ² is a solid support,

 m is an integer from 1 to 20,

 n is an integer of 1-20.

[0087] R ^S in the formula (XIS) is as defined for R ^S in the formula (IS).

 In the present invention, the hydroxyl-protecting group means a conventionally known primary alcohol protecting group, for example, 4,4′-dimethoxytrityl (DMTr), 4-monomethoxy known in the field of nucleoside chemistry. Trityl (MMTr), (9-phenol) xanthene-9-yl [pixy 1] and the like can be used as the protecting group.

 In the present invention, the solid phase carrier is not limited as long as it is a solid phase carrier suitable for synthesizing DNA, RNA, etc. with the solid phase carrier. For example, CPG (control pore glass ), HC P (highly cross-linked polystyrene).

In the above method, R ¹ can be removed by appropriately selecting an acid, an alkali, a catalyst or the like according to the type of R ¹ .

 [0091] In the method described above, in order to extend the nucleotide to the free hydroxyl group of the solid-phase synthesis unit compound, a nucleoside is used in accordance with the sequence of the modified oligonucleotide using a conventionally known technique in the field of oligonucleotide synthesis. Can be performed by sequentially coupling

[0092] As the nucleoside, coupling reagent, deprotection reagent, washing reagent, etc., those usually used for nucleic acid solid phase synthesis are used. The modified oligonucleotide on the obtained solid phase carrier is subjected to deprotection of the oligonucleotide side chain if necessary, and then the solid phase carrier force is cut out to obtain a crude modified oligonucleotide. The reagent used for excision can be appropriately selected from conventionally known reagents according to the structure of the solid phase carrier and the linker (the portion connecting the solid phase carrier and the modified oligonucleotide). The crude modified oligonucleotide may be purified by HPLC or the like, if necessary.

[0093] Next, production in the case where the modified oligonucleotide is double-stranded is described with an example. To do. For example, a single-stranded modified oligonucleotide is first produced according to the method as described above. A single-stranded natural oligonucleotide having a sequence complementary to the modified oligonucleotide is also separately produced according to a conventionally known method. The resulting single-stranded modified oligonucleotide is then placed in an annealing buffer (eg, a buffer containing lOOmM KOAc aqueous solution, 2 mM MgOAc solution, and 30 mM HEPES-KOH (pH 7.4)). The solubilized product and the single-stranded natural oligonucleotide dissolved in the annealing buffer are mixed, treated at 95 ° C for 5 minutes, and then gradually cooled to 25 ° C. Thus, a double-stranded modified oligonucleotide can be obtained. This double-stranded modified oligonucleotide can be isolated and purified by further performing phenol Z chromatography, ethanol precipitation or the like, if necessary.

 [0094] The solid-phase synthesis unity compound of formula (XIA) and the solid-phase synthesis unity compound of formula (XIS) used in the production method can be produced, for example, by the following method. .

First, the production of unitary compound (XIA) will be described with reference to Scheme 1. For example, a step of reacting a compound represented by the formula (XII) with R ^A —COOH and a peptide condensing agent to obtain a compound represented by the formula (ΧΙΠΑ); Removing the R ³ of the compound, and then reacting with a compound represented by the following formula (XIV) to obtain a compound represented by the following formula (XVA): And a solid phase carrier having an amino group are reacted to obtain a compound represented by the above formula (XIA).

[0096] [Chemical 20]

 (XII) (XIIIA)

Scheme 1 First, the production of unitary compound (XIA) will be described with reference to Scheme 1. The amine derivative of the formula (ΧΠ) and R ^A —COOH are condensed in the presence of a peptide condensing agent (eg WSC (1-ethyl-3-propyl (3-dimethylaminopropyl) -carbodiimide) hydrochloride). The amine derivative of formula (XII) may be obtained commercially or protected at the hydroxyl groups at positions 1 and 3 of glycerol, for example 1 -0- (4, 4, -Dimethoxytrityl) -3-O-tert-Butyldimethylsilylglycerol (see De Napoli, et. Al, Tetrahedron 1999, 55 (32), 9899-9914) Good.

Next, R ³ of the obtained compound of the formula (ΧΙΠΑ) is removed, and then an anhydride (XIV) is optionally added to a base (eg, pyridine, triethylamine, etc.), a catalyst (eg, 4-dimethylamino). Reaction is carried out in the presence of pyridine (DMAP, etc.) to obtain a compound of formula (XVA). Next, the obtained compound of the formula (XVA) and solid phase carrier R ² —NH having an amino group are cut

 2 Condensation in the presence of a pulling reagent (for example, WSC (1-ethyl 3-propyl (3 dimethylaminopropyl) carbopositimide 'hydrochloride)) gives unit compound (XIA).

[0099] Next, the production of the unitary compound (XIS) will be described with reference to Scheme 2. For example, a step of obtaining a compound represented by the formula (xms) by reacting a compound represented by the formula (XII) with —011 shobi 1, 1, -carbodiimidazole; removing the R ³ of the compound represented by xms), and obtaining the following, a compound represented by the following formula (XIV) represented by is reacted with compound of the following formula (XVS), the formula (XVS ) And a solid phase carrier having an amino group are reacted to obtain the compound represented by the formula (XIS).

 [0100] [Chemical 21]

 (XVS)

 (XIS)

Scheme 2 An amine derivative of the formula (XII) and R ^S —OH are condensed in the presence of 1,1, -carbodiimidazole to obtain a compound of the formula (XIIIS). The amine derivative of the formula (XII) may be obtained commercially, or protected with hydroxyl groups at positions 1 and 3 of glycerol, such as 1-O— (4,4, -dimethoxytrityl) -3-0. — 6-Butyldimethylsilylglycerol (see 06 Napoli, et. Al, Tetrahedron 1999, 55 (32), 9899-9914) should be made in-house using known literature.

[oioi] Next, R ³ of the obtained compound of formula (xms) is removed, and then anhydride (XIV) is optionally added.

, Reaction in the presence of a base (eg, pyridine, triethylamine, etc.), a catalyst (eg, 4-dimethylaminopyridin (DMAP), etc.) to obtain a compound of formula (XVS).

[0102] Next, the obtained compound of formula (XVS) and solid phase carrier R ² — NH having an amino group were cut into cuts.

 2 Condensation in the presence of a pulling reagent (for example, WSC (1-ethyl-3- (3 dimethylaminopropyl) carbopositimide 'hydrochloride)) gives unit compound (XIS).

[0103] In each step of Schemes 1 and 2, if necessary, a protecting group may be introduced into each functional group, the protecting group may be deprotected, or the protecting group may be changed. Selection of a protecting group according to the type of functional group, introduction of the protecting group, and removal of the protecting group can be carried out according to methods known in the art. For example, “protective group in organic synthesis” s in Organic synthesis j, T. Greene et al., John Wiley & ¾ons, Inc.

 Example

 [0104] The following abbreviations are used in the description of this specification.

 APS: ammonium peroxodisulfate

 CPG: controlled pore glass

 DMAP: 4-dimethylaminopyridine

 DMTrCl: 4,4'-Dimethoxytritylchloride (4,4 dimethoxytritylchloride)

 DMF: Dimethylformamide

EDTA: ethylenediamine- N, N, Ν ', Ν, —tetraacetic acid, aisodium salt, dehydrate) FAB: fast atom bombardment (fast atom bombardment) HRMS: high-resolution mass spectrometry

Im CO: 1-1 '—Carborubbis- 1H-imidazole (1-1'-Carbonylbis- 1

2

 H- 匪 dazole)

 mRNA: messenger ribonucleic acid

 NBA: 3--Trobenzylalchol (3-nitrobenzylalchol)

 PAGE: polyacrylamide gel electrophoresis

TBAF: tributylammoniumfluoride

 TBE: Tris-boric acid- EDTA

 TEAA: triethylamine-acetic acid

 TBDMSCl: tert-butyldimethylsilylchloride TEMED: N, N, Ν ', N, monotetramethyl monoethylenediamine (Ν, Ν, Ν', Ν'- tetr amethyl-ethylenediamine)

 THF: Tetrahydroforan

 TRIS: Tris (hydroxymethyl) aminomethane WSC: 1-Ethyl-3- (3-dimethylaminopropyl) -carbodimide 'hydrochloride (1- Ethyl-3- (3-dimethylaminopropyl) -carbodnmide, hydrochloride )

 The activity of the compound on CPG was calculated as follows.

[0105] Place 6 mg of dried CPG on a glass filter and add HCIO and ethanol

 Four

 The mixture (HCIO: EtOH = 3: 2) was poured. Absorbance of the obtained filtrate (wavelength 498nm

 Four

 (DMTr group absorption wavelength)) was measured, and the value was substituted into the following equation.

[0106] [Equation 1]

 Abs. (498mn) x b /. (Solution) (mL) x 14.3 _ activity, ^)

 Ife¾Ai (support) (mg)

 In the above formula, Abs. Is the absorbance of CPG at a wavelength of 498 nm,

 Vol. Is the measured volume of the filtrate,

 weight is the weight of the measured CPG.

[0107] 1. Monomer production for production of modified single-stranded oligonucleotide or modified double-stranded oligonucleotide [0108] [Chemical 22]

 (I) 1—O— (4,4, -Dimethoxytrityl) -glycerol (1-0- (4,4'-dimet hoxytrityl) -glycerol) (23)

 A small amount of DMAP was added to a solution of glycerol (1.0 g, 10.86 mmol) in pyridine (24 mL) and stirred. Thereto was added DMTrCl (3.68 g, 10.86 mmol, leq.) In pyridine (30 mL), and the mixture was stirred for 12 hours. Dilute the reaction solution with ethyl acetate, saturated aqueous NaHCO

 Extraction was performed with 3 liquids (X 2) and a saturated aqueous NaCl solution (X 1). The organic phase was dried over anhydrous sodium sulfate. The solvent of the organic phase was distilled off, and the resulting residue was isolated and purified by silica gel column chromatography (N-hexane: EtOAc = 5: 1 to 1: 1) to give the title compound as a yellow oil. (Yield 2.52 g, 6.39 mmol, 59% yield).

[0109] ^J H NMR (400MHz, DMSO— d) δ: 2.87-2.93 (2H, m, 1— H), 3.30—3.41 (2H, m, 3— H

 6

), 3.53-3.65 (1H, m, cross), 3.71 (6H, s, — OCH), 4.44 (1H, t, J = 5.6 Hz, 3—OH), 4.7 4 (1H, d, J = 4.8Hz, 2—OH), 6.85—7.40 (13H, m, DMTr).

[0110] (Il) l -O- (4,4, -Dimethoxytrityl) —3— O-tert-Butyldimethylsilylglyceronorole (1—0— (4,4'-dimethoxytrityl)-3— 0— tert — Production of (butyldimethylsilylglycerol) (24)

 1 -0- (4,4, -dimethyloxylyl) -glycerol (24) (2. 46 g, 6. 24 mmol) in DMF (5 mL) was added to imidazole (0.85 g, 12.5 mmol, 2 eq. ) Was dissolved. There, a solution of TBDMSC1 (0.94 g, 6.24 mmol, leq.) In DMF (15 mL) was added and stirred for 12 hours. Dilute the reaction solution with ethyl acetate, water (X3), NaHCO saturated aqueous solution

 Washed with 3 liquids (X 2) and NaCl saturated aqueous solution (X 1). The organic phase was dried over anhydrous sodium sulfate. The solvent of the organic phase was distilled off, and the resulting residue was isolated and purified by silica gel column chromatography (N hexane: EtOAc = 10: 1 to 1: 3) to give a yellow oily title compound. (Yield 2.60 g, 5. l mmol, yield 82%).

[0111] ^J H NMR (400 MHz, CDC1) δ: 0.12 (6H, s, TBDMS—CH 3), 0.94 (9H, s, t-butinole),

 3 3

 3.20-3.31 (2H, m, 1— H), 3.69—3.81 (2H, m, 3— H), 3.88 (7H, s, —OCH and cross), 7

 Three

 .25-7.53 (13H, m, DMTr).

[0112] (III) l—O— (4,4, -Dimethoxytrityl) —2—0— [N— (4 aminobutyl) carbamoyl] 3—O—tert butyldimethylsilylglycerol (l-0- ( 4,4'-dimetho xytrityl)-2-0- [N- (4-aminobutyl) carbamoyl]-3-O- tert-butyldimethyto siiylgiycero 1) (25)

 1—O— (4,4′-dimethoxytrityl) 3-O-tert-butyldimethylsilylglycerol (24) (2.60 g, 5. l lmmol) in pyridine (17 mL) was added to Im CO (l. 66g, 1

 2

 0.222 mmol, 2 eq.) Was dissolved and the reaction solution was stirred for 1 h. After confirming the disappearance of the raw materials, putrescine (3 mL, 30.66 mmol, 6 eq.) Was added dropwise to the reaction solution under ice cooling. After stirring for 3 hours, the reaction solution was diluted with black mouthform. The reaction solution was washed with water (X 3) and saturated aqueous NaCl solution (X 1) and then dried over anhydrous sodium sulfate. The solvent of the reaction solution was distilled off, and the resulting residue was isolated and purified by silica gel column chromatography (MeOH: CH C1 = 10-25%) to obtain a colorless oily title compound. (Yield 3.0

Three

Og ゝ 4.82 mmol, 94% yield). [0113] H NMR (400 MHz, DMSO— d) δ: 0.13 (6H, s, TBDMS— CH), 0.97 (9H, s, t-butyl)

 6 3

 ), 1.72 (4H, s, petitnore), 3.62 (8H, m, petitnore and 1- H and 3-H), 3.94 (7H, s,-O CH and cross), 6.96—7.61 (13H, m, DMTr), 8.36 (1H, m, NH).

 Three

¹³ C-NMR (lOOMHz, DMSO- d) δ: -5.58, 17.73, 25.58, 26.76, 27.83, 48.53, 54.97,

 6

 61.67, 62.26, 72.52, 85.22, 113.09, 126.58, 127.62, 127.74, 129.63, 135.51, 144.8 5, 149.28, 155.77, 158.03.

FAB— HRMS (NBA) calcd for CHNO Si (MH ⁺ ), 623.3516; found, 623.3508.

 35 51 2 6

 [0114] (lVa) l -0- (4, 4, Dimethoxytrityl) —2— O— [N-palmitoyl— N— (4-aminobutyl) force rubermoyl] 3— O— tert butyl dimethylsilyl glycerol, 丄0— (4,4 -dimethoxytrityl)-2—0— [N-palmitoyl- N— (4— aminobutyl) carbamoyl] — 3— O-tert-butyl-dimethylsilyl-glycerol) (26a)

 1—O— (4,4, -Dimethoxytrityl) 2—O— [N— (4 Aminobutyl) strength rubymoyl] 3—O-tert Butyldimethyl-silylglycerol (25) (0.90 g, 1.44 mm ol) in CH CI (14 mL) with nonremitic acid (0.55 g, 2.16 mmol, 1.5 eq.)

 twenty two

 And stirred. After stirring for 30 minutes, WSC (0.41 g, 2.16 mmol, 1.5 eq.) Was added to the reaction solution and stirred for 24 hours. The reaction solution was diluted with chloroform, washed with water (X 1) and saturated aqueous NaCl solution (X 1), and dried over anhydrous sodium sulfate. After evaporating the solvent of the reaction solution, the obtained residue was isolated and purified by silica gel column chromatography (N hexane: EtOAc = 9: 1 to 1: 1) to give the title compound as a yellow oil. (Yield 0.85 g, 0.99 mmol, 69% yield).

[0115] ^J H NMR (400 MHz, CDCl) δ: 0.01 (6H, s, TBDMS—CH 3), 0.82 (9H, s, t-butinole),

 3 3

 0.88-0.91 (3H, m, pal), 1.26 (14H, s, pal), 2.06-2.18 (4H, m, butinole), 3.24 (8H, m, petitenore and 1-H and 3-H), 3.76- 3.80 (7H, m, — OCH and cross), 6.81-7.46 (13H,

 Three

 m, DMTr).

¹³ C-NMR (lOOMHz, CDCl) δ: -5.44, 14.09, 25.73, 29.32, 29.47, 29.60, 29.61, 29.

 Three

 65, 31.88, 36.74, 38.99, 40.44, 55.14, 60.37, 62.14, 74.01, 85.74, 112.96, 126.63, 127.68, 128.11, 130.02, 130.03, 136.05, 136.11, 144.92, 156.24, 158.33, 173.26.

[0116] (Va) l -0- (4, 4, Dimethoxytrityl) —2— O— [N-palmitoyl— N— (4— (Aminobutyl) power rubermoyl] -glycerol (1-0- (4,4'-dimethoxytrityl)-2- 0- [N-palmitoyto-N- (4-aminobutyl) carbamoyl] -glycerol) (27a)

 1—O— (4,4, —Dimethoxytrityl) 2—O— [N—palmitoyl—N— (4 aminobutyl) force rubamoyl] 3—O—tert butyl dimethylsilyl glycerol (26 a) (0.775 g, TBAF (2 mL, 2 mmol, 2.3 eq.) Was added dropwise to a solution of 0.887 mmol) in THF (4.4 mL), and the mixture was stirred for 1 hour. The reaction solution is diluted with ethyl acetate, washed with saturated aqueous NaHCO 3 solution (X 2), saturated aqueous NaCl solution (X 1), and dried over anhydrous sodium sulfate.

Three

 Let The solvent of the reaction solution was distilled off, and the resulting residue was isolated and purified by silica gel column chromatography (EtOAc 100%) to give the title compound as a yellow oil (yield 0.52 g, 0.5%). 70 mmol, yield 80%).

[0117] ^J H NMR (400 MHz, CDCl) δ: 0.86—0.95 (3H, m, pal), 1.25 (14H, s, pal), 2.02-2.3

 Three

 0 (4H, m, Petinole), 3.22-3.30 (8H, m, Petinole and 1- H and 3- H), 3.79 (7H, m,-OC H and cross), 6.81—7.43 (13H, m, DMTr ).

 Three

¹³ C-NMR (lOOMHz, CDCl) δ: 13.85, 14.09, 20.49, 22.65, 25.74, 26.74, 27.18, 29.

 Three

 32, 29.47, 29.61, 29.65, 31.87, 36.75, 38.95, 40.55, 52.06, 55.17, 62.86, 63.22, 74. 57, 86.12, 113.09, 126.78, 127.80, 128.01, 129.97, 135.72, 144.61, 156.57, 158.45, 173.37.

 FAB— HRMS (NBA) calcd for C H N O (MH +), 747.49485; found, 747.49549.

 45 67 2 7

 [0118] (Via) CPG unit with palmitic acid bound (29a)

1—O— (4,4, -dimethoxytrityl) 2—O— [N-palmitoyl—N— (4 aminobutyl) force rubamoyl] —glycerol (27a) (0.52 g, 0.70 mmol) in pyridine (7 0 mL) solution was added DMAP (small amount) and succinic anhydride (0.21 g, 2.10 mmol, 3. Oeq.) And stirred for 1 day. The reaction solution was diluted with chloroform, washed with water (X 3), dried over anhydrous sodium sulfate, and the organic solvent was distilled off. The obtained residue was vacuum-dried for 12 hours, then dissolved in DMF (17.5 mL), and applied to CPG (0.30 g, 0.18 mmol, 222 / z mol / g) for 30 minutes. WSC (0.13 g, 0.70 mmol, 1. Oeq.) Was added to the reaction mixture and then shaken for 3 days. After removing the solvent from the reaction mixture, the resulting CPG was washed with pyridine. After drying, 0.1M DMAP solution (solvent Is a mixture of pyridine and acetic anhydride (pyridine: Ac 2 O = 9: l)).

 2

 It was made to shake. After removing the reaction solution, the CPG was washed with MeOH and then with acetone and dried. The activity of the obtained CPG was 28 .: molZg (yield 0.32 g).

 [0119] (IVb) l— O— (4,4, —Dimethoxytrityl) 2— O— [N-oleyl— N— (4 aminobutyl) force rubermoyl] 3— O— tertbutyl-dimethyl-silylglycerol ( 1- 0- (4,4 -dimethoxytrityl)-2-0-[N-oleoyto N- (4-aminobutyl) carbamoyl]-«3- O-ter tbutyl- dimethyl-silylglycerol) (26b)

 1—O— (4,4, -Dimethoxytrityl) 2—O— [N— (4 Aminobutyl) strength rubymoyl] 3—O-tert Butyldimethyl-silylglycerol (25) (1.5 g, 2. 41 mmol ) To a solution of CH C1 (12 mL) with oleic acid (1.15 mL, 3.62 mmol, 1.5 eq.)

twenty two

 Stir. After stirring for 30 minutes, WSC (0.69 g, 3.62 mmol, 1.5 eq.) Was added to the solution and stirred for a further 26 hours. The reaction solution was diluted with chloroform, washed with water (XI) and saturated aqueous NaCl solution (X1), and dried over anhydrous sodium sulfate. After distilling off the organic solvent, the resulting residue was isolated and purified by silica gel column chromatography (N hexane: EtO Ac = 2: l to l: l) to give a yellow oily title compound. (Yield 1.83 g, 2.06 mmol, 86% yield).

[0120] ^J H NMR (400 MHz, CDC1) δ: 0.04 (6H, s, TBDMS— CH), 0.86 (14H, s, t-butinole

 3 3

 and ole), 1.29-2.19 (32H, m, ole and petitore), 3.23-3.53 (8H, m, petitnoré and 1-Hand 3-H), 3.83 (7H, m, — OCH and cross), 5.37 —5.40 (2H, m, ole), 6.84-7.49 (13H,

 Three

 m, DMTr).

¹³ C-NMR (100 MHz, CDC1) δ: -5.44, 14.10, 18.14, 22.65, 25.74, 26.58, 27.15, 27.

 Three

 19, 27.51, 29.14, 29.25, 29.28, 29.49, 29.62, 29.69, 29.74, 31.87, 36.71, 39.00, 40. 44, 50.62, 55.15, 60.39, 62.15, 62.38, 74.03, 85.75, 112.98, 126.64, 127.70, 128.1 2 , 129.72, 129.95, 130.03, 136.06, 136.13, 144.93, 156.30, 158.35, 173.32.

 Elemental analysis Calced for C H N O Si-CHCl + 1 / 2CH OH: C, 71.74; H, 9.31; N, 3.61,

 53 82 2 7 3 3

 Found: C, 64.08; H, 8.30; N, 2.75.

[0121] (Vb) l -0- (4, 4, -Dimethoxytrityl) 2— O— [N-olyl— N— (4 (NOBUTYL) POWER RUBERMOYL] -glycerol (1-0- (4,4'-dimethoxytrityl)-2- 0- [N-oleo yl-N- (4-aminobutyl) carbamoyl] -glycerol) (27b)

 1- O- (4, 4, 1-dimethoxytrityl) 2- O- [N-oleyl 1 N- (4 aminobutyl) force rubamoyl] 3- O-tertbutyl-dimethyl-silylglycerol (26b) (1.66 g 1.88 mmol) in THF (18 mL) was added dropwise TBAF (2 mL, 3.76 mmol, 2 eq.). After 7 hours, the reaction solution was diluted with ethyl acetate and saturated aqueous NaHCO solution.

 Three

 It was washed with (X 2) and a saturated aqueous NaCl solution (X 1) and dried over anhydrous sodium sulfate. The reaction solvent was distilled off, and the resulting residue was isolated and purified by silica gel column chromatography (N hexane: EtOAc = 1: 2 to 0: 1) to give the title compound as a yellow oil ( Yield 1.19 g, 1.54 mmol, yield 82%).

[0122] ^J H NMR (400 MHz, CDC1) δ: 0.85 (3H, m, ole), 1.26—2.16 (32H, m, ole and

 Three

 ), 2.68 (1H, br, 3-OH), 3.21-3.29 (8H, m, Petinole and 1- H and 3-H), 3.78 (7H, m, — OCH and cross), 5.29—5.38 (2H , m, ole), 6.81—7.43 (13H, m, DMTr).

 Three

¹³ C-NMR (100 MHz, CDC1) δ: 14.05, 22.60, 25.70, 26.68, 27.14, 29.09, 29.24, 29.

 Three

 45, 29.65, 29.69, 31.83, 32.53, 36.67, 38.92, 40.50, 55.12, 62.84, 63.06, 74.53, 86. 06, 113.05, 126.74, 127.76, 127.98, 129.67, 129.93, 135.70, 144.60, 156.58, 158.4 0, 173.34 .

 Elemental analysis. Calced for C H N O-10/3 CHC1: C, 73.02; H, 8.87; N, 3.62, Found

 47 68 2 7 3

 : C, 51.79; H, 6.08; N, 2.48.

[0123] (VIb) CPG unit with oleic acid bound (29b)

1— O— (4, 4, 1-dimethoxytrityl) 2— O— [N-oleyl 1 N— (4 aminobutyl) force rubamoyl] -glycerol (27b) (0.77 g, 1. OOmmol) in pyridine (10 mL ) Solution [This, DMAP (small amount) and succinic anhydride (0.30 g, 3.00 mmol, 3. Oeq.) Were added and stirred for 1 day. The reaction solution was diluted with ethyl acetate, washed with water (X3), dried over anhydrous sodium sulfate, and the organic solvent was distilled off. The obtained residue was vacuum-dried for 12 hours, dissolved in DMF (12.5 mL), and applied to CPG (0.50 g, 0.25 mmol, 222 mol / g) for 30 minutes. The reaction mixture was charged with WSC (0.19 g, 1.00 mmol, 1. Oeq.) And then shaken for 3 days. After the reaction mixture force also removed the solvent, the resulting CPG Washed with pyridine. After drying, add 20 mL of 0.1 M DMAP solution (solvent is a mixture of pyridine and acetic anhydride (pyridine: Ac 2 O = 9: l)) to the CPG and shake at room temperature for about 1 day.

 2

 I let you go. The CPG was washed with MeOH and then with acetone and dried. The activity of the obtained CPG was 83.2 / z mol / g (yield 0.52 g).

[0124] (IVC) I-O- (4,4, -Dimethoxytrityl) 2 O— [N cholesteryloxyl N- (4 aminobutyl) force rubamoyl] 3— O— tert butyldimethylsilanol Glycerellore (1—0— (4,4'-dimethoxytrityl)-2—0— [N-cholesteryloxycarbonyl- N- (4-aminobutyl) carbamoyl]-3-0- tert-butyldimethylsilyl-glycerol) (26c): ^ Cholesterol (0.22g, 0.58mmol) in pyridine (3mL) solution with Im CO (0.10g

 2

 0.63 mmol, 1. leq.) Was added and stirred for 2 hours. In the reaction solution, 1—O— (4,4, -dimethoxytrityl) 2—O— [N— (4 aminobutyl) force rubermoyl] — 3—O-tert butyldimethyl-silylglycerol (25) (0 43 g, 0.70 mmol) was added and stirred for 12 hours. The reaction solution was diluted with ethyl acetate and saturated aqueous NaHCO 3 solution (X 2)

 Three

 And washed with a saturated aqueous NaCl solution (X 1) and dried over anhydrous sodium sulfate. The organic solvent was distilled off, and the resulting residue was isolated and purified by silica gel column chromatography (N hexane: EtO Ac = 10: 1) to give the title compound as a white foam (yield 0.39 g). 0.37 mmol, yield 64%).

[0125] ^J H NMR (400 MHz, CDC1) δ: 0.10 (6H, s, TBDMS— CH), 0.68—2.34 (58H, m, t-b

 3 3

 Chill and chol and butyl), 3.09-3.18 (4H, m, butyl), 3.76-3.79 (7H, m,-OCH a

 3 nd cross), 4.48-4.74 (2H, m, 1- H), 4.96-5.37 (2H, m, 2-H), 6.80-7.44 (13H, m, D MTr).

¹³ C-NMR (100 MHz, CDC1) δ: −5.40, −3.60, 11.84, 17.96, 18.69, 19.31, 21.01, 22.

 Three

 54, 22.81, 23.80, 24.26, 25.63, 25.76, 27.27, 27.99, 28.15, 28.21, 31.84, 31.87, 35. 77, 36.15, 36.52, 36.95, 38.54, 39.48, 39.71, 40.57, 42.28, 49.97, 55.17, 56.09, 56. 65, 62.13, 76.68, 85.76, 112.99, 112.45, 126.63, 127.70, 128.14, 130.05, 135.62, 1 36.13, 139.81, 144.94, 156.10, 158.34.

 Elemental analysis Calced for C H N O Si-2 H O: C, 73.07; H, 9.15; N, 2.71, Found: C,

 63 94 2 8 2

70.63; H, 9.40; N, 2.34. [0126] (Vc) l -O- (4,4, —Dimethoxytrityl) 2— O— [N cholesteryloxyborane N— (4 aminobutyl) force rubermoyl] -glycerol (1-0- (4 , 4'-dimethox ytrityl)-2-O- [N-cholesteryloxycarbonyl-N- (4-aminobutyl) carbamoyl] -glycerol) (27c)

 1—O— (4,4, —Dimethoxytrityl) 2—O— [N cholesteryloxycarbol N— (4 aminobutyl) force rubamoyl] 3—O— tert butyldimethylsilyl glycerol (26c) (0 (34 g, 0.33 mmol) in THF (5. OmL) was added dropwise TBAF (1 mL, 1 mmol, 3. Oeq.) And then stirred for 4 hours. After evaporating the reaction solvent, the obtained residue was isolated and purified by silica gel column chromatography (N-hexane: EtOAc = 5: 1 to 0: 1) to give the title oily compound as a colorless oil. (Yield 0.61 g, 0.18 mmol, 53% yield) o

[0127] ^J H NMR (400MHZ, CDCl) δ: 0.62-2.28 (49H, m, chol and butinole), 3.12-3.22 (4

 Three

 H, m, petitnore), 3.73 (8H, s, — OCH and cross), 4.42-4.61 (2H, m, 1- H), 4.83-5.30

 Three

 (2H, m, 2-H), 6.75-7.37 (13H, m, DMTr).

¹³ C-NMR (100 MHz, CDCl) δ: 11.83, 18.68, 19.30, 21.01, 22.54, 22.80, 23.80, 24.

 Three

 26, 27.08, 27.28, 27.98, 28.14, 28.21, 31.83, 31.87, 35.77, 36.14, 36.51, 36.93, 38. 52, 39.48, 39.70, 40.68, 42.27, 49.95, 55.19, 56.09, 56.65, 62.86, 63.37, 74.55, 86. 18, 113.12, 122.46, 126.82, 127.84, 128.02, 129.97, 130.01, 135.71, 135.73, 139.7 7, 144.59, 156.21, 158.47.

 Elemental analysis Calced for C H N O-1/3 H 2 O: C, 74.31; H, 8.75; N, 3.04, Found: C,

 57 80 2 8 2

 73.87; H, 8.68; N, 2.92.

[0128] (Vic) CPG unit with cholesterol bound (29c)

1—O— (4,4, —Dimethoxytrityl) 2—O— [N cholesteryloxycarbol —N— (4 aminobutyl) force rubamoyl] —glycerol (27c) (0.60 g, 0.17 mmol) To a solution of pyridine (1.7 mL) was added DMAP (small amount) and succinic anhydride (0.051 g, 0.51 mmol, 3. Oeq.) And stirred for 1 day. The reaction solution was diluted with chloroform, washed with water (X 3), dried over anhydrous sodium sulfate, and the organic solvent was distilled off. The obtained residue was vacuum-dried for 12 hours, then dissolved in DMF (2.2 mL), and CPG (0.19 g, 0.043 m mol, 222 / z mol / g) for 30 minutes. WSC (0.033 g, 0.17 mmol, 1. Oeq.) Was added to the reaction mixture, and then shaken for 3 days. After removing the solvent from the reaction mixture, the resulting CPG was washed with pyridine. After drying, add 0.1 mL of D MAP solution (solvent is a mixture of pyridine and acetic anhydride (pyridine: Ac 0 = 9: 1)) to the CPG.

 2

Yes, it was shaken at room temperature for 24 hours. The CPG was washed with MeOH and then with acetone and dried. The resulting activity of the CPG 88.3 _11101/8 Deatta (yield 0. 20 g).

[0129] 2. Production of single-stranded modified oligonucleotide

 The target protein is Homo sapiens ribonuclease L (2 ', 5' -oligoisoadenylate synthetase-dependent, RNase L) [gi: 30795246], and the target self-sequence is from 92 to 114 in the start codon. A base sequence consisting of SEQ ID NO: 1 was used. As a modified single-stranded oligonucleotide, a modified DNA sense strand in which palmitic acid, oleic acid or cholesterol is bound to the base sequence consisting of SEQ ID NO: 1 via a linker at the 3 'end (SEQ ID NO: 2) And a modified DNA antisense strand (SEQ ID NO: 3) in which palmitic acid, oleic acid or cholesterol was bound to the 3 ′ end via a linker. For comparison, siRNA (manufactured by Ambion) having a DNA sense strand (SEQ ID NO: 4) and a DNA antisense strand (SEQ ID NO: 5) with respect to the base sequence consisting of SEQ ID NO: 1 was used.

[0130] Example 1

 Production of a modified DNA sense strand in which palmitic acid is bound to the 3 'end via a linker. Nucleic acid synthesis using the CPG unit (29a) (amount equivalent to 1 μmol based on each activity) System (Expedite 8909 system, manufactured by Applied Biosystems)), and modified DNA sense strand with palmitic acid bound to the end via phosphoramidite method 3 according to SEQ ID NO: 2 on CPG according to phosphoramidite method. Was synthesized.

[0131] The condensation time was 15 minutes, and the synthesis was completed with the DMTr group removed. Modified DNA sense strand conjugated with CPG mixed with ethanol and ammonia (EtOH: NH = 1: 3) solution 2

 Three

mL was added and reacted at 55 ° C for 12 hours. After the reaction, the obtained filtrate was transferred to an Eppendorf tube and concentrated under reduced pressure. To the obtained residue, 1 mL of 1M TBAF in THF was added and shaken for 12 hours. The reaction solution is diluted with 0.1 M TEAA buffer, 30 mL. This reaction solution was mixed with a C-18 reverse phase column (Sep-Pak) (CH CN10mL, 0.1M).

 Three

 The TEAA buffer solution (10 mL) was passed through and equilibrated), and the modified DNA sense strand mixture was adsorbed onto the column. The column is washed with sterile water to remove salt, and then 80% CH C

 Three

Elute with 3 mL of aqueous N solution. The eluate was concentrated under reduced pressure. To the resulting residue, 100 μL of a loading solution (TBE in 1 × 90% formamide) was added, and the desired modified DNA sense strand was separated from the others by 20% PAGE (20 A, 6 hours). The target modified DNA sense strand band was excised from the PAGE, and 20 mL of 0.1 M EDTA aqueous solution was added to the band and allowed to stand. This solution was purified by C-18 reverse phase column chromatography (Sep-Pak) with 3 mL of 50% aqueous CH 3 CN to give the title compound.

 Three

 [0132] A method for preparing the solution will be described.

 [0133] A 0.1 M TEAA buffer was prepared as follows. First, water was added to 1 L in a mixture of 2N acetic acid (114. 38 mL) and triethylamine (277. 6 mL). The solution was prepared by adding acetic acid to adjust the pH to 7.0, and then diluting the solution 20 times.

 [0134] 20% PAGE was prepared as follows. First, 40% acrylamide (acrylamide: N, N, -methylenebisacrylamide = 19: 1) solution (45 mL), urea (37.8 g) and 1 O XTBE buffer solution (9 mL) are mixed and dissolved, and then water is added. Was adjusted to 90 mL. APS (62 mg) was added to the solution and dissolved, and then TEMED (45 μL) was added and shaken. The solution was poured between two glass plates fixed with a 1.5 mm spacer in between, and allowed to stand for 1 hour or more to solidify to obtain 20% PAGE.

 [0135] A 0.1 M aqueous EDTA solution was prepared by dissolving EDTA'4Na (l. 80 g) in water (40 mL).

 [0136] 1 X 90% formamide was prepared by mixing 10 XTBE buffer (1 ml) and formamide (9 ml).

[0137] Example 1P

The single-stranded 5 ′ end of SEQ ID NO: 2 is ³² P in the same manner as in Example 1 except that the 5 ′ end of the modified single-stranded oligonucleotide consisting of SEQ ID NO: 2 is labeled with the ³² P label. A labeled modified DNA sense strand was produced. [0138] Example 2

 Production of modified DNA antisense strand with palmitic acid bound to the 3 'end via a linker

 The title compound was obtained in the same manner as in Example 1 except that SEQ ID NO: 3 was used instead of SEQ ID NO: 2.

[0139] Example 3

 Production of a modified DNA sense strand in which oleic acid is linked to the 3 'end via a linker. Production was carried out in the same manner as in Example 1 except that CPG unit (29b) was used instead of CPG unit (29a). To give the title compound.

[0140] Example 4

 3. Preparation of modified DNA antisense strand with oleic acid attached to the end via a linker

 The title compound was obtained in the same manner as in Example 3 except that SEQ ID NO: 3 was used instead of SEQ ID NO: 2.

[0141] Reference Example 1

 Production of modified DNA sense strand in which cholesterol is bound to the 3 'end via a linker. Production is carried out in the same manner as in Example 1 except that CPG unit (29c) is used instead of CPG unit (29a). The title compound was obtained.

[0142] Reference Example 2

 3. Production of modified DNA antisense strand with cholesterol linked to the end via a linker

 The title compound was obtained in the same manner as in Reference Example 1 except that SEQ ID NO: 3 was used instead of SEQ ID NO: 2.

[0143] The obtained oligonucleotide was dissolved in water (lmL) and diluted 100 times with water to prepare a diluted solution. The absorbance (260 nm) of the diluted solution was measured, and the yield was calculated. The absorbance ε value, absorbance at 260 nm and yield are shown in Table 1. The molecular weight of the obtained oligonucleotide was confirmed by MALDI-TOFZMS. The results are also shown in Table 1. [0144] [Table 1]

The absorbance and yield of the oligonucleotide were measured as follows.

 [0145] The oligonucleotide was dissolved in water to prepare an aqueous solution, and the aqueous solution was diluted so that the absorbance (Abs) at a wavelength of 260 nm was within the effective range of the absorptiometer. Optical path length (

 260

 1) Abs was measured at room temperature using a lcm absorbance measurement quartz cell. Total OD value

 260 260 The calculation was performed using the following formula (1).

OD (ε -M-mL '^ Abs (ε -cm-M) XV " ¹ (mL) Xf ^ cm) (1)

 260 260

 In the formula (1), Abs is the absorbance of the oligonucleotide solution at a wavelength of 260 nm.

 260

 Degrees, V is the total volume of the solution, 1 is the optical path length, M is the molar concentration.

In the above formula (1), the molar extinction coefficient _ε 260 of the oligonucleotide was calculated using the following formula (2).

 ε = 2 {ε (Ν Ν) + ε (Ν Ν) + ... ε (Ν Ν)}-{ε (Ν) + ε (Ν) + +

 1ρ 2 2ρ 3 n-lp η 2 3

 ε (Ν)} (2)

 η-1

 In the formula (2), ε (Ν) represents ε of a certain nucleic acid Ν, and ε (Ν)) represents a nucleic acid dimer.

 η η 260 n-lp n

 Shows ε of N N. (11—s), (11—a), (12—s), (12—a), (13—s), (n-lp n 260

 For 13—a) (F12—s), (F12—a), (P-12—a), and (P13—a), ε was calculated using the modified TT sequence at the 3 ′ end as a UU sequence.

 260

 [0147] The concentration C (molZL) was calculated using the following equation (3).

C = Abs X ε "'ΧΓ ¹ (3)

 260 260

 In the formula (3), Abs ε and 1 are as described above.

 260 260

3. Production of modified double-stranded oligonucleotides Example 5

 Dissolve the modified DNA sense strand (lnmmol) produced in Example 1 and the modified antisense strand (lnmmol) produced in Example 2 in annealing buffer (10 mM Tris-HCl (pH 7.5) and lOO mM NaCl). I let you. The solution was incubated at 90 ° C for 1 minute, then at 37 ° C for 1 hour, and the modified DNA sense strand (SEQ ID NO: 2) and 3 ' A modified double-stranded oligonucleotide consisting of a modified DNA antisense strand (SEQ ID NO: 3) having normitic acid bound to the end via a linker was obtained.

[0148] Example 6

 Instead of the modified DNA sense strand produced in Example 1, the modified DNA sense strand produced in Example 3 was replaced by the modified DNA produced in Example 4 instead of the modified DNA antisense strand produced in Example 2. A modified DNA sense strand (SEQ ID NO: 2) in which oleic acid was bound to the 3 ′ end via a linker, and oleic acid at the 3 ′ end, in the same manner as in Example 5 except that the antisense strand was used. A modified double-stranded oligonucleotide with a modified DNA antisense strand (SEQ ID NO: 3) force bound via a linker was obtained.

[0149] Example 7

 Instead of the modified DNA sense strand produced in Example 1, the modified DNA sense strand produced in Reference Example 1 was replaced by the modified DNA produced in Reference Example 2 instead of the modified DNA antisense strand produced in Example 2. A modified DNA sense strand (SEQ ID NO: 2) in which cholesterol is bound to the 3 ′ end via a linker in the same manner as in Example 5 except that the antisense strand is used, and cholesterol at the end is linked to the linker. Thus, a modified double-stranded oligonucleotide having a modified DNA antisense strand (SEQ ID NO: 3) force bound thereto was obtained.

 [0150] The structure of the obtained antisense strand and sense strand is as shown in the following formula (anti) and formula (sens).

[0151] [Chemical 23] Antisense strand 5 '-GCUGUUCAAAACGAAGAUGTTX-3 (ant 0 sense strand 3' -XTTCGACAAGUUUUGCUUCUAUC -5 (sens) Example 5: X = palmitic acid

 Example 6: X = oleic acid

 Example 7: X = cholesterol Example 8

 Instead of the modified DNA antisense strand prepared in Example 2, a natural DNA antisense strand (SEQ ID NO: 5) (manufactured by Ambion) was used in the same manner as in Example 5 except that palmitic acid was added to the 3 ′ end. A modified double-stranded oligonucleotide consisting of a modified DNA sense strand (SEQ ID NO: 2) and a natural DNA antisense strand (SEQ ID NO: 5) bound to each other via a linker. The structure of the obtained antisense strand and sense strand is as shown in the following formula (anti) and formula (sen s).

 [0152] [Chemical 24] Antisense strand 5 '-GCUGUUCAAAACGAAGAUGTT-3' (ant i)

 Sense strand 3'-XTTCGACAAGUUUUGCUUCUAC -5 (sens) Example 8: X = palmitic acid Example 9

 A natural DNA sense strand (sequence) was prepared in the same manner as in Example 5 except that the natural DNA sense strand (SEQ ID NO: 4) (manufactured by Ambion) was used instead of the modified DNA sense strand prepared in Example 1. No. 4) and a modified double-stranded oligonucleotide having a modified DNA antisense strand (SEQ ID NO: 3) force having palmitic acid bound to the 3 ′ end via a linker. The structure of the resulting antisense strand and sense strand is as shown in the following formulas (anti) and (sens).

 [0153] [Chemical 25] Antisense strand 5 '-GCUGUUCAAAACGAAGAUGTTX-3 (ant i)

 Sense strand 3 '-TTCGACAAGUUUUGCUUCUAC-5 (sens) Example 9: X = palmitic acid

(Evaluation of modified double-stranded oligonucleotide) 1. Thermal stability

 For the modified double-stranded oligonucleotides prepared in Examples 5-7, 50%% melting temperature Tm was measured. Table 2 shows the results obtained.

 [Table 2]

The structure of natural siRNA is as follows:

 [0155] [Chemical 26] Natural type s i R N A:

 Antisense strand 5 '-GCUGUUCAAAACGAAGAUGTT-3' (ant i)

 Sense strand 3′-TTCGACAAGUUUUGCUUCUAC-5 ′ (sens) From Table 2 above, it was confirmed that the modified double-stranded oligonucleotide of the present invention was excellent in double-stranded forming ability.

[0156] 2. Evaluation of RNaseL expression in cells

 (1) Cell culture

 Cell culture HT1080 cells were grown at 37 ° C in RPMI 1640 medium supplemented with 10% FBS, penicillin (100 units Zml) and streptomycin (0.1 mgZml). Cells were passaged regularly to maintain exponential growth. HT1080 cells were trypsinized 24 hours before transfer at approximately 25% confluency, diluted with medium without antibiotics, and transferred to 35 ml dishes (2 ml Z dishes) ).

[0157] (2) Transfection of modified double-stranded oligonucleotide into cells

The modified double-stranded oligonucleotide was transfected to the adherent cell line using Lipofuectamine 2000 reagent (Invitrogen) by the method described below. First, double-stranded oligonucleotide (50, ΙΟΟηΜ or 200 nM per 250 1 of the medium) (modified double-stranded oligonucleotide obtained in Example 5) was dissolved in RPMI1640 medium (without antibiotics and FBS). And incubated for 5 minutes at room temperature. An equal volume of ribofactoramine 2000 reagent diluted 50 times was added to the solution, and then incubated at room temperature for 20 minutes. Of the resulting solution, 5001 was collected and transferred to the 35 millidish prepared in (1) above for transfection. The cells in the 35 millidish were incubated 24 hours and 48 hours after transfection. After that, the toxicity of the modified double-stranded oligonucleotide obtained in Example 6 or Example 7 to the! /, Shift was strong without confirming the toxicity to the transfected cells.

[0158] (3) Monitoring RNaseL expression by Western blotting

 HT1080 cells obtained after 24 hours incubation and 48 hours incubation from transfection were trypsinized and washed twice with cold PBS (—). The resulting cell pellet was then added to 2 volumes of low osmolarity buffer A (0.5% (vZv) Nonidet P-40, 20 mM Hepes (pH 7.5), 10 mM CH. CO K, 15mM (CH

 3 2

 CO) Mg, ImM dithiothreitol, ImM phenylmethylsulfonylfluora

3 2 2

 Id, 10 gZml of Aprotune).

 [0159] The obtained cell suspension was incubated on ice for 10 minutes, and then thirty times on ice using a tight 'fitting-cuffs' Dough ~~ 's' homomonizer ~~ (Ίlght—fitting glass dounce homogenizer). Stroke up and down to homogenize. The obtained homogenized solution was centrifuged at 4 ° C for 10 minutes using an ultracentrifuge (lOOOO X g). The centrifuged supernatant is then collected and an equal volume of 2X sample buffer (0.14M Tris-HCl pH 6.8, 0.2% (vZv) glycerol, 2. 8% (vZv) SDS, appropriate amount of bromophenol 'blue' was added and incubated in boiling water for 5 minutes. The incubated supernatant is separated by electrophoresis using 7.5% polyacrylamide gel (ATTO) at 20 mA for 70 minutes, and then transferred to a PVDF membrane (Millipore). did.

[0160] The PVDF membrane was incubated with 5% bovine serum albumin at 25 ° C for 1 hour. Thereafter, the PVDF membrane was rinsed twice with TBST (200 mM Tris pH 7.6, 1.37 M sodium chloride sodium salt, 0.1% Tween-20). The PVDF membrane was incubated with 0.5 gZml of anti-RNaseL monoclonal antibody at 4 ° C for 16 hours. After that, the PVDF membrane is placed in TBST once for 15 minutes and then 3 times for 5 minutes. Washed. The PVDF membrane was then incubated for 1 hour at 25 ° C. with anti-mouse IgG labeled with Horse radish peroxidase (500 μg ZmL diluted 100000 fold). Thereafter, the PVDF membrane was washed with TBST once for 15 minutes and then 3 times for 5 minutes.

 [0161] The detection of C. peroxidase labeling on the PVDF membrane was performed using an ECL plus 'Western' blotting detection kit (manufactured by Amersham). Quantification of C. peroxidase labeling on the PVDF membrane was performed using a densitometer (Kodak Digital Science DC 290 Zoom Digital Camera). The relative ratio of the RNase L intensity of cells treated with the double-stranded oligonucleotide was determined with the RNase L intensity of cells not treated with the double-stranded oligonucleotide as 1. The obtained values were standardized with the GAPDH intensity set to 1. The obtained results are shown in Fig. 1 (a).

 [0162] (4) In place of the modified double-stranded oligonucleotide obtained in Example 5, instead of the modified double-stranded oligonucleotide obtained in Example 6 and Example 7, respectively (1 In the same manner as in (3) to (3), the relative ratio of the RNase L intensity of cells treated with the modified double-stranded oligonucleotide was determined. The results are shown in Fig. 1 (b) and Fig. 1 (c), respectively.

 [0163] As shown in Figs. 1 (a) to (c), the modified double-stranded oligonucleotide of the present invention has an effect of suppressing the expression of RNaseL as compared to the unmodified double-stranded oligonucleotide. It was confirmed that the RNAi activity was improved significantly.

 [0164] 3. Measurement of IC for RNaseL in cells

 50

 (1) 2.The concentration of the modified double-stranded oligonucleotide used in (2) is the same as in 2. above (1) except that 1ηΜ, 5nM or ΙΟηΜ is used instead of 50, ΙΟΟηΜ or 200nM per 2501 of the medium In the same manner as in (4), the relative ratio of the RNase L intensity of the cells treated with the modified double-stranded oligonucleotide was determined.

[0165] (2) Further, instead of the modified double-stranded oligonucleotide obtained in Example 5, the modified double-stranded oligonucleotide obtained in Examples 8 and 9 was used, respectively. ) To obtain the relative ratio of RNaseL intensity of cells treated with double-stranded oligonucleotide. (3) IC was determined from the relative ratio obtained and shown in Table 3 below.

 50

 [0167] [Table 3]

 As shown in Table 3 above, the modified double-stranded oligonucleotide of the present invention has a markedly improved effect of suppressing the expression of RNaseL compared to the natural (unmodified) double-stranded oligonucleotide. It could be confirmed. This remarkable improvement is an effect superior to that expected from the fact that the modified double-stranded oligonucleotide of the present invention has excellent cell membrane permeability and improved nuclease resistance. In addition, in Example 9 in which the 3 ′ end of the antisense strand is modified, the effect of suppressing the expression of RNaseL is improved compared to Example 8 in which the 3 ′ end of the sense strand is modified. Was confirmed.

[0168] 4. Nuclease resistance

 Evaluation of exonuclease resistance of double-stranded modified oligonucleotides

 The exonuclease resistance of the double-stranded modified oligonucleotide and the natural double-stranded oligonucleotide was evaluated. As the exonuclease, snake venom phosphorodiesterase (SVP) was used. SVP selectively cleaves phosphodiester bonds and cleaves oligonucleotides into 5, monomonophosphate nucleotides.

[0169] Modified DNA sense strand (20 M, 4 produced in Example 1P, labeled with ³² P at the end)

 1) and natural DNA antisense strand (SEQ ID NO: 5) (Ambion) (8pmmol) were added to annealing buffer (250mM Tris-HCl (pH8.0) and 50mM MgCl).

2, 6 1) and water (26 μ 1). The solution for 5 min at 95 ° C the following, in and incubated for 1 hour at room temperature, the 3 'end palmitate linked through a linker one, 5, modifications terminally labeled with ³² P A modified double-stranded oligonucleotide was obtained that also possessed the ability of a DNA sense strand (SEQ ID NO: 2) and a natural DNA antisense strand (SEQ ID NO: 5). The resulting double-stranded structure is as shown in the following formula.

[0170] [Chemical 27] Antisense strand 5 '-GCUGUUCAAAACGAAGAUGTT-3' (ant i) Sense strand 3 '-XTTCGACAAGUUUUGCUUCMC -5' (sens)

X = palmitic acid SVP (0. 101) was added to the solution. After adding SVP, 0, 1, 3, 5

The reaction solution (51) was sampled into each Eppendorf tube after 10, 30, 60 minutes, and mixed with 8 M urea solution (51) to stop the reaction. Note that the sample after 0 minutes does not contain the SVP aqueous solution. The samples obtained at each time were annealed at 95 ° C for 5 minutes, and then analyzed by 20% denaturing PAGE (7M urea solution) (electrokinetic buffer: 1 XTBE, ~ 300V, 2 hours electric) Electrophoresis). The results obtained are shown in Table 4 below.

 [0171] [Table 4]

 From Table 4 above, it was confirmed that the modified oligonucleotide had improved exonuclease resistance compared to the natural oligonucleotide.

 Industrial applicability

[0172] The modified oligonucleotide of the present invention is useful, for example, as a disease therapeutic agent.

 Sequence listing free text

[0173] SEQ ID NO: 1 Target sequence of RNaseL

 SEQ ID NO: 2 Modified sense strand of siRNA sequence for target sequence of SEQ ID NO: 1 SEQ ID NO: 3 Modified antisense strand of siRNA sequence for target sequence of SEQ ID NO: 1 SEQ ID NO: 4 Sense strand of siRNA sequence for target sequence of SEQ ID NO: 1

 SEQ ID NO: 5 antisense strand of siRNA sequence against the target sequence of SEQ ID NO: 1

Claims

The scope of the claims

 [1] A modified oligonucleotide containing a modified nucleoside as a constituent component at the 3, terminal end of the oligonucleotide,

A modified oligonucleotide which is a modified nucleoside in which a carboxylic acid represented by the following formula (III) is bound to the 3, terminus of the modified nucleoside via a linker at the 3, terminus of the nucleoside. R ^A -COOH (III)

In the above formula, R ^A means a saturated aliphatic hydrocarbon group, an unsaturated aliphatic hydrocarbon group, an aryl group, or a heterocyclic group.

 [2] The modified oligonucleotide according to claim 1, which is represented by formula (IA).

 [Chemical 1]

 0

 H

R'— 0-P-0-L— N—— R ^A (IA)

 O-O in the above formula,

 R, —OH means oligonucleotide,

 The hydroxyl group in R′—OH means the hydroxyl group at the 3 ′ end of the modified nucleoside contained as a constituent component at the 3 ′ end of the oligonucleotide,

 L means a divalent group,

R ^A represents a saturated aliphatic hydrocarbon group, an unsaturated aliphatic hydrocarbon group, an aryl group, or a heterocyclic group.

 [3] The modified oligonucleotide according to [1] or [2], wherein the saturated aliphatic hydrocarbon group is a saturated aliphatic hydrocarbon group having 1 to 25 carbon atoms.

[4] The modified oligonucleotide according to claim 1 or 2, wherein the unsaturated aliphatic hydrocarbon group is an unsaturated aliphatic hydrocarbon group having 3 to 25 carbon atoms.

[5] R ^A is CH-(CH) — or CH-(CH) — CH = CH— (CH) —

 3 2 14 3 2 7 2 7 Modified oligonucleotide according to claim 1 or 2.

6. The modified oligonucleotide according to claim 2, wherein the divalent group is a divalent group represented by the following formula (IVA). -XMY- (IVA)

 In the above formula,

 —X— is — (CH) or (CH) —CH (CH OH) —,

 2 m 2 m 2

 —Y— is one (CH) or one (CH) -CH (CH OH) one;

 2 n 2 n 2

 —M is one 0—C (= 0) NH, one O—, one (= 0) —? << 1-or-? << 1-ji (= O)-

 m and n are each independently an integer of 1 to 20.

[7] The modified oligonucleotide according to [2], wherein the divalent group is a divalent group represented by the following formula (IVA-1).

 — CH— CH (CH 0H) -0-C (= 0) NH- (CH) I (IVA—1)

 2 2 2 n

 In the above formula, n is an integer of 1-20.

[8] A double-stranded modified oligonucleotide,

 A sense strand having the same base sequence as a part of the target mRNA, and an antisense strand against the sense strand,

 Among the antisense strand and the sense strand, at least the antisense strand is an oligonucleotide analogue containing a modified nucleoside as a constituent component at the 3, end of the oligonucleotide, and at the 3, end of the modified nucleoside force nucleoside. A double-stranded modified oligonucleotide which is a modified nucleoside to which a compound having a steroid skeleton represented by the following formula (Π) is bound via a linker.

R ^S -OH (II)

In the above formula, R ^s means a steroid skeleton optionally having one or more substituents, wherein one or more single bonds in the skeleton are arbitrarily replaced with double bonds.

[9] The double-stranded modified oligonucleotide according to [8], wherein the oligonucleotide analogue is represented by the formula (IS).

 [Chemical 2]

 0

 H

 R'-O— P-0-L— N Ding OR. (IS)

0- 0 In the above formula,

 R, 1 OH means oligonucleotide,

 The hydroxyl group in R′—OH means the hydroxyl group at the 3 ′ end of the modified nucleoside contained as a constituent component at the 3 ′ end of the oligonucleotide,

L means a divalent group,

R ^s means a steroid skeleton that optionally has one or more substituents, and one or more single bonds in the skeleton are optionally replaced with double bonds.

 HU R

[Chemical 3]

The double-stranded modified oligonucleotide according to claim 8, wherein the double-stranded modified oligonucleotide is selected.

 10. The double-stranded modified oligonucleotide according to claim 9, wherein the divalent group is a divalent group represented by the following formula (IVS).

-X-M-Y- (IVS)

In the above formula,

 —X— is — (CH) or (CH) —CH (CH OH) —,

 2 m 2 m 2

 —Y— is one (CH) or one (CH) -CH (CH OH) one;

 2 n 2 n 2

—M is one 0—C (= 0) NH, one O—, one (= 0) —? << 1-or-? << 1-ji (= O)- m and n are each independently an integer of 1 to 20.

 10. The double-stranded modified oligonucleotide according to claim 9, wherein the divalent group is a divalent group represented by the following formula (IVS-1).

 — CH— CH (CH OH) -0-C (= 0) NH- (CH) One (IVS— 1)

 2 2 2 n

In the above formula, n is an integer of 1-20.