WO2013077446A1

WO2013077446A1 - Single-stranded nucleic acid molecule for regulating expression of gene

Info

Publication number: WO2013077446A1
Application number: PCT/JP2012/080461
Authority: WO
Inventors: 忠明大木; 久男白水; 智洋濱崎
Original assignee: 株式会社ボナック
Priority date: 2011-11-26
Filing date: 2012-11-26
Publication date: 2013-05-30

Abstract

Provided is a novel nucleic acid molecule capable of suppressing the expression of a gene. A single-stranded nucleic acid molecule containing an expression-suppressing sequence for a target gene and a complementary sequence having a mismatch with the expression-suppressing sequence, wherein a 5'-side region (Xc), an internal region (Z) and a 3'-side region (Yc) are contained in this order from the 5'-side toward the 3'-side, the region (Z) is formed by linking an internal 5'-side region (X) to an internal 3'-side region (Y), the region (Xc) is complementary to the region (X), the region (Yc) is complementary to the region (Y), at least one of the region (Z), the region (Xc) and the region (Yc) contains the expression-suppressing sequence, a region complementary to the region containing the expression-suppressing sequence contains the complementary sequence, and a 5'-terminal nucleotide and a 3'-terminal nucleotide in the single-stranded nucleic acid molecule are not bound to each other. The single-stranded nucleic acid molecule can suppress the expression of the target gene.

Description

Single-stranded nucleic acid molecules for gene expression control

The present invention relates to a single-stranded nucleic acid molecule that suppresses gene expression, a composition containing the same, and use thereof.

For example, microRNA (miRNA) is known as a nucleic acid molecule that suppresses gene expression. In general, miRNAs have been reported to repress the transcription of proteins encoded by genes through the following production process. That is, first, a miRNA transcript (Pri-miRNA) having a cap structure at the 5 'end and poly (A) at the 3' end is generated in the nucleus. The Pri-miRNA is cleaved by RNase (Drosha) to generate a miRNA precursor (Pre-miRNA). The Pre-miRNA has a hairpin structure having a loop region and a stem region. This Pre-miRNA moves to the outside of the nucleus, and then is degraded by cytoplasmic RNase (Dicer) to cut out double-stranded miRNA having an overhang of 1 to 4 bases at the 3 'end. One strand of this double-stranded miRNA binds to a complex similar to RNA induced Silencing Complex (RISC). The miRNA / RISC complex binds to the 3 'untranslated region (3'UTR) of a specific mRNA, thereby suppressing protein translation from the mRNA.

It has become clear that miRNAs are deeply involved in life phenomena such as differentiation, cell proliferation and apoptosis, and many diseases such as viral infections and cancer (Patent Document 1, Non-Patent Document 1, Non-Patent Documents). 2). Therefore, application in the medical field is particularly expected.

WO 2010/056737 A2

Therefore, an object of the present invention is to provide a new nucleic acid molecule capable of suppressing gene expression.

In order to achieve the above object, the single-stranded nucleic acid molecule of the present invention is a single-stranded nucleic acid molecule comprising an expression suppressing sequence that suppresses the expression of a target gene and a complementary sequence that has a mismatch with the expression suppressing sequence. And
From the 5 ′ side to the 3 ′ side, the 5 ′ side region (Xc), the inner region (Z), and the 3 ′ side region (Yc) are included in the above order,
The internal region (Z) is configured by connecting an internal 5 ′ side region (X) and an internal 3 ′ side region (Y),
The 5 ′ side region (Xc) is complementary to the internal 5 ′ side region (X);
The 3 ′ side region (Yc) is complementary to the inner 3 ′ side region (Y);
At least one of the internal region (Z), the 5 ′ side region (Xc) and the 3 ′ side region (Yc) includes the expression suppression sequence,
A region complementary to the region containing the expression suppression sequence comprises the complementary sequence;
The 5 ′ terminal base and the 3 ′ terminal base of the single-stranded nucleic acid molecule are unbound.

The composition of the present invention is a composition for suppressing the expression of a target gene, and is characterized by containing the single-stranded nucleic acid molecule of the present invention.

The composition of the present invention is a pharmaceutical composition, and is characterized by containing the single-stranded nucleic acid molecule of the present invention.

The expression suppression method of the present invention is a method of suppressing the expression of a target gene, characterized in that the single-stranded nucleic acid molecule of the present invention is used.

The method for treating a disease of the present invention includes a step of administering the single-stranded nucleic acid molecule of the present invention to a patient, wherein the single-stranded nucleic acid molecule serves as the expression-suppressing sequence of a gene causing the disease. It has a sequence that suppresses expression.

The single-stranded nucleic acid molecule of the present invention can suppress gene expression, specifically, translation of a protein encoded by the gene.

The present inventors are the first to find that the structure of the single-stranded nucleic acid molecule of the present invention can suppress gene expression. The gene expression suppression effect of the single-stranded nucleic acid molecule of the present invention is presumed to be due to the same phenomenon as RNA interference, but gene expression suppression in the present invention is not limited or limited to RNA interference.

FIG. 1 is a schematic diagram showing an example of a single-stranded nucleic acid molecule of the present invention. FIG. 2 is a schematic diagram showing another example of the single-stranded nucleic acid molecule of the present invention. FIG. 3 is a schematic diagram showing another example of the single-stranded nucleic acid molecule of the present invention. FIG. 4 is a graph showing the amount of HMGA2 mRNA in Examples of the present invention. FIG. 5 is a graph showing the amount of mature miRNA of human let-7a-1 miRNA in the examples of the present invention.

Unless otherwise stated, terms used in this specification can be used in the meaning normally used in the technical field.

1. The ssNc molecule The single-stranded nucleic acid molecule of the present invention is a single-stranded nucleic acid molecule comprising an expression suppressing sequence that suppresses expression of a target gene and a complementary sequence that has a mismatch with the expression suppressing sequence, as described above. ,
From the 5 ′ side to the 3 ′ side, the 5 ′ side region (Xc), the inner region (Z), and the 3 ′ side region (Yc) are included in the above order,
The internal region (Z) is configured by connecting an internal 5 ′ side region (X) and an internal 3 ′ side region (Y),
The 5 ′ side region (Xc) is complementary to the internal 5 ′ side region (X);
The 3 ′ side region (Yc) is complementary to the inner 3 ′ side region (Y);
At least one of the internal region (Z), the 5 ′ side region (Xc) and the 3 ′ side region (Yc) includes the expression suppression sequence,
A region complementary to the region containing the expression suppression sequence comprises the complementary sequence;
The 5 ′ terminal base and the 3 ′ terminal base of the single-stranded nucleic acid molecule are unbound.

In the present invention, “target gene expression suppression” means, for example, suppression of translation of the target gene, that is, suppression of translation of a protein encoded by the target gene, and more specifically, mRNA of the target gene. Means suppression of translation of the protein from The suppression of the expression of the target gene can be achieved, for example, by reducing the production amount of the transcription product from the target gene, reducing the activity of the transcription product, reducing the production amount of the translation product from the target gene, or activity of the translation product. It can be confirmed by decrease of Examples of the protein include a mature protein or a precursor protein before undergoing processing or post-translational modification.

Hereinafter, the single-stranded nucleic acid molecule of the present invention is also referred to as “ssNc molecule” of the present invention. Since the ssNc molecule of the present invention can be used, for example, in vivo or in vitro to suppress target gene expression, it is also referred to as “target gene expression suppression ssNc molecule” or “target gene expression inhibitor”. Moreover, the ssNc molecule of the present invention can suppress side effects such as interferon induction.

The ssNc molecule of the present invention has an unlinked 5 'end and 3' end, and can also be referred to as a linear single-stranded nucleic acid molecule. In the ssNc molecule of the present invention, for example, in the inner region (Z), the inner 5 'region (X) and the inner 3' region (Y) are directly linked. In the nucleic acid molecule of this embodiment, for example, it is preferable that the 5 'end is a non-phosphate group in order to maintain unbonded at both ends.

In the ssNc molecule of the present invention, the 5 ′ side region (Xc) is complementary to the internal 5 ′ side region (X), and the 3 ′ side region (Yc) is the internal 3 ′ side region (Y ). Therefore, on the 5 ′ side, the region (Xc) is folded toward the region (X), and the region (Xc) and the region (X) can form a double chain by self-annealing. In addition, on the 3 ′ side, the region (Yc) is folded toward the region (Y), and the region (Yc) and the region (Y) can form a double chain by self-annealing. . The 5 'end of the 5' side region (Xc) and the 3 'end of the 3' side region (Yc) are also referred to as unbound ends.

In the ssNc molecule of the present invention, the expression suppression sequence only needs to have, for example, expression suppression activity, and a mature miRNA sequence is preferable. The production process of miRNA in vivo and the protein expression suppression process by miRNA are as follows. First, a single-stranded miRNA transcript (Pri-miRNA) having a cap structure at the 5 'end and a polyA structure at the 3' end is expressed in the nucleus. The Pri-miRNA has a hairpin structure. The Pri-miRNA is cleaved by RNase (Drosha) to generate a single-stranded precursor (Pre-miRNA). The Pre-miRNA has a hairpin structure having a stem region and a loop region. The Pre-miRNA is transported to the cytoplasm, then cleaved by RNase (Dicer), and from its stem structure, a double-stranded miRNA having an overhang of several bases (eg, 1 to 4 bases) at the 3 ′ end. Is generated. The single-stranded mature miRNA of the double-stranded miRNA binds to a complex similar to RISC (RNA induced Silencing Complex), and the mature miRNA / RISC complex binds to a specific mRNA. , Protein translation from the mRNA is suppressed. In the present invention, the mature miRNA sequence may be used as the expression suppression sequence. The expression suppression sequence can be referred to as a translation suppression sequence because it specifically suppresses translation of a protein.

The present invention relates to the structure of a nucleic acid molecule for allowing the expression suppression activity of the target gene by the expression suppression sequence to function in the cell, for example, not the point of the sequence information of the expression suppression sequence for the target gene. . Therefore, in the present invention, for example, in addition to the single-stranded mature miRNA sequence of the miRNA known at the time of filing, a sequence that will become apparent in the future can be used as the expression suppression sequence. Since the ssNc molecule of the present invention is loaded with miRNA as described above, it can also be referred to as a nucleic acid for miRNA supplementation.

The expression suppression sequence preferably has complementarity to a predetermined region of the target gene, for example. The expression suppression sequence preferably has, for example, a 5 ′ region having a sequence that is completely complementary to the target gene, more preferably a sequence from the second base to the 8th base at the 5 ′ end (Seed sequence). Also has a sequence that is completely complementary to the target gene. The complementarity of the expression suppression sequence is not particularly limited, and is preferably 90% or more, more preferably 95%, still more preferably 98%, and particularly preferably 100%. By satisfying such complementarity, for example, off-target can be sufficiently reduced.

A specific example is a mature miRNA of Human let-7a-1 miRNA that suppresses translation of HMGA2 (high mobility group AT-hook 2) protein. HMGA2 protein belongs to a family of non-histone chromosomal high mobility group (HMG) proteins. The Human let-7a-1 nature miRNA sequence is shown below.
UGAGGUAGUAGGUUGUAUAGUU (SEQ ID NO: 1)

The length of the expression suppression sequence is not particularly limited, and the lower limit is, for example, 18 base length, preferably 19 base length, more preferably 20 base length, and the upper limit is, for example, 25 base length, preferably 24. The base length is more preferably 23 bases.

The complementary sequence is a sequence having a mismatch when aligned with the expression suppression sequence. This means that the expression suppressing sequence is a sequence having a mismatch when aligned with the complementary sequence. The number of mismatches is not particularly limited.

As described above, the ssNc molecule of the present invention has a structure in which the expression suppression sequence is arranged in at least one of the internal region (Z), the 5 ′ side region (Xc), and the 3 ′ side region (Yc). By taking this, it is presumed that expression suppression occurs as in the case of Pre-miRNA. That is, it is speculated that the mature miRNA is generated from the ssNC molecule of the present invention, and that the translation of the protein from the target gene is suppressed by the mature miRNA. Note that the present invention is not limited by this mechanism.

In the ssNc molecule of the present invention, the expression suppressing sequence is contained in at least one of the internal region (Z), the 5 'side region (Xc), and the 3' side region (Yc) as described above. The ssNc molecule of the present invention may have, for example, one or more than two of the expression suppression sequences.

In the latter case, for example, the ssNc molecule of the present invention may have two or more of the same expression suppression sequences for the same target gene, or may have two or more different expression suppression sequences for the same target gene. Two or more different expression suppression sequences for different target genes may be included. When the ssNc molecule of the present invention has two or more expression suppression sequences, the location of each expression suppression sequence is not particularly limited, and the internal region (Z), the 5 ′ side region (Xc), and the Any one of the 3 ′ side regions (Yc) may be used, or a different region may be used. When the ssNc molecule of the present invention has two or more of the expression suppression sequences for different target genes, for example, the expression of two or more different target genes can be suppressed by the ssNc molecule of the present invention.

As described above, in the living body, the mature miRNA sequence is generated from one strand (also referred to as a guide strand) of the stem structure by cleaving the Pre-miRNA. At this time, a minor miRNA ^* sequence (also referred to as a passenger strand) is generated from the other strand of the structure of the stem structure. The minor miRNA ^* sequence is usually a complementary strand having a mismatch of 1 to several bases when aligned with the Pre-miRNA. The ssNc molecule of the present invention may further have the minor miRNA ^* sequence, for example.

The mature miRNA sequence and the minor miRNA ^* sequence may have non-complementary bases when they are aligned. The minor miRNA ^* has, for example, a complementation to the mature miRNA sequence of, for example, 60 to 100%. Each of the mature miRNA sequence and the minor miRNA ^* may have, for example, one or several bases that are non-complementary, and the several bases are, for example, 2 to 15 bases . The non-complementary bases may be, for example, continuous or non-continuous.

In the ssNc molecule of the present invention, the position of the minor miRNA ^* sequence is not particularly limited, and is preferably, for example, a position that can be annealed with the mature miRNA sequence to form a double strand. In the ssNc molecule of the present invention,
When the 5 ′ region (Xc) has the mature miRNA sequence, the internal 5 ′ region (X) preferably has the minor miRNA ^* sequence,
When the 3 ′ region (Yc) has the mature miRNA sequence, the internal 3 ′ region (Y) preferably has the minor miRNA ^* sequence,
When the internal 5 ′ region (X) has the mature miRNA sequence, the 5 ′ region (Xc) preferably has the minor miRNA ^* sequence,
When the internal 3 ′ region (Y) has the mature miRNA sequence, the 3 ′ region (Yc) preferably has the minor miRNA ^* sequence.

The length of the minor miRNA ^* sequence is not particularly limited, and the difference from the length of the mature miRNA sequence is, for example, 0 to 10 bases long, preferably 0 to 7 bases long, more preferably 0 to 5 bases long It is. The minor miRNA ^* sequence and the mature miRNA sequence may be, for example, the same length, the former may be longer, or the latter may be longer.

A specific example is the minor miRNA ^* sequence for Human let-7a-1 miRNA that suppresses translation of HMGA2 (high mobility group AT-hook 2) protein. An example of the arrangement is shown below.
CUAUACAAUCUACUGUCUUCUC (SEQ ID NO: 2)

As described above, the inner region (Z) is formed by connecting the inner 5 'region (X) and the inner 3' region (Y). The region (X) and the region (Y) are directly connected, for example, and do not have an intervening sequence therebetween. The inner region (Z) is defined as “the inner 5 ′ side region (X) and the inner 3 ′ side in order to indicate the arrangement relationship between the 5 ′ side region (Xc) and the 3 ′ side region (Xc)”. The region (Y) is connected to each other ”, and in the inner region (Z), the 5 ′ side region (Xc) and the 3 ′ side region (Yc) are, for example, The use of ssNc molecules is not limited to being a separate and independent region. That is, for example, when the internal region (Z) has the expression suppression sequence, the expression suppression sequence is arranged across the region (X) and the region (Y) in the internal region (Z). Also good.

In the ssNc molecule of the present invention, the 5 'side region (Xc) is complementary to the internal 5' side region (X). Here, the region (Xc) may have a sequence complementary to the entire region of the region (X) or a partial region thereof. Specifically, for example, the region (Xc) It is preferable that the entire region or a partial region thereof includes a complementary sequence or consists of the complementary sequence. The region (Xc) may be completely complementary to the entire region complementary to the region (X) or the complementary partial region, for example, or one or several bases may be non-complementary. . In the ssNc molecule of the present invention, the 3 'side region (Yc) is complementary to the internal 3' side region (Y). Here, the region (Yc) may have a sequence complementary to the entire region of the region (Y) or a partial region thereof. Specifically, for example, the entire region (Y) Alternatively, it preferably includes a sequence complementary to the partial region, or consists of the complementary sequence. The region (Yc) may be completely complementary to the entire region complementary to the region (Y) or the complementary partial region, for example, or one or several bases may be non-complementary. . The one base or several bases is, for example, 1 to 3 bases, preferably 1 base or 2 bases.

In the ssNc molecule of the present invention, the 5 'side region (Xc) and the internal 5' side region (X) may be directly connected or indirectly connected, for example. In the former case, direct linkage includes, for example, linkage by a phosphodiester bond. In the latter case, for example, a linker region (Lx) is provided between the region (Xc) and the region (X), and the region (Xc) and the region ( And X) are linked together.

In the ssNc molecule of the present invention, the 3 ′ side region (Yc) and the inner 3 ′ side region (Y) may be directly connected or indirectly connected, for example. In the former case, direct linkage includes, for example, linkage by a phosphodiester bond. In the latter case, for example, a linker region (Ly) is provided between the region (Yc) and the region (Y), and the region (Yc) and the region ( And Y) are linked.

The ssNc molecule of the present invention may have, for example, both the linker region (Lx) and the linker region (Ly), or one of them. In the latter case, for example, the linker region (Lx) is provided between the 5 ′ side region (Xc) and the inner 5 ′ side region (X), and the 3 ′ side region (Yc) and the inner 3 'The linker region (Ly) is not present between the side region (Y), that is, the region (Yc) and the region (Y) are directly linked. In the latter case, for example, the linker region (Ly) is provided between the 3 ′ side region (Yc) and the inner 3 ′ side region (Y), and the 5 ′ side region (Xc) and the The linker region (Lx) is not provided between the internal 5′-side region (X), that is, the region (Xc) and the region (X) are directly linked.

The linker region (Lx) and the linker region (Ly) each preferably have a structure that does not cause self-annealing within its own region.

When either the linker region (Lx) or the linker region (Ly) is adjacent to the 3 ′ end of the mature miRNA sequence, the linker region may be the same as the sequence of the loop region of the Pre-miRNA, for example. Alternatively, it may be a miniaturized arrangement of the loop region arrangement. Since the size of the linker region is smaller than that of the Pre-miRNA, the linker region is preferably a miniaturized sequence of the sequence or a non-nucleotide residue as described later, for example.

An example of the ssNc molecule having no linker region is shown in the schematic diagram of FIG. FIG. 1A is a schematic diagram showing an outline of the order of each region from the 5 ′ side to the 3 ′ side of the ssNc molecule. FIG. 1B shows the ssNc molecule. It is a schematic diagram which shows the state which forms the double chain | strand in the inside. As shown in FIG. 1B, the ssNc molecule is folded between the 5′-side region (Xc) and doubled between the 5′-side region (Xc) and the inner 5′-side region (X). A chain is formed, the 3 ′ side region (Yc) is folded, and a double chain is formed between the 3 ′ side region (Yc) and the inner 3 ′ side region (Y). FIG. 1 merely shows the order of connection of the regions and the positional relationship of the regions forming the double chain. For example, the length of each region is not limited to this.

An example of the ssNc molecule having the linker region is shown in the schematic diagram of FIG. FIG. 2 (A) is a schematic diagram showing an outline of the order of each region from the 5 ′ side to the 3 ′ side of the ssNc molecule as an example, and FIG. 2 (B) shows the ssNc molecule. FIG. 2 is a schematic diagram showing a state in which a double chain is formed in the molecule. As shown in FIG. 2 (B), the ssNc molecule is located between the 5 ′ side region (Xc) and the inner 5 ′ side region (X), and between the inner 3 ′ side region (Y) and the 3 ′ side. A double chain is formed with the side region (Yc), and the Lx region and the Ly region have a loop structure. FIG. 2 merely shows the connection order of the regions and the positional relationship of the regions forming the double chain. For example, the length of each region is not limited to this.

In the ssNc molecule of the present invention, the number of bases in the 5 ′ side region (Xc), the internal 5 ′ side region (X), the internal 3 ′ side region (Y) and the 3 ′ side region (Yc) is particularly For example, it is as follows. In the present invention, “the number of bases” means, for example, “length” and can also be referred to as “base length”.

As described above, the 5′-side region (Xc) may be complementary to the entire region of the inner 5′-side region (X), for example. In this case, for example, the region (Xc) has the same base length as the region (X), and is composed of a base sequence complementary to the entire region from the 5 ′ end to the 3 ′ end of the region (X). preferable. More preferably, the region (Xc) has the same base length as the region (X), and all bases in the region (Xc) are complementary to all bases in the region (X). That is, for example, it is preferably completely complementary. However, the present invention is not limited to this. For example, as described above, one or several bases may be non-complementary.

Further, as described above, the 5′-side region (Xc) may be complementary to a partial region of the inner 5′-side region (X), for example. In this case, the region (Xc) has, for example, the same base length as the partial region of the region (X), that is, consists of a base sequence having a base length shorter by one base or more than the region (X). preferable. More preferably, the region (Xc) has the same base length as the partial region of the region (X), and all the bases of the region (Xc) are included in the partial region of the region (X). It is preferred that it is complementary to all bases, that is, for example, completely complementary. The partial region of the region (X) is preferably, for example, a region (segment) having a base sequence continuous from the 5 ′ terminal base (first base) in the region (X).

As described above, the 3′-side region (Yc) may be complementary to the entire region of the inner 3′-side region (Y), for example. In this case, the region (Yc) has, for example, the same base length as the region (Y) and is composed of a base sequence complementary to the entire region from the 5 ′ end to the 3 ′ end of the region (Y). preferable. More preferably, the region (Yc) has the same base length as the region (Y), and all bases in the region (Yc) are complementary to all bases in the region (Y). That is, for example, it is preferable to be completely complementary. However, the present invention is not limited to this. For example, as described above, one or several bases may be non-complementary.

Further, as described above, the 3′-side region (Yc) may be complementary to a partial region of the inner 3′-side region (Y), for example. In this case, the region (Yc) has, for example, the same base length as the partial region of the region (Y), that is, consists of a base sequence having a base length shorter by one base or more than the region (Y). preferable. More preferably, the region (Yc) has the same base length as the partial region of the region (Y), and all the bases of the region (Yc) are included in the partial region of the region (Y). It is preferred that it is complementary to all bases, that is, for example, completely complementary. The partial region of the region (Y) is preferably, for example, a region (segment) having a base sequence continuous from the base at the 3 'end (first base) in the region (Y).

In the ssNc molecule of the present invention, the number of bases (Z) in the internal region (Z), the number of bases (X) in the internal 5 ′ side region (X), and the number of bases in the internal 3 ′ side region (Y) ( Y), the number of bases (Z) in the internal region (Z), the number of bases (Yc) in the 3′-side region (Yc), and the number of bases (Xc) in the 5′-side region (Xc) For example, the relationship satisfies the conditions of the following formulas (1) and (2).
Z = X + Y (1)
Z ≧ Xc + Yc (2)

In the ssNc molecule of the present invention, the relationship between the number of bases (X) in the inner 5 ′ side region (X) and the number of bases (Y) in the inner 3 ′ side region (Y) is not particularly limited, For example, any of the following formulas may be satisfied.
X = Y (19)
X <Y (20)
X> Y (21)

In the ssNc molecule of the present invention, the number of bases (X) in the internal 5 ′ side region (X), the number of bases (Xc) in the 5 ′ side region (Xc), the number of bases in the internal 3 ′ side region (Y) The relationship between (Y) and the number of bases (Yc) in the 3 ′ side region (Yc) satisfies, for example, the following conditions (a) to (d).
(A) The conditions of the following formulas (3) and (4) are satisfied.
X> Xc (3)
Y = Yc (4)
(B) The conditions of the following formulas (5) and (6) are satisfied.
X = Xc (5)
Y> Yc (6)
(C) The conditions of the following formulas (7) and (8) are satisfied.
X> Xc (7)
Y> Yc (8)
(D) The conditions of the following formulas (9) and (10) are satisfied.
X = Xc (9)
Y = Yc (10)

In (a) to (d), the difference between the number of bases (X) in the inner 5 ′ side region (X) and the number of bases (Xc) in the 5 ′ side region (Xc), the inner 3 ′ side region ( The difference between the number of bases (Y) of Y) and the number of bases (Yc) of the 3 ′ side region (Yc) preferably satisfies the following condition, for example.
(A) The conditions of the following formulas (11) and (12) are satisfied.
X−Xc = 1 to 10, preferably 1, 2, 3 or 4,
More preferably 1, 2 or 3 (11)
Y−Yc = 0 (12)
(B) The conditions of the following formulas (13) and (14) are satisfied.
X−Xc = 0 (13)
Y−Yc = 1 to 10, preferably 1, 2, 3 or 4,
More preferably 1, 2 or 3 (14)
(C) The conditions of the following formulas (15) and (16) are satisfied.
X−Xc = 1 to 10, preferably 1, 2 or 3,
More preferably 1 or 2 (15)
Y−Yc = 1 to 10, preferably 1, 2 or 3,
More preferably 1 or 2 (16)
(D) The conditions of the following formulas (17) and (18) are satisfied.
X−Xc = 0 (17)
Y−Yc = 0 (18)

An example of the structure of each of the ssNc molecules (a) to (d) is shown in the schematic diagram of FIG. FIG. 3 shows ssNc containing the linker region (Lx) and the linker region (Ly), (A) is the ssNc molecule of (a), (B) is the ssNc molecule of (b), ( C) is an example of the ssNc molecule of (c), and (D) is an example of the ssNc molecule of (d). In FIG. 3, a dotted line shows the state which has formed the double chain | strand by self-annealing. In the ssNc molecule of FIG. 3, the number of bases (X) in the inner 5 ′ side region (X) and the number of bases (Y) in the inner 3 ′ side region (Y) are expressed by “X <Y” in the formula (20). However, the present invention is not limited to this, and as described above, “X = Y” in the formula (19) or “X> Y” in the formula (21) may be used. Further, FIG. 3 is merely the relationship between the inner 5 ′ side region (X) and the 5 ′ side region (Xc), and the relationship between the inner 3 ′ side region (Y) and the 3 ′ side region (Yc). For example, the length and shape of each region are not limited thereto, and the presence or absence of the linker region (Lx) and the linker region (Ly) is not limited thereto.

The ssNc molecules of (a) to (c) include, for example, the 5 ′ side region (Xc) and the inner 5 ′ side region (X), and the 3 ′ side region (Yc) and the inner 3 ′ side. The region (Y) has a base that cannot be aligned with any of the 5 ′ side region (Xc) and the 3 ′ side region (Yc) in the internal region (Z) by forming a double chain, respectively. It can be said that the structure has a base that does not form a double chain. In the internal region (Z), the base that cannot be aligned (also referred to as a base that does not form a double chain) is hereinafter referred to as “free base”. In FIG. 3, the free base region is indicated by “F”. The number of bases in the region (F) is not particularly limited. The number of bases (F) in the region (F) is, for example, the number of bases “X—Xc” in the case of the ssNc molecule of (a), and “Y—Yc” in the case of the ssNc molecule of (b). In the case of the ssNc molecule of (c), it is the total number of bases “X-Xc” and “Y-Yc”.

On the other hand, the ssNc molecule of (d) is, for example, a structure in which the entire region of the internal region (Z) is aligned with the 5 ′ side region (Xc) and the 3 ′ side region (Yc), It can also be said that the entire region (Z) forms a double chain. In the ssNc molecule of (d), the 5 'end of the 5' side region (Xc) and the 3 'end of the 3' side region (Yc) are unlinked.

For the ssNc molecule of the present invention, the length of each region is exemplified below, but the present invention is not limited thereto. In the present invention, for example, the numerical range of the number of bases discloses all positive integers belonging to the range. For example, the description “1 to 4 bases” includes “1, 2, 3, 4 bases”. "Means all disclosures (the same applies hereinafter).

The total number of bases of the free base (F) in the 5 ′ side region (Xc), the 3 ′ side region (Yc), and the internal region (Z) is, for example, the number of bases in the internal region (Z) It becomes. For this reason, the lengths of the 5 ′ side region (Xc) and the 3 ′ side region (Yc) depend on, for example, the length of the internal region (Z), the number of free bases (F), and the position thereof. Can be determined as appropriate.

The number of bases in the internal region (Z) is, for example, 19 bases or more. The lower limit of the number of bases is, for example, 19 bases, preferably 20 bases, and more preferably 21 bases. The upper limit of the number of bases is, for example, 50 bases, preferably 40 bases, and more preferably 30 bases. Specific examples of the number of bases in the internal region (Z) include, for example, 19 bases, 20 bases, 21 bases, 22 bases, 23 bases, 24 bases, 25 bases, 26 bases, 27 bases, 28 bases, 29 bases, or , 30 bases.

When the internal region (Z) includes the expression suppression sequence, the internal region (Z) may be, for example, a region composed only of the expression suppression sequence or a region including the expression suppression sequence. The number of bases in the expression suppressing sequence is, for example, 19 to 30 bases, and preferably 19, 20 or 21 bases. When the internal region (Z) contains the expression suppression sequence, it may further have an additional sequence on the 5 'side and / or 3' side of the expression suppression sequence. The number of bases of the additional sequence is, for example, 1 to 31 bases, preferably 1 to 21 bases, more preferably 1 to 11 bases, and further preferably 1 to 7 bases.

The number of bases in the 5 ′ side region (Xc) is, for example, 1 to 29 bases, preferably 1 to 11 bases, more preferably 1 to 7 bases, and further preferably 1 to 4 bases. Particularly preferred are 1 base, 2 bases and 3 bases. When the internal region (Z) or the 3 'side region (Yc) includes the expression suppression sequence, for example, such a base number is preferable. As a specific example, when the number of bases in the internal region (Z) is 19 to 30 bases (for example, 19 bases), the number of bases in the 5 ′ side region (Xc) is, for example, 1 to 11 bases, The number is preferably 1 to 7 bases, more preferably 1 to 4 bases, and still more preferably 1 base, 2 bases, and 3 bases.

When the 5′-side region (Xc) includes the expression suppression sequence, the 5′-side region (Xc) may be, for example, a region composed only of the expression suppression sequence, or a region including the expression suppression sequence But you can. The length of the expression suppression sequence is, for example, as described above. When the 5 'region (Xc) contains the expression suppression sequence, it may further have an additional sequence on the 5' side and / or 3 'side of the expression suppression sequence. The number of bases of the additional sequence is, for example, 1 to 11 bases, and preferably 1 to 7 bases.

The number of bases in the 3 ′ side region (Yc) is, for example, 1 to 29 bases, preferably 1 to 11 bases, more preferably 1 to 7 bases, and further preferably 1 to 4 bases. Particularly preferred are 1 base, 2 bases and 3 bases. When the internal region (Z) or the 5 'side region (Xc) includes the expression suppression sequence, for example, such a base number is preferable. As a specific example, when the number of bases in the internal region (Z) is 19 to 30 bases (for example, 19 bases), the number of bases in the 3 ′ side region (Yc) is, for example, 1 to 11 bases, The number is preferably 1 to 7 bases, more preferably 1 to 4 bases, and still more preferably 1 base, 2 bases, and 3 bases.

When the 3 ′ side region (Yc) includes the expression suppression sequence, the 3 ′ side region (Yc) may be, for example, a region composed only of the expression suppression sequence, or a region including the expression suppression sequence But you can. The length of the expression suppression sequence is, for example, as described above. When the 3 'side region (Yc) includes the expression suppression sequence, it may further have an additional sequence on the 5' side and / or 3 'side of the expression suppression sequence. The number of bases of the additional sequence is, for example, 1 to 11 bases, and preferably 1 to 7 bases.

As described above, the number of bases in the internal region (Z), the 5′-side region (Xc), and the 3′-side region (Yc) is expressed by, for example, “Z ≧ Xc + Yc” in the formula (2). Can do. As a specific example, the number of bases “Xc + Yc” is, for example, the same as or smaller than the inner region (Z). In the latter case, “Z− (Xc + Yc)” is, for example, 1 to 10, preferably 1 to 4, more preferably 1, 2 or 3. The “Z− (Xc + Yc)” corresponds to, for example, the number of bases (F) in the free base region (F) in the internal region (Z).

In the ssNc molecule of the present invention, the length of the linker region (Lx) and the linker region (Ly) is not particularly limited. The linker region (Lx) preferably has, for example, a length that allows the internal 5 ′ side region (X) and the 5 ′ side region (Xc) to form a double chain, and the linker region (Ly) ) Is, for example, preferably a length such that the inner 3 ′ side region (Y) and the 3 ′ side region (Yc) can form a double chain. When the structural unit of the linker region (Lx) and the linker region (Ly) includes a base, the number of bases of the linker region (Lx) and the linker region (Ly) may be the same or different. The base sequence may be the same or different. The lower limit of the number of bases in the linker region (Lx) and the linker region (Ly) is, for example, 1 base, preferably 2 bases, more preferably 3 bases, and the upper limit thereof is, for example, 100 bases, preferably 80 bases, more preferably 50 bases. Specific examples of the number of bases in each linker region include 1 to 50 bases, 1 to 30 bases, 1 to 20 bases, 1 to 10 bases, 1 to 7 bases, and 1 to 4 bases. This is not a limitation.

In the ssNc molecule of the present invention, for example, as shown in FIGS. 1 to 3, when the 5 ′ side region (Xc) and the 3 ′ side region (Yc) are aligned with the internal region (Z), Examples of the position of the unbound end of the 5 ′ side region (Xc) with respect to the internal region (Z) include the following conditions. When the unbound end of the 5 ′ region (Xc) corresponds to the 5 ′ side of the center of the internal region (Z), the position of the unbound end is, for example, in the internal region (Z) The site is preferably 1/50 to 1/2 from the 5 ′ end, more preferably 1/50 to 1/3 or 1/50 to 1/4, and even more preferably 1/30 to 1 /. 2, 1/30 to 1/3 or 1/30 to 1/4. When the unbound end of the 3 ′ side region (Yc) corresponds to the 3 ′ side of the center of the internal region (Z), the position of the unbound end is, for example, the internal region (Z) In this case, it is preferably 1/50 to 1/2 of the 3 ′ end, more preferably 1/50 to 1/3 or 1/50 to 1/4, and even more preferably 1/30 to 1/2, 1/30 to 1/3 or 1/30 to 1/4.

The total length of the ssNc molecule of the present invention is not particularly limited. In the ssNc molecule of the present invention, the lower limit of the total number of bases (total number of bases) is, for example, 38 bases, preferably 42 bases, more preferably 50 bases, and even more preferably 51 bases. The upper limit is, for example, 300 bases, preferably 200 bases, more preferably 150 bases, still more preferably 100 bases, particularly preferably 80 bases. It is. In the ssNc molecule of the present invention, the lower limit of the total number of bases excluding the linker region (Lx) and the linker region (Ly) is, for example, 38 bases, preferably 42 bases, more preferably 50 bases. Yes, more preferably 51 bases, particularly preferably 52 bases, and the upper limit is, for example, 300 bases, preferably 200 bases, more preferably 150 bases, still more preferably 100 bases Particularly preferred is 80 bases.

The structural unit of the ssNc molecule of the present invention is not particularly limited, and examples thereof include nucleotide residues. Examples of the nucleotide residue include a ribonucleotide residue and a deoxyribonucleotide residue. Examples of the nucleotide residue include an unmodified unmodified nucleotide residue and a modified modified nucleotide residue. The ssNc molecule of the present invention can improve nuclease resistance and stability by including, for example, the modified nucleotide residue. The ssNc molecule of the present invention may further contain a non-nucleotide residue in addition to the nucleotide residue, for example. Details of the nucleotide residue and the non-nucleotide residue will be described later.

In the ssNc molecule of the present invention, each of the constituent units of the internal region (Z), the 5 ′ side region (Xc) and the 3 ′ side region (Yc) is preferably the nucleotide residue. Each region is composed of the following residues (1) to (3), for example.
(1) Unmodified nucleotide residue (2) Modified nucleotide residue (3) Unmodified nucleotide residue and modified nucleotide residue

In the ssNc molecule of the present invention, the structural units of the linker region (Lx) and the linker region (Ly) are not particularly limited, and examples thereof include the nucleotide residue and the non-nucleotide residue. The linker region may be composed of, for example, only the nucleotide residue, may be composed of only the non-nucleotide residue, or may be composed of the nucleotide residue and the non-nucleotide residue. . The linker region is composed of the following residues (1) to (7), for example.
(1) Unmodified nucleotide residues (2) Modified nucleotide residues (3) Unmodified nucleotide residues and modified nucleotide residues (4) Nonnucleotide residues (5) Nonnucleotide residues and unmodified nucleotide residues (6 ) Non-nucleotide residues and modified nucleotide residues (7) non-nucleotide residues, unmodified nucleotide residues and modified nucleotide residues

When the ssNc molecule of the present invention has both the linker region (Lx) and the linker region (Ly), for example, both structural units may be the same or different. Specific examples include, for example, a form in which the constituent units of both linker regions are the nucleotide residues, a form in which the constituent units of both linker regions are the non-nucleotide residues, and the constituent units of one region are the nucleotide residues. And the other linker region is a non-nucleotide residue.

Examples of the ssNc molecule of the present invention include a molecule composed only of the nucleotide residue, a molecule containing the non-nucleotide residue in addition to the nucleotide residue, and the like. In the ssNc molecule of the present invention, as described above, the nucleotide residue may be, for example, only the unmodified nucleotide residue, only the modified nucleotide residue, or the unmodified nucleotide residue and the modification. Both nucleotide residues may be used. When the ssNc molecule includes the unmodified nucleotide residue and the modified nucleotide residue, the number of the modified nucleotide residue is not particularly limited, and is, for example, “one or several”, specifically For example, 1 to 5, preferably 1 to 4, more preferably 1 to 3, and most preferably 1 or 2. When the ssNc molecule of the present invention includes the non-nucleotide residue, the number of the non-nucleotide residue is not particularly limited, and is, for example, “one or several”, specifically, for example, 1 to Eight, one to six, one to four, one, two or three.

In the ssNc molecule of the present invention, the nucleotide residue is preferably, for example, a ribonucleotide residue. In this case, the ssNc molecule of the present invention is also referred to as “RNA molecule” or “ssRNA molecule”, for example. Examples of the ssRNA molecule include a molecule composed only of the ribonucleotide residue, and a molecule containing the non-nucleotide residue in addition to the ribonucleotide residue. In the ssRNA molecule, as described above, the ribonucleotide residue may be, for example, only the unmodified ribonucleotide residue, only the modified ribonucleotide residue, or the unmodified ribonucleotide residue and Both of the modified ribonucleotide residues may be included.

When the ssRNA molecule includes, for example, the modified ribonucleotide residue in addition to the unmodified ribonucleotide residue, the number of the modified ribonucleotide residue is not particularly limited. For example, “1 or several” Specifically, for example, 1 to 5, preferably 1 to 4, more preferably 1 to 3, and most preferably 1 or 2. The modified ribonucleotide residue relative to the unmodified ribonucleotide residue may be, for example, the deoxyribonucleotide residue in which a ribose residue is replaced with a deoxyribose residue. For example, when the ssRNA molecule includes the deoxyribonucleotide residue in addition to the unmodified ribonucleotide residue, the number of the deoxyribonucleotide residue is not particularly limited, and is, for example, “one or several”. Specifically, for example, 1 to 5, preferably 1 to 4, more preferably 1 to 3, and most preferably 1 or 2.

The ssNc molecule of the present invention may contain, for example, a labeling substance and may be labeled with the labeling substance. The labeling substance is not particularly limited, and examples thereof include fluorescent substances, dyes, isotopes and the like. Examples of the labeling substance include fluorophores such as pyrene, TAMRA, fluorescein, Cy3 dye, and Cy5 dye, and examples of the dye include Alexa dye such as Alexa488. Examples of the isotope include a stable isotope and a radioactive isotope, and a stable isotope is preferable. For example, the stable isotope has a low risk of exposure and does not require a dedicated facility, so that it is easy to handle and the cost can be reduced. In addition, the stable isotope, for example, has no change in physical properties of the labeled compound, and is excellent in properties as a tracer. The stable isotope is not particularly limited, and examples thereof include ² H, ¹³ C, ¹⁵ N, ¹⁷ O, ¹⁸ O, ³³ S, ³⁴ S, and ³⁶ S.

As described above, the ssNc molecule of the present invention can suppress the expression of the target gene. Therefore, the ssNc molecule of the present invention can be used as a therapeutic agent for diseases caused by genes, for example. When the ssNc molecule of the present invention includes, for example, a sequence that suppresses the expression of a gene causing the disease as the expression suppression sequence, the disease can be treated by suppressing the expression of the target gene, for example. In the present invention, “treatment” includes, for example, the meanings of preventing the disease, improving the disease, and improving the prognosis. The disease is not particularly limited, and for example, the expression suppression sequence can be appropriately set according to the target disease. Examples of the disease include cancers such as breast cancer, lung cancer and stomach cancer.

The method of using the ssNc molecule of the present invention is not particularly limited, and for example, the ssNc molecule may be administered to an administration subject having the target gene.

Examples of the administration subject include cells, tissues, and organs. Examples of the administration target include non-human animals such as humans and non-human mammals other than humans. The administration can be, for example, in vivo or in vitro . The cells are not particularly limited, and examples thereof include various cultured cells such as HeLa cells, 293 cells, NIH3T3 cells, and COS cells, stem cells such as ES cells and hematopoietic stem cells, and cells isolated from living bodies such as primary cultured cells. can give.

In the present invention, the target gene to be subject to expression suppression is not particularly limited, and a desired gene can be set. Then, as described above, the expression suppression sequence may be appropriately designed according to the type of the target gene.

Referring to the use of the ssNc molecule of the present invention, reference can be made to the description of the composition of the present invention, expression suppression method, treatment method and the like described later.

Since the ssNc molecule of the present invention can suppress the expression of a target gene as described above, it is useful as a research tool for, for example, pharmaceuticals, diagnostic agents and agricultural chemicals, and agricultural chemicals, medicine, and life sciences.

2. Nucleotide residues The nucleotide residues include, for example, sugars, bases and phosphates as constituent elements. As described above, examples of the nucleotide residue include a ribonucleotide residue and a deoxyribonucleotide residue. The ribonucleotide residue has, for example, a ribose residue as a sugar, and has adenine (A), guanine (G), cytosine (C) and U (uracil) as bases, and the deoxyribose residue is For example, it has a deoxyribose residue as a sugar and has adenine (A), guanine (G), cytosine (C) and thymine (T) as bases.

The nucleotide residue includes an unmodified nucleotide residue and a modified nucleotide residue. In the unmodified nucleotide residue, each of the constituent elements is, for example, the same or substantially the same as that existing in nature, and preferably the same or substantially the same as that naturally occurring in the human body. .

The modified nucleotide residue is, for example, a nucleotide residue obtained by modifying the unmodified nucleotide residue. In the modified nucleotide residue, for example, any of the constituent elements of the unmodified nucleotide residue may be modified. In the present invention, “modification” refers to, for example, substitution, addition and / or deletion of the component, substitution, addition and / or deletion of atoms and / or functional groups in the component, and is referred to as “modification”. be able to. Examples of the modified nucleotide residue include naturally occurring nucleotide residues, artificially modified nucleotide residues, and the like. For example, Limbac et al. (Limbach et al., 1994, Summary: the modified nucleosides of RNA, Nucleic Acids Res. 22: 2183-2196) can be referred to as the naturally-occurring modified nucleotide residues. The modified nucleotide residue may be, for example, a residue of an alternative to the nucleotide residue

Examples of the modification of the nucleotide residue include modification of a ribose-phosphate skeleton (hereinafter referred to as ribophosphate skeleton).

In the ribophosphate skeleton, for example, a ribose residue can be modified. The ribose residue can be modified, for example, at the 2′-position carbon. Specifically, for example, a hydroxyl group bonded to the 2′-position carbon can be replaced with hydrogen or a halogen such as fluoro. By substituting the hydroxyl group at the 2'-position with hydrogen, the ribose residue can be replaced with deoxyribose. The ribose residue can be substituted with, for example, a stereoisomer, and can be substituted with, for example, an arabinose residue.

The ribophosphate skeleton may be substituted with a non-ribophosphate skeleton having a non-ribose residue and / or non-phosphate, for example. Examples of the non-ribophosphate skeleton include uncharged ribophosphate skeletons. Examples of the substitute for the nucleotide substituted with the non-ribophosphate skeleton include morpholino, cyclobutyl, pyrrolidine and the like. Other examples of the substitute include artificial nucleic acid monomer residues. Specific examples include PNA (peptide nucleic acid), LNA (Locked Nucleic Acid), ENA (2'-O, 4'-C-Ethylenebridged Nucleic Acid), and PNA is preferable.

In the ribophosphate skeleton, for example, a phosphate group can be modified. In the ribophosphate skeleton, the phosphate group closest to the sugar residue is called an α-phosphate group. The α-phosphate group is negatively charged, and the charge is evenly distributed over two oxygen atoms that are not bound to a sugar residue. Of the four oxygen atoms in the α-phosphate group, in the phosphodiester bond between nucleotide residues, the two oxygen atoms that are non-bonded to the sugar residue are hereinafter referred to as “non-linking oxygen”. Say. On the other hand, in the phosphodiester bond between the nucleotide residues, the two oxygen atoms bonded to the sugar residue are hereinafter referred to as “linking oxygen”. The α-phosphate group is preferably subjected to, for example, a modification that makes it uncharged or a modification that makes the charge distribution in the unbound oxygen asymmetric.

The phosphate group may replace the non-bonded oxygen, for example. The oxygen is, for example, one of S (sulfur), Se (selenium), B (boron), C (carbon), H (hydrogen), N (nitrogen), and OR (R is an alkyl group or an aryl group). And is preferably substituted with S. In the non-bonded oxygen, for example, both are preferably substituted, and more preferably, both are substituted with S. Examples of the modified phosphate group include phosphorothioate, phosphorodithioate, phosphoroselenate, boranophosphate, boranophosphate ester, phosphonate hydrogen, phosphoramidate, alkyl or arylphosphonate, and phosphotriester. Among them, phosphorodithioate in which the two non-bonded oxygens are both substituted with S is preferable.

The phosphate group may substitute, for example, the bonded oxygen. The oxygen can be substituted, for example, with any atom of S (sulfur), C (carbon) and N (nitrogen), and the modified phosphate group is, for example, a bridged phosphoramidate, S substituted with N Substituted bridged phosphorothioates, bridged methylene phosphonates substituted with C, and the like. The binding oxygen substitution is preferably performed, for example, on at least one of the 5 ′ terminal nucleotide residue and the 3 ′ terminal nucleotide residue of the ssNc molecule of the present invention, and in the case of the 5 ′ side, substitution with C is preferable. For the 'side, substitution with N is preferred.

The phosphate group may be substituted with, for example, the phosphorus-free linker. Examples of the linker include siloxane, carbonate, carboxymethyl, carbamate, amide, thioether, ethylene oxide linker, sulfonate, sulfonamide, thioformacetal, formacetal, oxime, methyleneimino, methylenemethylimino, methylenehydrazo, and methylenedimethyl. It contains groups such as hydrazo and methyleneoxymethylimino, and preferably contains a methylenecarbonylamino group and a methylenemethylimino group.

In the ssNc molecule of the present invention, for example, at least one nucleotide residue at the 3 'end and the 5' end may be modified. The modification may be, for example, either the 3 'end or the 5' end, or both. The modification is, for example, as described above, and is preferably performed on the terminal phosphate group. For example, the phosphate group may be modified entirely, or one or more atoms in the phosphate group may be modified. In the former case, for example, the entire phosphate group may be substituted or deleted.

Examples of the modification of the terminal nucleotide residue include addition of other molecules. Examples of the other molecule include functional molecules such as a labeling substance and a protecting group as described above. Examples of the protecting group include S (sulfur), Si (silicon), B (boron), ester-containing groups, and the like. The functional molecule such as the labeling substance can be used for, for example, detection of the ssNc molecule of the present invention.

The other molecule may be added to the phosphate group of the nucleotide residue, for example, or may be added to the phosphate group or the sugar residue via a spacer. The terminal atom of the spacer can be added or substituted, for example, to the binding oxygen of the phosphate group or O, N, S or C of the sugar residue. The binding site of the sugar residue is preferably, for example, C at the 3 'position or C at the 5' position, or an atom bonded thereto. The spacer can be added or substituted at a terminal atom of a nucleotide substitute such as PNA.

The spacer is not particularly limited. For example, — (CH ₂ ) _n —, — (CH ₂ ) _n N—, — (CH ₂ ) _n O—, — (CH ₂ ) _n S—, O (CH ₂ CH ₂ O) _n CH ₂ CH ₂ OH, including abasic sugar, amide, carboxy, amine, oxyamine, oxyimine, thioether, disulfide, thiourea, sulfonamide, morpholino, and the like, and biotin reagent and fluorescein reagent Good. In the above formula, n is a positive integer, and n = 3 or 6 is preferable.

In addition to these, the molecule to be added to the terminal includes, for example, a dye, an intercalating agent (for example, acridine), a crosslinking agent (for example, psoralen, mitomycin C), a porphyrin (TPPC4, texaphyrin, suffirin), a polycyclic Aromatic hydrocarbons (eg phenazine, dihydrophenazine), artificial endonucleases (eg EDTA), lipophilic carriers (eg cholesterol, cholic acid, adamantaneacetic acid, 1-pyrenebutyric acid, dihydrotestosterone, 1,3-bis- O (hexadecyl) glycerol, geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid, O3- (oleoyl) lithocholic acid, O3- (oleoyl) call , Dimethoxytrityl, or phenoxazine) and peptide conjugates (e.g., antennapedia peptide, Tat peptide), alkylating agents, phosphate, amino, mercapto, PEG (e.g., PEG-40K), MPEG, [MPEG] 2, Polyamino, alkyl, substituted alkyl, radiolabeled marker, enzyme, hapten (eg, biotin), transport / absorption enhancer (eg, aspirin, vitamin E, folic acid), synthetic ribonuclease (eg, imidazole, bisimidazole, histamine, imidazole cluster) , Acridine-imidazole complex, tetraaza macrocycle Eu ³⁺ complex) and the like.

In the ssNc molecule of the present invention, the 5 ′ end may be modified with, for example, a phosphate group or a phosphate group analog. The phosphate group is, for example, 5 ′ monophosphate ((HO) ₂ (O) PO-5 ′), 5 ′ diphosphate ((HO) ₂ (O) POP (HO) (O) —O— 5 '), 5' triphosphate ((HO) ₂ (O) PO- (HO) (O) POP (HO) (O) -O-5 '), 5'-guanosine cap (7-methylated or Unmethylated, 7m-GO-5 '-(HO) (O) PO- (HO) (O) POP (HO) (O) -O-5'), 5'-adenosine cap (Appp), optional Modified or unmodified nucleotide cap structure (NO-5 '-(HO) (O) PO- (HO) (O) POP (HO) (O) -O-5'), 5 'monothiophosphate (phosphorothioate: ( HO) ₂ (S) PO-5 ′), 5 ′ monodithiophosphate (phosphorodithioate: (HO) (HS) (S) PO-5 ′), 5′-phosphorothiolic acid ((HO) ₂ (O) PS-5 ′), sulfur-substituted monophosphate, diphosphate and triphosphate (eg, 5′-α-thiotriphosphate, 5′-γ-thiotriphosphate, etc.), 5 ′ Phosphoramidates ((HO) ₂ (O) P—NH-5 ′, (HO) (NH ₂ ) (O) PO-5 ′), 5′-alkylphosphonic acids (eg RP (OH) ( O) -O-5 ' _{(OH) 2 (O) P} -5'-CH 2, R is alkyl (e.g., methyl, ethyl, isopropyl, propyl, etc.)), 5'-alkyl ether phosphonic acid (e.g., RP (OH) (O) - O-5 ′ and R include alkyl ethers (for example, methoxymethyl, ethoxymethyl, etc.).

In the nucleotide residue, the base is not particularly limited. The base may be, for example, a natural base or a non-natural base. The base may be, for example, naturally derived or a synthetic product. As the base, for example, a general base or a modified analog thereof can be used.

Examples of the base include purine bases such as adenine and guanine, and pyrimidine bases such as cytosine, uracil and thymine. Other examples of the base include inosine, thymine, xanthine, hypoxanthine, nubalarine, isoguanisine, and tubercidine. Examples of the base include alkyl derivatives such as 2-aminoadenine and 6-methylated purine; alkyl derivatives such as 2-propylated purine; 5-halouracil and 5-halocytosine; 5-propynyluracil and 5-propynylcytosine; -Azouracil, 6-azocytosine and 6-azothymine; 5-uracil (pseudouracil), 4-thiouracil, 5-halouracil, 5- (2-aminopropyl) uracil, 5-aminoallyluracil; 8-halogenated, aminated, Thiolated, thioalkylated, hydroxylated and other 8-substituted purines; 5-trifluoromethylated and other 5-substituted pyrimidines; 7-methylguanine; 5-substituted pyrimidines; 6-azapyrimidines; N-2, N -6 and O-6 substituted purines (2-aminopropyladenyl 5-propynyluracil and 5-propynylcytosine; dihydrouracil; 3-deaza-5-azacytosine; 2-aminopurine; 5-alkyluracil; 7-alkylguanine; 5-alkylcytosine; 7-deazaadenine; N6 2,6-diaminopurine; 5-amino-allyl-uracil; N3-methyluracil; substituted 1,2,4-triazole; 2-pyridinone; 5-nitroindole; 3-nitropyrrole; -Methoxyuracil; uracil-5-oxyacetic acid; 5-methoxycarbonylmethyluracil; 5-methyl-2-thiouracil; 5-methoxycarbonylmethyl-2-thiouracil; 5-methylaminomethyl-2-thiouracil; 3- (3 -Amino-3-carboxypropyl) uracil 3-methylcytosine; 5-methylcytosine; N4-acetylcytosine; 2-thiocytosine; N6-methyladenine; N6-isopentyladenine; 2-methylthio-N6-isopentenyladenine; N-methylguanine; O-alkylated base Etc. Purines and pyrimidines are disclosed in, for example, U.S. Pat. No. 3,687,808, “Concise Encyclopedia Of Polymer And Engineering”, pages 858-859, edited by Kroschwitz JI & W. JI. Sons, 1990, and English et al., Angewante Chemie, International Edition, 1991, 30, p. 613 is included.

In addition to these, the modified nucleotide residue may include, for example, a residue lacking a base, that is, an abasic ribophosphate skeleton. The modified nucleotide residues are, for example, US Provisional Application No. 60 / 465,665 (filing date: April 25, 2003) and International Application No. PCT / US04 / 07070 (filing date: 2004/3). The residues described on the 8th of May) can be used, and the present invention can incorporate these documents.

3. Non-nucleotide residue The non-nucleotide residue is not particularly limited. The ssNc molecule of the present invention may have, for example, a non-nucleotide structure containing a pyrrolidine skeleton or a piperidine skeleton as the non-nucleotide residue. For example, the non-nucleotide residue is preferably present in at least one of the linker region (Lx) and the linker region (Ly). For example, the non-nucleotide residue may be included in the linker region (Lx), may be included in the linker region (Ly), or may be included in both the linker regions. The linker region (Lx) and the linker region (Ly) may be the same or different, for example.

The pyrrolidine skeleton may be, for example, a skeleton of a piperidine derivative in which one or more carbons constituting the five-membered ring of pyrrolidine are substituted. When the pyrrolidine skeleton is substituted, for example, a carbon atom other than C-2 carbon is used. It is preferable. The carbon may be substituted with, for example, nitrogen, oxygen, or sulfur. The pyrrolidine skeleton may include, for example, a carbon-carbon double bond or a carbon-nitrogen double bond in the 5-membered ring of pyrrolidine. In the pyrrolidine skeleton, the carbon and nitrogen constituting the 5-membered ring of pyrrolidine may be bonded to, for example, a hydrogen group or a substituent as described later. The linker region (Lx) may be bonded to the region (X) and the region (Xc) through, for example, any group of the pyrrolidine skeleton, preferably any one of the 5-membered rings Carbon atoms and nitrogen, preferably carbon (C-2) and nitrogen at the 2-position of the 5-membered ring. Examples of the pyrrolidine skeleton include a proline skeleton and a prolinol skeleton. The proline skeleton, prolinol skeleton, and the like are excellent in safety because they are, for example, in-vivo substances and their reduced forms.

The piperidine skeleton may be, for example, a skeleton of a piperidine derivative in which one or more carbons constituting the six-membered ring of piperidine are substituted, and when substituted, for example, a carbon atom other than C-2 carbon. It is preferable. The carbon may be substituted with, for example, nitrogen, oxygen, or sulfur. The piperidine skeleton may contain, for example, a carbon-carbon double bond or a carbon-nitrogen double bond in the six-membered ring of piperidine. In the piperidine skeleton, the carbon and nitrogen constituting the piperidine 6-membered ring may be bonded to, for example, a hydrogen group or a substituent as described later. The linker region (Lx) may be bonded to the region (X) and the region (Xc), for example, via any group of the piperidine skeleton, and preferably any one of the six-membered rings Of the 6-membered ring, and carbon (C-2) and nitrogen are preferred. The same applies to the linker region (Ly).

The linker region may be, for example, only a non-nucleotide residue consisting of the non-nucleotide structure, or may include a non-nucleotide residue consisting of the non-nucleotide structure and a nucleotide residue.

The linker region is represented by the following formula (I), for example.

In the formula (I),
X ¹ and X ² are each independently H ₂ , O, S or NH;
Y ¹ and Y ² are each independently a single bond, CH ₂ , NH, O or S;
R ³ is a hydrogen atom or substituent bonded to C-3, C-4, C-5 or C-6 on ring A;
The substituent is OH, OR ⁴ , NH ₂ , NHR ⁴ , NR ⁴ R ⁵ , SH, SR ⁴ or an oxo group (═O);
When R ³ is the substituent, the substituent R ³ may be one, plural or absent, and in plural cases, may be the same or different;
R ⁴ and R ⁵ are substituents or protecting groups and may be the same or different;
L ¹ is an alkylene chain consisting of n atoms, wherein the hydrogen atom on the alkylene carbon atom is substituted with OH, OR ^a , NH ₂ , NHR ^a , NR ^a R ^b , SH or SR ^a May not be substituted, or
L ¹ is a polyether chain in which one or more carbon atoms of the alkylene chain are substituted with an oxygen atom,
However, when Y ¹ is NH, O or S, the atom of L ¹ bonded to Y ¹ is carbon, the atom of L ¹ bonded to OR ¹ is carbon, and oxygen atoms are not adjacent to each other;
L ² is an alkylene chain consisting of m atoms, wherein the hydrogen atom on the alkylene carbon atom is substituted with OH, OR ^c , NH ₂ , NHR ^c , NR ^c R ^d , SH or SR ^c May not be substituted, or
L ² is a polyether chain in which one or more carbon atoms of the alkylene chain are substituted with an oxygen atom,
However, when Y ² is NH, O or S, the atom of L ² bonded to Y ² is carbon, the atom of L ² bonded to OR ² is carbon, and oxygen atoms are not adjacent to each other;
R ^a , R ^b , R ^c and R ^d are each independently a substituent or a protecting group;
l is 1 or 2;
m is an integer ranging from 0 to 30;
n is an integer ranging from 0 to 30;
In ring A, one carbon atom other than C-2 on ring A may be substituted with nitrogen, oxygen, or sulfur.
The ring A may contain a carbon-carbon double bond or a carbon-nitrogen double bond. When the linker region (Lx) is represented by the formula (1), the region (Xc) and the region (X) are each linked to the linker region (Lx) via —OR ¹ — or —OR ² —. ). When the linker region (Ly) is represented by the formula (1), the region (Yc) and the region (Y) are each linked to the linker region via —OR ¹ — or —OR ² —. It binds to (Ly). Here, R ¹ and R ² may be present or absent, and when present, R ¹ and R ² are each independently a nucleotide residue or the structure (I).

In the formula (I), X ¹ and X ² are each independently, for example, H ₂ , O, S or NH. In the formula (I), X ¹ being H ₂ means that X ¹ together with the carbon atom to which X ¹ is bonded forms CH ₂ (methylene group). The same is true for X ^2.

In the formula (I), in ring A, l is 1 or 2. When l = 1, ring A is a 5-membered ring, for example, the pyrrolidine skeleton. Examples of the pyrrolidine skeleton include a proline skeleton and a prolinol skeleton, and examples thereof include a bivalent structure. When l = 2, ring A is a 6-membered ring, for example, the piperidine skeleton. In ring A, one carbon atom other than C-2 on ring A may be substituted with nitrogen, oxygen or sulfur. Ring A may contain a carbon-carbon double bond or a carbon-nitrogen double bond in ring A. Ring A may be, for example, either L-type or D-type.

In the formula (I), Y ¹ and Y ² are each independently a single bond, CH ₂ , NH, O or S.

In the formula (I), R ³ is a hydrogen atom or a substituent bonded to C-3, C-4, C-5 or C-6 on the ring A. The substituent is OH, OR ⁴ , NH ₂ , NHR ⁴ , NR ⁴ R ⁵ , SH, SR ⁴ or an oxo group (═O). When R ³ is the above-described substituent, the substituent R ³ may be one, plural, or absent, and when plural, it may be the same or different. R ⁴ and R ⁵ are substituents or protecting groups, and may be the same or different.

Examples of the substituent include halogen, alkyl, alkenyl, alkynyl, haloalkyl, aryl, heteroaryl, arylalkyl, cycloalkyl, cycloalkenyl, cycloalkylalkyl, cyclylalkyl, hydroxyalkyl, alkoxyalkyl, aminoalkyl, heterocyclylalkenyl. , Heterocyclylalkyl, heteroarylalkyl, silyl, silyloxyalkyl and the like. The same applies hereinafter.

The protecting group is, for example, a functional group that converts a highly reactive functional group to be inert, and examples thereof include known protecting groups. For example, the description of the literature (J. F. W. McOmie, “Protecting Groups in Organic Chemistry”, Prenum Press, London and New York, 1973) can be used as the protecting group. The protecting group is not particularly limited, and examples thereof include tert-butyldimethylsilyl (TBDMS) group, bis (2-acetoxyethyloxy) methyl (ACE) group, triisopropylsilyloxymethyl (TOM) group, 1- (2 -Cyanoethoxy) ethyl (CEE) group, 2-cyanoethoxymethyl (CEM) group and tolylsulfonylethoxymethyl (TEM) group, dimethoxytrityl (DMTr) and the like. When R ³ is OR ⁴ , the protecting group is not particularly limited, and examples thereof include a TBDMS group, an ACE group, a TOM group, a CEE group, a CEM group, and a TEM group. In addition to this, silyl-containing groups described later are also included. The same applies hereinafter.

In the formula (I), L ¹ is an alkylene chain composed of n atoms. The hydrogen atom on the alkylene carbon atom may be substituted with, for example, OH, OR ^a , NH ₂ , NHR ^a , NR ^a R ^b , SH or SR ^a , or may not be substituted. Alternatively, L ¹ may be a polyether chain in which one or more carbon atoms of the alkylene chain are substituted with an oxygen atom. The polyether chain is, for example, polyethylene glycol. When Y ¹ is NH, O, or S, the L ¹ atom bonded to Y ¹ is carbon, the L ¹ atom bonded to OR ¹ is carbon, and oxygen atoms are not adjacent to each other. That is, for example, when Y ¹ is O, the oxygen atom and the oxygen atom of L ¹ are not adjacent, and the oxygen atom of OR ^{1 and} the oxygen atom of L ¹ are not adjacent.

In the formula (I), L ² is an alkylene chain composed of m atoms. The hydrogen atom on the alkylene carbon atom may be substituted with, for example, OH, OR ^c , NH ₂ , NHR ^c , NR ^c R ^d , SH or SR ^c , or may not be substituted. Alternatively, L ² may be a polyether chain in which one or more carbon atoms of the alkylene chain are substituted with an oxygen atom. When Y ² is NH, O, or S, the L ² atom bonded to Y ² is carbon, the L ² atom bonded to OR ² is carbon, and oxygen atoms are not adjacent to each other. That is, for example, when Y ² is O, the oxygen atom and the oxygen atom of L ² are not adjacent, and the oxygen atom of OR ^{2 and} the oxygen atom of L ² are not adjacent.

N in L ¹ and m in L ² are not particularly limited, and the lower limit is, for example, 0, and the upper limit is not particularly limited. n and m can be appropriately set depending on, for example, the desired length of the linker region (Lx). For example, n and m are each preferably 0 to 30, more preferably 0 to 20, and still more preferably 0 to 15 from the viewpoints of production cost and yield. n and m may be the same (n = m) or different. n + m is, for example, 0 to 30, preferably 0 to 20, and more preferably 0 to 15.

R ^a , R ^b , R ^c and R ^d are each independently a substituent or a protecting group. The substituent and the protective group are the same as described above, for example.

In the formula (I), hydrogen atoms may be independently substituted with halogens such as Cl, Br, F and I, for example.

In the case where the linker region (Lx) is represented by the formula (I), the region (Xc) and the region (X) are, for example, linked to the linker via —OR ¹ — or —OR ² —, respectively. Join to region (Lx). Here, R ¹ and R ² may or may not exist. When R ¹ and R ² are present, R ¹ and R ² are each independently a nucleotide residue or the structure of formula (I) above. When R ¹ and / or R ² is the nucleotide residue, the linker region (Lx) is, for example, the non-nucleotide residue having the structure of the formula (I) excluding the nucleotide residue R ¹ and / or R ^2. And a nucleotide residue. When R ¹ and / or R ² has the structure of the formula (I), the linker region (Lx) has, for example, two or more non-nucleotide residues having the structure of the formula (I) linked to each other. It becomes a structure. The structure of the formula (I) may include 1, 2, 3, or 4, for example. As described above, when a plurality of the structures are included, the structure (I) may be directly connected or may be bonded via the nucleotide residue. On the other hand, when R ¹ and R ² are not present, the linker region (Lx) is formed only from the non-nucleotide residue having the structure of the formula (I), for example. Further, when the linker region (Ly) is represented by the formula (I), for example, the region (Yc), the region (Y), and the linker region (Ly) are described in the linker region (Lx). Can be used.

The combination of the region (Xc) and the region (X), the region (Yc) and the region (Y), and the —OR ¹ — and —OR ² — is not particularly limited. One of the following conditions can be given.
Condition (1)
The region (Xc) is bonded to the structure of the formula (I) through —OR ² —, and the region (X) is bonded through —OR ¹ —.
The region (Yc) is bonded to the structure of the formula (I) through —OR ¹ —, and the region (Y) is bonded through —OR ² —.
Condition (2)
The region (Xc) is bonded to the structure of the formula (I) through —OR ² —, and the region (X) is bonded through —OR ¹ —.
The region (Yc) is bonded to the structure of the formula (I) through —OR ² —, and the region (Y) is bonded through —OR ¹ —.
Condition (3)
The region (Xc) is bonded to the structure of the formula (I) through —OR ¹ —, and the region (X) is bonded through —OR ² —.
The region (Yc) is bonded to the structure of the formula (I) through —OR ¹ —, and the region (Y) is bonded through —OR ² —.
Condition (4)
The region (Xc) is bonded to the structure of the formula (I) through —OR ¹ —, and the region (X) is bonded through —OR ² —.
The region (Yc) is bonded to the structure of the formula (I) through —OR ² —, and the region (Y) is bonded through —OR ¹ —.

Examples of the structure of the formula (I) include the following formulas (I-1) to (I-9), in which n and m are the same as those in the formula (I). In the following formula, q is an integer of 0 to 10.

In the above formulas (I-1) to (I-9), n, m and q are not particularly limited and are as described above. As a specific example, n = 8 in the formula (I-1), n = 3 in the (I-2), n = 4 or 8 in the formula (I-3), (I-4) N = 7 or 8, in the formula (I-5), n = 3 and m = 4, in the (I-6), n = 8 and m = 4, in the formula (I-7), In n = 8 and m = 4, in the above (I-8), n = 5 and m = 4, and in the formula (I-9), q = 1 and m = 4. An example (n = 8) of the formula (I-4) is represented by the following formula (I-4a), an example (n = 5, m = 4) of the formula (I-6) is represented by the following formula (I− This is shown in 6a).

In the present invention, “alkyl” includes, for example, a linear or branched alkyl group. The number of carbon atoms of the alkyl is not particularly limited, and is, for example, 1 to 30, preferably 1 to 6 or 1 to 4. Examples of the alkyl group include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, isohexyl, n-heptyl, Examples thereof include n-octyl, n-nonyl, n-decyl and the like. Preferred examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, isohexyl and the like.

In the present invention, “alkenyl” includes, for example, linear or branched alkenyl. Examples of the alkenyl include those having one or more double bonds in the alkyl. The number of carbon atoms of the alkenyl is not particularly limited, and is the same as, for example, the alkyl, preferably 2 to 8. Examples of the alkenyl include vinyl, 1-propenyl, 2-propenyl, 1-butenyl, 2-butenyl, 3-butenyl, 1,3-butadienyl, 3-methyl-2-butenyl and the like.

In the present invention, “alkynyl” includes, for example, linear or branched alkynyl. Examples of the alkynyl include those having one or more triple bonds in the alkyl. The number of carbon atoms of the alkynyl is not particularly limited, and is the same as, for example, the alkyl, preferably 2 to 8. Examples of the alkynyl include ethynyl, propynyl, butynyl and the like. The alkynyl may further have one or more double bonds, for example.

In the present invention, “aryl” includes, for example, a monocyclic aromatic hydrocarbon group and a polycyclic aromatic hydrocarbon group. Examples of the monocyclic aromatic hydrocarbon group include phenyl and the like. Examples of the polycyclic aromatic hydrocarbon group include 1-naphthyl, 2-naphthyl, 1-anthryl, 2-anthryl, 9-anthryl, 1-phenanthryl, 2-phenanthryl, 3-phenanthryl, 4-phenanthryl, 9- Examples include phenanthryl. Preferable examples include naphthyl such as phenyl, 1-naphthyl and 2-naphthyl.

In the present invention, “heteroaryl” includes, for example, a monocyclic aromatic heterocyclic group and a condensed aromatic heterocyclic group. Examples of the heteroaryl include furyl (eg, 2-furyl, 3-furyl), thienyl (eg, 2-thienyl, 3-thienyl), pyrrolyl (eg, 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl), Imidazolyl (eg, 1-imidazolyl, 2-imidazolyl, 4-imidazolyl), pyrazolyl (eg, 1-pyrazolyl, 3-pyrazolyl, 4-pyrazolyl), triazolyl (eg, 1,2,4-triazol-1-yl, 1,2,4-triazol-3-yl, 1,2,4-triazol-4-yl), tetrazolyl (eg 1-tetrazolyl, 2-tetrazolyl, 5-tetrazolyl), oxazolyl (eg 2-oxazolyl, 4-oxazolyl, 5-oxazolyl), isoxazolyl (eg 3-isoxazolyl, 4-isoxazolyl, 5-isoxazolyl) Thiazolyl (eg: 2-thiazolyl, 4-thiazolyl, 5-thiazolyl), thiadiazolyl, isothiazolyl (eg: 3-isothiazolyl, 4-isothiazolyl, 5-isothiazolyl), pyridyl (eg: 2-pyridyl, 3-pyridyl, 4- Pyridinyl), pyridazinyl (eg: 3-pyridazinyl, 4-pyridazinyl), pyrimidinyl (eg: 2-pyrimidinyl, 4-pyrimidinyl, 5-pyrimidinyl), furazanyl (eg: 3-furazinyl), pyrazinyl (eg: 2-pyrazinyl) Oxadiazolyl (eg 1,3,4-oxadiazol-2-yl), benzofuryl (eg 2-benzo [b] furyl, -benzo [b] furyl, 4-benzo [b] furyl, 5-benzo [B] furyl, 6-benzo [b] furyl, 7-benzo [b] furyl), benzothienyl (eg 2-benzo [b] thienyl, 3-benzo [b] thienyl, 4-benzo [b] thienyl, 5-benzo [b] thienyl, 6-benzo [b] thienyl, 7-benzo [b] thienyl), benz Imidazolyl (eg 1-benzimidazolyl, 2-benzimidazolyl, 4-benzoimidazolyl, 5-benzoimidazolyl), dibenzofuryl, benzoxazolyl, benzothiazolyl, quinoxalyl (eg 2-quinoxalinyl, 5-quinoxalinyl, 6-quinoxalinyl), cinnolinyl ( Examples: 3-cinnolinyl, 4-cinnolinyl, 5-cinnolinyl, 6-cinnolinyl, 7-cinnolinyl, 8-cinnolinyl), quinazolyl (eg 2-quinazolinyl, 4-quinazolinyl, 5-quinazolinyl, 6-quinazolinyl, 7-quinazolinyl) , 8-quinazolinyl) Quinolyl (eg, 2-quinolyl, 3-quinolyl, 4-quinolyl, 5-quinolyl, 6-quinolyl, 7-quinolyl, 8-quinolyl), phthalazinyl (eg, 1-phthalazinyl, 5-phthalazinyl, 6-phthalazinyl) , Isoquinolyl (eg, 1-isoquinolyl, 3-isoquinolyl, 4-isoquinolyl, 5-isoquinolyl, 6-isoquinolyl, 7-isoquinolyl, 8-isoquinolyl), prill, pteridinyl (eg, 2-pteridinyl, 4-pteridinyl, 6- Pteridinyl, 7-pteridinyl), carbazolyl, phenanthridinyl, acridinyl (eg 1-acridinyl, 2-acridinyl, 3-acridinyl, 4-acridinyl, 9-acridinyl), indolyl (eg 1-indolyl, 2-indolyl) , 3-indolyl, 4-indolyl, 5-i Drill, 6-indolyl, 7-indolyl), isoindolyl, phenazinyl (eg 1-phenazinyl, 2-phenazinyl) or phenothiazinyl (eg 1-phenothiazinyl, 2-phenothiazinyl, 3-phenothiadinyl, 4-phenothiazinyl) ) Etc.

In the present invention, “cycloalkyl” is, for example, a cyclic saturated hydrocarbon group, and the number of carbons is, for example, 3-15. Examples of the cycloalkyl include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, a bridged cyclic hydrocarbon group, a spiro hydrocarbon group, and the like, preferably cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl. And a bridged cyclic hydrocarbon group.

In the present invention, the “bridged cyclic hydrocarbon group” includes, for example, bicyclo [2.1.0] pentyl, bicyclo [2.2.1] heptyl, bicyclo [2.2.2] octyl and bicyclo [3. 2.1] octyl, tricyclo [2.2.1.0] heptyl, bicyclo [3.3.1] nonane, 1-adamantyl, 2-adamantyl and the like.

In the present invention, examples of the “spiro hydrocarbon group” include spiro [3.4] octyl and the like.

In the present invention, “cycloalkenyl” includes, for example, a cyclic unsaturated aliphatic hydrocarbon group, and has, for example, 3 to 7 carbon atoms. Examples of the group include cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclohexenyl, cycloheptenyl, and the like, preferably cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclohexenyl, and the like. The cycloalkenyl includes, for example, a bridged cyclic hydrocarbon group and a spiro hydrocarbon group having an unsaturated bond in the ring.

In the present invention, “arylalkyl” includes, for example, benzyl, 2-phenethyl, naphthalenylmethyl and the like, and “cycloalkylalkyl” or “cyclylalkyl” includes, for example, cyclohexylmethyl, adamantylmethyl and the like. Examples of the “hydroxyalkyl” include hydroxymethyl and 2-hydroxyethyl.

In the present invention, “alkoxy” includes the alkyl-O— group, and examples thereof include methoxy, ethoxy, n-propoxy, isopropoxy, and n-butoxy. “Alkoxyalkyl” includes, for example, methoxymethyl Examples of “aminoalkyl” include 2-aminoethyl and the like.

In the present invention, “heterocyclyl” is, for example, 1-pyrrolinyl, 2-pyrrolinyl, 3-pyrrolinyl, 1-pyrrolidinyl, 2-pyrrolidinyl, 3-pyrrolidinyl, pyrrolidinone, 1-imidazolinyl, 2-imidazolinyl, 4-imidazolinyl, 1 -Imidazolidinyl, 2-imidazolidinyl, 4-imidazolidinyl, imidazolidinone, 1-pyrazolinyl, 3-pyrazolinyl, 4-pyrazolinyl, 1-pyrazolidinyl, 3-pyrazolidinyl, 4-pyrazolidinyl, piperidinone, piperidinyl, 2-piperidinyl 4-piperidinyl, 1-piperazinyl, 2-piperazinyl, piperazinone, 2-morpholinyl, 3-morpholinyl, morpholino, tetrahydropyranyl, tetrahydrofuranyl and the like.

In the present invention, “heterocyclylalkyl” includes, for example, piperidinylmethyl, piperazinylmethyl and the like, and “heterocyclylalkenyl” includes, for example, 2-piperidinylethenyl and the like, and “heteroarylalkyl” Examples include pyridylmethyl and quinolin-3-ylmethyl.

In the present invention, “silyl” includes a group represented by the formula R ³ Si—, and R ³ can be independently selected from the above alkyl, aryl and cycloalkyl, such as trimethylsilyl group, tert-butyldimethyl Examples of the silyl group include silyloxy and the like. Examples of the “silyloxy” include trimethylsilyloxy and the like. Examples of the “silyloxyalkyl” include trimethylsilyloxymethyl and the like.

In the present invention, “alkylene” includes, for example, methylene, ethylene, propylene and the like.

In the present invention, the various groups described above may be substituted. Examples of the substituent include hydroxy, carboxy, halogen, alkyl halide (eg, CF ₃ , CH ₂ CF ₃ , CH ₂ CCl ₃ ), nitro, nitroso, cyano, alkyl (eg, methyl, ethyl, isopropyl, tert) -Butyl), alkenyl (eg vinyl), alkynyl (eg ethynyl), cycloalkyl (eg cyclopropyl, adamantyl), cycloalkylalkyl (eg cyclohexylmethyl, adamantylmethyl), cycloalkenyl (eg cyclopropenyl) , Aryl (eg phenyl, naphthyl), arylalkyl (eg benzyl, phenethyl), heteroaryl (eg pyridyl, furyl), heteroarylalkyl (eg pyridylmethyl), heterocyclyl (eg piperidyl), heterocyclylalkyl ( Example: Morpholylmethyl), alkoxy (eg methoxy, ethoxy, propoxy, butoxy), halogenated alkoxy (eg OCF ₃ ), alkenyloxy (eg vinyloxy, allyloxy), aryloxy (eg phenyloxy), alkyloxycarbonyl (eg : Methoxycarbonyl, ethoxycarbonyl, tert-butoxycarbonyl), arylalkyloxy (eg, benzyloxy), amino [alkylamino (eg, methylamino, ethylamino, dimethylamino), acylamino (eg, acetylamino, benzoylamino) Arylalkylamino (eg, benzylamino, tritylamino), hydroxyamino], alkylaminoalkyl (eg, diethylaminomethyl), sulfamoyl, oxo and the like.

4). Method for synthesizing ssNc molecule of the present invention The method for synthesizing the ssNc molecule of the present invention is not particularly limited, and conventionally known methods can be adopted. Examples of the synthesis method include a synthesis method using a genetic engineering technique, a chemical synthesis method, and the like. Examples of genetic engineering techniques include in vitro transcription synthesis, a method using a vector, and a method using a PCR cassette. The vector is not particularly limited, and examples thereof include non-viral vectors such as plasmids and viral vectors. The chemical synthesis method is not particularly limited, and examples thereof include a phosphoramidite method and an H-phosphonate method. For the chemical synthesis method, for example, a commercially available automatic nucleic acid synthesizer can be used. In the chemical synthesis method, amidite is generally used. The amidite is not particularly limited, and commercially available amidites include, for example, RNA Phosphoramidates (2′-O-TBDMSi, trade name, Michisato Pharmaceutical), ACE amidite, TOM amidite, CEE amidite, CEM amidite, TEM amidite, etc. Is given.

5). Composition As described above, the composition for suppressing expression of the present invention is a composition for suppressing the expression of a target gene, and is characterized by containing the ssNc molecule of the present invention. The composition of the present invention is characterized by containing the ssNc molecule of the present invention, and other configurations are not limited at all. The expression suppressing composition of the present invention can also be referred to as an expression suppressing reagent, for example.

According to the present invention, for example, the expression of the target gene can be suppressed by administering the composition for suppressing expression to a subject in which the target gene is present.

Further, as described above, the pharmaceutical composition of the present invention is characterized by containing the ssNc molecule of the present invention. The composition of the present invention is characterized by containing the ssNc molecule of the present invention, and other configurations are not limited at all. The pharmaceutical composition of the present invention can also be referred to as a pharmaceutical product, for example.

According to the present invention, for example, by administering the pharmaceutical composition to a patient having a disease caused by a gene, the expression of the gene can be suppressed and the disease can be treated. In the present invention, as described above, “treatment” includes, for example, the meanings of prevention of the above-mentioned diseases, improvement of the diseases, and improvement of the prognosis.

In the present invention, the disease to be treated is not particularly limited, and examples thereof include diseases caused by gene expression. According to the type of the disease, a gene that causes the disease is set as the target gene, and the expression suppression sequence may be set as appropriate according to the target gene.

The use method of the composition for suppressing expression and the pharmaceutical composition (hereinafter referred to as composition) of the present invention is not particularly limited. For example, if the ssNc molecule is administered to an administration subject having the target gene. Good.

The administration method is not particularly limited, and can be appropriately determined according to the administration subject, for example. When the administration subject is a cultured cell, examples thereof include a method using a transfection reagent and an electroporation method.

The composition of the present invention may contain, for example, only the ssNc molecule of the present invention, or may further contain other additives. The additive is not particularly limited, and for example, a pharmaceutically acceptable additive is preferable. The type of the additive is not particularly limited, and can be appropriately selected depending on, for example, the type of administration target.

In the composition of the present invention, the ssNc molecule may form a complex with the additive, for example. The additive can also be referred to as a complexing agent, for example. By the complex formation, for example, the ssNc molecule can be efficiently delivered. The bond between the ssNc molecule and the complexing agent is not particularly limited, and examples thereof include non-covalent bonds. Examples of the complex include an inclusion complex.

The complexing agent is not particularly limited, and examples thereof include a polymer, cyclodextrin, adamantine and the like. Examples of the cyclodextrin include a linear cyclodextrin copolymer and a linear oxidized cyclodextrin copolymer.

Examples of the additive include a carrier, a binding substance to a target cell, a condensing agent, a fusing agent, an excipient, and the like.

The carrier is preferably a polymer, and more preferably a biopolymer, for example. The carrier is preferably biodegradable, for example. Examples of the carrier include proteins such as human serum albumin (HSA), low density lipoprotein (LDL), and globulin; carbohydrates such as dextran, pullulan, chitin, chitosan, inulin, cyclodextrin, and hyaluronic acid; lipids and the like Can be given. As the carrier, for example, a synthetic polymer such as a synthetic polyamino acid can also be used. Examples of the polyamino acid include polylysine (PLL), poly L-aspartic acid, poly L-glutamic acid, styrene-maleic anhydride copolymer, poly (L-lactide-co-glycolide) copolymer, divinyl ether-maleic anhydride. Copolymer, N- (2-hydroxypropyl) methacrylamide copolymer (HMPA), polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane, poly (2-ethylacrylic acid), N-isopropylacrylamide polymer, or polyphosphazine ) Etc.

Examples of the binding substance include thyroid stimulating hormone, melanocyte stimulating hormone, lectin, glycoprotein, surfactant protein A, mucin carbohydrate, polyvalent lactose, polyvalent galactose, N-acetylgalactosamine, N-acetylglucosamine, polyvalent mannose. Multivalent fucose, glycosylated polyamino acid, polyvalent galactose, transferrin, bisphosphonate, polyglutamic acid, polyaspartic acid, lipid, cholesterol, steroid, bile acid, folate, vitamin B12, biotin, neproxin, RGD peptide, RGD peptide mimetic and the like.

Examples of the fusing agent and condensing agent include polyamino chains such as polyethyleneimine (PEI). PEI may be, for example, linear or branched, and may be either a synthetic product or a natural product. The PEI may be alkyl-substituted or lipid-substituted, for example. In addition, for example, polyhistidine, polyimidazole, polypyridine, polypropyleneimine, melittin, polyacetal substance (for example, cationic polyacetal) and the like can be used as the fusion agent. The fusion agent may have an α helical structure, for example. The fusion agent may be a membrane disrupting agent such as melittin.

The composition of the present invention can use, for example, US Pat. No. 6,509,323, US Patent Publication No. 2003/0008818, PCT / US04 / 07070, and the like for the formation of the complex.

Other examples of the additive include amphiphilic molecules. The amphiphilic molecule is, for example, a molecule having a hydrophobic region and a hydrophilic region. The molecule is preferably a polymer, for example. The polymer is, for example, a polymer having a secondary structure, and a polymer having a repetitive secondary structure is preferable. As a specific example, for example, a polypeptide is preferable, and an α-helical polypeptide is more preferable.

The amphiphilic polymer may be a polymer having two or more amphiphilic subunits, for example. Examples of the subunit include a subunit having a cyclic structure having at least one hydrophilic group and one hydrophobic group. The subunit may have, for example, a steroid such as cholic acid, an aromatic structure, or the like. The polymer may have both a cyclic structure subunit such as an aromatic subunit and an amino acid.

6). Expression suppression method As described above, the expression suppression method of the present invention is a method of suppressing the expression of a target gene, characterized by using the ssNc molecule of the present invention. The expression suppression method of the present invention is characterized by using the ssNc molecule of the present invention, and other processes and conditions are not limited at all.

In the expression suppression method of the present invention, the mechanism of gene expression suppression is not particularly limited, and examples thereof include expression suppression by mature miRNA.

The expression suppression method of the present invention includes, for example, a step of administering the ssNc molecule to a subject in which the target gene is present. By the administration step, for example, the ssNc molecule is brought into contact with the administration subject. Examples of the administration subject include cells, tissues, and organs. Examples of the administration target include non-human animals such as humans and non-human mammals other than humans. The administration can be, for example, in vivo or in vitro .

In the expression suppression method of the present invention, for example, the ssNc molecule may be administered alone, or the composition of the present invention containing the ssNc molecule may be administered. The administration method is not particularly limited, and can be appropriately selected depending on, for example, the type of administration target.

7). Treatment Method The treatment method for a disease of the present invention includes the step of administering the ssNc molecule of the present invention to a patient as described above, and the ssNc molecule serves as a gene causing the disease as the expression-suppressing sequence. It has a sequence that suppresses the expression of. The treatment method of the present invention is characterized by using the ssNc molecule of the present invention, and other steps and conditions are not limited at all.

For the treatment method of the present invention, for example, the expression suppression method of the present invention can be used. The administration method is not particularly limited, and may be, for example, oral administration or parenteral administration.

8). Use of ssNc molecule The use of the present invention is the use of the ssNc molecule of the present invention for suppressing the expression of the target gene.

The nucleic acid molecule of the present invention is a nucleic acid molecule for use in the treatment of a disease, wherein the nucleic acid molecule is the ssNc molecule of the present invention, and the ssNc molecule serves as the cause of the disease as the expression suppression sequence. It has a sequence that suppresses the expression of the gene to be

Hereinafter, the present invention will be described in detail with reference to examples, but the present invention is not limited thereto.

(Example A1)
(1) Synthesis of RNA Single-stranded RNA having the following sequences was synthesized by a nucleic acid synthesizer (trade name: ABI Expedite (registered trademark) 8909 Nucleic Acid Synthesis System, Applied Biosystems) based on the phosphoramidite method. In the synthesis, RNA Phosphoramidites (2′-O-TBDMSi, trade name, Michisato Pharmaceutical) was used as an RNA amidite (hereinafter the same). The deprotection of the amidite followed a conventional method. The synthesized RNA was purified by HPLC. Each purified RNA was lyophilized.

Examples of ssRNA are shown below. In each of the above sequences, the 5 ′ uppercase region is the mature miRNA sequence of human let-7a-1 miRNA (SEQ ID NO: 1), and the 3 ′ uppercase region is the human let-7a-1 miRNA sequence. minor miRNA ^* sequence (SEQ ID NO: 2), and the sequence surrounded by a square is a linker region (Lx) on the 5 ′ side and a linker region (Ly) on the 3 ′ side. In addition, two underlined portions via the 5′-side linker region (Lx) are regions that self-anneal with each other, and two underlined portions via the 3′-side linker region (Ly) self-anneal with each other. The bases marked with * are free bases.

In the PK-0021, PK-0022, PK-0023, PK-0024, and PK-0025, Lx and Ly surrounded by a square can be expressed by the following formula, and amidite of the scheme 3 shown in Example B: Synthesized by using compound 10 (L-proline diamide amidite).

In addition, the following NM-0001 was synthesized as a reference example ssRNA. The NM-0001 is a human let-7a-1 precursor miRNA. In the following sequences, the upper case underline is the mature miRNA sequence of human let-7a-1 miRNA, and the lower case underline is the minor miRNA ^* sequence of human let-7a-1 miRNA. In the arrangement, the area surrounded by a square is a loop area.

(2) Detection of HMGA2 mRNA The ssRNA was introduced into human lung adenocarcinoma epithelial cell line A549 cells, and HMGA2 mRNA targeted by human let-7a-1 miRNA was detected.

The ssRNA was dissolved in distilled water for injection (Otsuka Pharmaceutical, the same applies hereinafter) to prepare a 100 μmol / L RNA solution. As the cells, A549 cells (DS Pharma Biomedical) were used. As the medium, DMEM (Invitrogen) containing 10% FBS was used. The culture conditions were 37 ° C. and 5% CO ₂ .

First, the cells were cultured in the medium, and the culture solution was dispensed into a 24-well plate in 400 μL portions at 5 × 10 ⁴ cells / well. After culturing the cells in the well for 24 hours, the ssRNA was transfected using a transfection reagent HiperFect Reagent (trade name, QIAGEN) according to the attached protocol. For transfection, the composition per well was set as follows. In the following composition, (B) is Opti-MEM (trade name, Invitrogen), and (C) is the RNA solution, and 97 μL of both were added. In the wells, the final ssRNA concentrations were 1 nmol / L, 10 nmol / L, and 100 nmol / L. After transfection, the cells in the wells were cultured for 3 days.

And about the obtained cultured cell, RNA was collect | recovered according to the attached protocol using ISOGEN reagent (brand name, Nippon Gene).

Next, reverse transcriptase (trade name: M-MLV reverse transcriptase, Invitrogen) was used to synthesize cDNA from the RNA according to the attached protocol. Then, quantitative PCR was performed using the synthesized cDNA as a template, and the amount of HMGA2 cDNA was measured. The amount of GAPDH mRNA was used as an internal control, and the amount of cDNA was also measured.

In the quantitative PCR, FastStart Universal SYBR Green Master (trade name, Roche) was used as a reagent, MX3000P (trade name, Stratagene) was used as a thermocycler, and MxPro (trade name, Stratagene) was used as an analysis instrument (hereinafter the same). The following primer sets were used for amplification of the HMGA2 cDNA and GAPDH cDNA, respectively. The total amount of the reaction solution was 25 μL, and each measurement was performed three times.
Primer set for HMGA2 cDNA 5′-GAAGCCACTGGAGAAAAACG-3 ′ (SEQ ID NO: 12)
5'-CTTCGGCAGACTCTTGTGAG-3 '(SEQ ID NO: 13)
GAPDH cDNA primer set 5'-ATGGGGAAGGTGAAGGTCG-3 '(SEQ ID NO: 14)
5'-GGGTCATTGATGGCAATATC-3 '(SEQ ID NO: 15)

As a control, the same treatment and measurement were performed on the A549 cells to which the ssRNA had not been added. Then, when the amount of HMGA2 mRNA in the control (no ssRNA added) was 1, the relative value of HMGA2 mRNA in each transfected cell was calculated.

These results are shown in FIG. FIG. 4 shows the amount of HMGA2 mRNA when the final concentration of ssRNA at the time of transfection is 100 nmol / L. As shown in FIG. 4, when any of the ssRNAs was used, the amount of HMGA2 mRNA was decreased as compared with the control. Thus, since the amount of HMGA2 mRNA is reduced by the transfection of the ssRNA in the Example, it can be said that protein translation is also suppressed.

(3) Detection of mature miRNA sequence The mature miRNA sequence of the human let-7a-1 miRNA was quantified from the RNA recovered in (2). The quantification was performed using TaqMan ™ MicroRNA Assays (trade name, Applied Biosystems) according to the attached instructions. The total amount of the reaction solution was 25 μL, and each measurement was performed three times. The quantification of the mature miRNA sequence was normalized by the amount of RNU6B rRNA. The relative value in each sample was determined with the amount of endogenous feature let7-a-1 in the A549 cells not added with ssRNA being 1.

These results are shown in FIG. FIG. 5 is a graph showing the amount of mature miRNA of the human let-7a-1 miRNA in each transfected cell. In FIG. 5, the vertical axis represents the relative value of the amount of the character let7a-1. As shown in FIG. 5, mature miRNA was detected in the cells transfected with any of the ssRNAs of Examples.

(Example B1)
1. Synthesis of Prolinol Prolinol protected with a dimethoxytrityl group was synthesized according to Scheme 1 shown in the following formula.

(1) Fmoc-L-prolinol (compound 2)
L-prolinol (Compound 1) (0.61 g, 6.0 mmol) was dissolved in 70 mL of pure water to prepare an L-prolinol aqueous solution. N- (9-fluorenylmethoxycarbonyloxy) succinimide (Fmoc-OSu) (2.0 g, 6.0 mmol) was dissolved in 10 mL of THF. This THF solution was added to the L-prolinol aqueous solution and stirred for 1 hour to react both. This reaction solution was separated into a liquid fraction and a precipitate fraction, and each fraction was extracted with ethyl acetate, and the organic layer was recovered. And after match | combining each organic layer, the anhydrous sodium sulfate was added and the water | moisture content was absorbed (henceforth drying). The organic layer was filtered to collect the filtrate, and the filtrate was concentrated under reduced pressure. The obtained residue was purified by silica gel column chromatography (developing solvent hexane: ethyl acetate = 1: 1) to obtain Compound 2 (1.4 g, yield 74%). The NMR results of the compound are shown below.
¹ H-NMR (CDCl ₃ ): δ 7.77 (2H, d, J = 7.7 Hz, Ar—H), 7.60 (2H, d, J = 7.3 Hz, Ar—H), 7.40 (2H, t, J = 7.5 Hz, Ar—H), 7.31 (2H, t, J = 7.6 Hz, Ar—H), 4.40-4.50 (2H, m, COOCH ₂ ) , 4.22 (1H, t, J = 6.5 Hz, Ar—CH), 3.20-3.80 (5H, m, H-5, H-6), 1.75 (3H, m, H -3, H-4), 1.40 (1H, m, H-3).

(2) Fmoc-DMTr-L-prolinol (compound 3)
The Fmoc-L-prolinol (Compound 2) (1.4 g, 4.3 mmol) was dissolved in 20 mL of pyridine and azeotroped three times. The obtained residue was dissolved in 20 mL of pyridine. To this solution, 4,4′-dimethoxytrityl chloride (DMTr—Cl) (1.8 g, 5.3 mmol) was added with stirring in an ice bath under an argon atmosphere. The reaction was monitored by chloroform / methanol TLC and reacted for 4 hours until the Fmoc-L-prolinol spot disappeared. In order to quench excess DMTr-Cl, 3 mL of methanol was added to the reaction solution and stirred for 10 minutes. After further adding chloroform to the reaction solution, the organic layer was recovered. The collected organic layer was washed with a saturated saline solution, washed with a 5% aqueous sodium hydrogen carbonate solution, and again washed with a saturated saline solution. The organic layer after washing was dried over anhydrous sodium sulfate. The organic layer was filtered, and the resulting filtrate was concentrated under reduced pressure. The obtained residue was purified by silica gel column chromatography (developing solvent: chloroform, 1% pyridine) to obtain Compound 3 (2.0 g, yield 74%). The NMR results of the compound are shown below.
¹ H-NMR (CDCl ₃ ): δ 7.77 (2H, d, J = 7.7 Hz, Ar—H), 7.60 (2H, d, J = 7.3 Hz, Ar—H), 7.40 −7.18 (13H, m, Ar—H), 6.89 (4H, d, J = 8.6 Hz, Ar—H), 4.20-4.40 (2H, m, COOCH ₂ ), 4 .02 (1H, t, J = 6.5 Hz, Ar—CH), 3.80-3.10 (5H, m, H-5, H-6), 3.73 (s, 6H, OCH ₃ ) , 1.84 (3H, m, H-3, H-4), 1.58 (1H, m, H-3).

(3) DMTr-L-prolinol (compound 4)
The Fmoc-DMTr-L-prolinol (compound 3) (2.0 g, 3.2 mmol) was dissolved in 25 mL of DMF solution containing 20% piperidine and stirred for 12 hours. The solution was concentrated under reduced pressure, and the resulting residue was purified by silica gel column chromatography (chloroform: methanol = 85: 15, containing 1% pyridine) to obtain Compound 4 (1.0 g, yield 78%). . The NMR results of the compound are shown below.
¹ H-NMR (CDCl ₃ ): δ 7.40-7.14 (9H, m, Ar—H), 6.82 (4H, d, J = 8.6 Hz, Ar—H), 3.78 (6H) , S, OCH ₃ ), 3.31 (1H, m, H-6), 3.07 (2H, m, H-2, H-6), 2.90 (2H, m, H-5), 1.84 (3H, m, H-3, H-4), 1.40 (1H, m, H-3).

2. Synthesis of Amidite Derivative Next, an amidite derivative having prolinol was synthesized according to Scheme 2 shown in the following formula. Hereinafter, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride is referred to as “EDC”, and N, N-dimethylaminopyridine (4-dimethylaminopyridine) is referred to as “DMAP”.

(1) DMTr-amide-L-prolinol (compound 5)
DMTr-L-prolinol (compound 4) (0.80 g, 2.0 mmol), EDC (0.46 g, 2.4 mmol) and DMAP (0.29 g, 2.4 mmol) were dissolved in 20 mL of dichloromethane. Stir. To this solution, 10-hydroxydecanoic acid (0.45 g, 2.4 mmol) was added and stirred. The reaction was monitored by TLC of ethyl acetate and allowed to react for 20 hours until the DMTr-L-prolinol spot disappeared. And after adding dichloromethane to the said reaction liquid, the organic layer was collect | recovered. The collected organic layer was washed with saturated brine, and then dried over anhydrous sodium sulfate. The organic layer was filtered, and the resulting filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (containing ethyl acetate and 1% pyridine) to obtain Compound 5 (0.71 g, yield). 62%). The NMR results of the compound are shown below.
¹ H-NMR (CDCl ₃ ): δ 7.40-7.14 (9H, m, Ar—H), 6.82 (4H, d, J = 8.6 Hz, Ar—H), 3.78 (6H) , S, OCH ₃ ), 3.68-1.93 (7H, m, H-2, H-5, H-6), 2.27-1.72 (6H, m, alkyl, H-3, H-4), 1.58 (4H, s, alkyl), 1.30 (10H, s, alkyl).

(2) DMTr-alkyl-L-prolinol (Compound 6)
The DMTr-L-prolinol (compound 4) (0.80 g, 2.0 mmol) was dissolved in 15 mL of methanol, and 5-hydroxypentanal (0.31 g, 3.0 mmol) was added and stirred. To this solution, sodium cyanoborohydride (0.25 g, 4.0 mmol) was added and further stirred. The reaction was monitored by TLC of ethyl acetate / hexane and allowed to react for 24 hours until the DMTr-L-prolinol spot disappeared. And the ethyl acetate was added to the said reaction liquid, and the organic layer was collect | recovered. The collected organic layer was washed with saturated brine, and then dried over anhydrous sodium sulfate. The organic layer was filtered, the obtained filtrate was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (hexane: ethyl acetate = 1: 1, containing 1% pyridine) to obtain Compound 6 ( 0.62 g, yield 63%). The NMR results of the compound are shown below.
¹ H-NMR (CDCl ₃ ): δ 7.40-7.14 (9H, m, Ar—H), 6.82 (4H, d, J = 8.6 Hz, Ar—H), 3.78 (6H) , S, OCH ₃ ), 3.70-2.86 (4H, m, CH ₂ OH, H-6), 2.06-1.79 (5H, m, alkyl, H-2, H-5) , 1.74-1.49 (6H, m, alkyl, H-3, H-4), 1.45-1.27 (4H, m, alkyl).

(3) DMTr-urethane-L-prolinol (compound 7)
1,4-butanediol (0.90 g, 10 mmol) was dissolved in 30 mL of dichloromethane, carbonyldiimidazole (1.4 g, 8.6 mmol) was further added, and the mixture was stirred for 3 hours. The organic layer of this reaction solution was washed with saturated brine, and then dried over anhydrous sodium sulfate. The organic layer was filtered, the obtained filtrate was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (chloroform: methanol = 9: 1). As a result, a compound in which one end of 1,4-butanediol was activated with carbonyldiimidazole was obtained (0.25 g, 1.5 mmol). This compound was dissolved in 15 mL of dichloromethane, and DMTr-L-prolinol (Compound 4) (0.6 g, 1.5 mmol) was added thereto, followed by stirring for 24 hours. To this mixture, ethyl acetate was further added, and the organic layer was recovered. The collected organic layer was washed with saturated brine, and then dried over anhydrous sodium sulfate. The organic layer was filtered, the obtained filtrate was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (hexane: ethyl acetate = 1: 1, containing 1% pyridine) to obtain Compound 7 ( 0.61 g, yield 77%). The NMR results of the compound are shown below.
¹ H-NMR (CDCl ₃ ): δ 7.40-7.14 (9H, m, Ar—H), 6.82 (4H, d, J = 8.6 Hz, Ar—H), 4.24-3 .94 (2H, m, COOCH ₂ ), 3.78 (s, 6H, OCH ₃ ), 3.72-2.96 (7H, m, alkyl, H-2, H-5, H-6), 2.10-1.30 (8H, m, alkyl, H-3, H-4).

(4) DMTr-Ureido-L-prolinol (Compound 8)
The DMTr-L-prolinol (compound 4) (0.50 g, 1.2 mmol) and triphosgene (0.12 g, 0.40 mmol) were dissolved in 8 mL of dichloromethane and stirred in an ice bath under an argon atmosphere. . Then, N, N-diisopropylethylamine (0.31 g, 2.4 mmol) was added to the solution and stirred for 1 hour. Further, 8-amino-1-octanol (0.17 g, 1.2 mmol) was added to the solution, and the mixture was similarly stirred in an ice bath for 30 minutes and then at room temperature for 20 hours. Dichloromethane was added to the solution, and the organic layer was recovered. The collected organic layer was washed with saturated brine, and then dried over anhydrous sodium sulfate. The organic layer was filtered, and the resulting filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (hexane: ethyl acetate = 4: 1, containing 1% triethylamine) to obtain Compound 8 ( 0.44 g, 62% yield). The NMR results of the compound are shown below.
¹ H-NMR (CDCl ₃ ): δ 7.40-7.14 (9H, m, Ar—H), 6.82 (4H, m, Ar—H), 3.78 (s, 6H, OCH ₃ ) _{_{, 3.68-3.25 (9H, m, CH}} 2 NH, CH 2 OH, H-2, H-5, H-6), 1.74-1.18 (16H, m, alkyl, H- 3, H-4).

(5) Amidite derivatives having prolinol (compounds 9 to 12)
Compounds 9 to 12 were synthesized by the following method using the modified prolinol (compounds 5 to 8) as raw materials. The modified prolinol and 5-benzylthio-1H-tetrazole were dissolved in 3 mL of acetonitrile. The amount of the modified prolinol used is 0.69 g (1.2 mmol) in the case of Compound 5, 0.60 g (1.2 mmol) in the case of Compound 6, and 0.60 g (1.2 mmol) in the case of Compound 7. In the case of Compound 8, the amount was 0.25 g (0.43 mmol). The amount of 5-benzylthio-1H-tetrazole used was 0.15 g (0.78 mmol) for compounds 5 to 7 and 54 mg (0.15 mmol) for compound 8. 2-Cyanoethyl N, N, N ′, N′-tetraisopropyl phosphorodiamidite was added to the solution under an argon atmosphere and stirred for 2 hours. The addition amount of the 2-cyanoethyl N, N, N ′, N′-tetraisopropyl phosphorodiamidite is 0.54 g (1.8 mmol) in the system using the compounds 5 to 7, and the compound 8 is used. In this system, it was 0.19 g (0.64 mmol). And the saturated sodium hydrogencarbonate aqueous solution was added to the said solution, and also it extracted with dichloromethane, and collect | recovered the organic layers. The collected organic layer was dried over anhydrous sodium sulfate. The organic layer was filtered, and the resulting filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (hexane: ethyl acetate = 1: 1, containing 1% triethylamine) to obtain compounds 9 to 12. It was. The NMR results of each compound are shown below.

DMTr-amide-L-prolinol amidite (Compound 9, 0.60 g, yield 55%)
¹ H-NMR (CDCl ₃ ) δ 7.40-7.14 (9H, m, Ar—H), 6.82 (4H, d, J = 8.6 Hz, Ar—H), 3.78 (6H, s, OCH ₃ ), 3.62-2.93 (11H, m, CH ₂ O, POCH ₂ , CHCH ₃ , H-2, H-5, H-6), 2.58 (2H, m, CH ₂ CN), 2.27-1.72 (6H, m, alkyl, H-3, H-4), 1.58 (4H, s, alkyl), 1.30 (22H, s, alkyl), CHCH ₃ ).

DMTr-alkyl-L-prolinol amidite (Compound 10, 0.71 g, 60% yield)
¹ H-NMR (CDCl ₃ ): δ 7.40-7.14 (9H, m, Ar—H), 6.82 (4H, d, J = 8.6 Hz, Ar—H), 3.78 (6H) , S, OCH ₃ ), 3.70-2.86 (8H, m, CH ₂ O, POCH ₂ , CHCH ₃ , H-6), 2.58 (2H, m, CH ₂ CN), 2.06 -1.79 (5H, m, alkyl, H-2, H-5), 1.74-1.49 (6H, m, alkyl, H-3, H-4), 1.37-1.10 (16H, m, alkyl, CHCH _3).

DMTr-urethane-L-prolinol amidite (Compound 11, 0.67 g, Yield 52%)
¹ H-NMR (CDCl ₃ ): δ 7.40-7.14 (9H, m, Ar—H), 6.82 (4H, d, J = 8.6 Hz, Ar—H), 4.24-3 .94 (2H, m, COOCH ₂ ), 3.78 (s, 6H, OCH ₃ ), 3.72-2.96 (11H, m, CH ₂ O, POCH ₂ , CHCH ₃ , H-2, H -5, H-6), 2.58 (2H, m, CH ₂ CN), 2.10-1.46 (8H, m, alkyl, H-3, H-4), 1.34-1. _{10 (12H, m, CHCH 3} ).

DMTr-ureido-L-prolinol amidite (compound 12, 0.20 g, 61% yield)
¹ H-NMR (CDCl ₃ ): δ 7.40-7.14 (9H, m, Ar—H), 6.82 (4H, m, Ar—H), 3.78 (s, 6H, OCH ₃ ) 3.65-3.25 (13H, m, CH ₂ O, POCH ₂ , CHCH ₃ , H-2, CH ₂ NH, CH ₂ OH, H-2, H-5, H-6), 2. _{73 (2H, m, CH 2} CN), 2.10-1.48 (16H, m, alkyl, H-3, H-4 ), 1.35-1.10 (12H, m, CHCH 3).

(Example B2)
Next, an amidite derivative having L-proline was synthesized according to Scheme 3 shown in the following formula.

(1) DMTr-hydroxyamidoamino-L-proline (Compound 11)
To an ethanol solution (7 mL) containing DMTr-amido-L-proline (Compound 6) (1.00 g, 2.05 mmol) and 5-hydroxypentanal (0.33 g, 3.07 mmol) under ice cooling, acetate buffer Liquid (7 mL) was added. The mixture was stirred for 20 minutes under ice-cooling, sodium cyanoborohydride (0.77 g, 12.28 mmol) was added, and the mixture was further stirred at room temperature for 7 hours. The mixture was diluted with dichloromethane, washed with water, and further washed with saturated brine. The organic layer was collected and dried over anhydrous sodium sulfate. The organic layer was filtered, and the solvent was distilled off from the filtrate under reduced pressure. The obtained residue was subjected to silica gel column chromatography (developing solvent CH ₂ Cl ₂ : CH ₃ OH = 98: _2, containing 0.05% pyridine). Subsequently, the obtained product was subjected to silica gel column chromatography (developing solvent CH ₂ Cl ₂ : CH ₃ OH = 98: _2, containing 0.05% pyridine), and the obtained product was further subjected to a silica gel column. Chromatography (developing solvent: dichloromethane: acetone = 7: 3, containing 0.05% pyridine) was used. This gave colorless syrupy compound 11 (0.49 g, 41% yield).
Ms (FAB +): m / z 575 (M ⁺ ), 303 (DMTr ⁺ )

(2) DMTr-amidoamino-L-proline amidite (compound 12)
The obtained DMTr-hydroxyamidoamino-L-proline (Compound 11) (0.50 g, 0.87 mmol) was mixed with anhydrous acetonitrile and azeotropically dried at room temperature. Diisopropylammonium tetrazolide (178 mg, 1.04 mmol) was added to the obtained residue, degassed under reduced pressure, and filled with argon gas. Anhydrous acetonitrile (1 mL) was added to the mixture, and an anhydrous acetonitrile solution (1 mL) of 2-cyanoethoxy-N, N, N ′, N′-tetraisopropyl phosphorodiamidite (313 mg, 1.04 mmol) was further added. added. The mixture was stirred for 4 hours at room temperature under an argon atmosphere. The mixture was diluted with dichloromethane and washed successively with saturated aqueous sodium hydrogen carbonate and saturated brine. The organic layer was collected and dried over anhydrous sodium sulfate, and then the organic layer was filtered. About the obtained filtrate, the solvent was distilled off under reduced pressure. The obtained residue was subjected to column chromatography using aminosilica as a filler (developing solvent hexane: acetone = 7: 3, containing 0.05% pyridine), and colorless syrupy compound 12 (0.57 g, purity) 93%, yield 79%). The purity was measured by HPLC (hereinafter the same). The NMR results of the compound are shown below.
¹ H-NMR (CDCl ₃ ): δ 7.41-7.43 (m, 2H, Ar—H), 7.28-7.32 (m, 4H, Ar—H), 7.25-7.27 (M, 2H, Ar-H), 7.18-7.21 (m, 1H, Ar-H), 6.80-6.84 (m, 4H, Ar-H), 3.73-3. 84 (m, 1H), 3.79 (s, 6H, OCH ₃ ), 3.47-3.64 (m, 3H), 3.12-3.26 (m, 2H), 3.05 (t , J = 6.4 Hz, 2H, CH ₂ ), 2.98-2.02 (m, 2H), 2.61 (t, J = 5.8 Hz, 2H, CH ₂ ), 2.55-2. 63 (m, 2H), 2.27-2.42 (m, 1H, CH), 2.31 (t, 7.8 Hz, 2H, CH ₂ ), 2.03-2.19 (m, 1H, CH), 1.40-1.9 0 (m, 8H), 1.23-1.33 (m, 5H), 1.14-1.20 (m, 12H, CH ₃ );
P-NMR (CDCl ₃ ): δ 146.91;
^{Ms (FAB +): m /} z 774 (M +), 303 (DMTr +), 201 (C 8 H 19 N 2 OP +).

(3) DMTr-hydroxyamidocarbamoyl-L-proline (Compound 13)
To an anhydrous acetonitrile solution (10 mL) in which DMTr-amido-L-proline (Compound 6) (1.00 g, 2.05 mmol) was dissolved, 1-imidazocarbonyloxy-8-hydroxyoctane (1.12 g, 4.92 mmol) An anhydrous acetonitrile solution (20 mL) in which was dissolved was added at room temperature under an argon atmosphere. The mixture was heated at 40-50 ° C. for 2 days and then allowed to stand at room temperature for 5 days. About the said liquid mixture, the solvent was distilled off under reduced pressure. The obtained residue was subjected to silica gel column chromatography (developing solvent: dichloromethane: acetone = 4: 1, containing 0.05% pyridine). This gave colorless syrup-like compound 13 (0.68 g, yield 50%). The NMR results of the compound are shown below.
¹ H-NMR (CDCl ₃ ): δ 7.40-7.42 (m, 2H, Ar—H), 7.27-7.31 (m, 6H, Ar—H), 7.17-7.21 (M, 1H, Ar-H), 6.79-6.82 (m, 4H, Ar-H), 4.23-4.30 (m, 1H), 4.05-4.10 (m, 2H), 3.79 (s, 6H, OCH ₃ ), 3.60-3.65 (m, 2H), 3.32-3.55 (m, 2H), 3.16-3.29 (m) , 2H), 3.01-3.07 (m, 2H), 2.38-2.40 (m, 1H, CH), 1.83-1.90 (m, 2H), 1.57-1 .69 (m, 8H), 1.26-1.36 (m, 2H);
Ms (FAB +): m / z 602 (M ^<+> ), 303 (DMTr ^<+> ).

(4) DMTr-amidocarbamoyl-L-proline amidite (Compound 14)
The obtained DMTr-hydroxyamidocarbamoyl-L-proline (Compound 13) (0.63 g, 1.00 mmol) was mixed with anhydrous pyridine and azeotropically dried at room temperature. Diisopropylammonium tetrazolide (206 mg, 1.20 mmol) was added to the resulting residue, degassed under reduced pressure, and filled with argon gas. Anhydrous acetonitrile (1 mL) was added to the mixture, and an anhydrous acetonitrile solution (1 mL) of 2-cyanoethoxy-N, N, N ′, N′-tetraisopropyl phosphorodiamidite (282 mg, 1.12 mmol) was further added. added. The mixture was stirred for 4 hours at room temperature under an argon atmosphere. The mixture was diluted with dichloromethane and washed successively with saturated aqueous sodium hydrogen carbonate and saturated brine. The organic layer was collected and dried over anhydrous sodium sulfate, and then the organic layer was filtered. About the obtained filtrate, the solvent was distilled off under reduced pressure. The obtained residue was subjected to column chromatography (developing solvent hexane: acetone = 7: 3, containing 0.5% pyridine) using amino silica as a filler, and colorless syrup-like compound 14 (0.74 g, purity 100) %, Yield 87%). The NMR results of the compound are shown below.
P-NMR (CDCl ₃ ): δ 147.19;
Ms (FAB +): m / z 860 (M ⁺ ), 303 (DMTr ⁺ ), 201 (C ₈ H ₁₉ N ₂ OP ⁺ ).

(5) DMTr-t-butyldimethylsiloxyamide ureido-L-proline (Compound 15)
Anhydrous tetrahydrofuran solution (10 mL) was added to triphosgene (1.22 g, 4.10 mmol) under an argon atmosphere and ice cooling. An anhydrous tetrahydrofuran solution (DMr-amido-L-proline (Compound 6) (1.00 g, 2.05 mmol)) and DIEA (9.80 g, 75.8 mmol) dissolved in an argon atmosphere and ice-cooled in this mixed solution ( 10 mL) was added dropwise over 30 minutes, followed by stirring at room temperature for 1 hour. An anhydrous tetrahydrofuran solution in which 10-amino-1-t-butyldimethylsiloxydecane (2.66 g, 10.25 mmol) and DIEA (3.20 g, 24.76 mmol) were dissolved in the mixed solution under an argon atmosphere and ice-cooling. (20 mL) was added dropwise over 45 minutes. The mixture was stirred overnight at room temperature under an argon atmosphere. The mixture was diluted with ethyl acetate (200 mL), and the organic layer was collected. The organic layer was washed with saturated aqueous sodium hydrogen carbonate, and then further washed with saturated brine. The organic layer was recovered and dried over anhydrous sodium sulfate. The organic layer was filtered, and the solvent was distilled off from the filtrate under reduced pressure. The obtained residue was subjected to silica gel column chromatography (developing solvent: dichloromethane: acetone = 4: 1, containing 0.05% pyridine). This gave colorless syrupy compound 15 (0.87 g, 55% yield).

(6) DMTr-hydroxyamidoureido-L-proline (Compound 16)
To the obtained DMTr-t-butyldimethylsiloxyamidoureido-L-proline (Compound 15) (0.87 g, 1.12 mmol), an anhydrous tetrahydrofuran dichloromethane solution (10 mL) was added at room temperature under an argon atmosphere. A 1 mol / L tetrabutylammonium fluoride-containing tetrahydrofuran solution (4.69 mL, Tokyo Kasei) was added to the mixture under an argon atmosphere, and the mixture was stirred at room temperature for 3 days. The mixture was diluted with dichloromethane (150 mL), washed with water, and further washed with saturated brine. The organic layer was collected and dried over anhydrous sodium sulfate, and then the organic layer was filtered. About the obtained filtrate, the solvent was distilled off under reduced pressure. The obtained residue was subjected to silica gel column chromatography (developing solvent: dichloromethane: acetone = 1: 1, containing 0.05% pyridine) to obtain colorless syrup-like compound 16 (0.68 g, yield 92%). . The NMR results of the compound are shown below.
¹ H-NMR (CDCl ₃ ): δ 7.41-7.43 (m, 2H, Ar—H), 7.27-7.31 (m, 4H, Ar—H), 7.19-7.26 (M, 2H, Ar-H), 7.19-7.21 (m, 1H, Ar-H), 6.80-6.83 (m, 4H, Ar-H), 4.34 (t, 2H, CH ₂ ), 3.79 (s, 6H, OCH ₃ ), 3.63 (d, 1H, J = 6.4 Hz, CH ₂ ), 3.61 (d, 1H, J = 6.4 Hz, _{CH 2), 3.34-3.37 (m,} 1H, CH), 3.16-3.27 (m, 5H), 3.04 (t, J = 5.9Hz, 2H, CH 2), 2.38-2.45 (m, 1H, CH), 1.83 to 2.05 (m, 3H), 1.45 to 1.64 (m, 8H), 1.25 to 1.38 (m , 7H).

(7) DMTr-amidoureido-L-proline amidite (Compound 17)
The obtained DMTr-hydroxyamidoureido-L-proline (Compound 16) (0.62 g, 0.94 mmol) was mixed with anhydrous acetonitrile and azeotropically dried at room temperature. Diisopropylammonium tetrazolide (192 mg, 1.12 mmol) was added to the resulting residue, degassed under reduced pressure, and filled with argon gas. Anhydrous acetonitrile (1 mL) was added to the mixture, and 2-cyanoethoxy-N, N, N ′, N′-tetraisopropyl phosphorodiamidite (282 mg, 1.12 mmol) in anhydrous acetonitrile (1 mL) was further added. Was added. The mixture was stirred for 4 hours at room temperature under an argon atmosphere. The mixture was diluted with dichloromethane and washed successively with saturated aqueous sodium hydrogen carbonate and saturated brine. The organic layer was collected and dried over anhydrous sodium sulfate, and then the organic layer was filtered. About the obtained filtrate, the solvent was distilled off under reduced pressure. The obtained residue was subjected to column chromatography (developing solvent hexane: acetone = 1: 1, containing 0.05% pyridine) using amino silica as a filler to obtain colorless syrup-like compound 17 (0.77 g). , Purity 88%, yield 84%). The NMR results of the compound are shown below.
P-NMR (CDCl ₃ ): δ 147.27;
Ms (FAB +): m / z 860 (M ⁺ +1), 303 (DMTr ⁺ ), 201 (C ₈ H ₁₉ N ₂ OP ⁺ ).

Example B3 Synthesis of Proline Diamide Amidite L-proline diamide amidite and D-proline diamide amidite were synthesized according to Scheme 3 above to generate a nucleic acid molecule of the present invention containing a linker having a proline skeleton.

(B3-1) L-proline diamide amidite (1) Fmoc-hydroxyamide-L-proline (compound 4)
Compound 2 (Fmoc-L-proline) in Scheme 3 was used as a starting material. Compound 2 (10.00 g, 29.64 mmol), 4-amino-1-butanol (3.18 g, 35.56 mmol) and 1-hydroxybenzotriazole (10.90 g, 70.72 mmol) were mixed, and the mixture On the other hand, it was deaerated under reduced pressure and filled with argon gas. To the mixture was added anhydrous acetonitrile (140 mL) at room temperature, and further an anhydrous acetonitrile solution (70 mL) of dicyclohexylcarbodiimide (7.34 g, 35.56 mmol) was added, followed by stirring at room temperature for 15 hours under an argon atmosphere. After completion of the reaction, the produced precipitate was filtered off, and the solvent was distilled off from the collected filtrate under reduced pressure. Dichloromethane (200 mL) was added to the resulting residue and washed with saturated aqueous sodium hydrogen carbonate (200 mL). And after collect | recovering an organic layer and drying with magnesium sulfate, the said organic layer was filtered. About the obtained filtrate, the solvent was distilled off under reduced pressure, and diethyl ether (200 mL) was added to the residue to form a powder. The resulting powder was collected by filtration to obtain colorless powdered compound 4 (10.34 g, yield 84%). The NMR results of the above compound are shown below.
¹ H-NMR (CDCl ₃ ): δ 7.76-7.83 (m, 2H, Ar—H), 7.50-7.63 (m, 2H, Ar—H), 7.38-7.43 (M, 2H, Ar-H), 7.28-7.33 (m, 2H, Ar-H), 4.40-4.46 (m, 1H, CH), 4.15-4.31 ( m, 2H, CH ₂ ), 3.67-3.73 (m, 2H, CH ₂ ), 3.35-3.52 (m, 2H, CH ₂ ), 3.18-3.30 (m, _{2H, CH 2), 2.20-2.50 (} m, 4H), 1.81-2.03 (m, 3H), 1.47-1.54 (m, 2H);
Ms (FAB +): m / z 409 (M + H ^< + ^> ).

(2) DMTr-amide-L-proline (Compound 6)
Fmoc-hydroxyamide-L-proline (compound 4) (7.80 g, 19.09 mmol) was mixed with anhydrous pyridine (5 mL) and azeotropically dried twice at room temperature. To the resulting residue, 4,4′-dimethoxytrityl chloride (8.20 g, 24.20 mmol), DMAP (23 mg, 0.19 mmol) and anhydrous pyridine (39 mL) were added. The mixture was stirred at room temperature for 1 hour, methanol (7.8 mL) was added, and the mixture was stirred at room temperature for 30 minutes. The mixture was diluted with dichloromethane (100 mL), washed with saturated aqueous sodium hydrogen carbonate (150 mL), and the organic layer was separated. The organic layer was dried over anhydrous sodium sulfate, and then the organic layer was filtered. About the obtained filtrate, the solvent was distilled off under reduced pressure. To the resulting crude residue, anhydrous dimethylformamide (39 mL) and piperidine (18.7 mL, 189 mmol) were added and stirred at room temperature for 1 hour. After completion of the reaction, the solvent was distilled off from the mixed solution at room temperature under reduced pressure. The obtained residue was subjected to silica gel column chromatography (trade name Wakogel C-300, developing solvent CH ₂ Cl ₂ : CH ₃ OH = 9: 1, containing 0.05% pyridine, to give pale yellow oily compound 6 (9. 11 g, yield 98%) The NMR results of the above compound are shown below.
¹ H-NMR (CDCl ₃ ): δ 7.39-7.43 (m, 2H, Ar—H), 7.30 (d, J = 8.8 Hz, 4H, Ar—H), 7, 21 (tt , 1H, 4.9, 1.3 Hz, Ar—H), 6.81 (d, J = 8.8 Hz, 4H, Ar—H), 3.78 (s, 6H, OCH ₃ ), 3.71 (Dd, H, J = 6.3 Hz, 5.4 Hz, CH), 3.21 (2H, 12.9, 6.3 Hz, 2H, CH ₂ ), 3.05 (t, J = 6.3 Hz, 2H, CH ₂ ), 2.85-2.91 (m, 2H, CH ₂ ), 2.08-2.17 (m, 1H, CH), 1.85-2.00 (m, 3H), 1.55-1.65 (m, 5H):
Ms (FAB +); m / z 489 (M + H ^< + ^> ), 303 (DMTr ^<+> ).

(3) DMTr-hydroxydiamide-L-proline (Compound 8)
The obtained DMTr-amide-L-proline (Compound 6) (6.01 g, 12.28 mmol), EDC (2.83 g, 14.74 mmol), 1-hydroxybenzotriazole (3.98 g, 29.47 mmol) And triethylamine (4.47 g, 44.21 mmol) in anhydrous dichloromethane (120 mL) were mixed. To this mixture was further added 6-hydroxyhexanoic acid (1.95 g, 14.47 mmol) at room temperature under an argon atmosphere, and then stirred at room temperature for 1 hour under an argon atmosphere. The mixture was diluted with dichloromethane (600 mL) and washed 3 times with saturated brine (800 mL). The organic layer was collected, and the organic layer was dried over anhydrous sodium sulfate, and then the organic layer was filtered. About the obtained filtrate, the solvent was distilled off under reduced pressure. As a result, pale yellow foamy compound 8 (6.29 g, yield 85%) was obtained. The NMR results of the above compound are shown below.
¹ H-NMR (CDCl ₃ ): δ 7.41-7.43 (m, 2H, Ar—H), 7.27-7.31 (m, 4H, Ar—H), 7.19-7.26 (M, 2H, Ar-H), 7.17-7.21 (m, 1H, Ar-H), 6.79-6.82 (m, 4H, Ar-H), 4.51-4. 53 (m, 1H, CH), 3.79 (s, 6H, OCH ₃ ), 3.61 (t, 2H, J = 6.4 Hz, CH ₂ ), 3.50 to 3.55 (m, 1H) , CH), 3.36-3.43 (m, 1H, CH), 3.15-3.24 (m, 2H, CH ₂ ), 3.04 (t, J = 6.3 Hz, 2H, CH ₂ ), 2.38-2.45 (m, 1H, CH), 2.31 (t, 6.8 Hz, 2H, CH ₂ ), 2.05-2.20 (m, 1H, CH), 1 .92-2.00 ( , 1H, CH), 1.75-1.83 ( m, 1H, CH), 1.48-1.71 (m, 8H), 1.35-1.44 (m, 2H, CH 2);
Ms (FAB +): m / z 602 (M ^<+> ), 303 (DMTr ^<+> ).

(4) DMTr-Diamide-L-proline amidite (Compound 10)
The obtained DMTr-hydroxydiamide-L-proline (Compound 8) (8.55 g, 14.18 mmol) was mixed with anhydrous acetonitrile and dried azeotropically three times at room temperature. Diisopropylammonium tetrazolide (2.91 g, 17.02 mmol) was added to the resulting residue, degassed under reduced pressure, and filled with argon gas. Anhydrous acetonitrile (10 mL) was added to the mixture, and 2-cyanoethoxy-N, N, N ′, N′-tetraisopropyl phosphorodiamidite (5.13 g, 17.02 mmol) in anhydrous acetonitrile (7 mL) was added. ) Was added. The mixture was stirred at room temperature for 2 hours under an argon atmosphere. The mixture was diluted with dichloromethane, washed 3 times with saturated aqueous sodium hydrogen carbonate (200 mL), and then washed with saturated brine (200 mL). The organic layer was collected and dried over anhydrous sodium sulfate, and then the organic layer was filtered. About the obtained filtrate, the solvent was distilled off under reduced pressure. The obtained residue was subjected to column chromatography using amino silica gel as a filler (developing solvent hexane: ethyl acetate = 1: 3, containing 0.05% pyridine) to give colorless syrup-like compound 10 (10.25 g, 92% purity, 83% yield). The NMR results of the above compound are shown below.
¹ H-NMR (CDCl ₃ ): δ 7.40-7.42 (m, 2H, Ar—H), 7.29-7.31 (m, 4H, Ar—H), 7.25-7.27 (M, 2H, Ar-H), 7.17-7.21 (m, 1H, Ar-H), 6.80-6.82 (m, 4H, Ar-H), 4.51-4. 53 (m, 1H, CH), 3.75-3.93 (m, 4H), 3.79 (s, 6H, OCH ₃ ), 3.45-3.60 (m, 4H), 3.35 −3.45 (m, 1H, CH), 3.20−3.29 (m, 1H), 3.04 (t, J = 6.4 Hz, 2H, CH ₂ ), 2.62 (t, J = 5.8 Hz, 2 H, CH ₂ ), 2.40-2.44 (m, 1 H, CH), 2.31 (t, 7.8 Hz, 2 H, CH ₂ ), 2.03-2.19 ( m, 1H, CH). 2-2.02 (m, 1H, CH), 1.70-1.83 (m, 1H, CH), 1.51-1.71 (m, 8H), 1.35-1.44 (m , 2H, CH ₂ ), 1.18 (d, J = 6.8 Hz, 6H, CH ₃ ), 1.16 (d, J = 6.8 Hz, 6H, CH ₃ );
P-NMR (CDCl ₃ ): Msδ 147.17;
^{Ms (FAB +): m /} z 802 (M +), 303 (DMTr +), 201 (C 8 H 19 N 2 OP +).

(B3-2) D-proline diamide amidite (1) Fmoc-hydroxyamide-D-proline (compound 3)
Compound 1 (Fmoc-D-proline) in Scheme 3 was used as a starting material. The mixture of Compound 1 (1.5 g, 4.45 mmol), dicyclohexylcarbodiimide (1.1 g, 5.34 mmol) and 1-hydroxybenzotriazole (1.5 g, 10.69 mmol) was degassed under reduced pressure. And filled with argon gas. Anhydrous acetonitrile (24 mL) was added to the mixture at room temperature, and further 4-amino-1-butanol (0.48 g, 5.34 mmol) in anhydrous acetonitrile (6 mL) was added. Stir for hours. After completion of the reaction, the produced precipitate was filtered off, and the solvent was distilled off from the collected filtrate under reduced pressure. Dichloromethane was added to the resulting residue, and the mixture was washed 3 times with an acetic acid buffer (pH 4.0) and 3 times with a saturated aqueous sodium bicarbonate solution. And after collect | recovering an organic layer and drying with magnesium sulfate, the said organic layer was filtered. About the obtained filtrate, the solvent was distilled off under reduced pressure, and diethyl ether (50 mL) was added to the residue to form a powder. The resulting powder was collected by filtration to obtain Compound 3 as a white powder. The NMR results of the above compound are shown below.
¹ H-NMR (400 MHz, CDCl ₃ ): δ 7.77 (d, J = 7.3 Hz, 2H); 7.58 (br, 2H); 7.41 (t, J = 7.3 Hz, 2H); 7.32 (t, J = 7.3 Hz, 2H); 4.25-4.43 (m, 4H); 3.25-3.61 (m, 6H); 1.57-1.92 (m , 8H).
MS (FAB +): m / z 409 (M + H ^< + ^> ).

(2) DMTr-amide-D-proline (compound 5)
Fmoc-hydroxyamide-D-proline (compound 3) (1.0 g, 2.45 mmol) was mixed with anhydrous pyridine (5 mL) and dried azeotropically twice at room temperature. To the obtained residue, 4,4′-dimethoxytrityl chloride (1.05 g, 3.10 mmol), DMAP (3 mg, 0.024 mmol) and anhydrous pyridine (5 mL) were added. The mixture was stirred at room temperature for 1 hour, methanol (1 mL) was added, and the mixture was stirred at room temperature for 30 minutes. The mixture was diluted with dichloromethane and washed with saturated aqueous sodium hydrogen carbonate, and the organic layer was separated. The organic layer was dried over anhydrous sodium sulfate, and then the organic layer was filtered. About the obtained filtrate, the solvent was distilled off under reduced pressure. To the resulting crude residue, anhydrous dimethylformamide (5 mL) and piperidine (2.4 mL, 24 mmol) were added and stirred at room temperature for 1 hour. After completion of the reaction, the solvent was distilled off from the mixed solution at room temperature under reduced pressure. The obtained residue was subjected to silica gel column chromatography (trade name: Wakogel C-300, developing solvent CH ₂ Cl ₂ : CH ₃ OH = 9: 1, containing 0.05% pyridine) to obtain Compound 5 (1.26 g, yield 96%) as a pale yellow oil. The NMR results of the above compound are shown below.
¹ H-NMR (400 MHz, CDCl ₃ ): δ 7.62 (br, 1H); 7.41-7.44 (m, 2H); 7.26-7.33 (m, 6H); 7.17- 7.22 (m, 1H); 6.80-6.84 (m, 4H); 3.78 (s, 6H); 3.71 (dd, J = 8.8, 5.4 Hz, 1H); 3.22 (q, 6.5 Hz, 2H); 3.07 (t, J = 6.1 Hz, 2H); 2.97-3.03 (m, 1H); 2.85-2.91 (m , 1H); 1.85-2.15 (m, 3H); 1.55-1.73 (m, 6H).
MS (FAB +): m / z 489 (M + H ^< + ^> ), 303 (DMTr ^<+> ).

(3) DMTr-hydroxydiamide-D-proline (Compound 7)
The obtained DMTr-amide-D-proline (compound 5) (1.2 g, 2.45 mmol), EDC (566 mg, 2.95 mmol), 1-hydroxybenzotriazole (796 mg, 5.89 mmol), and triethylamine ( 1.2 mL, 8.84 mmol) in anhydrous dichloromethane (24 mL) was mixed. To this mixture was further added 6-hydroxyhexanoic acid (390 mg, 2.95 mmol) at room temperature under an argon atmosphere, and then stirred at room temperature for 1 hour under an argon atmosphere. The mixture was diluted with dichloromethane and washed 3 times with saturated aqueous sodium bicarbonate. The organic layer was collected, and the organic layer was dried over anhydrous sodium sulfate, and then the organic layer was filtered. About the obtained filtrate, the solvent was distilled off under reduced pressure. This gave compound 7 (1.4 g, yield 95%) as a pale yellow oil. The NMR results of the above compound are shown below.
¹ H-NMR (400 MHz, CDCl ₃ ): δ 7.40-7.43 (m, 2H); 7.25-7.32 (m, 6H); 7.17-7.22 (m, 1H); 6.79-6.83 (m, 4H); 3.79 (s, 6H); 3.58-3.63 (m, 2H); 3.49-3.55 (m, 1H); 15-3.26 (m, 2H); 3.02-3.07 (m, 2H); 2.30-2.33 (m, 2H); 2.11-2.20 (m, 1H); 1.50-1.99 (m, 13H); 1.36-1.43 (m, 2H).
MS (FAB +): m / z 602 (M ^<+> ), 303 (DMTr ^<+> ).

(4) DMTr-Diamide-D-proline amidite (Compound 9)
The obtained DMTr-hydroxydiamide-D-proline (compound 7) (1.2 g, 1.99 mmol) was mixed with anhydrous acetonitrile and dried azeotropically three times at room temperature. Diisopropylammonium tetrazolide (410 mg, 2.40 mmol) was added to the obtained residue, degassed under reduced pressure, and filled with argon gas. To the mixture was added anhydrous acetonitrile (2.4 mL), and 2-cyanoethoxy-N, N, N ′, N′-tetraisopropyl phosphorodiamidite (722 mg, 2.40 mmol) was further added. The mixture was stirred at room temperature for 2 hours under an argon atmosphere. The mixture was diluted with dichloromethane, washed 3 times with saturated aqueous sodium hydrogen carbonate, and then washed with saturated brine. The organic layer was collected and dried over anhydrous sodium sulfate, and then the organic layer was filtered. About the obtained filtrate, the solvent was distilled off under reduced pressure. The obtained residue was subjected to column chromatography (developing solvent hexane: ethyl acetate = 1: 3) using amino silica gel as a filler to give colorless oily compound 9 (1.4 g, purity 95%, yield 83%). ) The NMR results of the above compound are shown below.
¹ H-NMR (400 MHz, CDCl ₃ ): δ 7.40-7.43 (m, 2H); 7.25-7.32 (m, 6H); 7.14-7.21 (m, 1H); 6.80-6.83 (m, 4H); 3.80-3.85 (m, 2H); 3.79 (s, 6H); 3.49-3.65 (m, 5H); 02-3.06 (m, 2H); 2.60-2.63 (m, 2H); 2.29-2.33 (m, 2H); 1.77-1.82 (m, 2H); 1.56-1.68 (m, 8H); 1.38-1.43 (m, 2H); 1.15-1.29 (m, 18H).
³¹ P-NMR (162 MHz, CDCl ₃ ): δ146.94.
^{MS (FAB +): m /} z 802 (M +), 303 (DMTr +), 201 (C 8 H 19 N 2 OP +).

(Example B4)
In order to generate a nucleic acid molecule of the present invention containing a linker having a proline skeleton, L-proline diamide amidite type B was synthesized according to Scheme 4 below.

(1) Fmoc-t-butyl-dimethylsiloxyamide-L-proline (Compound 18)
Fmoc-hydroxyamide-L-proline (compound 4) (2.00 g, 30 mmol), t-butyl-dimethylsilyl chloride (1.11 g, 35 mmol) and imidazole (10.90 g, 71 mmol) were mixed. The mixture was degassed under reduced pressure and filled with argon gas. To the mixture was added anhydrous acetonitrile (20 mL) at room temperature, and the mixture was stirred at room temperature overnight under an argon atmosphere. After completion of the reaction, dichloromethane (150 mL) was added to the mixture, washed 3 times with water, and washed with saturated brine. The organic layer was collected and dried over magnesium sulfate, and then the organic layer was filtered. About the obtained filtrate, the solvent was distilled off under reduced pressure, and the residue was subjected to silica gel column chromatography (developing solvent CH ₂ Cl ₂ : CH ₃ OH = 95: 5) to give colorless syrup-like compound 18 (2. 35 g, yield 92%). The NMR results of the above compound are shown below.
¹ H-NMR (CDCl ₃ ): δ 7.76-7.78 (m, 2H, Ar—H), 7.50-7.63 (m, 2H, Ar—H), 7.38-7.42 (m, 2H, Ar-H ), 7.29-7.34 (m, 2H, Ar-H), 4.10-4.46 (m, 4H, CH 2), 3.47-3.59 (M, 4H, CH ₂ ), 3.20-3.26 (m, 2H, CH), 1.85-1.95 (m, 2H), 1.42-1.55 (m, 6H), 0.96 (s, 9H, t-Bu), 0.02 (s, 6H, SiCH ₃ );
Ms (FAB +): m / z 523 (M + H ^< + ^> ).

(2) t-butyl-dimethylsiloxyamide-L-proline (Compound 19)
To the obtained Fmoc-t-butyl-dimethylsiloxyamide-L-proline (compound 18) (1.18 g, 2.5 mmol), anhydrous acetonitrile (5 mL) and piperidine (2.4 mL) were added, and at room temperature. Stir for 1 hour. After completion of the reaction, acetonitrile (50 mL) was added to the mixture, and insolubles were filtered off. About the obtained filtrate, the solvent was distilled off under reduced pressure, and the resulting residue was subjected to silica gel column chromatography (developing solvent CH ₂ Cl ₂ : CH ₃ OH = 9: 1) to give colorless syrup-like compound 19 ( 0.61 g, yield 90%) was obtained. The NMR results of the above compound are shown below.
¹ H-NMR (CDCl ₃ ): δ 3.71 (dd, 1H, J = 9.0 Hz, 5.2 Hz, CH), 3.61-3.64 (m, 2H, CH ₂ ), 3.22- 3.28 (m, 2H, CH ₂ ), 2.98-3.04 (m, 1H, CH), 2.86-2.91 (m, 1H, CH), 2.08-2.17 ( m, 1H, CH), 1.86-1.93 (m, 1H, CH), 1.66-1.75 (m, 2H, CH 2), 1.52-1.57 (m, 4H) , 0.89 (s, 9H, t-Bu), 0.05 (s, 6H, SiCH ₃ ):
Ms (FAB +); m / z 301 (M + H ⁺ ).

(3) t-butyl-dimethylsiloxyamide hydroxyamide-L-proline (Compound 20)
The resulting t-butyl-dimethylsiloxyamide-L-proline (Compound 19) (550 mg, 1.8 mmol), 6-hydroxyhexanoic acid (300 mg, 2.3 mmol), EDC (434 mg, 2.3 mmol), and An anhydrous dichloromethane solution (20 mL) of 1-hydroxybenzotriazole (695 mg, 4.5 mmol) was mixed. Triethylamine (689 mg, 6.8 mmol) was added to the mixture at room temperature under an argon atmosphere, and then the mixture was stirred overnight at room temperature under an argon atmosphere. The mixture was washed with saturated brine. The organic layer was collected and dried over anhydrous sodium sulfate, and then the organic layer was filtered. About the obtained filtrate, the solvent was distilled off under reduced pressure. The resulting residue was subjected to silica gel column chromatography (developing solvent CH ₂ Cl ₂ : CH ₃ OH = 9: 1) to obtain colorless syrup-like compound 20 (696 mg, yield 92%). The NMR results of the above compound are shown below.
¹ H-NMR (CDCl ₃ ): δ 4.54 (d, 1H, CH), 3.58-3.67 (m, 5H), 3.52-3.56 (m, 1H, CH), 3. 32-3.39 (m, 1H), 3.20-3.25 (m, 2H), 2.40-2.43 (m, 1H, CH), 2.33 (t, J = 7.3 Hz) , 2H, CH ₂ ), 2.05-2.25 (m, 2H), 1.93-2.03 (m, 1H, CH), 1.75-1.85 (m, 1H, CH), 1.50-1.73 (m, 8H), 1.37-1.46 (m, 2H, CH ₂ ), 0.87 (s, 9H, t-Bu), 0.04 (s, 6H, SiCH ₃ );
Ms (FAB +): m / z 415 (M ⁺⁺ 1).

(4) DMTr-hydroxydiamide-L-proline type B (Compound 21)
The obtained t-butyl-dimethylsiloxyamide hydroxyamide-L-proline (Compound 20) (640 mg, 1.54 mmol) was mixed with anhydrous pyridine (1 mL) and azeotropically dried at room temperature. To the obtained residue, 4,4′-dimethoxytrityl chloride (657 mg, 1.85 mmol), DMAP (2 mg) and anhydrous pyridine (5 mL) were added, and the mixture was stirred at room temperature for 4 hours, and then methanol (1 mL) was added. And stirred for 30 minutes at room temperature. The mixture was diluted with dichloromethane and washed with saturated aqueous sodium hydrogen carbonate. The organic layer was collected and dried over anhydrous sodium sulfate, and then the organic layer was filtered. About the obtained filtrate, the solvent was distilled off under reduced pressure. To the obtained residue were added anhydrous acetonitrile (5 mL) and 1 mol / L tetrabutylammonium fluoride-containing tetrahydrofuran solution (1.42 mL, tetrabutylammonium fluoride 1.42 mmol), and the mixture was stirred at room temperature overnight. After completion of the reaction, ethyl acetate (100 mL) was added to the mixture, washed with water, and then washed with saturated brine. The organic layer was collected and dried over anhydrous sodium sulfate, and then the organic layer was filtered. About the obtained filtrate, the solvent was distilled off under reduced pressure. The obtained residue was subjected to silica gel column chromatography (developing solvent CH ₂ Cl ₂ : CH ₃ OH = 95: 5, containing 0.05% pyridine) to give colorless syrup-like compound 21 (680 mg, yield 73%). Got. The NMR results of the above compound are shown below.
¹ H-NMR (CDCl ₃ ): δ 7.41-7.44 (m, 2H, Ar—H), 7.26-7.33 (m, 4H, Ar—H), 7.18-7.21 (M, 2H, Ar-H), 7.17-7.21 (m, 1H, Ar-H), 6.80-6.84 (m, 4H, Ar-H), 4.51-4. 53 (d, 6.8 Hz, 1 H, CH), 3.79 (s, 6 H, OCH ₃ ), 3.61 (dd, 2 H, J = 11 Hz, 5.4 Hz, CH ₂ ), 3.50-3 .54 (m, 1H, CH), 3.36-3.43 (m, 1H, CH), 3.20-3.26 (m, 2H, CH ₂ ), 3.05 (t, J = 6 .4 Hz, 2 H, CH ₂ ), 2.38-2.45 (m, 1 H, CH), 2.30 (t, J = 7.8 Hz, 2 H, CH ₂ ), 2.05-2.25 ( m, 1H, C H), 1.92-2.00 (m, 1H, CH), 1.75-1.83 (m, 1H, CH), 1.52-1.67 (m, 8H), 1.35 _{1.45 (m, 2H, CH 2} );
Ms (FAB +): m / z 602 (M ^<+> ), 303 (DMTr ^<+> ).

(5) DMTr-Diamide-L-proline amidite type B (Compound 22)
The obtained DMTr-hydroxydiamide-L-proline type B (Compound 21) (637 mg, 1.06 mmol) was mixed with anhydrous acetonitrile and azeotropically dried at room temperature. Diisopropylammonium tetrazolide (201 mg, 1.16 mmol) was added to the obtained residue, degassed under reduced pressure, and filled with argon gas. Anhydrous acetonitrile (1 mL) was added to the mixture, and an anhydrous acetonitrile solution (1 mL) of 2-cyanoethoxy-N, N, N ′, N′-tetraisopropyl phosphorodiamidite (350 mg, 1.16 mmol) was further added. added. The mixture was stirred for 4 hours at room temperature under an argon atmosphere. The mixture was diluted with dichloromethane and washed with saturated aqueous sodium hydrogen carbonate and saturated brine. The organic layer was collected and dried over anhydrous sodium sulfate, and then the organic layer was filtered. About the obtained filtrate, the solvent was distilled off under reduced pressure. The obtained residue was subjected to column chromatography using amino silica gel as a filler (developing solvent hexane: acetone = 7: 3) to give colorless syrup-like compound 22 (680 mg, purity 95%, yield 76%). Obtained. The NMR results of the above compound are shown below.
¹ H-NMR (CDCl ₃ ): δ 7.41-7.43 (m, 2H, Ar—H), 7.25-7.32 (m, 4H, Ar—H), 7.17-7.22 (M, 2H, Ar-H), 6.80-6.83 (m, 4H, Ar-H), 4.53 (d, J = 7.8 Hz, 1H, CH), 3.75-3. 93 (m, 3H), 3.79 (s, 6H, OCH ₃ ), 3.46-3.68 (m, 5H), 3.34-3.41 (m, 1H, CH), 3.10 −3.31 (m, 1H, CH), 3.05 (t, J = 6.3 Hz, 2H, CH ₂ ), 2.62 (t, J = 6.3 Hz, 2H, CH ₂ ), 2. 39-2.46 (m, 1 H, CH), 2.29 (t, 7.3 Hz, 2 H, CH ₂ ), 2.03-2.19 (m, 1 H, CH), 1.90-2. 00 (m, 1H, CH , 1.70-1.83 (m, 1H, CH ), 1.51-1.71 (m, 8H), 1.35-1.45 (m, 2H, CH 2), 1.18 (d _{, J = 6.4Hz, 6H, CH} 3), 1.16 (d, J = 6.4 Hz, 6H, CH 3);
P-NMR (CH ₃ CN): δ 146.90;
Ms (FAB +): m / z 803 (M ⁺ +1), 303 (DMTr ⁺ ).

(Example B5)
In order to generate the nucleic acid molecule of the present invention containing a linker having a proline skeleton, DMTr-amidoethyleneoxyethylamino-L-proline amidite (hereinafter referred to as PEG spacer type) was synthesized according to Scheme 5 below.

(1) DMTr-amidohydroxyethoxyethylamino-L-proline (Compound 23)
DMTr-amide-L-proline (Compound 6) (1.00 g, 2.05 mmol), 4-toluenesulfonic acid 2- (2-hydroxyethoxy) ethyl ester (3.10 g, 12.30 mmol), and potassium carbonate ( 0.85 g, 6.15 mmol) in anhydrous dimethylformamide solution (10 mL) was mixed and stirred at room temperature for 4 days under an argon atmosphere. About the said mixture, after depressurizing and distilling a solvent off at room temperature, the dichloromethane (20 mL) was added and it filtered. The filtrate was concentrated and the obtained residue was subjected to silica gel column chromatography. As an additive solvent for the silica gel column chromatography, first, ethyl acetate containing 0.05% pyridine was used, and then a mixed solution of CH ₂ Cl ₂ and CH ₃ OH containing 0.05% pyridine (CH ₂ Cl ₂ : CH ₃ OH = 9 :: 1) was used. As a result, colorless syrup-like compound 23 (1.15 g, yield 97%) was obtained. The NMR results of the above compound are shown below.
¹ H-NMR (CDCl ₃ ): δ 7.41-7.45 (m, 2H, Ar—H), 7.27-7.31 (m, 6H, Ar—H), 7.17-7.21 (M, 1H, Ar—H), 6.79-6.82 (m, 4H, Ar—H), 3.79 (s, 6H, OCH ₃ ), 3.60-3.70 (m, 2H) ), 3.39-3.57 (m, 4H), 3.13-3.27 (m, 3H), 3.07-3.08 (m, 2H), 2.71-2.84 (m) , 1H), 2.38-2.46 (m, 1H), 2.14-2.19 (m, 1H), 1.84-1.87 (m, 1H), 1.57-1.76. (M, 8H).

(2) DMTr-amidoethyleneoxyethylamino-L-proline amidite (Compound 24)
The obtained DMTr-amidohydroxyethoxyethylamino-L-proline (Compound 23) (0.63 g, 1.00 mmol) was mixed with anhydrous pyridine and azeotropically dried at room temperature. Diisopropylammonium tetrazolide (206 mg, 1.20 mmol) was added to the obtained residue, degassed under reduced pressure, and filled with argon gas. Anhydrous acetonitrile (1 mL) was added to the mixture, and an anhydrous acetonitrile solution (1 mL) of 2-cyanoethoxy-N, N, N ′, N′-tetraisopropyl phosphorodiamidite (282 mg, 1.12 mmol) was further added. added. The mixture was stirred for 4 hours at room temperature under an argon atmosphere. The mixture was diluted with dichloromethane and washed with saturated aqueous sodium hydrogen carbonate and saturated brine. The organic layer was collected and dried over anhydrous sodium sulfate, and then the organic layer was filtered. About the obtained filtrate, the solvent was distilled off under reduced pressure. The obtained residue was subjected to column chromatography (developing solvent hexane: acetone = 7: 3, containing 0.05% pyridine) using amino silica gel as a filler, and colorless syrup-like compound 24 (0.74 g, purity) 100%, yield 87%). The NMR results of the above compound are shown below.
¹ H-NMR (CD ₃ CN): δ 7.41-7.43 (m, 2H, Ar—H), 7.28-7.31 (m, 6H, Ar—H), 7.18-7. 22 (m, 1H, Ar—H), 6.84-6.86 (m, 4H, Ar—H), 3.73-3.84 (m, 2H, CH ₂ ), 3.79 (s, 6H, OCH ₃ ), 3.47-3.64 (m, 7H), 3.15-3.23 (m, 1H), 3.11 (t, J = 6.4 Hz, 2H, CH ₂ ), 3.01 (t, J = 5.9 Hz, 2H, CH ₂ ), 2.95-2.99 (m, 1H), 2.58-2.63 (m, 2H), 2.31-2. 35 (m, 1H, CH), 2.03-2.19 (m, 1H, CH), 1.48-1.78 (m, 10H), 1.12-1.57 (m, 12H, CH) ₃ );
P-NMR (CD ₃ CN): δ 148.00;
^{Ms (FAB +): m /} z 776 (M +), 303 (DMTr +) 201 (C 8 H 19 N 2 OP +).

(Example B6)
1. Synthesis of Protected Prolinol Prolinol (compound 3) protected with a dimethoxytrityl group was synthesized according to Scheme 6 shown below.

(1) Trifluoroacetyl-L-prolinol (Compound 1)
L-prolinol (2.0 g, 20 mmol) was dissolved in 20 mL of THF. On the other hand, ethyl trifluoroacetate (3.0 g, 21 mmol) was dissolved in 20 mL of THF. The latter THF solution was added dropwise to the former L-prolinol-containing THF solution and stirred for 12 hours. The reaction solution was concentrated under reduced pressure to obtain Compound 1 (3.7 g, yield 97%). The NMR results of the above compound are shown below.
¹ H-NMR (CDCl ₃ ): δ 4.28-4, 23 (1.0 H, m, OH), 3.90-3.41 (5H, H-2, H-5, H-6, m) , 2.27-1.77 (4H, H-3, H-4, m).

(2) Trifluoroacetyl-DMTr-L-prolinol (Compound 2)
The obtained trifluoroacetyl-L-prolinol (Compound 1) (3.7 g, 19 mmol) was dissolved in pyridine and dried azeotropically three times at room temperature. The obtained residue was dissolved in 15 mL of pyridine, and while stirring in an ice bath under an argon atmosphere, 4,4′-dimethoxytrityl chloride (DMTr-Cl) (8.1 g, 24 mmol) was added, and further at room temperature. The reaction was performed for 4 hours. Then, in order to quench excess DMTr-Cl, 10 mL of methanol was further added to the reaction solution and stirred for 10 minutes. Thereafter, dichloromethane was added to the reaction solution, and the mixture was washed with a saturated aqueous sodium hydrogen carbonate solution and saturated brine. The collected organic layer after washing was dried over anhydrous sodium sulfate. The organic layer was filtered, the obtained filtrate was concentrated under reduced pressure, and the residue was subjected to silica gel column chromatography (developing solvent CH ₂ Cl ₂ : CH ₃ OH = 95: 5, containing 0.1% pyridine). Purified compound 2 was obtained (8.5 g, yield 89%). The NMR results of the above compound are shown below.
¹ H-NMR (CDCl ₃ ): δ 7.39-7.18 (9H, m, Ar—H), 6.82 (4H, d, J = 8.6 Hz, Ar—H), 3.78 (6H) , S, OCH ₃ ), 3.70-3.41 (5H, H-2, H-5, H-6, m), 2.19-1.85 (4H, H-3, H-4, m).

(3) DMTr-L-prolinol (Compound 3)
The obtained trifluoroacetyl-DMTr-L-prolinol (compound 2) (5 g, 10 mmol) was dissolved in 100 mL of THF. To this THF solution, 100 mL of 5% aqueous sodium hydroxide solution was added and stirred. To this solution, 5 mL of a 1 mol / L tetra-n-butylammonium fluoride (TBAF) solution was added and stirred at room temperature for 12 hours. The reaction solution was washed with a saturated aqueous sodium hydrogen carbonate solution and saturated brine. The collected organic layer after washing was dried over anhydrous sodium sulfate. The organic layer was filtered, and the obtained filtrate was concentrated under reduced pressure to obtain Compound 3 (3.6 g, yield 90%). The NMR results of the above compound are shown below.
¹ H-NMR (CDCl ₃ ): δ 7.40-7.14 (9H, m, Ar—H), 6.82 (4H, d, J = 8.6 Hz, Ar—H), 3.78 (6H) , S, OCH3), 3.31 (1H, m, H-6), 3.07 (2H, m, H-2, H-6), 2.90 (2H, m, H-5), 1 .84 (3H, m, H-3, H-4), 1.40 (1H, m, H-3).

2. Synthesis of Amidite Derivatives Using the protected prolinol (compound 3) synthesized in the above “1.”, amidite derivatives having prolinol with different binding formats were synthesized according to Scheme 7 below.

(1) DMTr-urethane-L-prolinol (compound 4)
1,8-octanediol (9.0 g, 62 mmol) was dissolved in 90 mL of THF and placed under an argon atmosphere. On the other hand, carbonyldiimidazole (2.0 g, 12 mmol) was dissolved in 10 mL of THF. The latter THF solution was added to the former THF solution and stirred at room temperature for 1 hour. The reaction was washed with water until the 1,8-octanediol TLC spot disappeared. Furthermore, the organic layer collected after washing was washed with saturated saline, and the collected organic layer was dried over anhydrous sodium sulfate. The organic layer was filtered, and the resulting filtrate was concentrated under reduced pressure. The residue was subjected to silica gel column chromatography (developing solvent CH ₂ Cl ₂ : CH ₃ OH = 95: 5) to obtain a purified compound. This compound was a compound in which one end of 1,8-octanediol was activated with carbonyldiimidazole (2.3 g, yield 77%).

0.9 g of the compound was dissolved in 10 mL of acetonitrile and placed in an argon atmosphere. On the other hand, DMTr-L-prolinol (Compound 3) (1.9 g, 4.8 mmol) was dissolved in 20 mL of acetonitrile. The latter acetonitrile solution was added to the former acetonitrile solution and stirred at room temperature for 24 hours. And this reaction liquid was wash | cleaned by saturated sodium hydrogencarbonate aqueous solution and a saturated salt solution, and the collect | recovered organic layer was dried with anhydrous sodium sulfate. The organic layer was filtered, and the resulting filtrate was concentrated under reduced pressure. The residue was subjected to silica gel column chromatography (developing solvent dichloromethane: acetone = 9: 1, containing 0.1% pyridine) to obtain purified compound 4 (prolinol urethane amidite) (1.5 g, yield 65). %). The NMR results of the above compound are shown below.
¹ H-NMR (CDCl ₃ ): δ 7.40-7.14 (9H, m, Ar—H), 6.82 (4H, d, J = 8.6 Hz, Ar—H), 4.24-3 .94 (2H, m, COOCH ₂ ), 3.78 (s, 6H, OCH ₃ ), 3.72-2.96 (7H, m, alkyl, H-2, H-5, H-6), 2.10-1.30 (16H, m, alkyl, H-3, H-4).
FAB-MS: 576 [M + H] ⁺ .

(2) DMTr-ureido-L-prolinol (compound 5)
Under an argon atmosphere, triphosgene (2.0 g, 6.7 mmol) was dissolved in 10 mL of THF and stirred at 0 ° C. On the other hand, DMTr-L-prolinol (compound 3) (1.3 g, 3.2 mmol) and N, N-diisopropylethylamine (16 g, 124 mmol) were dissolved in 10 mL of THF and added dropwise to the THF solution of triphosgene. The reaction was stirred at 0 ° C. for 1 hour followed by 2 hours at room temperature. 8-Amino-1-octanol (2.3 g, 16 mmol) and N, N-diisopropylethylamine (5.0 g, 38 mmol) were dissolved in 30 mL of THF. The reaction solution after stirring was added dropwise to the THF solution, and the mixture was stirred at 0 ° C. for 1 hour and then at room temperature for 48 hours. The reaction solution was concentrated under reduced pressure, and the residue was dissolved in dichloromethane. This solution was washed with a saturated aqueous sodium hydrogen carbonate solution and saturated brine, and the collected organic layer was dried over anhydrous sodium sulfate. The organic layer was filtered, the obtained filtrate was concentrated under reduced pressure, and the residue was purified by reverse phase silica gel column chromatography. At this time, as the developing solvent, a mixed solvent of acetone and water containing 0.1% pyridine was used, and the mixing ratio of the acetone and water was stepwise. Specifically, the molar ratio of acetone: water The ratio was changed in the order of 2: 8, 3: 7, 4: 6 and 5: 5. The fraction containing the target compound 5 was extracted with dichloromethane, and the organic layer was dried over anhydrous sodium sulfate. The organic layer was filtered, and the obtained filtrate was concentrated under reduced pressure to obtain Compound 5 (prolinol urea amidite) (0.9 g, yield 49%). The NMR results of the above compound are shown below.
¹ H-NMR (CDCl ₃ ): δ 7.40-7.14 (9H, m, Ar—H), 6.82 (4H, m, Ar—H), 3.78 (s, 6H, OCH ₃ ) _{_{, 3.68-3.25 (9H, m, CH}} 2 NH, CH 2 OH, H-2, H-5, H-6), 1.74-1.18 (16H, m, alkyl, H- 3, H-4).
FAB-MS: 575 [M + H] ⁺ .

(3) Amidite derivatives having prolinol (compounds 6 and 7)
As the modified prolinol, the obtained compound 4 (0.80 g, 1.4 mmol) was dissolved in acetonitrile and dried azeotropically three times at room temperature. The resulting residue was dissolved in 1 mL of acetonitrile and placed under an argon atmosphere. Diisopropylammonium tetrazolide (0.24 g, 1.4 mmol) was added to the acetonitrile solution to prepare a reaction solution. On the other hand, 2-cyanoethyl N, N, N ′, N′-tetraisopropyl phosphorodiamidite (0.50 g, 1.7 mmol) was dissolved in 1 mL of acetonitrile. This was added to the reaction solution and stirred at room temperature for 4 hours. Dichloromethane was added to the reaction solution, and the mixture was washed with a saturated aqueous sodium bicarbonate solution and saturated brine. The collected organic layer after washing was dried over anhydrous sodium sulfate, the organic layer was filtered, and the obtained filtrate was concentrated under reduced pressure. The residue was subjected to amino silica gel column chromatography (developing solvent hexane: acetone = 10: 1, containing 0.1% pyridine) and purified compound 6 (DMTr-urethane-L-prolinol amidite) (0.90 g, Yield 83%) was obtained. The NMR results of the above compound are shown below.
¹ H-NMR (CDCl ₃ ): δ 7.40-7.14 (9H, m, Ar—H), 6.82 (4H, d, J = 8.6 Hz, Ar—H), 4.24-3 .94 (2H, m, COOCH ₂ ), 3.78 (s, 6H, OCH ₃ ), 3.72-2.96 (11H, m, CH ₂ O, POCH ₂ , CHCH ₃ , H-2, H -5, H-6), 2.58 (2H, m, CH ₂ CN), 2.10-1.46 (16H, m, alkyll, H-3, H-4), 1.34-1. _{10 (12H, m, CHCH 3} ). 31P-NMR (CD ₃ CN) δ 146.82.
FAB-MS: 776 [M + H] ⁺ .

Compound 7 (DMTr-ureido-L-prolinol amidite) (0.80 g, yield) was processed and purified in the same manner except that Compound 5 was used instead of Compound 4 as the modified prolinol. 74%). The NMR results of the above compound are shown below.
¹ H-NMR (CDCl ₃ ): δ 7.40-7.14 (9H, m, Ar—H), 6.82 (4H, m, Ar—H), 3.78 (s, 6H, OCH ₃ ) _{_{_{, 3.65-3.25 (13H, m, CH}}} 2 O, POCH 2, CHCH 3, H-2, CH 2 NH, CH 2 OH, H-2, H-5, H-6), 2. _{73 (2H, m, CH 2} CN), 2.10-1.48 (16H, m, alkyl, H-3, H-4), 1.35-1.10 (12H, m, CHCH 3).
31P-NMR (CD ₃ CN) δ 146.83.
FAB-MS: 775 [M + H] ⁺ .

As mentioned above, although this invention was demonstrated with reference to embodiment, this invention is not limited to the said embodiment. Various changes that can be understood by those skilled in the art can be made to the configuration and details of the present invention within the scope of the present invention.

The single-stranded nucleic acid molecule of the present invention can suppress the expression of a gene, specifically, the translation of a protein encoded by the gene. Since the ssNc molecule of the present invention can suppress the expression of the target gene as described above, it is useful as, for example, pharmaceuticals, diagnostic agents and agricultural chemicals, and research tools for agricultural chemicals, medicine, life sciences and the like.

Claims

A single-stranded nucleic acid molecule comprising an expression suppressing sequence that suppresses expression of a target gene and a complementary sequence having a mismatch with the expression suppressing sequence,
From the 5 ′ side to the 3 ′ side, the 5 ′ side region (Xc), the inner region (Z), and the 3 ′ side region (Yc) are included in the above order,
The internal region (Z) is configured by connecting an internal 5 ′ side region (X) and an internal 3 ′ side region (Y),
The 5 ′ side region (Xc) is complementary to the internal 5 ′ side region (X);
The 3 ′ side region (Yc) is complementary to the inner 3 ′ side region (Y);
At least one of the internal region (Z), the 5 ′ side region (Xc) and the 3 ′ side region (Yc) includes the expression suppression sequence,
A region complementary to the region containing the expression suppression sequence comprises the complementary sequence;
A single-stranded nucleic acid molecule, wherein the 5 ′ terminal base and the 3 ′ terminal base of the single-stranded nucleic acid molecule are unbound.
The single-stranded nucleic acid molecule according to claim 1, wherein the expression suppression sequence is a mature miRNA sequence.
Between the 5 ′ side region (Xc) and the inner 5 ′ side region (X), a linker region (Lx) is provided,
The single-stranded nucleic acid molecule according to claim 1 or 2, wherein the 5'-side region (Xc) and the internal 5'-side region (X) are linked via the linker region (Lx).
Between the 3 ′ side region (Yc) and the inner 3 ′ side region (Y), it has a linker region (Ly),
The single piece according to any one of claims 1 to 3, wherein the 3'-side region (Yc) and the internal 3'-side region (Y) are linked via the linker region (Ly). Strand nucleic acid molecule.
Between the 5 ′ side region (Xc) and the inner 5 ′ side region (X), a linker region (Lx) is provided,
The 5 ′ side region (Xc) and the inner 5 ′ side region (X) are linked via the linker region (Lx),
Between the 3 ′ side region (Yc) and the inner 3 ′ side region (Y), it has a linker region (Ly),
The single-stranded nucleic acid molecule according to claim 1 or 2, wherein the 3'-side region (Yc) and the internal 3'-side region (Y) are linked via the linker region (Ly).
The number of bases (Z) in the internal region (Z), the number of bases (X) in the internal 5 ′ side region (X), the number of bases (Y) in the internal 3 ′ side region (Y), the 5 ′ side region The number of bases (Xc) of (Xc) and the number of bases (Yc) of the 3 ′ side region (Yc) satisfy the conditions of the following formulas (1) and (2): A single-stranded nucleic acid molecule according to 1.
Z = X + Y (1)
Z ≧ Xc + Yc (2)
The number of bases (X) in the inner 5 ′ side region (X), the number of bases (Xc) in the 5 ′ side region (Xc), the number of bases (Y) in the inner 3 ′ side region (Y) and the 3 ′ The single-stranded nucleic acid molecule according to any one of claims 1 to 6, wherein the number of bases (Yc) in the side region (Yc) satisfies any of the following conditions (a) to (d):
(A) The conditions of the following formulas (3) and (4) are satisfied.
X> Xc (3)
Y = Yc (4)
(B) The conditions of the following formulas (5) and (6) are satisfied.
X = Xc (5)
Y> Yc (6)
(C) The conditions of the following formulas (7) and (8) are satisfied.
X> Xc (7)
Y> Yc (8)
(D) The conditions of the following formulas (9) and (10) are satisfied.
X = Xc (9)
Y = Yc (10)
In (a) to (d), the difference between the number of bases (X) in the inner 5 ′ side region (X) and the number of bases (Xc) in the 5 ′ side region (Xc), the inner 3 ′ side region ( The single-stranded nucleic acid molecule according to claim 7, wherein a difference between the number of bases (Y) of Y) and the number of bases (Yc) of the 3 'side region (Yc) satisfies the following condition.
(A) The conditions of the following formulas (11) and (12) are satisfied.
X−Xc = 1, 2 or 3 (11)
Y−Yc = 0 (12)
(B) The conditions of the following formulas (13) and (14) are satisfied.
X−Xc = 0 (13)
Y−Yc = 1, 2 or 3 (14)
(C) The conditions of the following formulas (15) and (16) are satisfied.
X−Xc = 1, 2 or 3 (15)
Y−Yc = 1, 2 or 3 (16)
(D) The conditions of the following formulas (17) and (18) are satisfied.
X−Xc = 0 (17)
Y−Yc = 0 (18)
The single-stranded nucleic acid molecule according to any one of claims 1 to 8, wherein the number of bases (Z) in the internal region (Z) is 19 bases or more.
The single-stranded nucleic acid molecule according to claim 9, wherein the internal region (Z) has a base number (Z) of 19 to 30 bases.
The single-stranded nucleic acid molecule according to any one of claims 1 to 10, wherein the 5'-side region (Xc) has a base number (Xc) of 1 to 11 bases.
The single-stranded nucleic acid molecule according to claim 11, wherein the number of bases (Xc) in the 5 'side region (Xc) is 1 to 7 bases.
The single-stranded nucleic acid molecule according to claim 11, wherein the number of bases (Xc) in the 5 'side region (Xc) is 1 to 3 bases.
The single-stranded nucleic acid molecule according to any one of claims 1 to 13, wherein the number of bases (Yc) in the 3 'side region (Yc) is 1 to 11 bases.
The single-stranded nucleic acid molecule according to claim 14, wherein the number of bases (Yc) in the 3 'side region (Yc) is 1 to 7 bases.
The single-stranded nucleic acid molecule according to claim 14, wherein the number of bases (Yc) in the 3 'side region (Yc) is 1 to 3 bases.
17. A single-stranded nucleic acid molecule according to any one of claims 1 to 16, comprising at least one modified residue.
The single-stranded nucleic acid molecule according to any one of claims 1 to 17, comprising a labeling substance.
The single-stranded nucleic acid molecule according to any one of claims 1 to 18, comprising a stable isotope.
The single-stranded nucleic acid molecule according to any one of claims 1 to 19, which is an RNA molecule.
The linker region (Lx) and / or the linker region (Ly),
The single-stranded nucleic acid molecule according to any one of claims 3 to 20, which is composed of at least one of a nucleotide residue and a non-nucleotide residue.
The single-stranded nucleic acid molecule according to claim 21, wherein the nucleotide residue is an unmodified nucleotide residue and / or a modified nucleotide residue.
The single-stranded nucleic acid molecule according to claim 21 or 22, wherein the linker region (Lx) and / or the linker region (Ly) is composed of any one of the following residues (1) to (7).
(1) Unmodified nucleotide residues (2) Modified nucleotide residues (3) Unmodified nucleotide residues and modified nucleotide residues (4) Nonnucleotide residues (5) Nonnucleotide residues and unmodified nucleotide residues (6 ) Non-nucleotide residues and modified nucleotide residues (7) non-nucleotide residues, unmodified nucleotide residues and modified nucleotide residues
The single-stranded nucleic acid molecule according to any one of claims 1 to 23, wherein the total number of bases in the single-stranded nucleic acid molecule is 50 bases or more.
A composition for suppressing the expression of a target gene,
An expression-suppressing composition comprising the single-stranded nucleic acid molecule according to any one of claims 1 to 24.
25. A pharmaceutical composition comprising a single-stranded nucleic acid molecule according to any one of claims 1 to 24.
27. The pharmaceutical composition according to claim 26, which is for cancer treatment.
A method for suppressing the expression of a target gene,
25. A method for suppressing expression, comprising using the single-stranded nucleic acid molecule according to any one of claims 1 to 24.
29. The expression suppression method according to claim 28, comprising a step of administering the single-stranded nucleic acid molecule to a cell, tissue or organ.
30. The expression suppression method according to claim 29, wherein the single-stranded nucleic acid molecule is administered in vivo or in vitro .
A method of treating a disease,
Administering the single stranded nucleic acid molecule of any one of claims 1 to 24 to a patient,
The treatment method, wherein the single-stranded nucleic acid molecule has a sequence that suppresses the expression of a gene causing the disease as the expression suppression sequence.
Use of the single-stranded nucleic acid molecule according to any one of claims 1 to 24 for suppressing expression of a target gene.
A nucleic acid molecule for use in the treatment of a disease,
The nucleic acid molecule is a single-stranded nucleic acid molecule according to any one of claims 1 to 24,
The nucleic acid molecule, wherein the single-stranded nucleic acid molecule has a sequence that suppresses the expression of a gene causing the disease as the expression suppressing sequence.