HK1181423B

HK1181423B - Single-strand nucleic acid molecule for controlling gene expression

Info

Publication number: HK1181423B
Application number: HK13108676.9A
Authority: HK
Inventors: 大木忠明; 林宏刚; 白水久男; 滨崎智洋; 伊藤彰浩; 铃木宽
Original assignee: 株式会社博纳克
Priority date: 2010-07-08
Filing date: 2011-07-08
Publication date: 2016-07-08

Description

Single-stranded nucleic acid molecules for controlling gene expression

Technical Field

The present invention relates to a single-stranded nucleic acid molecule that inhibits gene expression, a composition comprising the single-stranded nucleic acid molecule, and uses thereof.

Background

As a technique for suppressing gene expression, for example, RNA interference (RNAi) is known (non-patent document 1). Inhibition of expression of genes by RNA interference is generally performed by administering short double-stranded RNA molecules, for example, in cells or the like. Such double-stranded RNA molecules are generally referred to as siRNA (small interfering RNA). In addition, a circular RNA molecule that partially forms a double strand by intramolecular annealing and that can suppress gene expression has also been reported (patent document 1). However, these methods have the following problems with RNA molecules that induce suppression of gene expression.

First, in the case of producing the above siRNA, the following steps are required: after synthesis of the sense and antisense strands, respectively, these strands are finally hybridized. Therefore, there is a problem that the manufacturing efficiency is poor. In addition, when the siRNA is administered into a cell, since it needs to be administered into the cell in a state in which dissociation into single-stranded RNA is suppressed, setting of the operation conditions also requires labor. Next, in the case of a circular RNA molecule, there is a problem that synthesis is difficult.

Documents of the prior art

Patent document

Patent document 1: japanese patent laid-open No. 2008 278784

Non-patent document

Non-patent document 1: fire et al, Nature volume 391, p.806-811, 1998

Disclosure of Invention

Accordingly, an object of the present invention is to provide a novel nucleic acid molecule capable of suppressing gene expression and capable of being produced easily and efficiently.

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 expression of a target gene,

comprises a5 'side region (Xc), an inner region (Z) and a 3' side region (Yc) in this order from the 5 'side to the 3' side,

the inner region (Z) is formed by connecting an inner 5 'side region (X) and an inner 3' side region (Y),

the 5 '-side region (Xc) is complementary to the inner 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-suppressing sequence.

The composition of the present invention is a composition for inhibiting expression of a target gene, which comprises the above-described single-stranded nucleic acid molecule of the present invention.

The composition of the present invention is a pharmaceutical composition comprising the single-stranded nucleic acid molecule of the present invention.

The method for suppressing expression of the present invention is a method for suppressing expression of a target gene, and uses the single-stranded nucleic acid molecule of the present invention.

The method for treating a disease of the present invention is characterized by comprising a step of administering the single-stranded nucleic acid molecule of the present invention having a sequence that suppresses expression of a gene that causes the disease as the expression suppression sequence to a patient.

The single-stranded nucleic acid molecule of the present invention can suppress gene expression, can be easily synthesized because it is not circular, and can be efficiently produced because it is single-stranded and does not have a double-strand annealing step.

The present inventors have found for the first time that the structure of the single-stranded nucleic acid molecule of the present invention can inhibit gene expression. The effect of suppressing gene expression of the single-stranded nucleic acid molecule of the present invention is assumed to be the same phenomenon as that based on RNA interference, but the suppression of gene expression in the present invention is not limited to RNA interference.

Drawings

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 relative values of the expression amounts of GAPDH genes in examples of the present invention.

FIG. 5 is a graph showing the relative values of the expression amounts of GAPDH genes in examples of the present invention.

Fig. 6 is a graph showing the relative values of GAPDH gene expression amounts of a549 cells in the examples of the present invention.

FIG. 7 is a graph showing the relative values of GAPDH gene expression levels in 293 cells in examples of the present invention.

FIG. 8 is a graph showing the relative values of the expression levels of TGF-. beta.1 genes in examples of the present invention.

FIG. 9 is a graph showing the expression level of TGF-. beta.1 gene per weight of lung in each administration group in the example of the present invention.

Fig. 10 is a graph showing the number of cells in a BAFL sample in each administration group in the example of the present invention.

Fig. 11 is a graph showing the cell number of neutrophils in the BALF sample in each administration group in the example of the present invention.

FIG. 12 is a photograph showing the results of Giemsa staining of cells in BALF samples in the examples of the present invention, FIG. 12 (A) shows the results of LPS (+)/RNA (-) administration group 4, FIG. 12 (B) shows the results of LPS (+)/negative control NK-0035(+) administration group 6, and FIG. 12 (C) shows the results of LPS (+)/NK-0033(+) example administration group 5.

FIG. 13 is a photograph showing HE staining results of lung tissues in examples of the present invention, FIG. 13 (A) shows the results of LPS (+)/RNA (-) administration group 4, FIG. 13 (B) shows the results of LPS (+)/negative control NK-0035(+) administration group 6, and FIG. 13 (C) shows the results of LPS (+)/NK-0033(+) administration group 5.

FIG. 14 (A) shows the results of the expression level of a TGF- β 1 gene in an example of the invention, FIG. 14(B) shows the results of the expression level of an IFN- α gene in an example of the invention, and FIG. 14(C) shows the results of the expression level of an IFN- β gene in an example of the invention.

FIG. 15 is a graph showing the relative values of the expression levels of TGF-. beta.1 genes in examples of the present invention.

FIG. 16 is a graph showing the relative values of the expression levels of TGF-. beta.1 genes in examples of the present invention.

FIG. 17 is a graph showing the amount of TGF- β 1 expression per weight of lung in each administration group in an example of the present invention.

Fig. 18 (a) is a graph showing the amount of TNF- α in the BALF sample of each administration group in the example of the present invention, and fig. 18 (B) is a graph showing the amount of IFN- β in the BALF sample of each administration group in the example of the present invention.

FIG. 19 is a graph showing the relative values of the expression levels of the LAMA gene in 293 cells in examples of the present invention.

Fig. 20 is a graph showing relative values of the expression amounts of LMNA genes of a549 cells in the example of the present invention.

Fig. 21 is a diagram showing ssrnas used in an example of the present invention.

FIG. 22 is a graph showing the relative values of the expression amounts of GAPDH genes in examples of the present invention.

Fig. 23 is a diagram showing ssrnas used in an example of the present invention.

FIG. 24 is a graph showing the relative values of the expression amounts of GAPDH genes in examples of the present invention.

Fig. 25 is a diagram showing ssrnas used in an example of the present invention.

FIG. 26 is a graph showing the relative values of the expression amounts of GAPDH genes in examples of the present invention.

Fig. 27 is a diagram showing ssrnas used in an example of the present invention.

FIG. 28 is a graph showing the relative values of the expression amounts of GAPDH genes in examples of the present invention.

FIG. 29 is a graph showing the relative values of the expression amounts of GAPDH genes in examples of the present invention.

Fig. 30 is a graph showing the relative values of GAPDH gene expression levels of HCT116 cells in the examples of the present invention.

FIG. 31 is an electrophoretogram showing RNase resistance in the examples of the present invention.

Fig. 32 is an electrophoretogram showing S7 nuclease resistance in the examples of the present invention.

Detailed Description

Unless otherwise mentioned, terms used in the present specification can be used in the meaning generally used in the technical field.

ssNc molecules

As described above, the single-stranded nucleic acid molecule of the present invention is characterized by comprising an expression-suppressing sequence that suppresses expression of a target gene,

the 5 '-side region (Xc) is complementary to the inner 5' -side region (X),

the 3 '-side region (Yc) is complementary to the inner 3' -side region (Y),

In the present invention, "suppression of expression of a target gene" means, for example, inhibition of expression of the target gene. The mechanism of the above-mentioned inhibition is not particularly limited, and may be, for example, down regulation or silencing. The inhibition of the expression of the target gene can be confirmed, for example, by a decrease in the amount of the transcription product produced from the target gene, a decrease in the activity of the transcription product, a decrease in the amount of the translation product produced from the target gene, or a decrease in the activity of the translation product. Examples of the protein include a mature protein and a precursor protein before being processed or post-translationally modified.

Hereinafter, the single-stranded nucleic acid molecule of the present invention is also referred to as "ssNc molecule" of the present invention. The ssNc molecule of the present invention can be used for the inhibition of expression of a target gene, for example, in vivo (invivo) or in vitro (invitro), and is also referred to as "ssNc molecule for inhibition of expression of a target gene" or "inhibitor of expression of a target gene". The ssNc molecule of the present invention can suppress the expression of the target gene by RNA interference, and is therefore also referred to as "ssNc molecule for RNA interference", "RNA interference-inducing molecule", "RNA interfering agent", or "RNA interference-inducing agent". In addition, the ssNc molecules of the present invention are capable of inhibiting side effects such as interferon induction.

The 5 '-end and 3' -end of the ssNc molecule of the present invention are not linked, and can also be referred to as a linear single-stranded nucleic acid molecule. In the internal region (Z), the ssNc molecule of the present invention is, for example, such that the internal 5 'region (X) and the internal 3' region (Y) are directly linked.

In the ssNc molecule of the present invention, the 5 '-side region (Xc) is complementary to the inner 5' -side region (X), and the 3 '-side region (Yc) is complementary to the inner 3' -side region (Y). Therefore, on the 5 'side, the region (Xc) is folded back to the region (X), and the region (Xc) and the region (X) can form a double strand by self-annealing, and on the 3' side, the region (Yc) is folded back to the region (Y), and the region (Yc) and the region (Y) can form a double strand by self-annealing. The ssNc molecule of the present invention can form a double strand in the molecule in this way, and has a structure that is significantly different from a double-stranded RNA formed by annealing two separated single-stranded RNAs, such as siRNA used in conventional RNA interference.

In the ssNc molecule of the present invention, for example, when the ssNc molecule of the present invention is introduced into a cell in vivo or in vitro, the expression inhibitory sequence is a sequence exhibiting an activity of inhibiting the expression of the target gene. The expression-suppressing sequence is not particularly limited and can be appropriately set according to the type of target gene of interest. The expression inhibitory sequence can be suitably used, for example, as a sequence related to siRNA-based RNA interference. RNA interference is typically the following phenomenon: long double-stranded RNA (dsRNA) is cleaved by a nicking enzyme into double-stranded RNA (siRNA: small interfering RNA) of about 19 to 21 base pairs protruding from the 3' -end in a cell, and one of the single-stranded RNAs binds to a target mRNA to decompose the mRNA, thereby inhibiting the translation of the mRNA. The sequence of the single-stranded RNA in the siRNA that binds to the target mRNA is reported in various types, for example, depending on the type of the target gene. In the present invention, a sequence of a single-stranded RNA such as the siRNA described above can be used as the expression suppressing sequence described above.

The key point of the present invention is not the sequence information of the expression suppressing sequence for the target gene, but relates to the structure of a nucleic acid molecule for functioning the expression suppressing activity of the target gene by the expression suppressing sequence in, for example, a cell. Therefore, in the present invention, for example, in addition to the single-stranded RNA sequence of the siRNA known at the time of application, a sequence known in the future can be used as the expression suppressing sequence.

The expression suppressing sequence preferably has 90% or more complementarity to a predetermined region of the target gene, more preferably 95%, even more preferably 98%, and particularly preferably 100%, for example. By satisfying such complementarity, for example, off-target can be sufficiently reduced.

Specifically, in the case where the target gene is the GAPDH gene, for example, a 19-base long sequence shown in SEQ ID NO. 1; when the target gene is TGF-. beta.1, the expression-suppressing sequence may be, for example, a sequence 21 bases long as shown in SEQ ID NO. 16; when the target gene is LAMA1 gene, a sequence 19 bases long as shown in sequence No. 5 can be used as the expression suppressing sequence; when the target gene is the LMNA gene, a 19-base long sequence shown in SEQ ID NO. 6 can be used, for example.

5'-GUUGUCAUACUUCUCAUGG-3' (Serial number 1)

5'-AAAGUCAAUGUACAGCUGCUU-3' (Serial number 16)

5'-AUUGUAACGAGACAAACAC-3' (Serial number 5)

5'-UUGCGCUUUUUGGUGACGC-3' (Serial number 6)

The inhibition of the expression of the target gene by the ssNc molecule of the present invention is presumed to be due to the following reasons: for example, the phenomenon of RNA interference or the similar phenomenon to RNA interference (RNA interference-like phenomenon) occurs by a configuration in which the expression suppressing sequence is arranged in at least one of the internal region (Z), the 5 '-side region (Xc), and the 3' -side region (Yc). The present invention is not limited to this mechanism. The ssNc molecule of the present invention is not introduced into a cell or the like as dsRNA composed of two single-stranded RNAs, as in so-called siRNA, for example, and the expression-suppressing sequence is not necessarily cut out in the cell. Therefore, the ssNc molecule of the present invention can also be said to have, for example, an RNA interference-like function.

In the ssNc molecule of the present invention, as described above, the expression suppressing sequence is included in at least one of the internal region (Z), the 5 '-side region (Xc), and the 3' -side region (Yc). The ssNc molecule of the present invention may have, for example, 1 or 2 or more of the expression suppression sequences.

In the latter case, the ssNc molecule of the present invention may have, for example, 2 or more identical expression suppressing sequences for the same target gene, 2 or more different expression suppressing sequences for the same target gene, or 2 or more different expression suppressing sequences for different target genes. When the ssNc molecule of the present invention has 2 or more expression suppression sequences, the position of the expression suppression sequences is not particularly limited, and may be any one of the internal region (Z), the 5 '-side region (Xc), and the 3' -side region (Yc), or may be different regions. In the case where the ssNc molecule of the present invention has 2 or more of the above-described expression suppression sequences for different target genes, for example, the expression of 2 or more different target genes can be suppressed by the ssNc molecule of the present invention.

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) may be directly connected to each other without intervening sequences therebetween, for example. In order to show the sequence relationship between the internal region (Z) and the 5 'side region (Xc) and the 3' side region (Xc), the internal region (Z) is defined as "the internal 5 'side region (X) and the internal 3' side region (Y) are connected to each other", and the 5 'side region (Xc) and the 3' side region (Xc) in the internal region (Z) are not limited to separate regions when the ssNc molecule is used, for example. That is, for example, in the case where the internal region (Z) has the expression suppression sequence, the expression suppression sequence may be arranged in the internal region (Z) so as to span the region (X) and the region (Y).

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 or a partial region of the region (X), and specifically, for example, preferably includes a sequence complementary to the entire region or a partial region of the region (X) or is composed of the complementary sequence. The region (Xc) may be completely complementary to the entire region or the partial region complementary to the region (X), or may be non-complementary to 1 or several bases, but is preferably completely complementary. In the ssNc molecule of the present invention, the 3 '-side region (Yc) is complementary to the inner 3' -side region (Y). Here, the region (Yc) may have a sequence complementary to the entire region or a partial region of the region (Y), and specifically, for example, preferably includes a sequence complementary to the entire region or a partial region of the region (Y) or is composed of the complementary sequence. The region (Yc) may be completely complementary to the entire region or the partial region complementary to the region (Y), or may be non-complementary to 1 or several bases, but is preferably completely complementary. The 1 base or several bases are, 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 inner 5' -side region (X) may be directly or indirectly linked, for example. In the former case, the direct linkage includes, for example, linkage based on a phosphodiester bond. In the latter case, for example, the following modes can be mentioned: a connection sub-region (Lx) is provided between the region (Xc) and the region (X), and the region (Xc) and the region (X) are connected by the connection sub-region (Lx).

In the ssNc molecule of the present invention, the 3 '-side region (Yc) and the inner 3' -side region (Y) may be directly or indirectly connected, for example. In the former case, the direct linkage includes, for example, linkage based on a phosphodiester bond. In the latter case, for example, the following modes can be mentioned: a connecting sub-region (Ly) is provided between the region (Yc) and the region (Y), and the region (Yc) and the region (Y) are connected by the connecting sub-region (Ly).

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

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

An example of ssNc molecules of the present invention that do not have the linker region is shown schematically in fig. 1. Fig. 1 (a) is a schematic diagram showing the sequence of the regions from the 5 'side to the 3' side of the ssNc molecule, and fig. 1 (B) is a schematic diagram showing a state in which the ssNc molecule forms a double strand in the molecule. As shown in fig. 1 (B), the 5 'side region (Xc) is folded back to form a double strand between the 5' side region (Xc) and the inner 5 'side region (X), the 3' side region (Yc) is folded back to form a double strand between the 3 'side region (Yc) and the inner 3' side region (Y), and the ssNc molecule is labeled as ssNc. Fig. 1 is a diagram showing only the connection order of the regions and the positional relationship of the regions forming a double strand, and the length of each region and the like are not limited thereto.

An example of the ssNc molecule having the linker region of the ssNc molecule of the present invention is shown in the schematic diagram of fig. 2. Fig. 2 (a) is a schematic diagram showing, as an example, the sequence of the regions from the 5 'side to the 3' side of the ssNc molecule, and fig. 2 (B) is a schematic diagram showing a state in which the ssNc molecule forms a double strand in the molecule. As shown in fig. 2 (B), in the ssNc molecule, a double strand is formed between the 5 'side region (Xc) and the inner 5' side region (X), a double strand is formed between the inner 3 'side region (Y) and the 3' side region (Yc), and the Lx region and the Ly region form a loop structure. Fig. 2 is a diagram showing only the connection order of the regions and the positional relationship of the regions forming a double strand, and the length of each region and the like are not limited thereto.

In the ssNc molecule of the present invention, the number of bases in the 5 '-side region (Xc), the inner 5' -side region (X), the inner 3 '-side region (Y), and the 3' -side region (Yc) is not particularly limited, and examples thereof are as follows. In the present invention, the "number of bases" refers to, for example, the "length" and may be referred to as the "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, the region (Xc) is preferably composed of a base sequence having the same base length as the region (X) and complementary to all regions from the 5 '-end to the 3' -end of the region (X), for example. The region (Xc) is more preferably the same base length as the region (X) and all bases of the region (Xc) are complementary to all bases of the region (X), that is, preferably, completely complementary, for example. For example, as described above, the non-complementary base may be 1 base or several bases.

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) is preferably composed of a base sequence having the same base length as that of a partial region of the region (X), that is, a base length shorter than the region (X) by 1 base or more. The region (Xc) is more preferably the same base length as the partial region of the region (X) and all bases of the region (Xc) are complementary to all bases of the partial region of the region (X), that is, preferably, for example, completely complementary. The partial region of the region (X) is preferably a region (fragment) composed of a base sequence continuous from the 5' -end base (base at position 1) in the region (X), for example.

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) is preferably composed of a base sequence having the same base length as that of the region (Y) and complementary to all regions from the 5 '-end to the 3' -end of the region (Y). The region (Yc) is more preferably the same base length as the region (Y) and all bases of the region (Yc) are complementary to all bases of the region (Y), that is, preferably, for example, completely complementary. For example, as described above, the non-complementary base may be 1 base or several bases.

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) is preferably composed of a base sequence having the same base length as that of a partial region of the region (Y), that is, a base length shorter than the region (Y) by 1 base or more. More preferably, the region (Yc) is the same base length as the partial region of the region (Y) and all bases of the region (Yc) are complementary to all bases of the partial region of the region (Y), that is, preferably, for example, completely complementary. The partial region of the region (Y) is preferably a region (fragment) composed of a base sequence continuous from the base at the 3' -end (base at the 1 st position) in the region (Y), for example.

In the ssNc molecule of the present invention, the relationship between the number of bases (Z) in the inner region (Z), 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), and the relationship between the number of bases (Z) in the inner region (Z), the number of bases (X) in the inner 5 'side region (X), and the number of bases (Xc) in the 5' side region (Xc) satisfy, for example, the conditions of the following expressions (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 internal 5 'side region (X) and the length of the number of bases (Y) in the internal 3' side region (Y) is not particularly limited, and for example, any condition in the following formula is satisfied.

X=Y…(19)

X＜Y…(20)

X＞Y…(21)

In the ssNc molecule of the present invention, the relationship between the number of bases (X) in the internal 5 'region (X), the number of bases (Xc) in the 5' region (Xc), the number of bases (Y) in the internal 3 'region (Y), and the number of bases (Yc) in the 3' region (Yc) satisfies, for example, any one of the following conditions (a) to (d).

(a) The following conditions (3) and (4) are satisfied.

X＞Xc…(3)

Y=Yc…(4)

(b) The following conditions (5) and (6) are satisfied.

X=Xc…(5)

Y＞Yc…(6)

(c) The following conditions (7) and (8) are satisfied.

X＞Xc…(7)

Y＞Yc…(8)

(d) The following conditions (9) and (10) are satisfied.

X=Xc…(9)

Y=Yc…(10)

In the above (a) to (d), the difference between the number of bases (X) in the internal 5 'side region (X) and the number of bases (Xc) in the 5' side region (Xc), and the difference between the number of bases (Y) in the internal 3 'side region (Y) and the number of bases (Yc) in the 3' side region (Yc) preferably satisfy the following conditions, for example.

(a) The following conditions (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 following conditions (13) and (14) are satisfied.

X-Xc=0…(13)

Y-Yc = 1-10, preferably 1,2, 3 or 4,

more preferably 1,2 or 3 … (14)

(c) The following conditions (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 following conditions (17) and (18) are satisfied.

X-Xc=0…(17)

Y-Yc=0…(18)

Examples of the structures of the ssNc molecules (a) to (d) are shown schematically in fig. 3. Fig. 3 shows examples of ssNc molecules including the linker region (Lx) and the linker region (Ly), where (a) is the ssNc molecule of (a), where (B) is the ssNc molecule of (B), where (C) is the ssNc molecule of (C), and where (D) is the ssNc molecule of (D). In fig. 3, the dotted line indicates a state in which a double strand is formed by self-annealing. The ssNc molecule in fig. 3 represents the number of bases (X) in the internal 5 'side region (X) and the number of bases (Y) in the internal 3' side region (Y) as "X < Y" in the above formula (20), and is not limited thereto, and may be "X = Y" in the above formula (19) or "X > Y" in the above formula (21), as described above. Fig. 3 is a schematic diagram showing only the relationship between the internal 5 'side region (X) and the 5' side region (Xc) and the relationship between the internal 3 'side region (Y) and the 3' side region (Yc), and for example, the length, shape, and the like of each region are not limited thereto, and the presence or absence of the connecting sub-region (Lx) and the connecting sub-region (Ly) are not limited thereto.

The ssNc molecules (a) to (c) have the following structures, 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 region (Y) form a double strand, respectively, and the inner region (Z) has a base that is not aligned with both the 5 '-side region (Xc) and the 3' -side region (Yc), and is also referred to as having a base structure that does not form a double strand. Hereinafter, the nonalignable base (also referred to as a base that does not form a double strand) in the internal region (Z) is referred to as a "free base". In FIG. 3, the region of the free base is represented by "F". The number of bases in the above-mentioned 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 ssNc molecules in the above (a); the number of bases of "Y-Yc" in the case of ssNc molecule (b) above; in the case of ssNc molecules of the above-mentioned group (c), the total number of bases of "X-Xc" and "Y-Yc" is defined as the number of bases.

On the other hand, the ssNc molecule of the above (d) has a structure in which, for example, the entire region of the above internal region (Z) is aligned with the 5 '-side region (Xc) and the 3' -side region (Yc), and may be referred to as a structure in which the entire region of the above internal region (Z) forms a double strand. In the ssNc molecule (d), the 5 '-end of the 5' -side region (Xc) and the 3 '-end of the 3' -side region (Yc) are not linked.

The length of each region of the ssNc molecule of the present invention is shown below by way of example, but the present invention is not limited thereto. In the present invention, the numerical range of the number of bases includes, for example, all positive integers belonging to the range, and the description of "1 to 4 bases" refers to all disclosures of "1, 2, 3, and 4 bases" (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). Therefore, the length of the 5 '-side region (Xc) and the 3' -side region (Yc) can be determined as appropriate, for example, depending on the length of the internal region (Z), the number (F) of free bases, and the position thereof.

The number of bases in the inner 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 19 bases, 20 bases, 21 bases, 22 bases, 23 bases, 24 bases, 25 bases, 26 bases, 27 bases, 28 bases, 29 bases, and 30 bases.

When the internal region (Z) includes the expression-suppressing sequence, the internal region (Z) may be a region composed of only the expression-suppressing sequence, or may be a region including the expression-suppressing sequence. The number of bases of the expression-suppressing sequence is, for example, 19 to 30 bases, preferably 19, 20 or 21 bases. When the internal region (Z) contains the expression-suppressing sequence, it may further have an additional sequence on the 5 'side and/or 3' side of the expression-suppressing sequence. The number of bases in 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, still more preferably 1 to 4 bases, and particularly preferably 1 base, 2 bases, or 3 bases. When the internal region (Z) or the 3' -side region (Yc) contains the expression-suppressing sequence, the number of bases is preferably as large as that. Specifically, when the number of bases in the inner 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, preferably 1 to 7 bases, more preferably 1 to 4 bases, and further preferably 1 base, 2 bases, or 3 bases.

When the 5 'region (Xc) includes the expression-suppressing sequence, the 5' region (Xc) may be a region composed of only the expression-suppressing sequence, or may be a region including the expression-suppressing sequence. The length of the expression-suppressing sequence is, for example, as described above. When the 5 ' -side region (Xc) contains the expression-suppressing sequence, the 5 ' -side and/or 3 ' -side of the expression-suppressing sequence may further contain an additional sequence. The number of bases in the additional sequence is, for example, 1 to 11 bases, 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, still more preferably 1 to 4 bases, and particularly preferably 1 base, 2 bases, or 3 bases. When the internal region (Z) or the 5' -side region (Xc) includes the expression-suppressing sequence, the number of bases is preferably as large as this. Specifically, when the number of bases in the inner 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, preferably 1 to 7 bases, more preferably 1 to 4 bases, and further preferably 1 base, 2 bases, or 3 bases.

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

As described above, the number of bases in the inner region (Z), the 5 '-side region (Xc) and the 3' -side region (Yc) can be represented by, for example, "Z.gtoreq.Xc + Yc" in the formula (2). Specifically, the number of bases of "Xc + Yc" is, for example, the same as or smaller than the internal region (Z). In the latter case, "Z- (Xc + Yc)" is, for example, 1 to 10, preferably 1 to 4, and more preferably 1,2 or 3. The "Z- (Xc + Yc)" corresponds to, for example, the number of bases (F) in the region (F) of the free base in the internal region (Z).

In the ssNc molecule of the present invention, the lengths of the linker region (Lx) and the linker region (Ly) are not particularly limited. The linker region (Lx) is preferably a length that allows the internal 5 'region (X) and the 5' region (Xc) to form a double strand, and the linker region (Ly) is preferably a length that allows the internal 3 'region (Y) and the 3' region (Yc) to form a double strand. When the structural units of the linker region (Lx) and the linker region (Ly) comprise bases, the number of bases of the linker region (Lx) and the number of bases of the linker region (Ly) may be the same or different, and the base sequences may be the same or different. The number of bases in the linker region (Lx) and the linker region (Ly) is, for example, 1 base, preferably 2 bases, and more preferably 3 bases at the lower limit, and 100 bases, preferably 80 bases, and more preferably 50 bases at the upper limit. Specific examples of the number of bases in each linker region include, but are not limited to, 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.

The overall length of the ssNc molecule of the present invention is not particularly limited. In the ssNc molecule of the present invention, the total number of bases (the number of bases in the entire length) has a lower limit of, for example, 38 bases, preferably 42 bases, more preferably 50 bases, further preferably 51 bases, and particularly preferably 52 bases, and an upper limit of, for example, 300 bases, preferably 200 bases, more preferably 150 bases, further preferably 100 bases, and particularly preferably 80 bases. In the ssNc molecule of the present invention, the total number of bases other than the linker region (Lx) and the linker region (Ly) has a lower limit of, for example, 38 bases, preferably 42 bases, more preferably 50 bases, further preferably 51 bases, and particularly preferably 52 bases, and an upper limit of, for example, 300 bases, preferably 200 bases, more preferably 150 bases, further preferably 100 bases, and particularly preferably 80 bases.

The constitutional 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 nucleotide residue and a modified nucleotide residue. The ssNc molecule of the present invention can improve nuclease resistance and stability by including the modified nucleotide residue. In addition, the ssNc molecules of the present invention may comprise non-nucleotide residues in addition to, for example, the nucleotide residues described above. The nucleotide residues and the non-nucleotide residues are described in detail below.

In the ssNc molecule of the present invention, the constituent units of the internal region (Z), the 5 '-side region (Xc), and the 3' -side region (Yc) are preferably the nucleotide residues, respectively. The respective regions are composed of, for example, the following residues (1) to (3).

(1) Non-modified nucleotide residue

(2) Modified nucleotide residues

(3) Non-modified nucleotide residue and modified nucleotide residue

In the ssNc molecule of the present invention, the constituent units of the linker region (Lx) and the linker region (Ly) are not particularly limited, and examples thereof include the nucleotide residues and the non-nucleotide residues. The linker region may be composed of, for example, only the nucleotide residues, only the non-nucleotide residues, or both the nucleotide residues and the non-nucleotide residues. The linker region is composed of, for example, the following residues (1) to (7).

(1) Non-modified nucleotide residue

(2) Modified nucleotide residues

(3) Non-modified nucleotide residue and modified nucleotide residue

(4) Non-nucleotide residues

(5) Non-nucleotide residues and non-modified nucleotide residues

(6) Non-nucleotide residues and modified nucleotide residues

(7) Non-nucleotide residue, non-modified nucleotide residue and modified nucleotide residue

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

Examples of the ssNc molecule of the present invention include a molecule composed of only the above-mentioned nucleotide residues, and a molecule containing the above-mentioned non-nucleotide residues in addition to the above-mentioned nucleotide residues. In the ssNc molecule of the present invention, as described above, the nucleotide residue may be, for example, only the non-modified nucleotide residue, only the modified nucleotide residue, or both the non-modified nucleotide residue and the modified nucleotide residue. When the ssNc molecule includes the unmodified nucleotide residue and the modified nucleotide residue, the number of the modified nucleotide residues is not particularly limited, and is, for example, "1 or several", specifically, 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 contains the above-mentioned non-nucleotide residues, the number of the above-mentioned non-nucleotide residues is not particularly limited, and is, for example, "1 or several", specifically, 1 to 8, 1 to 6, 1 to 4, 1,2 or 3.

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

When the ssRNA molecule includes the modified ribonucleotide residue in addition to the non-modified ribonucleotide residue, the number of the modified ribonucleotide residues is not particularly limited, and is, for example, "1 or several", specifically, 1 to 5, preferably 1 to 4, more preferably 1 to 3, and most preferably 1 or 2. The modified ribonucleotide residue that is opposite to the non-modified ribonucleotide residue can be, for example, the deoxyribonucleotide residue in which a ribonucleotide residue is substituted with a deoxyribose residue. When the ssRNA molecule includes the deoxyribonucleotide residues in addition to the non-modified ribonucleotide residue, the number of the deoxyribonucleotide residues is not particularly limited, and is, for example, "1 or several", specifically, 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 contains, for example, a labeling substance, and can be labeled with the labeling substance. The labeling substance is not particularly limited, and examples thereof include a fluorescent substance, a dye, and an isotope. 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 Alexa 488. Examples of the isotope include a stable isotope and a radioactive isotope, and a stable isotope is preferable. The stable isotope is excellent in operability because of its low risk of exposure and no need for dedicated facilities, and can be reduced in cost. In addition, the stable isotope is excellent in properties as a tracer, for example, because there is no change in physical properties of a labeled compound. The stable isotope is not particularly limited, and examples thereof include²H、¹³C、¹⁵N、¹⁷O、¹⁸O、³³S、³⁴S and³⁶S。

as described above, the ssNc molecule of the present invention can inhibit the expression of the above 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 contains, for example, a sequence that suppresses the expression of the gene responsible for the disease as the expression suppression sequence, the disease can be treated by, for example, suppression of the expression of the target gene. In the present invention, "treatment" includes, for example, prevention of the above-mentioned diseases, improvement of diseases, and improvement of prognosis, and may be any of them.

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 a subject having the target gene.

Examples of the subject to be administered include cells, tissues and organs. Examples of the subject to be administered include humans, and non-human animals such as non-human mammals other than humans. The administration may be in vivo or in vitro, for example. The above cells are not particularly limited, and examples thereof include various cultured cells such as HeLa cells, 293 cells, NIH3T3 cells, COS cells, and the like; stem cells such as ES cells and hematopoietic stem cells; cells isolated from organisms such as primary cultured cells; and so on.

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

Specific examples of ssNc molecules of the present invention are shown below, but the present invention is not limited to these. The base sequence of the ssNc molecule may be, for example, the base sequence of seq id nos. 2, 7,8, 13, 14, 29 to 35, 37, 43, 44, 47, 48 and 51 to 80, or may be, for example, a base sequence in which 1 or more bases are deleted, substituted and/or added to the base sequence. Examples of the target gene include nucleotide sequences of SEQ ID Nos. 2, 7,8, 13, 37 and 51 to 80 when GAPDH gene is used as the target gene, nucleotide sequences of SEQ ID Nos. 14 and 29 to 35 when TGF-. beta.1 is used as the target gene, nucleotide sequences of SEQ ID Nos. 43 and 44 when LAMA1 gene is used as the target gene, and nucleotide sequences of SEQ ID Nos. 47 and 48 when LMNA gene is used as the target gene.

The use of the ssNc molecule of the present invention can be described with reference to the composition, expression inhibition method, treatment method, and the like of the present invention described below.

As described above, the ssNc molecule of the present invention can inhibit the expression of a target gene, and thus is useful as a research tool for pharmaceuticals, diagnostic agents, agricultural chemicals, medicine, life sciences, and the like.

2. Nucleotide residues

The nucleotide residue includes, for example, a sugar, a base and a phosphate as constituent elements. As described above, examples of the nucleotide residues include ribonucleotide residues and deoxyribonucleotide residues. The ribonucleotide residue has, for example, a ribose residue as a sugar, and adenine (a), guanine (G), cytosine (C), and U (uracil) as bases, and the deoxyribose residue has, for example, a deoxyribose residue as a sugar, and adenine (a), guanine (G), cytosine (C), and thymine (T) as bases.

Examples of the nucleotide residue include an unmodified nucleotide residue and a modified nucleotide residue. In the unmodified nucleotide residue, the respective components are, for example, the same as or substantially the same as those of naturally occurring components, and preferably the same as or substantially the same as those of naturally occurring components in a human body.

The modified nucleotide residue is, for example, a nucleotide residue obtained by modifying the unmodified nucleotide residue. The modified nucleotide residue may be, for example, one of the constituent elements of the unmodified nucleotide residue described above. In the present invention, "modification" includes, for example, substitution, addition and/or deletion of the above-mentioned constituent elements; substitution, addition and/or deletion of atoms and/or functional groups in the above-described constituent elements can be referred to as "alteration". Examples of the modified nucleotide residue include naturally occurring nucleotide residues and artificially modified nucleotide residues. The naturally-derived modified nucleotide residues can be referred to, for example, as in Limbach et al (Limbache., 1994, summer: the modified nucleotides of RNA, nucleic acids of Res.22: 2183-2196). The modified nucleotide residue may be, for example, a residue of a substitute for the nucleotide.

Examples of the modification of the nucleotide residue include modification of a ribose-phosphate backbone (hereinafter, ribose-phosphate backbone).

The ribose phosphate backbone can be modified with a ribose residue, for example. The ribose residue can be modified, for example, at the 2 '-position carbon, and specifically, for example, the hydroxyl group bonded to the 2' -position carbon can be substituted with a halogen such as hydrogen or fluorine. The hydroxyl group at the 2' -carbon can be substituted with hydrogen to substitute a ribose residue with deoxyribose. The ribose residue may be substituted with, for example, a stereoisomer, and may be substituted with, for example, an arabinose residue.

The ribose phosphate backbone may be substituted with a non-ribose phosphate backbone having a non-ribose residue and/or a non-phosphate, for example. Examples of the non-ribose phosphate skeleton include the non-charged bodies of the ribose phosphate skeleton. Examples of the substitution of the nucleotide substituted for the non-ribose phosphate skeleton include morpholino, cyclobutyl and pyrrolidine. In addition, the above-mentioned substitute includes, for example, an artificial nucleic acid monomer residue. Specific examples thereof include PNA (peptide nucleic acid), LNA (locked nucleic acid), ENA (2 '-O, 4' -C-ethylene bridged nucleic acid, 2 '-O, 4' -C-ethylene bridged nucleic acid), and the like, and PNA is preferable.

In the ribose phosphate backbone, for example, a phosphate group can be modified. In the above ribose phosphate backbone, the phosphate group closest to the sugar residue is referred to as an α phosphate group. The above-mentioned alpha phosphate group is negatively charged, and its charge is uniformly distributed over 2 oxygen atoms not bonded to the sugar residue. Among the 4 oxygen atoms of the above-mentioned α -phosphate group, 2 oxygen atoms not bonded to the sugar residue in the phosphodiester bond between nucleotide residues are hereinafter referred to as "non-bonding oxygen". On the other hand, the 2 oxygen atoms bonded to the sugar residue in the phosphodiester bond between the above-mentioned nucleotide residues are hereinafter referred to as "bonding oxygen". The α phosphate group is preferably modified to be uncharged or to have asymmetric charge distribution in the non-bonded oxygen, for example.

In the above-mentioned phosphoric acid group, the above-mentioned non-bonded oxygen may be substituted, for example. The oxygen may be substituted with any atom selected from S (sulfur), Se (selenium), B (boron), C (carbon), H (hydrogen), N (nitrogen) and OR (R is an alkyl group OR an aryl group), for example, and is preferably substituted with S. The non-bonded oxygen is preferably substituted with, for example, S. Examples of the modified phosphoric acid group include phosphorothioate, phosphorodithioate, selenothioate, boranophosphate, hydrogenphosphonate, phosphoroamidate, alkylphosphonate or arylphosphonate, and phosphotriester, and among them, phosphorodithioate in which both of the 2 non-bonded oxygens are substituted with S is preferable.

In the above-mentioned phosphoric acid group, the above-mentioned bonded oxygen may be substituted, for example. The above oxygen may be substituted with, for example, any one atom of S (sulfur), C (carbon) and N (nitrogen). Examples of the modified phosphate group include a crosslinked phosphoramidate substituted with N, a crosslinked phosphorothioate substituted with S, and a crosslinked methylenephosphonate substituted with C. The substitution of the bonding oxygen is preferably performed, for example, at least at 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, the substitution is preferably performed with C, and in the case of the 3' -side, the substitution is preferably performed with N.

The phosphate group may be substituted with the non-phosphorus-containing linker. The above linkers include, for example, siloxane, carbonate, carboxymethyl, carbamate, amide, thioether, ethylene oxide linker, sulfonate, sulfonamide, thioacetal, methylal, oxime, methyleneimino, methylenemethylimino, methylenehydrazino, methylenedimethylhydrazino, methyleneoxymethylimino, etc., preferably methylenecarbonylamino and methylenemethylimino.

The ssNc molecules of the present invention may be modified, for example, at nucleotide residues at least one of the 3 'terminus and the 5' terminus. The modification may be, for example, either the 3 '-terminus or the 5' -terminus, or both. As described above, the modification is preferably performed, for example, on the terminal phosphate group. The phosphate group may be modified as a whole, or 1 or more atoms in the phosphate group may be modified. In the former case, for example, the phosphate group may be entirely substituted or may be deleted.

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

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

The spacer is not particularly limited and may include, for example, - (CH)₂)_n-、-(CH₂)_nN-、-(CH₂)_nO-、-(CH₂)_nS-、O(CH₂CH₂O)_nCH₂CH₂OH, alkali-free sugars, amides, carboxyl, amines, hydroxyamines, hydroxyimines, thioethers, disulfides, thioureas, sulfonamides, morpholinos, and the like, as well as biotin and fluorescein agents, and the like. In the above formula, n is a positive integer, preferably n =3 or 6.

In addition to these, examples of the molecule added to the above-mentioned terminal include a dye, a chimera (e.g., acridine), a crosslinking agent (e.g., psoralen, mitomycin C), porphyrin (TPPC4, Texophyrin, Sapphyrin), a polycyclic aromatic hydrocarbon (e.g., phenazine, dihydrophenazine), an artificial endonuclease (e.g., EDTA), a lipophilic carrier (e.g., cholesterol, cholic acid, adamantane acid, 1-pyrenebutanoic acid, dihydrotestosterone, 1, 3-bis-O (hexadecyl) glycerol, geranylhexyl, hexadecaneGlycerol, borneol, menthol, 1, 3-propanediol, heptadecyl, palmitic acid, myristic acid, O3- (oleoyl) lithocholic acid, O3- (oleoyl) cholic acid, dimethoxytrityl or phenoxazine) and peptide complexes (e.g., drosophila melanogaster podophyllum peptide, Tat peptide), alkylating agents, phosphoric acid, amino groups, mercapto groups, PEG (e.g., PEG-40K), MPEG, [ MPEG ] -40K]₂Polyamino groups, alkyl groups, substituted alkyl groups, radioactive labels, enzymes, haptens (e.g., biotin), transport/absorption enhancers (e.g., aspirin, vitamin E, folic acid), synthetic ribonucleases (e.g., imidazole, diimidazole, histamine, imidazole clusters, acridine-imidazole complexes, tetraazamacrocycle Eu)³⁺Complex), and the like.

The above-mentioned 5' end of the ssNc molecule of the present invention may be modified with, for example, a phosphate group or a phosphate group analog. Examples of the phosphate group include 5' -monophosphate ((HO)₂(O) P-O-5 '), 5' diphosphonic acid ((HO)₂(O) P-O-P (HO) (O) O-5 '), 5' triphosphate ((HO)₂(O) P-O- (HO) (O) P-O-P (HO) (O) (O) -O-5 '), 5' -guanosine cap (7-methylated or unmethylated, 7m-G-O-5 '- (HO) (O) P-O- (HO) (O) P-O-P (HO) (O)) (O) -O-5'), 5 '-adenosine cap (Appp), optionally modified or unmodified nucleotide cap structure (N-O-5' - (HO) (O) P-O- (HO) (O) P-O-P (HO) (O)) (O) -O-5 '), 5' -phosphorothioate (phosphorothioate: (HO)₂(S) P-O-5 '), 5' -dithiophosphoric acid (dithiophosphoric acid esters: (HO) (HS) (S) P-O-5 '), 5' -thiophosphate ((HO)₂(O) P-S-5 '), sulfur-substituted monophosphates, diphosphates and triphosphates (e.g., 5' -alpha-thiotriphosphate, 5 '-gamma-thiotriphosphate, etc.), 5' -phosphoramidates ((HO)₂(O)P-NH-5’、(HO)(NH₂) (O) P-O-5 '), 5 ' -alkylphosphonic acids (e.g., RP (OH) (O) -O-5 ', (OH)₂(O)P-5’-CH₂R is an alkyl group (e.g., methyl, ethyl, isopropyl, propyl, etc.)), 5 '-alkyletherphosphonic acid (e.g., RP (OH) (O) -O-5', R is an alkylether (e.g., methoxymethyl, ethoxymethyl, etc.)), and the like.

In the nucleotide residue, the base is not particularly limited. The base may be, for example, a natural base or an unnatural base. The base may be, for example, a natural one or a synthetic one. Examples of the base include a general base and a modified analog thereof.

Examples of the base include purine bases such as adenine and guanine; cytosine bases such as cytosine, uracil and thymine. In addition, examples of the above base include inosine, thymine, xanthine, hypoxanthine, nubularine, isogluaniine, tubercidin (tubercidine), and the like. Examples of the base include alkyl derivatives such as 2-aminoadenine and 6-methylated purine; alkyl derivatives such as 2-propylated purine; 5-halouracils and 5-halocytosines; 5-propynyluracil and 5-propynylcytosine; 6-azouracil, 6-azacytosine, and 6-azothymine; 5-uracil (pseudouracil), 4-thiouracil, 5-halouracil, 5- (2-aminopropyl) uracil, 5-aminoallyl uracil; 8-halogenated, aminated, thiolated, thioalkylated, hydroxylated and other 8-substituted purines; 5-trifluoromethylated and other 5-substituted pyrimidines; 7-methylguanine; 5-substituted pyrimidines; 6-azapyrimidine; n-2, N-6 and O-6 substituted purines (including 2-aminopropyladenine); 5-propynyluracil and 5-propynylcytosine; a dihydrouracil; 3-deaza-5-azacytosine; 2-aminopurine; 5-alkyluracils; 7-alkylguanine; 5-alkylcytosine; 7-deazaadenine; n6, N6-dimethyladenine; 2, 6-diaminopurine; 5-amino-allyl-uracil; n3-methyluracil; substituted 1,2, 4-triazoles; 2-pyridone; 5-nitroindole; 3-nitropyrrole; 5-methoxy uracil; 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 bases, and the like. In addition, purines and pyrimidines include, for example, U.S. Pat. No. 3,687,808, "ConsiseEncyclopedia of Polymer science and engineering", pp.858-859, eds Kroschwitz J.I., John Wiley & Sons, 1990; and Englisch et al, Angewandte Chemie, International edition, 1991, Vol.30, p.613.

In addition to these, the modified nucleotide residues may also include, for example, a missing base residue, i.e., an abasic ribose phosphate backbone. Further, as the modified nucleotide residue, for example, those described in U.S. provisional application No. 60/465,665 (application date: 4/25/2003) and International application No. PCT/US04/07070 (application date: 3/8/2004) can be used, and these documents can be cited in the present invention.

3. Non-nucleotide residues

The above-mentioned non-nucleotide residues are not particularly limited. The ssNc molecule of the present invention may have a non-nucleotide structure including a pyrrolidine skeleton or a piperidine skeleton as the non-nucleotide residue. Preferably, for example, at least one of the linker region (Lx) and the linker region (Ly) has the non-nucleotide residue. The non-nucleotide residue may be present in the linker region (Lx), the non-nucleotide residue may be present in the linker region (Ly), or the non-nucleotide residues may be present in both of 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 pyrrolidine derivative in which 1 or more carbon atoms constituting the five-membered ring of pyrrolidine are substituted, and when substituted, for example, a carbon atom other than C-2 carbon atoms is preferable. The above carbons may be substituted with, for example, nitrogen, oxygen or sulfur. The aforementioned pyrrolidine skeleton may, for example, contain, for example, a carbon-carbon double bond or a carbon-nitrogen double bond within the five-membered ring of pyrrolidine. In the pyrrolidine skeleton, hydrogen may be bonded to a carbon and a nitrogen constituting a five-membered ring of pyrrolidine, or a substituent described later may be bonded thereto. The linker region (Lx) may be bonded to the region (X) and the region (Xc) via any group of the pyrrolidine skeleton, and is preferably any 1 carbon atom and nitrogen of the five-membered ring, and is preferably carbon (C-2) and nitrogen at the 2-position of the five-membered ring. Examples of the pyrrolidine skeleton include a proline skeleton and a prolinol skeleton. The proline skeleton, prolinol skeleton, and the like are, for example, substances in vivo and reduction products thereof, and therefore are also excellent in safety.

The piperidine skeleton may be, for example, a skeleton of a piperidine derivative in which 1 or more carbon atoms constituting the six-membered ring of the piperidine are substituted, and when substituted, for example, a carbon atom other than the carbon atom of C-2 is preferable. The above carbons may be substituted with, for example, nitrogen, oxygen or sulfur. The piperidine skeleton may, for example, contain, for example, a carbon-carbon double bond or a carbon-nitrogen double bond within a six-membered ring of piperidine. In the piperidine skeleton, a carbon and a nitrogen constituting a six-membered ring of the piperidine may be bonded to a hydrogen group, for example, or may be bonded to a substituent described later. The linker region (Lx) may be bonded to the region (X) and the region (Xc) via any group of the piperidine skeleton, and is preferably any 1 carbon atom and nitrogen in the six-membered ring, and is preferably carbon (C-2) and nitrogen at the 2-position of the six-membered ring. The same applies to the linker region (Ly) described above.

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

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

[ chemical formula 1]

In the above-mentioned formula (I),

X¹and X²Each independently is H₂O, S or NH;

Y¹and Y²Each independently is a single bond, CH₂NH, O or S;

R³is a hydrogen atom or a substituent bonded to C-3, C-4, C-5 or C-6 on the ring A,

the above substituents are OH and OR⁴、NH₂、NHR⁴、NR⁴R⁵、SH、SR⁴Or oxy (= O);

R³in the case of the above-mentioned substituent, the substituent R³May be 1, plural or none, and in the case of plural, the same or different;

R⁴and R⁵Are substituents or protecting groups, which may be the same or different;

L¹is an alkylene chain composed of n atoms, where the hydrogen atoms on the alkylene carbon atoms may be replaced by OH, OR^a、NH₂、NHR^a、NR^aR^bSH or SR^aMay be unsubstituted or, alternatively,

L¹a polyether chain in which 1 or more carbon atoms of the alkylene chain are replaced with oxygen atoms,

wherein, Y¹In the case of NH, O or S, with Y¹Bonded L¹Is carbon, and OR¹Bonded L¹The atoms of (b) are carbon, and the oxygen atoms are not adjacent to each other;

L²is an alkylene chain of m atoms, where the hydrogen atoms on the alkylene carbon atoms may be replaced by OH, OR^c、NH₂、NHR^c、NR^cR^dSH or SR^cMay be unsubstituted or, alternatively,

L²a polyether chain in which 1 or more carbon atoms of the alkylene chain are replaced with oxygen atoms,

wherein, Y²In the case of NH, O or S, with Y²Bonded L²Atom of (2)Is carbon, with OR²Bonded L²The atoms of (b) are carbon, and the oxygen atoms are not adjacent to each other;

R^a、R^b、R^cand R^dEach independently is 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;

ring A may be a ring wherein 1 carbon atom other than C-2 on the above ring A is substituted with nitrogen, oxygen or sulfur,

the above ring A may contain a carbon-carbon double bond or a carbon-nitrogen double bond. When the connecting sub-region (Lx) is represented by the formula (1), the region (Xc) and the region (X) are each defined by-OR¹-OR-OR²-binding to the above-mentioned linker region (Lx). When the connecting domain (Ly) is represented by the formula (1), the domain (Yc) and the domain (Y) are each represented by-OR¹-OR-OR²-binding to the above-mentioned linker region (Ly). Here, R¹And R²May or may not be present, R¹And R²In the presence of R¹And R²Each independently a nucleotide residue or the structure (I) above.

In the above formula (I), X¹And X²For example each independently of the other is H₂O, S or NH. In the above formula (I), X¹Is H₂Means X¹And X¹The bonded carbon atoms together forming CH₂(methylene group). With respect to X²The same applies.

In the above formula (I), in the ring A, l is 1 or 2. When l =1, the ring a is a five-membered ring, for example, the aforementioned pyrrolidine skeleton. Examples of the pyrrolidine skeleton include a proline skeleton and a prolinol skeleton, and divalent structures thereof are exemplified. When l =2, ring a is a six-membered ring, for example, the piperidine skeleton described above. Ring A may be a ring A wherein 1 carbon atom other than C-2 is replaced by nitrogen, oxygen or sulfur. In addition, ring a may contain a carbon-carbon double bond or a carbon-nitrogen double bond within ring a. Ring A may be, for example, any of L-type and D-type.

In the above formula (I), Y¹And Y²Each independently is a single bond, CH₂NH, O or S.

In the above formula (I), R³Is a hydrogen atom or substituent bonded to C-3, C-4, C-5 or C-6 on ring A. The above substituents are OH and OR⁴、NH₂、NHR⁴、NR⁴R⁵、SH、SR⁴Or oxy (= O). R³In the case of the above-mentioned substituent, the substituent R³The number of the organic compounds may be 1, plural or none, and in the case of plural, the organic compounds may be the same or different. R⁴And R⁵The substituents or protecting groups may be the same or different.

Examples of the substituent include halogen, alkyl, alkenyl, alkynyl, haloalkyl, aryl, heteroaryl, aralkyl, cycloalkyl, cycloalkenyl, cycloalkylalkyl, hydroxyalkyl, alkoxyalkyl, aminoalkyl, heterocyclylalkyl, heteroaralkyl, silyl, siloxyalkyl, and the like. The same applies below.

The protective group is, for example, a functional group which converts a highly reactive functional group into an inactive functional group, and known protective groups and the like can be mentioned. Examples of the above-mentioned protecting group include those described in the literature (J.F.W.McOmie, "protecting group organic chemistry," PrenumPress, London and dNewYork, 1973). The protecting group is not particularly limited, and examples thereof include tert-butyldimethylsilyl (TBDMS), bis (2-acetoxyethoxy) methyl (ACE), Triisopropylsiloxymethyl (TOM), 1- (2-cyanoethoxy) ethyl (CEE), 2-Cyanoethoxymethyl (CEM), Tosylethoxymethyl (TEM), and dimethoxytrityl (DMTr). R³Is OR⁴In the case of (2), the protecting group is not particularly limited, and examples thereof include a TBDMS group, an ACE group, a TOM group and a CEE groupCEM-based and TEM-based, and the like. In addition to these, the silyl group-containing group described later can be mentioned. The same applies to the following.

In the above formula (I), L¹Is an alkylene chain consisting of n atoms. The hydrogen atoms on the above-mentioned alkylene carbon atoms may be replaced by, for example, OH, OR^a、NH₂、NHR^a、NR^aR^bSH or SR^aAnd may be unsubstituted. Or, L¹It may be a polyether chain in which 1 or more carbon atoms of the alkylene chain are replaced with an oxygen atom. The polyether chain is, for example, polyethylene glycol. In addition, Y is¹In the case of NH, O or S, with Y¹Bonded L¹Is carbon, and OR¹Bonded L¹Is carbon and the oxygen atoms are not adjacent to each other. I.e. for example Y¹In the case of O, the oxygen atom is bonded to L¹Are not adjacent to an oxygen atom of, OR¹Oxygen atom of (A) and L¹Are not adjacent.

In the above formula (I), L²Is an alkylene chain consisting of m atoms. The hydrogen atoms on the above-mentioned alkylene carbon atoms may be replaced by, for example, OH, OR^c、NH₂、NHR^c、NR^cR^dSH or SR^cAnd may be unsubstituted. Or, L²It may be a polyether chain in which 1 or more carbon atoms of the alkylene chain are replaced with an oxygen atom. In addition, Y is²In the case of NH, O or S, with Y²Bonded L²Is carbon, and OR²Bonded L²Is carbon and the oxygen atoms are not adjacent to each other. That is, for example, Y²In the case of O, the oxygen atom is bonded to L²Are not adjacent to an oxygen atom of, OR²Oxygen atom of (A) and L²Are not adjacent.

L¹N and L of²The lower limit of m is, for example, 0, and the upper limit is not particularly limited. n and m can be set as appropriate, for example, according to the desired length of the connecting sub-region (Lx). For example, n and m are each preferably the same in view of production cost, yield and the like0 to 30, preferably 0 to 20, and more preferably 0 to 15. 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^cAnd R^dSubstituents or protecting groups may be mentioned independently of each other. The substituents and the protecting groups are, for example, the same as those described above.

In the above formula (I), the hydrogen atoms may be each independently substituted with a halogen such as Cl, Br, F or I.

When the connecting sub-region (Lx) is represented by the formula (I), the region (Xc) and the region (X) are each, for example, through-OR¹-OR-OR²-binding to the above-mentioned linker region (Lx). Here, R¹And R²May or may not be present. R¹And R²In the presence of R¹And R²Each independently a nucleotide residue or the structure of formula (I) above. R¹And/or R²In the case of the above-mentioned nucleotide residue, the above-mentioned linker region (Lx) is obtained by, for example, removing the nucleotide residue R¹And/or R²The non-nucleotide residue and the nucleotide residue constituting the structure of the formula (I) above. R¹And/or R²In the case of the structure of the formula (I), the linker region (Lx) has, for example, a structure in which 2 or more non-nucleotide residues consisting of the structure of the formula (I) are linked. The structure of formula (I) above may comprise, for example, 1,2, 3 or 4. In this way, when a plurality of the structures are contained, the structures (I) may be linked directly or may be linked via the nucleotide residue. In another aspect, R is absent¹And R²In the case of (2), the linker region (Lx) is formed only by the non-nucleotide residue composed of the structure of the formula (I). In addition, when the connecting sub-region (Ly) is represented by the formula (I), for example, the region (Yc), the region (Y), and the connecting sub-region (Ly) can be described with reference to the connecting sub-region (Lx).

The region (Xc) and the region (X) are in contact with the-OR¹-and-OR²A combination of the above-mentioned region (Yc) and the above-mentioned region (Y) with the above-mentioned-OR¹-and-OR²The combination of (a) and (b) is not particularly limited, and examples thereof include any of the following conditions.

Condition (1)

The region (Xc) is defined by-OR²-in combination with the structure of formula (I), the region (X) being through-OR¹In combination with the structure of formula (I) above,

the region (Yc) is defined by-OR¹-in combination with the structure of formula (I), the region (Y) being through-OR²-in combination with the structure of formula (I) above.

Condition (2)

the region (Yc) is defined by-OR²-in combination with the structure of formula (I), the region (Y) being through-OR¹-in combination with the structure of formula (I) above.

Condition (3)

The region (Xc) is defined by-OR¹-in combination with the structure of formula (I), the region (X) being through-OR²In combination with the structure of formula (I) above,

Condition (4)

the region (Yc) is defined by-OR²-andthe structure of the formula (I) is combined, and the region (Y) is through-OR¹-in combination with the structure of formula (I) above.

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

[ chemical formula 2]

In the above formulae (I-1) to (I-9), n, m and q are not particularly limited, as described above. Specific examples thereof include: in the above formula (I-1), n = 8; in the above (I-2), n = 3; in the above formula (I-3), n =4 or 8; in the above (I-4), n =7 or 8; in the above formula (I-5), n =3 and m = 4; in the above (I-6), n =8 and m = 4; in the above formula (I-7), n =8 and m = 4; in the above (I-8), n =5 and m = 4; in the formula (I-9), q =1 and m = 4. An example of the formula (I-4) (n =8) is shown in the following formula (I-4a), and an example of the formula (I-6) (n =5, m =4) is shown in the following formula (I-6 a).

[ chemical formula 3]

In the present invention, the "alkyl group" includes, for example, a linear or branched alkyl group. The number of carbon atoms of the alkyl group 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 a methyl group, an ethyl group, an n-propyl group, an isopropyl group, an n-butyl group, an isobutyl group, a sec-butyl group, a tert-butyl group, an n-pentyl group, an isopentyl group, a neopentyl group, an n-hexyl group, an isohexyl group, an n-heptyl group, an n-octyl group, an n-nonyl group, and an n-decyl group. Preferred examples thereof include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, and isohexyl groups.

In the present invention, the "alkenyl group" includes, for example, a straight-chain or branched-chain alkenyl group. Examples of the alkenyl group include alkenyl groups having 1 or more double bonds in the alkyl group. The number of carbon atoms of the alkenyl group is not particularly limited, and is, for example, the same as that of the alkyl group, preferably 2 to 8. Examples of the alkenyl group include an ethenyl group, a 1-propenyl group, a 2-propenyl group, a 1-butenyl group, a 2-butenyl group, a 3-butenyl group, a1, 3-butadienyl group, and a 3-methyl-2-butenyl group.

In the present invention, the "alkynyl group" includes, for example, a straight-chain or branched-chain alkynyl group. Examples of the alkynyl group include alkynyl groups having 1 or more triple bonds in the alkyl group. The number of carbon atoms of the alkynyl group is not particularly limited, and is, for example, the same as that of the alkyl group, preferably 2 to 8. Examples of the alkynyl group include an ethynyl group, a propynyl group, and a butynyl group. The alkynyl group may further have 1 or more double bonds, for example.

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

In the present invention, "heteroaryl" includes, for example, monocyclic aromatic heterocyclic groups and fused aromatic heterocyclic groups. Examples of the heteroaryl group include furyl (e.g., 2-furyl, 3-furyl), thienyl (e.g., 2-thienyl, 3-thienyl), pyrrolyl (e.g., 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl), imidazolyl (e.g., 1-imidazolyl, 2-imidazolyl, 4-imidazolyl), pyrazolyl (e.g., 1-pyrazolyl, 3-pyrazolyl, 4-pyrazolyl), triazolyl (e.g., 1,2, 4-triazol-1-yl, 1,2, 4-triazol-3-yl, 1,2, 4-triazol-4-yl), tetrazolyl (e.g., 1-tetrazolyl, 2-tetrazolyl, 5-tetrazolyl), oxazolyl (e.g., 2-oxazolyl, etc, 4-oxazolyl, 5-oxazolyl), isoxazolyl (for example: 3-isoxazolyl, 4-isoxazolyl, 5-isoxazolyl), thiazolyl (for example: 2-thiazolyl, 4-thiazolyl, 5-thiazolyl), thiadiazolyl, isothiazolyl (e.g.: 3-isothiazolyl, 4-isothiazolyl, 5-isothiazolyl), pyridyl (e.g.: 2-pyridyl, 3-pyridyl, 4-pyridyl), pyridazinyl (e.g.: 3-pyridazinyl, 4-pyridazinyl), pyrimidinyl (for example: 2-pyrimidinyl, 4-pyrimidinyl, 5-pyrimidinyl), furazanyl (e.g.: 3-furoazine), pyrazinyl (for example: 2-pyrazinyl), oxadiazolyl (for example: 1,3, 4-oxadiazol-2-yl), benzofuranyl (for example: 2-benzo [ b ] furyl group, 3-benzo [ b ] furyl group, 4-benzo [ b ] furyl group, 5-benzo [ b ] furyl group, 6-benzo [ b ] furyl group, 7-benzo [ b ] furyl group), benzothienyl group (for example: 2-benzo [ b ] thienyl, 3-benzo [ b ] thienyl, 4-benzo [ b ] thienyl, 5-benzo [ b ] thienyl, 6-benzo [ b ] thienyl, 7-benzo [ b ] thienyl), benzimidazolyl (for example: 1-benzimidazolyl, 2-benzimidazolyl, 4-benzimidazolyl, 5-benzimidazolyl), dibenzofuranyl, benzoxazolyl, benzothiazolyl, quinoxalinyl (for example: 2-quinoxalinyl, 5-quinoxalinyl, 6-quinoxalinyl), cinnolinyl (for example: 3-cinnolinyl, 4-cinnolinyl, 5-cinnolinyl, 6-cinnolinyl, 7-cinnolinyl, 8-cinnolinyl), quinazolinyl (for example: 2-quinazolinyl, 4-quinazolinyl, 5-quinazolinyl, 6-quinazolinyl, 7-quinazolinyl, 8-quinazolinyl), quinolinyl (e.g.: 2-quinolyl, 3-quinolyl, 4-quinolyl, 5-quinolyl, 6-quinolyl, 7-quinolyl, 8-quinolyl), phthalazinyl (for example: 1-phthalazinyl group, 5-phthalazinyl group, 6-phthalazinyl group), an isoquinolinyl group (for example: 1-isoquinolinyl, 3-isoquinolinyl, 4-isoquinolinyl, 5-isoquinolinyl, 6-isoquinolinyl, 7-isoquinolinyl, 8-isoquinolinyl), purinyl, pteridinyl (for example: 2-pteridinyl, 4-pteridinyl, 6-pteridinyl, 7-pteridinyl), carbazolyl, phenanthridinyl, acridinyl (for example: 1-acridinyl, 2-acridinyl, 3-acridinyl, 4-acridinyl, 9-acridinyl), indolyl (for example: 1-indolyl, 2-indolyl, 3-indolyl, 4-indolyl, 5-indolyl, 6-indolyl, 7-indolyl), isoindolyl, phenazinyl (for example: 1-phenazinyl, 2-phenazinyl) or phenothiazinyl (for example: 1-phenothiazinyl group, 2-phenothiazinyl group, 3-phenothiazinyl group, 4-phenothiazinyl group), and the like.

In the present invention, the "cycloalkyl group" is, for example, a cyclic saturated hydrocarbon group, and the number of carbon atoms is, for example, 3 to 15. Examples of the cycloalkyl group include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, bridged hydrocarbon groups, and spiro hydrocarbon groups, and preferable examples thereof include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and bridged hydrocarbon groups.

In the present invention, examples of the "bridged cyclic hydrocarbon group" include bicyclo [2.1.0] pentyl, bicyclo [2.2.1] heptyl, bicyclo [2.2.2] octyl, bicyclo [3.2.1] octyl, tricyclo [2.2.1.0] heptyl, bicyclo [3.3.1] nonyl, 1-adamantyl, and 2-adamantyl groups.

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

In the present invention, the "cycloalkenyl group" includes, for example, a cyclic unsaturated aliphatic hydrocarbon group, and the number of carbon atoms is, for example, 3 to 7. Examples of the above group include cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclohexenyl and cycloheptenyl, and cyclopropenyl, cyclobutenyl, cyclopentenyl and cyclohexenyl are preferable. The above-mentioned cycloalkenyl group also includes, for example, bridged cyclic hydrocarbon groups and spiro cyclic hydrocarbon groups having an unsaturated bond in the ring.

In the present invention, "aralkyl" includes, for example, benzyl, 2-phenylethyl, naphthylmethyl, etc., and "cycloalkylalkyl" or "cycloalkylcycloalkyl" includes, for example, cyclohexylmethyl, adamantylmethyl, etc., and "hydroxyalkyl" includes, for example, hydroxymethyl, 2-hydroxyethyl, etc.

In the present invention, "alkoxy" includes the above-mentioned alkyl-O-group, and examples thereof include methoxy, ethoxy, n-propoxy, isopropoxy, and n-butoxy, and examples thereof, "alkoxyalkyl" includes, for example, methoxymethyl, and examples thereof, "aminoalkyl" includes, for example, 2-aminoethyl.

In the present invention, examples of the "heterocyclic group" include 1-pyrrolinyl, 2-pyrrolinyl, 3-pyrrolinyl, 1-pyrrolidinyl, 2-pyrrolidinyl, 3-pyrrolidinyl, pyrrolidone, 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, piperidone, 2-piperidyl, 3-piperidyl, 4-piperidyl, 1-piperazinyl, 2-piperazinyl, piperazinone, 2-morpholinyl, 3-morpholinyl, morpholino, and the like, Tetrahydropyranyl, tetrahydrofuranyl, and the like.

In the present invention, "heterocyclylalkyl" includes, for example, piperidinylmethyl, piperazinylmethyl and the like, "heterocyclylalkenyl" includes, for example, 2-piperidinylvinyl and the like, and "heteroaralkyl" includes, for example, pyridylmethyl, quinolin-3-ylmethyl and the like.

In the present invention, "silyl" includes the formula R₃R in the group represented by Si-is independently selected from the group consisting of the above alkyl group, aryl group and cycloalkyl group, and examples thereof include trimethylsilyl group, t-butyldimethylsilyl group and the like, "siloxy group" includes, for example, trimethylsiloxy group and the like, and "siloxyalkyl group" includes, for example, trimethylsiloxymethyl group and the like.

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

In the present invention, the above-mentioned various groups may be substituted. Examples of the substituent include a hydroxyl group, a carboxyl group, a halogen, a haloalkyl group (e.g., CF)₃、CH₂CF₃、CH₂CCl₃) Nitro, nitroso, cyano, alkyl (for example: methyl, ethyl, isopropyl, tert-butyl), alkenyl (for example: vinyl), alkynyl (e.g.: ethynyl), cycloalkyl (for example: cyclopropyl, adamantyl), cycloalkylalkyl (for example: cyclohexylmethyl, adamantylmethyl), cycloalkenyl (for example: cyclopropenyl), aryl (e.g.: phenyl, naphthyl), aralkyl (for example: benzyl, phenethyl), heteroaryl (e.g.: pyridyl, furyl), heteroaralkyl (e.g.: pyridylmethyl), heterocyclic groups (for example: piperidinyl), heterocyclylalkyl (e.g.: morpholinomethyl), alkoxy(e.g., methoxy, ethoxy, propoxy, butoxy), haloalkoxy (e.g., OCF)₃) Alkenyloxy (for example: vinyloxy, allyloxy), aryloxy (for example: phenoxy), alkoxycarbonyl (for example: methoxycarbonyl, ethoxycarbonyl, tert-butoxycarbonyl), aralkyloxy (for example: benzyloxy), amino [ alkylamino (for example: methylamino, ethylamino, dimethylamino), acylamino (e.g.: acetylamino, benzoylamino), aralkylamino (for example: benzylamino, tritylamino), hydroxyamino]Alkylaminoalkyl (e.g., diethylaminomethyl), sulfamoyl, oxo, and the like.

4. Synthesis method of ssNc molecule of the invention

The method for synthesizing ssNc molecules of the present invention is not particularly limited, and conventionally known methods can be used. Examples of the above synthesis method include a synthesis method based on a genetic engineering method, a chemical synthesis method, and the like. Examples of genetic engineering methods include in vitro transcription synthesis methods, methods using vectors, and methods based on PCR cassettes. The vector is not particularly limited, and examples thereof include a non-viral vector such as a plasmid, a viral vector and the like. The chemical synthesis method is not particularly limited, and examples thereof include a phosphoramidite method and an H-phosphonate method. The chemical synthesis method can use, for example, a commercially available automatic nucleic acid synthesizer. Amidite is generally used for the above chemical synthesis. The amidite is not particularly limited, and commercially available amidites include, for example, RNA phosphoramidite (2' -O-TBDMSi, trade name, Sanqianli pharmaceutical), ACEamidite, TOMamidite, CEEamidite, CEMamidite, TEMamidite, and the like.

5. Composition comprising a metal oxide and a metal oxide

As described above, the expression suppressing composition of the present invention is a composition for suppressing expression of a target gene, and includes the ssNc molecule of the present invention. The composition of the present invention is characterized by containing the above-described ssNc molecule of the present invention, and the other constituents 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, by administering the target gene to a subject in which the target gene is present, the expression of the target gene can be suppressed.

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 above-described ssNc molecule of the present invention, and the other constituents are not limited at all. The pharmaceutical composition of the present invention can also be referred to as a pharmaceutical, for example.

According to the present invention, for example, by administering the gene to a patient suffering from a disease causing the 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, prevention of the above-mentioned diseases, improvement of the diseases, and improvement of prognosis, and may be any of them.

In the present invention, the disease to be treated is not particularly limited, and examples thereof include diseases caused by gene expression. The target gene may be a gene responsible for the disease according to the type of the disease, and the expression suppressing sequence may be appropriately set according to the target gene.

Specifically, if the target gene is the TGF- β 1 gene and an expression suppressing sequence for the gene is disposed in the ssNc molecule, the TGF- β 1 gene can be used for the treatment of inflammatory diseases, specifically, acute lung injury, for example.

The method of using the composition for expression suppression and the pharmaceutical composition (hereinafter referred to as composition) of the present invention is not particularly limited, for example, as long as the above-described ssNc molecule is administered to an administration subject having the above-described target gene.

Examples of the subject to be administered include cells, tissues and organs. Examples of the subject to be administered include non-human animals such as humans and non-human mammals other than humans. The administration may be in vivo or in vitro, for example. The above cells are not particularly limited, and examples thereof include various cultured cells such as HeLa cells, 293 cells, NIH3T3 cells, COS cells, and the like; stem cells such as ES cells and hematopoietic stem cells; cells isolated from organisms such as primary cultured cells; and so on.

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

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 may be appropriately selected depending on the type of the subject to be administered, for example.

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 forming the complex, for example, the ssNc molecule can be efficiently transported. The binding of the ssNc molecule to the complexing agent is not particularly limited, and examples thereof include non-covalent binding. Examples of the complex include an inclusion complex.

The complexing agent is not particularly limited, and examples thereof include polymers, cyclodextrins, and amantadine. Examples of the cyclodextrin include linear cyclodextrin copolymers and linear acidified cyclodextrin copolymers.

In addition, examples of the additive include a carrier, a binding substance to a target cell, a condensing agent, a fusion agent, and an excipient.

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; saccharides such as dextran, pullulan, chitin, chitosan, inulin, cyclodextrin, hyaluronic acid, etc.; lipids, and the like. The carrier may be a synthetic polymer such as a synthetic polyamino acid. 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, and polyphosphazine (polyphosphazine).

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

Examples of the fluxing agent and the condensing agent include a polyurethane chain such as Polyethyleneimine (PEI). The PEI may be, for example, either linear or branched, or may be either synthetic or natural. The PEI may be substituted with an alkyl group or a lipid, for example. In addition, for example, polyhistidine, polyimidazole, polypyridine, polypropyleneimine, melittin, polyacetal (e.g., cationic polyacetal), and the like can be used as the fusion agent. The fusogenic agent may have, for example, an alpha-helical structure. The fusogenic agent may be, for example, a membrane-disintegrating agent such as melittin.

For example, U.S. Pat. No. 6,509,323, U.S. Pat. No. 2003/0008818, and PCT/US04/07070 can be cited as examples of the composition of the present invention for the formation of the above-mentioned complex.

In addition, the additive may be, for example, an amphiphilic molecule. The amphipathic 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 preferably a polymer having a repeating secondary structure. Specifically, for example, a polypeptide is preferable, and an α -helical polypeptide is more preferable.

The amphiphilic polymer may be, for example, a polymer having two or more amphiphilic subunits. Examples of the above-mentioned subunit include, for example, a subunit having a cyclic structure including 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 inhibition method

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

In the method for suppressing expression of the present invention, the mechanism of suppressing expression of the gene is not particularly limited, and examples thereof include suppression of expression by RNA interference or an RNA interference-like phenomenon. Here, the expression suppression method of the present invention is, for example, a method of inducing RNA interference that suppresses expression of the target gene, and can also be referred to as an expression induction method characterized by using the ssNc molecule of the present invention.

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. In the administration step, for example, the ssNc molecule is brought into contact with the administration target. Examples of the subject to be administered include cells, tissues and organs. Examples of the subject to be administered include non-human animals such as humans and non-human mammals other than humans. The administration may be in vivo or in vitro, for example.

The expression suppressing method of the present invention may be, for example, a method of administering the ssNc molecule alone or a method of administering the composition of the present invention containing the ssNc molecule. The administration method is not particularly limited, and may be appropriately selected depending on the kind of the subject to be administered, for example.

7. Method of treatment

As described above, the method for treating a disease of the present invention includes a step of administering the ssNc molecule of the present invention to a patient, wherein the ssNc molecule has a sequence that suppresses expression of a gene that causes the disease as the expression suppression sequence. The treatment method of the present invention is characterized by using the ssNc molecule of the present invention described above, and other steps and conditions are not limited at all.

The therapeutic method of the present invention may be, for example, the expression suppressing method of the present invention described above. The administration method is not particularly limited, and for example, oral administration and parenteral administration may be used.

Use of ssNc molecules

The use of the present invention is the use of the ssNc molecule of the present invention described above for inhibiting the expression of the target gene described above. In addition, the use of the present invention is the use of the ssNc molecule of the present invention described above for inducing RNA interference.

The nucleic acid molecule of the present invention is a nucleic acid molecule for treating a disease, wherein the nucleic acid molecule is the ssNc molecule of the present invention, and the ssNc molecule has a sequence that suppresses expression of a gene that causes the disease as the expression suppression sequence.

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

Examples

Example A1 Synthesis of RNA

As RNA (Ex) in the examples, ssRNA (NK-0016) shown below was synthesized. The NK-0016 gene has an expression-suppressing sequence (SEQ ID NO: 1) having a length of 19 bases for suppressing the expression of the GAPDH gene. In the sequence of NK-0016, a linker region (Lx) is located between region (Xc) and region (X), and a linker region (Ly) is located between region (Y) and region (Yc). (the same applies hereinafter). In the sequence, the 5 'region (Xc) and the 3' region (Yc) are indicated by lower case letters, respectively (the same applies hereinafter).

GAPDH gene expression inhibitory sequence (SEQ ID NO: 1)

5’-GUUGUCAUACUUCUCAUGG-3’

[ chemical formula 4]

Ex: NK-0016 (sequence number 2)

As comparative RNA, dsRNA (NI-0011) was synthesized as an RNAi positive control (Pc) shown below. The 3' -end of each single strand of NI-0011 has a 2-base overhang, and the single strand of SEQ ID NO. 4 has the expression-suppressing sequence having the above-mentioned length of 19 bases as in the case of NK-0016.

[ chemical formula 5]

Pc：NI-0011

5'-CCAUGAGAAGUAUGACAACAG-3' (SEQ ID NO. 3)

3 '-UUGGUACUCUUCAUACUGUUG-5' (SEQ ID NO. 4)

The RNA was synthesized by the phosphoramidite method using a nucleic acid synthesizer (trade name ABIExpedicte (registered trademark) 8909nucleic acid synthesis System, applied biosystems). In the above synthesis, RNAphosporamides (2' -O-TBDMSi, trade name, Sanqianli pharmaceutical) was used as RNAamides (the same applies hereinafter). Deprotection of the amidites described above is according to conventional methods. The synthesized RNA was purified by HPLC. The purified RNAs were separately freeze-dried. In the following examples, unless otherwise specified, RNA synthesis was performed in the same manner as in the present examples.

The lyophilized RNA was dissolved in distilled water for injection (Otsuka pharmaceutical, the same shall apply hereinafter) to obtain a desired concentration.

Example A2 expression inhibitory Effect of GAPDH Gene in HCT116 cells

Using the ssRNA of the present invention, in vitro inhibition of expression of the GAPDH gene was confirmed.

(1) Materials and methods

As RNA (Ex) in the examples, the ssRNA (NK-0016) of example A1 described above was used.

[ chemical formula 6]

Ex: NK-0016 (sequence number 2)

The RNA was dissolved in distilled water for injection to obtain a desired concentration, and an RNA solution was prepared. HCT116 cells (DSPharmabiomedicalal) were used as cells, and 10% FBS-containing McCoy's 5A (Invitrogen) was used as medium, and the culture conditions were 37 ℃ and 5% CO₂The following steps.

First, HCT116 cells were cultured in the above-mentioned medium, and the culture medium was filled with 400. mu.L of the culture medium and 2X 10 cells of the culture medium⁴Cell/well format was dispensed into 24-well plates. Further, after culturing the cells in the wells for 24 hours, the RNA was transfected using a transfection reagent Lipofectamine2000 (trade name, Invitrogen) according to the attached protocol. Specifically, transfection was performed by setting the composition of each well as described above as follows. The final concentration of RNA in the wells was 5nmol/L, 10nmol/L, 20nmol/L, or 40 nmol/L.

[ Table 1]

After transfection, the cells in the wells were cultured for 48 hours, and then RNA was recovered according to the attached protocol using RNeasyMiniKit (trade name, Qiagen). Subsequently, cDNA was synthesized from the above RNA using reverse transcriptase (trade name SuperScriptIII, Invitrogen) according to the attached protocol. Then, PCR was performed using the synthesized cDNA as a template to measure the expression level of GAPDH gene and the expression level of β -actin gene as an internal standard. The expression level of the GAPDH gene was corrected by the expression level of the β -actin gene.

In the above PCR, LightCyclerFastStartDNAastSYBRGreenI (trade name, Roche) was used as a reagent, and LightCyclerDX400 (trade name, Roche) was used as a device (the same applies hereinafter). The following primer sets were used for amplification of the GAPDH gene and the β -actin gene, respectively.

Primer set for GAPDH gene

5'-GGAGAAGGCTGGGGCTCATTTGC-3' (Serial number 9)

5'-TGGCCAGGGGTGCTAAGCAGTTG-3' (Serial number 10)

Primer pair for beta-actin gene

5'-GCCACGGCTGCTTCCAGCTCCTC-3' (Serial number 11)

5'-AGGTCTTTGCGGATGTCCACGTCAC-3' (Serial number 12)

As control 1, the gene expression level (-) was also measured in the cells to which only 100. mu.L of the above-mentioned (B) had been added. In addition, as control 2, the gene expression level (mock) was measured also on the obtained cells by treating the cells in the same manner except that the RNA solution was not added during transfection and 1.5. mu.L of the (A) and the (B) were added in a total amount of 100. mu.L.

The corrected expression level of the GAPDH gene was determined as a relative value of the expression level of the cells into which each RNA was introduced, with the expression level of the control (-) set to 1.

(2) Results

These results are shown in fig. 4. Fig. 4 is a graph showing relative values of expression amounts of GAPDH genes. As shown in FIG. 4, it was found that NK-0016 was expressed in a lower amount than the control (-) to which RNA was not added, and the expression of the GAPHD gene was suppressed. As shown in fig. 4, it is also found that the gene expression-suppressing effect is exhibited depending on the administration amount.

Example A3 expression inhibitory Effect of GAPDH Gene in HCT116 cells

(1) Materials and methods

As RNA (Ex) in the examples, the ssRNA (NK-0016) of example A1 described above was used. As the RNA of comparative example, the dsRNA (NI-0011) as RNAi positive control (Pc) was used. The RNA was dissolved in distilled water for injection to 40. mu. mol/L to prepare an RNA solution.

The expression level of GAPDH gene in HCT116 cells was confirmed in the same manner as in example a2, except that the RNA solution was used. The RNA concentration at transfection was 40 nmol/L.

(2) Results

These results are shown in fig. 5. FIG. 5 is a graph showing the relative values of the expression levels of the GAPDH gene, and the vertical axis shows the relative gene expression levels. As shown in FIG. 5, NK-0016 of the above example showed a very strong gene expression inhibitory activity as compared with NI-0011 of the comparative example.

Example A4 expression inhibitory Effect of GAPDH Gene in A549 cells and 293 cells

(1) Materials and methods

NK-0016 of the above example A1 and Ex-ssRNA (PK-0004) below were used as RNA (Ex) of the examples. In PK-0004 described above, the linker region (Lx) and the linker region (Ly) were bound between Xc and X and between Yc and Y using Compound 10 (L-proline diamide amides) of scheme 3 shown in example B. The chemical formulas of the two linkers are shown below. The NK-0016 and PK-0004 have the same sequence, except that the linker region (Lx) between Xc and X and the linker region (Ly) between Yc and Y are different.

[ chemical formula 7]

Ex: NK-0016 (sequence number 2)

Ex: PK-0004 (SEQ ID NO: 13)

The RNA was dissolved in distilled water for injection to give a solution of 20. mu. mol/L RNA. A549 cells and 293 cells (DSPharmabiomedicalal) were used as the cells. The former medium was dmem (invitrogen) medium containing 10% FBS, and the latter medium was mem (invitrogen) medium containing 10% FBS. The culture conditions were 37 ℃ and 5% CO₂The following steps.

First, cells were cultured in the above-mentioned medium, and the culture medium was used in an amount of 400. mu.L and 5X 10, respectively⁴Cell/well format was dispensed into 24-well plates. Further, after culturing the cells in the wells for 24 hours, the RNA was transfected using a transfection reagent Lipofectamine2000 (trade name, Invitrogen) according to the attached protocol. Specifically, the groups per well were set as follows for A549 cells and 293 cells, respectivelyIn this case, transfection was performed. In the following composition, (B) was Opti-MEM (trade name, Invitrogen) and (C) was 20. mu. mol/L of the above RNA solution, and 98.5. mu.L or 99. mu.L of both were added. The final concentration of RNA in the wells was 1nmol/L, 3nmol/L, or 10 nmol/L.

[ Table 2]

After transfection, the cells were cultured for 48 hours, and RNA was recovered in the same manner as in example A2, and cDNA synthesis and PCR were carried out to measure the relative expression level of the GAPDH gene.

(2) Results

These results are shown in fig. 6 and 7. Fig. 6 shows the results of a549 cells, and fig. 7 shows the results of 293 cells. Fig. 6 and 7 are graphs showing relative values of GAPDH gene expression levels. As shown in FIGS. 6 and 7, it is understood that NK-0016 and PK-0004 of the examples showed strong gene expression inhibitory activity and showed effects in a concentration-dependent manner.

Example A5 Effect of inhibiting expression of TGF-. beta.1 Gene in Hepa1-6 cells

The ssRNA of the present invention was confirmed to have an effect of inhibiting the expression of the TGF-. beta.1 gene in vitro.

(1) Materials and methods

As RNA (Ex) in the examples, ssRNA (NK-0033) shown below was used. The NK-0033 has the following sequence of 21 bases long which inhibits the expression of TGF-. beta.1 gene. The sequence was designed based on siRNA used by Cheng et al (mol. Pharm.,2009,6, 772-779).

TGF-. beta.1 Gene expression-suppressing sequence (SEQ ID NO: 16)

5’-AAAGUCAAUGUACAGCUGCUU-3’

As comparative examples, ssRNA (NK-0035) as an RNAi negative control (Nc) shown below was used. NK-0035 is not the expression inhibition sequence, but with the incorporation of expression inhibition unrelated random sequence.

[ chemical formula 8]

Ex: NK-0033 (SEQ ID NO. 80)

Nc: NK-0035 (SEQ ID NO. 15)

The RNA was dissolved in distilled water for injection to prepare an RNA solution. The cells used were Hepa1-6 cells (center for Bioresources of institute of physicology), and the medium used was DMEM (Invitrogen) containing 10% FBS, and the culture conditions were 37 ℃ and 5% CO₂The following steps.

First, Hepa1-6 cells were cultured in the above-mentioned medium, and the culture medium was filled at 400. mu.L and 3X 10 cells, respectively⁴Cell/well format was dispensed into 24-well plates. Further, after culturing the cells in the wells for 24 hours, the ssRNA was transfected using a transfection reagent Lipofectamine2000 (trade name, Invitrogen) according to the attached protocol. Specifically, transfection was performed by setting the composition of each well as described above as follows. In the following composition, (B) was Opti-MEM (trade name, Invitrogen) and (C) was 20. mu. mol/L of the above RNA solution, and 98.5. mu.L of both solutions were added. The final concentrations of the RNA in the wells were 10nmol/L, 25nmol/L, 50nmol/L, and 100 nmol/L.

[ Table 3]

After transfection, the cells in the wells were cultured for 48 hours, and then RNA was recovered according to the attached protocol using RNeasyMiniKit (trade name, Qiagen). Subsequently, cDNA was synthesized from the RNA using reverse transcriptase (trade name SuperScriptIII (Invitrogen)) according to the protocol attached thereto, and PCR was performed in the same manner as in example A2 except that the following PCR primer set for the TGF- β 1 gene and the following primer set for the β -actin gene were used, and the expression level of the TGF- β 1 gene and the expression level of the β -actin gene as an internal standard were measured, and the expression level of the TGF- β 1 gene was corrected by the expression level of the β -actin gene.

Primer set for TGF-beta 1 gene

5'-CCATTGCTGTCCCGTGCAGAGCTG-3' (Serial number 17)

5'-ATGGTAGCCCTTGGGCTCGTGGATC-3' (Serial number 18)

Primer pair for beta-actin gene amplification

5'-GTCGTACCACAGGCATTGTGATGG-3' (Serial number 19)

5'-GCAATGCCTGGGTACATGGTGG-3' (Serial number 20)

Further, with respect to the control (-) and the control (mock), the gene expression level was measured in the same manner as in example A2. Then, the corrected expression level of the TGF-. beta.1 gene was determined as a relative value of the expression level of the cells into which each RNA had been introduced, with the expression level of the cells of the control (-) set at 1.

(2) Results

These results are shown in fig. 8. FIG. 8 is a graph showing relative values of the expression levels of TGF-. beta.1 genes. As shown in FIG. 8, NK-0033 of the above examples suppressed the expression of TGF-. beta.1 gene in vitro. On the other hand, NK-0035 as a negative control did not inhibit the expression of the TGF-. beta.1 gene.

(example A6) inhibitory Effect on TGF-. beta.1 Gene expression in vivo and inhibitory Effect on acute Lung injury

The ssRNA of the present invention was confirmed to have effects of inhibiting gene expression and acute lung injury in vivo. The above-mentioned effects were confirmed by the method described in Takagi et al (J.Thromb Hemost 2009; 7: 2053-.

(1) Materials and methods

(1.1) administration of ssRNA to mice with acute Lung injury

RNA (Ex) of example the ssRNA (NK-0033) described in example A5 was used. As RNA of comparative example, ssRNA (NK-0035) was used as the RNAi negative control (Nc) shown in example A5.

100. mu.g of the above-mentioned RNA was dissolved in 80. mu.L of sterilized physiological saline (Nippon chemical Co., Ltd., the same shall apply hereinafter) to prepare an RNA solution. On the other hand, 100. mu.g of Lipopolysaccharide (LPS) was dissolved in 50. mu.L of sterilized physiological saline to prepare an LPS solution.

First, 80. mu.L of the RNA solution was added dropwise into the trachea of the mouse. After 1 hour of the addition, 50. mu.L of the LPS solution was added dropwise to the trachea of the mouse to induce lung injury.

As a negative control for LPS, 50. mu.L of sterilized physiological saline to which LPS was not added was used in place of the above LPS solution. In addition, as a negative control for the RNA solution, 80. mu.L of sterilized physiological saline was used.

Each administration group is shown below. In each administration group, 4 to 6 mice were used.

Administration group 1

80. mu.L of sterilized physiological saline 1 hours later, 50. mu.L of sterilized physiological saline was administered

Administration group 2

80. mu.L of the RNA solution (NK-0033) was administered for 1 hours, and 50. mu.L of sterilized physiological saline was administered

Administration group 3

80. mu.L of the RNA solution (NK-0035) was administered for 1 hours, and 50. mu.L of sterilized physiological saline was administered

Administration group 4

80. mu.L of sterilized physiological saline 1 hours later, 50. mu.L of the above LPS solution was administered

Administration group 5

80. mu.L of the RNA solution (NK-0033) was administered for 1 hours, and 50. mu.L of the LPS solution was administered

Administration group 6

80. mu.L of the RNA solution (NK-0035) was administered for 1 hours, and 50. mu.L of the LPS solution was administered

(1.2) sampling of bronchoalveolar lavage fluid (BALF)

24 hours after the addition of the LPS solution, an excess amount of pentobarbital was administered to the abdominal cavity of the mouse and euthanized to prepare biochemical and histological analysis samples. The negative control was supplemented with sterilized physiological saline instead of the above LPS solution.

The heart of the mouse was punctured, and a blood sample was collected and added to a test tube containing a 3.8% sodium citrate aqueous solution. The amount (volume) of the above sodium citrate aqueous solution was 1/10 of the above blood sample. BALF samples were recovered from the mixtures according to Yasui et al (AmJRespirCritCareMed 2001: 163: 1660-8). Then, the total cell number in the above BALF sample was measured using a Nucleocounter (trade name, Chemometed Co.).

The BALF sample was subjected to centrifugation, and the supernatant of the BALF sample was collected and stored at-80 ℃ until biochemical analysis was performed. In addition, in order to count different kinds of cells contained in the BALF sample, the BALF sample was centrifuged using a cytospin smear machine, and the separated cells were subjected to Giemsa staining using May-Grunwald-Giemsa (trade name, Merck). In addition, lung tissue was collected from the above mice and HE staining was performed.

(2) Results

(2.1) inhibition of TGF-. beta.1 Gene expression in Lung

For the lung samples of the mice, the amount of TGF-. beta.1 expression per weight of lung was measured by TGF-. beta.1 QuantikineCoolometric SandwichELISA (trade name, R & DSsystems).

The results are shown in FIG. 9. FIG. 9 is a graph showing the expression level of TGF-. beta.1 gene per unit weight of lung in each administration group. The expression level of the above gene was increased as a result of LPS treatment in LPS (+)/RNA (-) administration group 4, as compared to LPS (-)/RNA (-) administration group 1. Furthermore, the increase in the expression level of the genes in LPS (+)/NK-0033(+) administered in example 5 was suppressed as compared with the LPS (+)/RNA (-) administered group 4. This inhibitory effect was not observed in LPS (+)/negative control NK-0035(+) administered group 6. From these results, it was found that the expression of the TGF-. beta.1 gene can be effectively suppressed by NK-0033 of the above example.

(2.2) inhibitory Effect on acute Lung injury

Inflammation in acute lung injury is caused by infiltration of cells such as neutrophils in the lung. Therefore, drugs that inhibit the infiltration of cells such as neutrophils into the lung are therapeutic agents for inflammation caused by acute lung injury. Therefore, the pharmacological effect of the ssrnas of the present invention was confirmed by using the number of cells in bronchoalveolar lavage fluid (BALF) as an index of the number of cells infiltrating into the lung.

The results of measuring the number of cells in the BALF sample are shown in FIG. 10. Fig. 10 is a graph showing the number of cells in BAFL samples in each administration group. The number of cells in the BALF sample was increased as a result of LPS treatment in LPS (+)/RNA (-) administration group 4, as compared with LPS (-)/RNA (-) administration group 1. This indicates that inflammation was induced by LPS and as a result, the cells infiltrated the lung. Furthermore, the administration group 5 of the example of LPS (+)/NK-0033(+) inhibited the increase in the number of cells as compared with the administration group 4 of LPS (+)/RNA (-). This shows that inflammation in acute lung injury is suppressed due to NK-0033 described above. This inhibitory effect was not observed in LPS (+)/negative control NK-0035(+) administered group 6. From these results, it is understood that inflammation caused by acute lung injury can be effectively suppressed by NK-0033 of the above example.

The results of measuring the number of neutrophils in the BALF sample are shown in FIG. 11. Fig. 11 is a graph showing the cell number of neutrophils in the BALF sample in each administration group. The number of neutrophils in the BALF sample was increased as a result of LPS treatment in LPS (+)/RNA (-) administration group 4, compared to LPS (-)/RNA (-) administration group 1. This indicates that inflammation was induced by LPS, and as a result, neutrophils infiltrated the lung. Furthermore, the administration group 5 of the example of LPS (+)/NK-0033(+) inhibited the increase in the number of neutrophils in the BALF sample as compared with the administration group 4 of LPS (+)/ssRNA (-). This shows that inflammation in acute lung injury is suppressed due to NK-0033 described above. This inhibitory effect was not observed in LPS (+)/negative control NK-0035(+) administered group 6. From these results, it is understood that inflammation caused by acute lung injury can be effectively suppressed by NK-0033 of the above example.

(2.3) histological observation: giemsa staining

The results of giemsa staining are shown in figure 12. Fig. 12 is a photograph (magnification 100 times) showing the results of giemsa staining of cells in the BALF sample. In FIG. 12, (A) shows the results of LPS (+)/RNA (-) administration group 4, (B) shows the results of LPS (+)/negative control NK-0035(+) administration group 6, and (C) shows the results of LPS (+)/NK-0033(+) administration group 5 of the examples.

As shown in FIG. 12, the number of cells infiltrating into the lung was significantly reduced in the case of the LPS (+)/NK-0033(+) example administration group 5(C) compared to the case of the LPS (+)/RNA (-) administration group 4(A) and the case of the LPS (+)/negative control NK-0035(+) administration group 6 (B). This histological observation is consistent with the results of the number of cells in the BALF samples described above.

(2.4) histological observation: HE staining

The results of HE staining are shown in fig. 13. Fig. 13 is a photograph (magnification: 10 times) showing the results of HE staining of the lung tissue. In FIG. 13, (A) shows the results of LPS (+)/RNA (-) administration group 4, (B) shows the results of LPS (+)/negative control NK-0035(+) administration group 6, and (C) shows the results of LPS (+)/NK-0033(+) administration group 5 of the examples. As is clear from fig. 13, infiltration of cells such as neutrophils into the perivascular, alveolar cavity, alveolar wall and the vicinity of bronchi was reduced, and the lung tissue had decreased damage.

Example A7 evaluation of side effects by Interferon Induction

As a side effect of iRNA agents of the prior art, it is known to induce interferon independently of the sequence, and this side effect is regarded as a problem. Therefore, interferon-induced side effects were evaluated for the ssrnas of the present invention.

(1) Materials and methods

Administration of ssRNA was performed in mice with acute lung injury by the same method and conditions as described in example A6 above. In the same manner as in example a6, 24 hours after dropping the LPS solution or sterile physiological saline (negative control with respect to LPS), the mice were euthanized and lung tissues were collected.

[ Table 4]

Administration set	RNA	LPS
			1	-	-
2	Ex：NK-0033	-
			4	-	+
5	Ex：NK-0033	+

EX: example RNAs

In order to measure the expression level of each gene from the lung tissue, RNA was isolated using TRIZOL (trade name, Invitrogen). Subsequently, cDNA was synthesized from the above RNA using reverse transcriptase (trade name SuperScriptII, Invitrogen) according to the attached protocol. Then, PCR was performed using the synthesized cDNA as a template to measure the expression levels of the TGF- β 1 gene, IFN- α gene, and IFN- β gene.

For the above PCR, GoldAmpliTaq (trade name, applied biosystem, usa) was used as a reagent, and abapplied biosystem7600 (trade name, applied biosystem) was used as an assay device. The following primer sets were used for amplification of the TGF- β 1 gene, IFN- α gene, and IFN- β gene, respectively.

Primer set for GAPDH gene

5'-CCCTTATTGACCTCAACTACATGGT-3' (Serial number 21)

5'-GAGGGGCCATCCACAGTCTTCTG-3' (Serial number 22)

Primer set for TGF-beta 1 gene

5'-ACTCCACGTGGAAATCAACGG-3' (Serial number 23)

5'-TAGTAGACGATGGGCAGTGG-3' (Serial number 24)

Primer pair for IFN-alpha gene

5 '-ATGGCTAGRCTCTGTGCTTCCT-3' (SEQ ID NO: 25)

5 '-AGGGCTCTCCAGAYTTCTGCTCTG-3' (SEQ ID NO: 26)

Primer set for IFN-beta gene

5'-CATCAACTATAAGCAGCTCCA-3' (Serial number 27)

5' -TTCAAGTGGAGAGCAGTTCAG-3 (SEQ ID NO. 28)

Then, the resulting PCR product was subjected to agarose electrophoresis. In addition, density analysis was performed on agarose after electrophoresis using NIHimagingsystem, and the expression level of each gene was confirmed. The expression levels of the TGF- β 1 gene, the IFN- α gene, and the IFN- β gene were relatively evaluated using the expression level of the GAPDH gene as a standard. Specifically, the relative values of the measured intensities of the PCR products using the primer pairs for the respective genes were determined and evaluated, using the measured intensities of the PCR products using the primer pairs for the GAPDH gene as standard 1.

(2) Results and investigation

The results of the quantitative analyses of the expression levels of the TGF- β 1 gene, IFN- α gene, and IFN- β gene are shown in the graphs (A) to (C) of FIG. 14, respectively. Each administration group is as follows in the same manner as in example a6 described above.

FIG. 14 (A) shows the expression level of TGF-. beta.1 gene. As shown in FIG. 14 (A), the administration group 5 of the example in which NK-0033 was administered inhibited the increase in the expression of TGF-. beta.1 gene induced by LPS, as compared with the administration group 4 of RNA (-). The results are related to the results of measuring the expression level of TGF-. beta.1 shown in FIG. 8 of example A5.

FIG. 14(B) shows the results of the expression level of IFN-. alpha.gene, and FIG. 14(C) shows the results of the expression level of IFN-. beta.gene. As shown in FIG. 14(B) and FIG. 14(C), in the case where no LPS was added, the expression of IFN-. alpha.gene and IFN-. beta.gene of type I interferon was not induced by the addition of ssRNA in the group 1 to which RNA (-) was administered, as compared with the group 2 to which RNA (+) was administered. As shown in FIGS. 14(B) and 14(C), when LPS was added, the group 4 administered with RNA (-) did not induce the expression of IFN-. alpha.gene and IFN-. beta.gene by the addition of ssRNA in comparison with the group 5 administered with NK-0033 (+).

This result is comparable to the result of the conventional siRNA which produces the induced side effect of type I interferon. That is, it was actually verified that the ssRNA of the present invention unexpectedly does not cause the side effect of interferon induction, which has been a problem in the siRNA of the conventional method.

Example A8 Effect of inhibiting expression of TGF-. beta.1 Gene in Hepa1-6 cells

(1) Materials and methods

NK-0033 of the above-mentioned example A5, NK-0061, NK-0055 and NK-0062 shown below were used as RNA (Ex) of the examples. In the following sequences, "+" indicates a free base.

[ chemical formula 9]

Ex: NK-0033 (SEQ ID NO. 80)

Ex: NK-0061 (SEQ ID NO. 29)

Ex: NK-0055 (SEQ ID NO: 30)

Ex: NK-0062 (SEQ ID NO. 31)

Further, as RNA (Ex) of the examples, the following PK-0007, PK-0026, PK-0027 and PK-0028 were used. In these ssRNAs, linker regions (Lx) and (Ly) were bound between Xc and X, and between Yc and Y using Compound 10 (L-proline diamide amides) of scheme 3 shown in example B.

[ chemical formula 10]

Ex: PK-0007 (SEQ ID NO: 32)

Ex: PK-0026 (SEQ ID NO. 33)

Ex: PK-0027 (SEQ ID NO: 34)

Ex: PK-0028 (SEQ ID NO: 35)

NK-0033, NK-0061, NK-0055 and NK-0062 are identical to PK-0007, PK-0026, PK-0027 and PK-0028 except that the 1 st linker (L1) and the 2 nd linker (L2) are different from each other, and any of them has a sequence (SEQ ID NO: 16) for inhibiting the expression of TGF-. beta.1 gene.

(1.2) suppression of expression of Gene

The RNA stored in the frozen state was dissolved in distilled water for injection to a concentration of 20. mu. mol/L to prepare an RNA solution. Transfection of the aforementioned ssRNA into Hepal-6 cells, RNA recovery, cDNA synthesis, and PCR were performed in the same manner as in example A5, except that the RNA solution was used. The relative expression level of the TGF-. beta.1 gene was determined. The RNA concentration at the time of transfection was 1 nmol/L.

(2) Results

These results are shown in fig. 15 and 16. FIGS. 15 and 16 are graphs each showing the relative values of the expression levels of TGF-. beta.1 gene. FIG. 15 shows the results obtained using NK-0033, NK-0061, NK-0055 and NK-0062, and FIG. 16 shows the results obtained using PK-0007, PK-0026, PK-0027 and PK-0028. As shown in fig. 15 and 16, either ssRNA showed strong gene expression inhibitory activity.

(example A9)

In vivo TGF-beta 1 gene expression-inhibiting effect and acute lung injury-inhibiting effect

(A9-1) Effect of inhibiting expression of TGF-. beta.1 Gene in vivo

Using the ssRNA of the present invention, the effect of inhibiting the expression of the TGF-. beta.1 gene in vivo was confirmed.

(1) Materials and methods

Unless otherwise stated, administration of RNA to mice with acute lung injury was performed in the same manner as in example a6 described above.

As RNA (Ex) in the examples, PK-0007 and NK0033 of the above-mentioned example A8 were used. As RNAs of comparative examples, PK-0008 and NK-0035 as RNAi negative controls (Nc), dsRNA (NI-0030) as RNAi positive controls (Pc), and dsRNA (NI-0031) as RNAi negative controls (Nc) were used as shown below. PK-0008 as a negative control had linkers Lx and Ly derived from the above amidites (the above Compound 10: L-proline diamide amidites in scheme 3) in the same manner as PK-0007.

[ chemical formula 11]

Ex: PK-0007 (SEQ ID NO: 32)

Nc: PK-0008 (SEQ ID NO: 36)

Ex: NK-0033 (SEQ ID NO. 80)

Nc: NK-0035 (SEQ ID NO. 15)

Pc：NI-0030

5'-GCAGCUGUACAUUGACUUUAG-3' (Serial number 39)

3 '-UUCGUCGACAUGUAACUGAAA-5' (SEQ ID NO: 40)

Nc：NI-0031

5'-GUGUCAGUGCUCAUUUACAAG-3' (Serial number 41)

3 '-UUCACAGUCACGAGUAAAUGU-5' (SEQ ID NO: 42)

An RNA solution was prepared by dissolving 100. mu.g of the above-mentioned RNA in 75. mu.L of sterilized physiological saline. On the other hand, 100. mu.g of Lipopolysaccharide (LPS) was dissolved in 50. mu.L of sterilized physiological saline to prepare an LPS solution.

Each administration group is shown below. Administration was carried out in the same manner as in example a6, unless otherwise specified. In each administration group, 4 to 6 mice were used.

Administration group 1

After 75. mu.l of 5 minutes of sterilized physiological saline, 50. mu.l of sterilized physiological saline was administered.

Administration group 2

After 75. mu.l 5 minutes of sterilized physiological saline, 50. mu.l of LPS solution was administered.

Administration group 3

After 75. mu.l 5 minutes of RNA solution (PK-0007) was administered, 50. mu.l of LPS solution was administered.

Administration group 4

After 75. mu.l 5 minutes of RNA solution (PK-0008) was administered, 50. mu.l of LPS solution was administered.

Administration group 5

After 75. mu.L of 5 minutes of the RNA solution (NK-0033) was administered, 50. mu.L of the LPS solution was administered.

Administration group 6

After 75. mu.l of 5 minutes of the RNA solution (NK-0035) was administered, 50. mu.l of the LPS solution was administered.

Administration group 7

After 75. mu.l 5 minutes of RNA solution (NI-0030) was administered, 50. mu.l of LPS solution was administered.

Administration group 8

After 50. mu.l of 5 minutes of the RNA solution (NI-0031) was administered, 50. mu.l of the LPS solution was administered.

Then, a lung sample was prepared and the amount of TGF-. beta.1 expression per unit weight of lung was measured in the same manner as in example A6.

The results are shown in FIG. 17. FIG. 17 is a graph showing the amount of TGF-. beta.1 expression per unit weight of lung in each administration group. The administration group 3 of LPS (+)/PK-0007(+) and the administration group 5 of LPS (+)/NK-0033(+) inhibited the expression level of TGF-. beta.1 gene, respectively, as compared with the administration group 2 of LPS (+)/ssRNA (-). It was confirmed that the inhibitory effect was stronger than that of LPS (+)/positive control NI-0030-administered group 7. In particular, the administration group 3 of LPS (+)/PK-0007(+) showed a significant inhibitory effect. In addition, no inhibitory effect was observed in the groups 4(PK-0008), 6(NK-0035) and 8(NI-0031) to which RNA was administered as a negative control.

(A9-2) in vivo off-target Effect

Using the ssRNA of the present invention, the off-target effect in vivo was confirmed, and side effects were evaluated.

RNA of example the ssRNA described above in example A8 (PK-0007) was used. As the RNA of the comparative example, ssRNA (PK-0008) as the RNAi negative control (Nc) shown in example A9-1, dsRNA (NI-0030) as the RNAi positive control (Pc), and dsRNA (NI-0031) as the RNAi negative control were used. Then, 100. mu.g of the RNA was dissolved in 75. mu.L of sterilized physiological saline to prepare an RNA solution.

Each administration group is shown below. In each administration group, 2 to 4 mice were used.

Administration group 1

Administering sterilized physiological saline 75 μ l

Administration group 2

Administration of 75. mu.L of RNA solution (PK-0007)

Administration group 3

Administration of 75. mu.L of RNA solution (PK-0008)

After 24 hours of administration, a BALF sample was collected from the mouse in the same manner as in example a6, and the supernatant of the BALF sample was obtained. For the above supernatants, the amount of TNF-. alpha.and the amount of IFN-. beta.were determined. The amount of TNF-. alpha.described above was quantified using the trade name MouseTNFSetII (Beckton Dickinson and company) according to the instructions for its use. In addition, the amount of IFN-. beta.was quantified using ELISA plates manufactured under the trade names of Rabbitanti-MouseImterferon. beta. (PBLInterferon Source) and Biotin LabelingKit-NH2 (Dojindo laboratories) according to their instructions.

These results are shown in fig. 18. FIG. 18 (A) is a graph showing the amount of TNF- α in BALF samples of each administration group, and FIG. 18 (B) is a graph showing the amount of IFN- β in BALF samples of each administration group. In fig. 18, the horizontal axis represents each amount. The administration group 2 of PK-0007(+) did not cause the expression of TNF-. alpha.and IFN-. beta.as compared with the administration group 1 of RNA (-).

(example A10) expression-suppressing Effect of LAMA1 Gene in 293 cells

In vitro inhibition of LAMA1 gene expression was confirmed using the ssRNA of the present invention.

(1) Materials and methods

As RNA (Ex) in the examples, NK-0043 and NK-0064 shown below were used. In the following sequences, "+" indicates a free base (the same applies hereinafter).

[ chemical formula 12]

Ex: NK-0043 (SEQ ID NO. 43)

Ex: NK-0064 (SEQ ID NO. 44)

Transfection into 293 cells was performed in the same manner as in example A4, except that the RNA was used, and the cells were cultured for 48 hours. The RNA concentration at the time of transfection was 10 nmol/L. RNA recovery, cDNA synthesis, and PCR were performed in the same manner as in example a2 except that the following primer set for LAMA1 gene was used as primers, and the expression level of LAMA1 gene and the expression level of β -actin gene as an internal standard were measured. The expression level of the LAMA1 gene was corrected by the expression level of the β -actin gene as an internal standard.

Primer set for LAMA1 gene

5'-AAAGCTGCCAATGCCCCTCGACC-3' (Serial number 45)

5'-TAGGTGGGTGGCCCTCGTCTTG-3' (Serial number 46)

In addition, the expression level was measured for control 1(-) and control 2(mock) in the same manner as in example A2. Then, the corrected expression level of LAMA1 gene was determined as a relative value of the expression level of the cells into which each RNA was introduced, with the expression level of the control (-) cells set to 1.

(2) Results

These results are shown in fig. 19. Fig. 19 is a graph showing relative values of the expression amount of LAMA1 gene in 293 cells. As shown in FIG. 19, NK-0043 and NK-0064 of the examples showed strong gene expression inhibitory activity.

Example A11 expression inhibitory Effect of LMNA Gene in A549 cells

In vitro inhibition of LMNA gene expression based on the RNA interference effect was confirmed using the ssRNA of the present invention.

(1) Materials and methods

As RNA (Ex) in the examples, NK-0063 and NK-0066 shown below were used. In the following sequences, "+" indicates a free base.

[ chemical formula 13]

Ex: NK-0063 (SEQ ID NO. 47)

Ex: NK-0066 (Serial number 48)

Transfection into a549 cells was performed in the same manner as in example a4 above except that the RNA was used, and the cells were cultured for 48 hours. The RNA concentration at the time of transfection was 3 nmol/L. RNA recovery, cDNA synthesis, and PCR were performed in the same manner as in example a2 above, except that the following LMNA gene primer set was used as the primers, and the expression level of the LMNA gene and the expression level of the β -actin gene as an internal standard were measured. The expression level of the LMNA gene was corrected by the expression level of the β -actin gene as an internal standard.

Primer set for LMNA gene

5'-CTGGACATCAAGCTGGCCCTGGAC-3' (Serial number 49)

5'-CACCAGCTTGCGCATGGCCACTTC-3' (Serial number 50)

In addition, the expression level was measured for control 1(-) and control 2(mock) in the same manner as in example A2. Then, the corrected expression level of the LMNA gene was determined as a relative value of the expression level of the cells into which each RNA was introduced, with the expression level of the cells of the control (-) set to 1.

(2) Results

These results are shown in fig. 20. Fig. 20 is a graph showing relative values of expression amounts of LMNA genes in a549 cells. As shown in FIG. 20, NK-0063 and NK-0066 of the examples showed strong gene expression inhibitory activity.

(example A12) the lengths of Xc and Yc

In the ssRNA of the present invention, the length of the 5 '-side region (Xc) complementary to the internal 5' -side region (X) and the length of the 3 '-side region (Yc) complementary to the internal 3' -side region (Y) are varied to confirm the inhibition of GAPDH gene expression in vitro.

(1) Materials and methods

As RNA of the examples, ssRNA shown in FIG. 21 was used. In fig. 21, the right-hand number represents a serial number. In fig. 21, from the 5' side, a region underlined in lower case letters indicates the region (Xc), a region underlined in upper case letters indicates the internal region (Z), and a region underlined in lower case letters indicates the region (Yc). Between Xc and Z is a linker region (Lx), and between Z and Yc is a linker region (Ly). Further, "Xc/Yc" represents the ratio of the base length (Xc) of the region (Xc) to the base length (Yc) of the region (Yc). In FIG. 21, ". sup." indicates a free base.

Each ssRNA had an internal region (Z) with a base length of 26 bases, a linker region (Lx) with a base length of 7 bases, and a linker region (Ly) with a base length of 4 bases. Further, the total number of bases (Xc + Yc) in the region (Xc) and the region (Yc) of NK-0036 and NK-0040 is 26 bases, and the total number of bases (Xc + Yc) in the region (Xc) and the region (Yc) is 25 bases. Under these conditions, the base lengths of the region (Xc) and the region (Yc) are changed. Thus, NK-0036 and NK-0040 were molecules having no free base. In addition, for each ssRNA other than these, all free bases in the internal region (Z) that do not form a double strand are 1 base, and the position of the free base in the internal region (Z) is shifted from the 3 'side to the 5' side.

Transfection into HCT116 cells, culture, RNA recovery, cDNA synthesis, and PCR were performed in the same manner as in example a2, except that the RNA was used, and the relative expression level of the GAPDH gene was measured. The RNA concentration at the time of transfection was 10 nmol/L.

(2) Results and investigation

These results are shown in fig. 22. FIG. 22 is a graph showing the relative values of the expression amounts of GAPDH gene when RNA was used at a final concentration of 10 nmol/L. As shown in FIG. 22, the inhibition of the expression of GAPDH gene was confirmed in both of the ssRNAs with the changed lengths of the 5 '-side region (Xc) and the 3' -side region (Yc).

In particular, it was confirmed that as the difference between the base length of the region (Xc) and the base length of the region (Yc) becomes larger, the gene expression level is relatively decreased and the expression inhibitory activity is increased. That is, the expression inhibitory activity can be improved as the position of the free base in the internal region (Z) is located on the 5 'side or the 3' side relative to the center of the internal region.

In the above example A2, it was confirmed that NK-0016 has a very strong expression inhibitory activity. In this example, it was confirmed that NK-0025 and NK-0037 have activities further exceeding those of NK-0016.

The same effects as in example A8 (TGF-. beta.1 gene), example A10(LAMA1 gene), and example A11(LMNA gene) for the positions of free bases can be obtained for the genes different from those in this example.

That is, in example A8, as shown by the above sequences, both NK-0033 and NK-0055 have 1-base free base, the former being located at the 4 th position from the 3 'end of the internal region (Z), and the latter being located at the 2 nd position from the 3' end of the internal region (Z). Further, as shown in FIG. 15, NK-0055 with the free base position closer to the 3' -terminal side shows high expression inhibition. The same applies to NK-0061 and NK-0062, in which the free base is 2 bases. In examples a9 and 10, the position of the free base was changed under the same conditions as in example 8, and similarly, ssRNA whose free base position was closer to the 3' -end side showed high expression inhibition.

From these results, it was also clear that the same trends were exhibited regardless of the type of the target gene and the expression suppressing sequence therefor. Therefore, it can be said that the ssRNA of the present invention is a tool that can be used independently of the kind of the target gene.

(example A13) the lengths of X, Xc, Y and Yc

In the ssRNA of the present invention, the lengths of the internal 5 'region (X), the 5' region (Xc), the internal 3 'region (Y), and the 3' region (Yc) were varied, and inhibition of GAPDH gene expression in vitro was confirmed.

(1) Materials and methods

As RNA of the example, ssRNA shown in fig. 23 was used. In fig. 23, the right-hand number represents a serial number. In fig. 23, from the 5' side, a region underlined in lower case letters indicates the region (Xc), a region underlined in upper case letters indicates the internal region (Z), and a region underlined in lower case letters indicates the region (Yc). Further, "Xc + Yc/X + Y" represents a ratio of the total of the base lengths of the region (Xc) and the region (Yc) to the total of the base lengths of the region (X) and the region (Y). In FIG. 23, ". sup." indicates a free base.

In each ssRNA, the base length of the linker region (Lx) was 7 bases, the base length of the linker region (Ly) was 4 bases, the base length of the region (Yc) was 1 base, and the base at the 2 nd position from the 3' side of the internal region (Z) was a free base. And, the base length of the internal region (Z) and the base length of the region (Xc) are changed.

Unless otherwise specified, transfection of the above RNA into HCT116 cells, culture, RNA recovery, cDNA synthesis, and PCR were performed in the same manner as in example a2 to measure the expression level of GAPDH gene. With respect to the conditions for the above transfection, the composition of each well described above was set as follows. In the following composition, (B) was Opti-MEM (trade name, Invitrogen) and (C) was 20. mu. mol/L of the above RNA solution, and 98.5. mu.L of both solutions were added. The final concentration of RNA in the wells was 1 nmol/L. The correction based on the internal standard and the calculation of the relative value of the expression level were also performed in the same manner as in example a2 described above.

[ Table 5]

(2) Results and investigation

These results are shown in fig. 24. FIG. 24 is a graph showing the relative values of the expression amounts of GAPDH gene when RNA was used at a final concentration of 1 nmol/L. As shown in fig. 24, the expression inhibition of GAPDH gene was confirmed for all ssrnas having the modified lengths of the region (X), the region (Xc), the region (Y), and the region (Yc). Further, in example A2, it was confirmed that NK-0016 has a very strong expression inhibitory activity. In this example, it was confirmed that ssRNAs other than NK-0016 had activities further exceeding those of NK-0016.

(example A14) length of Xc

In the ssRNA of the present invention, the length of the 5 '-side region (Xc) complementary to the internal 5' -side region (X) is changed to confirm the inhibition of GAPDH gene expression in vitro.

(1) Materials and methods

As RNA of the example, ssRNA shown in fig. 25 was used. In fig. 25, from the 5 'side, a region underlined in lower case letters indicates the region (Xc), a region underlined in upper case letters indicates the internal 5' region (X), and a region underlined in lower case letters indicates the region (Yc). Further, "Xc/Yc" represents the ratio of the base length (Xc) of the region (Xc) to the base length (X) of the region (X). In FIG. 25, ". sup." denotes a free base. The sequences of the following RNAs are shown in SEQ ID Nos. 74 to 76.

Each ssRNA had an internal region (Z) of 26 bases in length, the region (X) of 25 bases in length, the region (Y) of 1 base in length, the region (Yc) of 1 base in length, the linker region (Lx) of 7 bases in length, and the linker region (Ly) of 4 bases in length. Under these conditions, the base length of the region (Xc) is changed. This changes the presence and number of free bases in the internal region (Z) of each ssRNA that do not form a double strand. Note that NK-0001 has no free base.

Transfection into HCT116 cells, culture, RNA recovery, cDNA synthesis, and PCR were performed in the same manner as in example a13, except that the RNA was used, and the expression level of the GAPDH gene was measured. The correction based on the internal standard and the calculation of the relative value of the expression level were also performed in the same manner as in example a13 described above.

(2) Results and investigation

These results are shown in fig. 26. FIG. 26 is a graph showing the relative values of GAPDH gene expression levels when RNA was used at a final concentration of 1 nmol/L. As shown in FIG. 26, the inhibition of the expression of GAPDH gene was confirmed in any ssRNA in which the length of the 5' -side region (Xc) was changed. In particular, it is considered that the expression inhibitory activity is improved as the number of free bases is smaller. Further, in example A2, it was confirmed that NK-0016 has a very strong expression inhibitory activity. In this example, it was confirmed that ssRNAs other than NK-0016 had activities further exceeding those of NK-0016.

The same effects as in example A8 (TGF-. beta.1 gene) for example, which is a gene different from that of this example, can be obtained with respect to the number of free bases. That is, in example A8, as shown in the above sequences, the former is 1 base and the latter is 2 base with respect to the free base of NK-0033 and NK-0061. Further, as shown in FIG. 15, NK-0033 having a small number of free bases showed high expression inhibition. Further, the positions of free bases in NK-0055 and NK-0062 were varied further toward the 3' -side of the inner region (Z) than in NK-0033 and NK-0061, thereby showing high expression suppression as in example A12. From these results, it was also clear that the same trends were shown regardless of the types of target genes and expression suppressing sequences therefor. Therefore, it can be said that the ssRNA of the present invention is a tool that can be used independently of the kind of the target gene.

(example A15) interchangeability of linkers

In the ssRNA of the present invention, the linker region (Lx) between the internal 5 'region (X) and the 5' region (Xc) and the linker region (Ly) between the internal 3 'region (Y) and the 3' region (Yc) were changed to confirm the inhibition of GAPDH gene expression in vitro.

(1) Materials and methods

As RNA of the example, ssRNA shown in fig. 27 was used. In fig. 27, from the 5 ' side, the area underlined in lower case letters indicates the 5 ' side area (Xc), the area underlined in upper case letters indicates the internal area (Z), and the area underlined in lower case letters indicates the 3 ' side area (Yc). The sequence between X and Xc is a linker region (Lx), and the sequence between Y and Yc is a linker region (Ly). In addition, the ratio (Lx/Ly) of the base length (Lx) of the linker region (Lx) to the base length (Ly) of the linker region (Ly) is shown for each RNA. In FIG. 27, ". sup." indicates a free base.

(2) Results and investigation

These results are shown in fig. 28. FIG. 28 is a graph showing the relative values of GAPDH gene expression levels when RNA was used at a final concentration of 1 nmol/L. As shown in fig. 28, expression inhibition of GAPDH gene was similarly confirmed for any ssRNA having the modified conditions of the linker regions (Lx) and (Ly), i.e., the modified length ratio between the two, the modified sequence, and the like. From this result, it is understood that the conditions for the linker regions (Lx) and (Ly) are not particularly limited, and the linker regions can be designed to have various lengths, sequences, and the like.

Example A16 expression inhibitory Effect of GAPDH Gene in HCT116 cells

The effect of GAPDH expression inhibition in HCT116 cells was confirmed using ssRNA substituted with a linker with proline or prolinol.

(1) Materials and methods

RNA (Ex) shown below was synthesized as RNA (ExssrRNA) of the examples. Further, as RNA of comparative example, NcssRNA as negative control (Nc) of RNAi shown below was synthesized. In the following formula, linker regions (Lx) and linker regions (Ly) were bound between Xc and X and Yc and Y using amidites (see example B) in the following table having proline or prolinol.

[ chemical formula 14]

ExssrRNA (SEQ ID NO: 13)

NcssRNA (SEQ ID NO: 38)

[ Table 6]

(2) Results

These results are shown in fig. 29. Fig. 29 is a graph showing relative values of expression amounts of GAPDH genes. As shown in fig. 29, it was confirmed that any of the exssrnas containing proline or prolinol as the linker region (Lx) and the linker region (Ly) had a strong gene expression inhibitory activity and exhibited an effect depending on the concentration. On the other hand, no inhibitory effect was observed with ssRNA as a negative control.

Example A17 expression inhibitory Effect of GAPDH Gene in HCT116 cells

The effect of inhibiting GAPDH expression in HCT116 cells was confirmed using ssRNA substituted with a linker with proline.

(1) Materials and methods

[ chemical formula 15]

ExssrRNA (SEQ ID NO: 13)

NcssRNA (SEQ ID NO: 38)

[ Table 7]

(2) Results

These results are shown in fig. 30. Fig. 30 is a graph showing relative values of GAPDH gene expression levels in HCT116 cells. As shown in fig. 30, the ExssRNA containing proline as the linker region (Lx) and linker region (Ly) showed strong gene expression inhibitory activity and showed effects in a concentration-dependent manner. On the other hand, no inhibitory effect was observed with ssRNA as a negative control.

Example A18 ribonuclease resistance

Regarding the ssRNA of the present invention, ribonuclease resistance was confirmed.

(1) Materials and methods

As RNA (Ex) of the examples, NK-0033 of example A5 and PK-0007 of example A8 were used. In addition, as the RNA of the comparative example, dsRNA (NI-0030) as the positive control (Pc) of example A9 was used.

First, 60pmol of the above RNA and 5X 10 cells were mixed in 20mmol/L of LTris-HCl (pH8)^-5Units of RNaseA (Roche) and 5X 10^-5Units of RNase T1(Roche) were incubated at 37 ℃. After 10 minutes, 20 minutes and 30 minutes from the initiation of the incubation, the RNase reaction was stopped according to the conventional method. Then, the reaction mixture was electrophoresed on a 15% polyacrylamide gel, stained with SYBRGreenII (Lonza, Switzerland), and then E-BOX-VX2 (M) was used&SInstruments, tokyo).

(2) Results

The results are shown in fig. 31. FIG. 31 is an electrophoresis photograph showing RNase resistance. In FIG. 31, lane "M" is a molecular weight marker, and min represents the incubation time.

As shown in FIG. 31, NI-0030 of comparative example was decomposed substantially completely after 10 minutes of incubation. In contrast, NK-0033 and PK-0007) of the examples remained after incubation for 10 minutes. From these results, it is clear that the ssRNA of the present invention is more excellent in ribonuclease resistance than dsRNA.

(example A19) nuclease resistance

Regarding the ssRNA of the present invention, nuclease resistance was confirmed.

(1) Materials and methods

The same RNA as in example A18 was used. First, in a solution containing 5mmol/LCaCl₂60pmol of the above RNA and 0.5 unit of S7 nuclease (Roche) were mixed with 50mmol/L of LTris-HCl (pH8), and incubated at 37 ℃. After 0.5 hour from the start (0 hour) of the incubation, the reaction of S7 nuclease was stopped according to a conventional method. Then, the reaction solution was electrophoresed on 7M urea-15% polyacrylamide gel according to a conventional method, stained with SYBRGreenII (trade name, Lonza), and then E-BOX-VX2 (trade name, M)&SInstruments) were performed.

(2) Results

The results are shown in fig. 32. Fig. 32 is an electrophoresis photograph showing nuclease resistance of S7. In FIG. 32, lane "M" is a molecular weight marker. Further, h represents an incubation time.

As shown in FIG. 32, NI-0030 of comparative example was decomposed substantially completely after 0.5 hour incubation. In contrast, NK-0033 and PK-0007 of the examples remained after 0.5 hour of incubation. From these results, it was found that the ssRNA of the present invention is superior in S7 nuclease resistance to dsRNA.

From the results of example A above, it is clear that the construction of ssRNA of the present invention can be independent of the type of target gene, for example. Specifically, it is found that the ssRNA can change, for example, the length of a double strand formed by the region (X) and the region (Xc), the length of a double strand formed by the region (Y) and the region (Yc), the presence, number, and position of free bases in the internal region (Z) that do not form a double strand, the presence, type, and length of the linker region (Lx) and the linker region (Ly), and the like. From this, it can be said that the ssRNA of the present invention is a highly versatile and novel tool that can be used for suppressing gene expression regardless of the type of target gene.

(example B1)

1. Prolinol synthesis

Proinol protected with dimethoxytrityl group was synthesized according to scheme 1 shown below.

[ chemical formula 16]

Scheme 1

(1) Fmoc-L-prolinol (Compound 2)

An aqueous solution of L-prolinol (compound 1) (0.61g, 6.0mmol) was prepared by dissolving in 70mL of pure water. N- (9-Fmoc-OSu) (2.0g, 6.0mmol) was dissolved in THF10 mL. The THF solution was added to the above L-prolinol aqueous solution, and the mixture was stirred for 1 hour to react the two solutions. The reaction solution was separated into a liquid fraction and a precipitate fraction, each fraction was extracted with ethyl acetate, and the organic layer was recovered separately. Then, the organic layers were combined, and anhydrous sodium sulfate was added to absorb water (hereinafter, referred to as drying). The organic layer was filtered, the filtrate was collected, 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(1.4g, yield 74%). The results of NMR of the compound are shown below.

¹H-NMR(CDCl₃)：7.77(2H，d，J＝7.7Hz，Ar-H)，7.60(2H，d，J＝7.3Hz，Ar-H)，7.40(2H，t，J＝7.5Hz，Ar-H)，7.31(2H，t，J＝7.6Hz，Ar-H)，4.40-4.50(2H，m，COOCH₂)，4.22(1H，t，J＝6.5Hz，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)

Fmoc-L-prolinol (Compound 2) (1.4g, 4.3mmol) was dissolved in 20mL of pyridine and azeotroped 3 times. The resulting residue was dissolved in 20mL of pyridine. While the solution was stirred in an ice bath under argon, 4' -dimethoxytrityl chloride (DMTr-Cl) (1.8g, 5.3mmol) was added. With respect to the reaction solution, the reaction was followed by chloroform/methanol TLC until the point of Fmoc-L-prolinol disappeared for 4 hours. Then, 3mL of methanol was added to the reaction solution to quench the excess DMTr-Cl, and the mixture was stirred for 10 minutes. Chloroform was further added to the reaction solution, and then the organic layer was collected. The recovered organic layer was washed with saturated brine, washed with a 5% sodium bicarbonate aqueous solution, and washed again with saturated brine. The washed organic layer was dried over anhydrous sodium sulfate. The organic layer was filtered, and the obtained 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(2.0g, yield 74%). The results of NMR of the compound are shown below.

¹H-NMR(CDCl₃)：7.77(2H，d，J＝7.7Hz，Ar-H)，7.60(2H，d，J＝7.3Hz，Ar-H)，7.40-7.18(13H，m，Ar-H)，6.89(4H，d，J＝8.6Hz，Ar-H)，4.20-4.40(2H，m，COOCH₂)，4.02(1H，t，J＝6.5Hz，Ar-CH)，3.80-3.10(5H，m，H-5，H-6)，3.73(s，6H，OCH₃)，1.84(3H，m，H-3，H-4)，1.58(1H，m，H-3)。

(3) DMTr-L-prolinol (Compound 4)

Fmoc-DMTr-L-prolinol (compound 3) (2.0g, 3.2mmol) was dissolved in 25mL of a DMF solution containing 20% piperidine, and stirred for 12 hours. The solution was concentrated under reduced pressure, and the obtained residue was purified by silica gel column chromatography (chloroform: methanol = 85: 15, containing 1% pyridine) to obtain compound 4(1.0g, yield 78%). The results of NMR of the compound are shown below.

¹H-NMR(CDCl₃)：7.40-7.14(9H，m，Ar-H)，6.82(4H，d，J＝8.6Hz，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)。

Synthesis of amidites derivatives

Next, an amidite derivative having prolinol was synthesized according to scheme 2 shown below. Hereinafter, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride is referred to as "EDC", and N, N-dimethylaminopyridine (4-dimethylaminopyridine) is referred to as "DMAP".

[ chemical formula 17]

Scheme 2

(1) DMTr-amide-L-prolinol (Compound 5)

The above DMTr-L-prolinol (compound 4) (0.80g, 2.0mmol), EDC (0.46g, 2.4mmol) and DMAP (0.29g, 2.4mmol) were dissolved in 20mL of dichloromethane and stirred. To the solution was added 10-hydroxydecanoic acid (0.45g, 2.4mmol) and stirred. The reaction was followed by ethyl acetate TLC for 20 hours until the point of DMTr-L-prolinol disappeared. Then, dichloromethane was added to the reaction solution, and the organic layer was collected. The recovered organic layer was washed with saturated brine and dried over anhydrous sodium sulfate. The organic layer was filtered, and the obtained filtrate was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (ethyl acetate, containing 1% pyridine) to obtain compound 5(0.71g, yield 62%). The results of NMR of the compound are shown below.

¹H-NMR(CDCl₃)：7.40-7.14(9H，m，Ar-H)，6.82(4H，d，J＝8.6Hz，Ar-H)，3.78(6H，s，OCH₃) 3.68-2.93(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) (0.80g, 2.0mmol) was dissolved in 15mL of methanol, and 5-hydroxypentanal (0.31g, 3.0mmol) was added thereto and stirred. To the solution was added sodium cyanoborohydride (0.25g, 4.0mmol), and the mixture was stirred. With respect to the reaction solution, the reaction was followed by ethyl acetate/hexane TLC until the point of DMTr-L-prolinol disappeared for 24 hours. Then, ethyl acetate was added to the reaction solution to recover an organic layer. The recovered organic layer was washed with saturated brine and dried over anhydrous sodium sulfate. The organic layer was filtered, and 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(0.62g, yield 63%). The results of NMR of the compound are shown below.

¹H-NMR(CDCl₃)：7.40-7.14(9H，m，Ar-H)，6.82(4H，d，J＝8.6Hz，Ar-H)，3.78(6H，s，OCH₃)，3.70-2.86(4H，m，CH₂OH, 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-carbamate-L-prolinol (Compound 7)

1, 4-butanediol (0.90g, 10mmol) was dissolved in 30mL of dichloromethane, and carbonyldiimidazole (1.4g, 8.6mmol) was added thereto and stirred for 3 hours. The organic layer of the reaction mixture was washed with saturated brine and 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). Thus, a compound (0.25g, 1.5mmol) in which one end of 1, 4-butanediol was activated with carbonyldiimidazole was obtained. This compound was dissolved in 15mL of methylene chloride, and the above DMTr-L-prolinol (compound 4) (0.6g, 1.5mmol) was added thereto and stirred for 24 hours. Ethyl acetate was further added to the mixture to recover an organic layer. The recovered organic layer was washed with saturated brine and dried over anhydrous sodium sulfate. The organic layer was filtered, and 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(0.61g, yield 77%). The results of NMR of the compound are shown below.

¹H-NMR(CDCl₃)：7.40-7.14(9H，m，Ar-H)，6.82(4H，d，J＝8.6Hz，Ar-H)，4.24-3.94(2H，m，COOCH₂)，3.78(s，6H，OCH₃) 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) (0.50g, 1.2mmol) and triphosgene (0.12g, 0.40mmol) were dissolved in 8mL of dichloromethane, and the mixture was stirred in an ice bath under argon. Then, N-diisopropylethylamine (0.31g, 2.4mmol) was added to the above solution and stirred for 1 hour. Further, 8-amino-1-octanol (0.17g, 1.2mmol) was added to the solution, and the mixture was stirred in an ice bath for 30 minutes and then stirred at room temperature for 20 hours. Methylene chloride was added to the above solution to recover an organic layer. The recovered organic layer was washed with saturated brine and dried over anhydrous sodium sulfate. The organic layer was filtered, and the obtained filtrate was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (hexane: ethyl acetate = 4: 1, containing 1% triethylamine) to obtain compound 8(0.44g, yield 62%). The results of NMR of the compound are shown below.

¹H-NMR(CDCl₃)：7.40-7.14(9H，m，Ar-H)，6.82(4H，m，Ar-H)，3.78(s，6H，OCH₃)，3.68-3.25(9H，m，CH₂NH，CH₂OH, H-2, H-5, H-6), 1.74-1.18(16H, m, alkyl, H-3, H-4).

(5) Amidites derivatives with prolinol (Compounds 9 to 12)

The modified prolinol compounds (compounds 5 to 8) were used as raw materials, and compounds 9 to 12 were synthesized by the following methods. The modified prolinol and 5-benzylthio-1H-tetrazole described above were dissolved in 3mL of acetonitrile. The amount of the modified prolinol used was 0.69g (1.2mmol) in the case of compound 5, 0.60g (1.2mmol) in the case of compound 6, 0.60g (1.2mmol) in the case of compound 7, and 0.25g (0.43mmol) in the case of compound 8. The amount of 5-benzylthio-1H-tetrazole used was 0.15g (0.78mmol) of compound 5 to 7 and 54mg (0.15mmol) of compound 8. To the above solution was added 2-cyanoethyl-N, N' -tetraisopropylphosphorodiamidite under argon and stirred for 2 hours. The amount of 2-cyanoethyl-N, N, N ', N' -tetraisopropylphosphorodiamidite added was 0.54g (1.8mmol) in the system using the above-mentioned compounds 5 to 7 and 0.19g (0.64mmol) in the system using the above-mentioned compound 8. Then, a saturated aqueous sodium bicarbonate solution was added to the solution, followed by extraction with dichloromethane, and the organic layer was collected. The recovered 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 silica gel column chromatography (hexane: ethyl acetate = 1: 1, containing 1% triethylamine) to obtain compounds 9 to 12. The results of NMR of each compound are shown below.

DMTr-amide-L-prolinol amidite (Compound 9, 0.60g, yield 55%)

¹H-NMR(CDCl₃)7.40-7.14(9H，m，Ar-H)，6.82(4H，d，J＝8.6Hz，Ar-H)，3.78(6H，s，OCH₃)，3.68-2.93(11H，m，CH₂O，POCH₂，CHCH₃，H-2，H-5，H-6)，2.58(2H，m，CH₂CN), 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, 0.71g, yield 60%)

¹H-NMR(CDCl₃)：7.40-7.14(9H，m，Ar-H)，6.82(4H，d，J＝8.6Hz，Ar-H)，3.78(6H，s，OCH₃)，3.70-2.86(8H，m，CH₂O，POCH₂，CHCH₃，H-6)，2.58(2H，m，CH₂CN), 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-carbamate-L-prolinol amidite (Compound 11, 0.67g, yield 52%)

¹H-NMR(CDCl₃)：7.40-7.14(9H，m，Ar-H)，6.82(4H，d，J＝8.6Hz，Ar-H)，4.24-3.94(2H，m，COOCH₂)，3.78(s，6H，OCH₃)，3.72-2.96(11H，m，CH₂O，POCH₂，CHCH₃，H-2，H-5，H-6)，2.58(2H，m，CH₂CN), 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, 0.20g, yield 61%)

¹H-NMR(CDCl₃)：7.40-7.14(9H，m，Ar-H)，6.82(4H，m，Ar-H)，3.78(s，6H，OCH₃)，3.65-3.25(13H，m，CH₂O，POCH₂，CHCH₃，H-2，CH₂NH，CH₂OH，H-2，H-5，H-6)，2.73(2H，m，CH₂CN), 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 below.

[ chemical formula 18]

Scheme 3

(1) DMTr-hydroxyamidoamino-L-proline (Compound 11)

To an ethanol solution (7mL) containing DMTr-amide-L-proline (compound 6) (1.00g, 2.05mmol) and 5-hydroxypentanal (0.33g, 3.07mmol), an acetic acid buffer (7mL) was added under ice-cooling conditions. After the mixture was stirred for 20 minutes under ice-cooling, sodium cyanoborohydride (0.77g, 12.28mmol) was added thereto, 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. Then, recoveringThe organic layer was dried over 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)₂Cl₂：CH₃OH = 98: 2. containing 0.05% pyridine). Then, the obtained product was subjected to silica gel column chromatography (developing solvent CH)₂Cl₂：CH₃OH = 98: 2. containing 0.05% pyridine), and the obtained product was subjected to silica gel column chromatography (developing solvent dichloromethane: acetone = 7: 3. containing 0.05% pyridine). Thus, compound 11(0.49g, yield 41%) was obtained as a colorless syrup.

Ms(FAB+)：m/z575(Mw)，303(DMTr⁺)

(2) DMTr-amidoamino-L-proline amidites (Compound 12)

The obtained DMTr-hydroxyamidoamino-L-proline (compound 11) (0.50g, 0.87mmol) was mixed with anhydrous acetonitrile and azeotropically dried at room temperature. To the resulting residue, tetrazolediisopropylamine (178mg, 1.04mmol) was added, degassed under reduced pressure, and filled with argon. To the mixture was added anhydrous acetonitrile (1mL), and further added a solution of 2-cyanoethoxy-N, N, N ', N' -tetraisopropylphosphoramidite (313mg, 1.04mmol) in anhydrous acetonitrile (1 mL). The mixture was stirred at room temperature for 4 hours under an argon atmosphere. Then, the above mixture was diluted with dichloromethane and washed with a saturated aqueous sodium bicarbonate solution and saturated brine in this order. The organic layer was collected, dried over sodium sulfate, and the organic layer was filtered. The solvent was distilled off under reduced pressure from the obtained filtrate. The obtained residue was subjected to column chromatography using an amino silica gel as a packing material (developing solvent hexane: acetone = 7: 3, containing 0.05% pyridine) to obtain compound 12(0.57g, purity 93%, yield 79%) as a colorless syrup. The purity was measured by HPLC (the same shall apply hereinafter). The results of NMR of the compound are shown below.

¹H-NMR(CDCl₃)：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，OCH₃)、3.47-3.64(m，3H)、3.12-3.26(m，2H)、3.05(t，J＝6.4Hz，2H，CH₂)、2.98-2.02(m，2H)、2.61(t，J＝5.8Hz，2H，CH₂)、2.55-2.63(m，2H)、2.27-2.42(m，1H，CH)、2.31(t，7.8Hz，2H，CH₂)、2.03-2.19(m，1H，CH)、1.40-1.90(m，8H)、1.23-1.33(m，5H)、1.14-1.20(m，12H，CH₃)；

P-NMR(CDCl₃)：146.91；

Ms(FAB+)：m/z774(M⁺)、303(DMTr⁺)，201(C₈H₁₉N₂OP⁺)。

(3) DMTr-hydroxyamide carbamoyl-L-proline (Compound 13)

To an anhydrous acetonitrile solution (10mL) in which DMTr-amide-L-proline (compound 6) (1.00g, 2.05mmol) was dissolved at room temperature was added an anhydrous acetonitrile solution (20mL) in which 1-imidazolylcarbonyloxy-8-hydroxyoctane (1.12g, 4.92mmol) was dissolved under an argon atmosphere. The mixture was heated at 40 to 50 ℃ for 2 days, and then allowed to stand at room temperature for 5 days. The solvent was distilled off from the mixed solution under reduced pressure. The obtained residue was subjected to silica gel column chromatography (developing solvent dichloromethane: acetone = 4: 1, containing 0.05% pyridine). Thus, compound 13(0.68g, yield 50%) was obtained as a colorless syrup. The results of NMR of the compound are shown below.

¹H-NMR(CDCl₃)：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，OCH₃)、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/z602(M⁺)、303(DMTr⁺)。

(4) DMTr-amide carbamoyl-L-proline amidites (Compound 14)

The obtained DMTr-hydroxyamide carbamoyl-L-proline (compound 13) (0.63g, 1.00mmol) was mixed with anhydrous pyridine and azeotropically dried at room temperature. To the resulting residue, tetrazolediisopropylamine (206mg, 1.20mmol) was added, and the mixture was degassed under reduced pressure and purged with argon. To the mixture was added anhydrous acetonitrile (1mL), and further added a solution of 2-cyanoethoxy-N, N, N ', N' -tetraisopropylphosphoramidite (282mg, 1.12mmol) in anhydrous acetonitrile (1 mL). The mixture was stirred at room temperature for 4 hours under an argon atmosphere. Then, the mixture was diluted with dichloromethane, and washed with a saturated aqueous sodium bicarbonate solution and saturated brine in this order. The organic layer was collected, dried over sodium sulfate, and the organic layer was filtered. With respect to the obtained filtrate, the solvent was distilled off under reduced pressure. The obtained residue was subjected to column chromatography using an amino silica gel as a packing material (developing solvent hexane: acetone = 7: 3, containing 0.5% pyridine) to obtain compound 14(0.74g, purity 100%, yield 87%) as a colorless syrup. The results of NMR of the compound are shown below.

P-NMR(CDCl₃)：147.19；

Ms(FAB+)：m/z860(M⁺)、303(DMTr⁺)，201(C₈H₁₉N₂OP⁺)。

(5) DMTr-tert-butyldimethylsilyloxyamide ureido-L-proline (Compound 15)

To triphosgene (1.22g, 4.10mmol) was added an anhydrous tetrahydrofuran solution (10mL) under an argon atmosphere and ice-cooling. To the mixture was added dropwise a solution (10mL) of DMTr-amide-L-proline (compound 6) (1.00g, 2.05mmol) and DIEA (9.80g, 75.8mmol) in anhydrous tetrahydrofuran dissolved under an argon atmosphere and ice-cooling for 30 minutes, and the mixture was stirred at room temperature for 1 hour. A solution (20mL) of 10-amino-1-tert-butyldimethylsilyloxydecane (2.66g, 10.25mmol) and DIEA (3.20g, 24.76mmol) in anhydrous tetrahydrofuran was added dropwise to the above mixture over 45 minutes under an argon atmosphere and ice-cooling. Then, the mixture was stirred at room temperature overnight under an argon atmosphere. The mixture was diluted with ethyl acetate (200mL) to collect the organic layer. The organic layer was washed with a saturated aqueous sodium bicarbonate solution and then with saturated brine. Then, the organic layer was recovered and dried over 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). Thus, compound 15(0.87g, yield 55%) was obtained as a colorless syrup.

(6) DMTr-hydroxyamide ureido-L-proline (16)

To the resulting DMTr-t-butyldimethylsilyloxyamidoureido-L-proline (15) (0.87g, 1.12mmol) was added anhydrous tetrahydrofuran dichloromethane solution (10mL) at room temperature under an argon atmosphere. A tetrahydrofuran solution (4.69mL, manufactured by Tokyo chemical Co., Ltd.) containing 1mol/L tetrabutylammonium fluoride was added to the above mixture under an argon atmosphere, and the mixture was stirred at room temperature for 3 days. The mixture was diluted with dichloromethane (150mL), washed with water, and further washed with saturated brine. The organic layer was collected, dried over sodium sulfate, and the organic layer was filtered. With respect to 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 compound 16(0.68g, yield 92%) as a colorless syrup. The results of NMR of the compound are shown below.

¹H-NMR(CDCl₃)：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，CH₂)、3.79(s，6H，OCH₃)、3.63(d，1H，J＝6.4Hz，CH2)、3.61(d，1H，J＝6.4Hz，CH₂)、3.34-3.37(m，1H，CH)、3.16-3.27(m，5H)，3.04(t，J＝5.9Hz，2H，CH₂)、2.38-2.45(m，1H，CH)、1.83-2.05(m，3H)、1.45-1.64(m，8H)、1.25-1.38(m，7H)。

(7) DMTr-amide ureido-L-proline amidite (Compound 17)

The thus-obtained DMTr-hydroxyamide ureido-L-proline (compound 16) (0.62g, 0.94mmol) was mixed with anhydrous acetonitrile and azeotropically dried at room temperature. To the obtained residue, tetrazolediisopropylamine (192mg, 1.12mmol) was added, and the mixture was degassed under reduced pressure and then purged with argon. To the mixture was added anhydrous acetonitrile (1mL), and further added an anhydrous acetonitrile solution (1mL) of 2-cyanoethoxy-N, N, N ', N' -tetraisopropylphosphoramidite (282mg, 1.12 mmol). The mixture was stirred at room temperature for 4 hours under an argon atmosphere. Then, the above mixture was diluted with dichloromethane, and washed with a saturated aqueous sodium bicarbonate solution and saturated brine in this order. The organic layer was collected, dried over sodium sulfate, and the organic layer was filtered. The solvent was distilled off under reduced pressure from the obtained filtrate. The obtained residue was subjected to column chromatography using an amino silica gel as a packing material (developing solvent hexane: acetone = 1: 1, containing 0.05% pyridine) to obtain compound 17(0.77g, purity 88%, yield 84%) as a colorless syrup. The results of NMR of the compound are shown below.

P-NMR(CDCl₃)：147.27；

Ms(FAB+)：m/z860(M⁺+1)、303(DMTr⁺)，201(C₈H₁₉N₂OP⁺)。

Example B3 Synthesis of proline diamide amidites

To generate the nucleic acid molecule of the present invention comprising a linker having a proline backbone, L-proline diamide amidite and D-proline diamide amidite were synthesized by scheme 3 above.

(B3-1) L-proline diamide amidites

(1) Fmoc-hydroxyamide-L-proline (Compound 4)

Compound 2 (Fmoc-L-proline) of scheme 3 above was used as the starting material. Compound 2(10.00g, 29.64mmol), 4-amino-1-butanol (3.18g, 35.56mmol) and 1-hydroxybenzotriazole (10.90g, 70.72mmol) were mixed, and the mixture was degassed under reduced pressure and filled with argon. To the mixture was added anhydrous acetonitrile (140mL) at room temperature, and further added a solution of dicyclohexylcarbodiimide (7.34g, 35.56mmol) in anhydrous acetonitrile (70mL), followed by stirring at room temperature for 15 hours under an argon atmosphere. After completion of the reaction, the formed precipitate was filtered off, and the recovered filtrate was subjected to distillation under reduced pressure to remove the solvent. Methylene chloride (200mL) was added to the resulting residue, which was washed with a saturated aqueous solution of sodium hydrogencarbonate (200 mL). Then, the organic layer was recovered, dried over magnesium sulfate, and then filtered. The solvent was distilled off from the obtained filtrate under reduced pressure, and diethyl ether (200mL) was added to the residue to pulverize it. The resulting powder was collected by filtration, whereby compound 4(10.34g, yield 84%) was obtained as a colorless powder. The results of NMR of the above compound are shown below.

¹H-NMR(CDCl₃)：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，CH₂)，3.67-3.73(m，2H，CH₂)、3.35-3.52(m，2H，CH₂)、3.18-3.30(m，2H，CH₂)、2.20-2.50(m，4H)、1.81-2.03(m，3H)、1.47-1.54(m，2H)；

Ms(FAB+)：m/z409(M+H⁺)。

(2) DMTr-amide-L-proline (Compound 6)

Fmoc-hydroxyamide-L-proline (compound 4) (7.80g, 19.09mmol) was mixed with anhydrous pyridine (5mL) and azeotropically dried 2 times at room temperature. To the resulting residue were added 4, 4' -dimethoxytrityl chloride (8.20g, 24.20mmol), DMAP (23mg, 0.19mmol) and anhydrous pyridine (39 mL). After the mixture was stirred at room temperature for 1 hour, methanol (7.8mL) was added and the mixture was stirred at room temperature for 30 minutes. The mixture was diluted with dichloromethane (100mL) and washed with saturated aqueous sodium bicarbonate (150mL), and the organic layer was separated. After the organic layer was dried over sodium sulfate,the organic layer was filtered. With respect to the obtained filtrate, the solvent was distilled off under reduced pressure. To the obtained crude residue were added anhydrous dimethylformamide (39mL) and piperidine (18.7mL, 189mmol), and the mixture was stirred at room temperature for 1 hour. After completion of the reaction, the solvent was distilled off from the mixture at room temperature under reduced pressure. The obtained residue was subjected to silica gel column chromatography (trade name: Wakogel C-300, developing solvent CH)₂Cl₂：CH₃OH = 9: 1. containing 0.05% pyridine) to give compound 6(9.11g, yield 98%) as a pale yellow oil. The results of NMR of the above compound are shown below.

¹H-NMR(CDCl₃)：7.39-7.43(m，2H，Ar-H)、7.30(d，J＝8.8Hz，4H，Ar-H)、7，21(tt，1H，4.9，1.3Hz，Ar-H)、6.81(d，J＝8.8Hz，4H，Ar-H)、3.78(s，6H，OCH₃)、3.71(dd，H，J＝6.3Hz，5.4Hz，CH)、3.21(2H，12.9，6.3Hz，2H，CH₂)、3.05(t，J＝6.3Hz，2H，CH₂)、2.85-2.91(m，2H，CH₂)、2.08-2.17(m，1H，CH)、1.85-2.00(m，3H)、1.55-1.65(m，5H)；

Ms(FAB+)；m/z489(M+H⁺)、303(DMTr⁺)。

(3) DMTr-Hydroxydiamide-L-proline (Compound 8)

The thus-obtained DMTr-amide-L-proline (compound 6) (6.01g, 12.28mmol), EDC (2.83g, 14.74mmol), 1-hydroxybenzotriazole (3.98g, 29.47mmol) and triethylamine (4.47g, 44.21mmol) in dry dichloromethane (120mL) were mixed. To the mixture was further added 6-hydroxycaproic acid (1.95g, 14.47mmol) under an argon atmosphere at room temperature, and then the mixture was stirred under an argon atmosphere at room temperature for 1 hour. The mixture was diluted with dichloromethane (600mL) and washed 3 times with saturated brine (800 mL). The organic layer was collected, dried over sodium sulfate, and then filtered. With respect to the obtained filtrate, the solvent was distilled off under reduced pressure. Thus, Compound 8(6.29g, yield 85%) was obtained as pale yellow bubbles. The results of NMR of the above compound are shown below.

¹H-NMR(CDCl₃)：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，OCH₃)、3.61(t，2H，J＝6.4Hz，CH₂)、3.50-3.55(m，1H，CH)、3.36-3.43(m，1H，CH)，3.15-3.24(m，2H，CH₂)，3.04(t，J＝6.3Hz，2H，CH₂)、2.38-2.45(m，1H，CH)、2.31(t，6.8Hz，2H，CH₂)、2.05-2.20(m，1H，CH)、1.92-2.00(m，1H，CH)、1.75-1.83(m，1H，CH)、1.48-1.71(m，8H)、1.35-1.44(m，2H，CH₂)；

Ms(FAB+)：m/z602(M⁺)、303(DMTr⁺)。

(4) DMTr-diamide-L-proline amidites (Compound 10)

The thus-obtained DMTr-hydroxydiamide-L-proline (compound 8) (8.55g, 14.18mmol) was mixed with anhydrous acetonitrile and azeotropically dried at room temperature for 3 times. To the obtained residue, tetrazolediisopropylamine (2.91g, 17.02mmol) was added, and the mixture was degassed under reduced pressure and then purged with argon. To the mixture was added anhydrous acetonitrile (10mL), and further added a solution of 2-cyanoethoxy-N, N, N ', N' -tetraisopropylphosphoramidite (5.13g, 17.02mmol) in anhydrous acetonitrile (7 mL). The mixture was stirred at room temperature for 2 hours under an argon atmosphere. Then, the above mixture was diluted with dichloromethane and washed with saturated aqueous sodium bicarbonate (200mL) 3 times, followed by washing with saturated brine (200 mL). The organic layer was collected, dried over sodium sulfate, and the organic layer was filtered. The solvent was distilled off under reduced pressure from the obtained filtrate. The obtained residue was subjected to column chromatography using an amino silica gel as a packing material (developing solvent hexane: ethyl acetate = 1: 3, containing 0.05% pyridine) to obtain compound 10(10.25g, purity 92%, yield 83%) as a colorless syrup. The results of NMR of the above compound are shown below.

¹H-NMR(CDCl₃)：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，OCH₃)、3.45-3.60(m，4H)、3.35-3.45(m，1H，CH)、3.20-3.29(m，1H)、3.04(t，J＝6.4Hz，2H，CH₂)、2.62(t，J＝5.8Hz，2H，CH₂)、2.40-2.44(m，1H，CH)、2.31(t，7.8Hz，2H，CH₂)、2.03-2.19(m，1H，CH)、1.92-2.02(m，1H，CH)、1.70-1.83(m，1H，CH)、1.51-1.71(m，8H)、1.35-1.44(m，2H，CH₂)、1.18(d，J＝6.8Hz，6H，CH₃)、1.16(d，J＝6.8Hz，6H，CH₃)

P-NMR(CDCl₃)Ms147.17；

Ms(FAB+)：m/z802(M⁺)、303(DMTr⁺)，201(C₈H₁₉N₂OP⁺)。

(B3-2) D-proline diamide amidites

(1) Fmoc-hydroxyamide-D-proline (Compound 3)

Compound 1 (Fmoc-D-proline) of scheme 3 above was used as the starting material. A mixture of the above-mentioned compound 1(1.5g, 4.45mmol), dicyclohexylcarbodiimide (1.1g, 5.34mmol) and 1-hydroxybenzotriazole (1.5g, 10.69mmol) was degassed under reduced pressure and filled with argon. To the mixture was added anhydrous acetonitrile (24mL) at room temperature, and further added an anhydrous acetonitrile solution (6mL) of 4-amino-1-butanol (0.48g, 5.34mmol), followed by stirring at room temperature under an argon atmosphere for 15 hours. After completion of the reaction, the formed precipitate was filtered off, and the recovered filtrate was subjected to distillation under reduced pressure to remove the solvent. Methylene chloride was added to the obtained residue, and the mixture was washed 3 times with an acetic acid buffer solution (pH4.0) and 3 times with a saturated aqueous sodium bicarbonate solution. Then, the organic layer was recovered, dried over magnesium sulfate, and then filtered. The solvent was distilled off from the obtained filtrate under reduced pressure, and diethyl ether (50mL) was added to the residue to pulverize it. The resulting powder was collected by filtration to obtain compound 3 as a white powder. The results of NMR of the above compound are shown below.

¹H-NMR(400MHz，CDCl₃)：7.77(d，J＝7.3Hz，2H)；7.58(br，2H)；7.41(t，J＝7.3Hz，2H)；7.32(t，J＝7.3Hz，2H)；4.25-4.43(m，4H)；3.25-3.61(m，6H)；1.57-1.92(m，8H)。

MS(FAB+)：m/z409(M+H⁺)。

(2) DMTr-amide-D-proline (Compound 5)

Fmoc-hydroxyamide-D-proline (compound 3) (1.0g, 2.45mmol) was mixed with anhydrous pyridine (5mL) and azeotropically dried 2 times at room temperature. To the resulting residue were added 4, 4' -dimethoxytrityl chloride (1.05g, 3.10mmol), DMAP (3mg, 0.024mmol) and anhydrous pyridine (5 mL). After the mixture was stirred at room temperature for 1 hour, methanol (1mL) 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 bicarbonate, and the organic layer was separated. The organic layer was dried over sodium sulfate, and the organic layer was filtered. With respect to the obtained filtrate, the solvent was distilled off under reduced pressure. To the obtained crude residue were added anhydrous dimethylformamide (5mL) and piperidine (2.4mL, 24mmol), and the mixture was stirred at room temperature for 1 hour. After completion of the reaction, the solvent was distilled off from the mixture at room temperature under reduced pressure. The obtained residue was subjected to silica gel column chromatography (trade name: Wakogel C-300, developing solvent CH)₂Cl₂：CH₃OH = 9: 1. containing 0.05% pyridine) to give compound 5(1.26g, yield 96%) as a pale yellow oil. The results of NMR of the above compound are shown below.

¹H-NMR(400MHz，CDCl₃)：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＝8.8，5.4Hz，1H)；3.22(q，6.5Hz，2H)；3.07(t，J＝6.1Hz，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/z489(M+H⁺)，303(DMTr⁺)。

(3) DMTr-Hydroxydiamide-D-proline (Compound 7)

The thus-obtained DMTr-amide-D-proline (compound 5) (1.2g, 2.45mmol), EDC (566mg, 2.95mmol), 1-hydroxybenzotriazole (796mg, 5.89mmol) and triethylamine (1.2mL, 8.84mmol) in dry dichloromethane (24mL) were mixed. To the mixture was further added 6-hydroxyhexanoic acid (390mg, 2.95mmol) under an argon atmosphere at room temperature, and then the mixture was stirred under an argon atmosphere at room temperature for 1 hour. The mixture was diluted with dichloromethane and washed 3 times with saturated aqueous sodium bicarbonate. The organic layer was collected, dried over sodium sulfate, and then filtered. With respect to the obtained filtrate, the solvent was distilled off under reduced pressure. Thus, compound 7 was obtained as a pale yellow oil (1.4g, yield 95%). The results of NMR of the above compound are shown below.

¹H-NMR(400MHz，CDCl₃)：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)；3.15-3.26(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/z602(M⁺)，303(DMTr⁺)。

(4) DMTr-diamide-D-proline amidites (Compound 9)

The thus-obtained DMTr-hydroxydiamide-D-proline (compound 7) (1.2g, 1.99mmol) was mixed with anhydrous acetonitrile and azeotropically dried at room temperature for 3 times. To the resulting residue, tetrazolediisopropylamine (410mg, 2.40mmol) was added, and the mixture was degassed under reduced pressure and then purged with argon. To the mixture was added anhydrous acetonitrile (2.4mL), and further added 2-cyanoethoxy-N, N, N ', N' -tetraisopropylphosphoramidite (722mg, 2.40 mmol). The mixture was stirred at room temperature for 2 hours under an argon atmosphere. Then, the above mixture was diluted with dichloromethane and washed with saturated aqueous sodium bicarbonate solution 3 times, followed by washing with saturated brine. The organic layer was collected, dried over sodium sulfate, and the organic layer was filtered. With respect to the obtained filtrate, the solvent was distilled off under reduced pressure. The obtained residue was subjected to column chromatography using an amino silica gel as a packing material (development solvent hexane: ethyl acetate = 1: 3), whereby compound 9(1.4g, purity 95%, yield 83%) was obtained as a colorless oil. The results of NMR of the above compound are shown below.

¹H-NMR(400MHz，CDCl₃)：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)；3.02-3.06(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)。

³¹P-NMR(162MHz，CDCl₃)：146.94.

MS(FAB+)：m/z802(M⁺)，303(DMTr⁺)，201(C₈H₁₉N₂OP⁺)。

(example B4)

To generate the nucleic acid molecule of the present invention comprising a linker having a proline backbone, L-proline diamide amidite type b was synthesized by scheme 4 below.

[ chemical formula 19]

Scheme 4

(1) Fmoc-tert-butyl-dimethylsiloxyamide-L-proline (Compound 18)

Fmoc-hydroxyamide-L-proline (compound 4) (2.00g, 30mmol), tert-butyldimethylsilyl chloride (1.11g, 35mmol) and imidazole (10.90g, 71mmol) were combined. For the above mixture, degassed under reduced pressure and filled with argon. To the above mixture was added anhydrous acetonitrile (20mL) at room temperature, and stirred at room temperature overnight under an argon atmosphere. After completion of the reaction, methylene chloride (150mL) was added to the above mixture, and the mixture was washed with water 3 times and saturated brine. The organic layer was collected, dried over magnesium sulfate, and then filtered. The solvent was distilled off from the obtained filtrate under reduced pressure, and the residue was subjected to silica gel column chromatography (developing solvent CH)₂Cl₂：CH₃OH = 95: 5) compound 18(2.35g, yield 92%) was obtained as a colorless syrup. The results of NMR of the above compound are shown below.

¹H-NMR(CDCl₃)：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，CH₂)，3.47-3.59(m，4H，CH₂)、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，SiCH₃)；

Ms(FAB+)：m/z523(M+H⁺)。

(2) t-butyldimethylsilyloxyamide-L-proline (Compound 19)

To the obtained Fmoc-t-butyl-dimethylsilyloxyamide-L-proline (compound 18) (1.18g, 2.5mmol) was added anhydrous acetonitrile (5mL) and piperidine (2.4mL), and the mixture was stirred at room temperature for 1 hour. After completion of the reaction, acetonitrile (50mL) was added to the above mixture, and insoluble matter was filtered off. The solvent was distilled off from the obtained filtrate under reduced pressure, and the obtained residue was subjected to silica gel column chromatography (developing solvent CH)₂Cl₂：CH₃OH = 9: 1) compound 19(0.61g, yield 90%) was obtained as a colorless syrup. The results of NMR of the above compound are shown below.

¹H-NMR(CDCl₃)：3.71(dd，1H，J＝9.0Hz，5.2Hz，CH)、3.61-3.64(m，2H，CH₂)、3.22-3.28(m，2H，CH₂)、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，CH₂)、1.52-1.57(m，4H)、0.89(s，9H，t-Bu)、0.05(s，6H，SiCH₃)：

Ms(FAB+)；m/z301(M+H⁺)。

(3) Tert-butyldimethylsilyloxyamide hydroxyamide-L-proline (Compound 20)

The obtained tert-butyldimethylsilyloxyamide-L-proline (compound 19) (550mg, 1.8mmol), 6-hydroxyhexanoic acid (300mg, 2.3mmol), EDC (434mg, 2.3mmol) and a solution of 1-hydroxybenzotriazole (695mg, 4.5mmol) in dry dichloromethane (20mL) were mixed. To the above mixture was added triethylamine (689mg, 6.8mmol) at room temperature under an argon atmosphere, and then, the mixture was stirred at room temperature overnight under an argon atmosphere. The mixture was washed with saturated brine. The organic layer was collected, dried over sodium sulfate, and then filtered. With respect to the obtained filtrate, the solvent was distilled off under reduced pressure. The obtained residue was subjected to silica gel column chromatography (developing solvent CH)₂Cl₂：CH₃OH = 9: 1) compound 20(696mg, yield 92%) was obtained as a colorless syrup. The results of NMR of the above compound are shown below.

¹H-NMR(CDCl₃)：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＝7.3Hz，2H，CH₂)、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，CH₂)、0.87(s，9H，t-Bu)、0.04(s，6H，SiCH₃)；

Ms(FAB+)：m/z415(M⁺+1)。

(4) DMTr-Hydroxydiamide-L-proline type B (Compound 21)

The obtained tert-butyldimethylsilyloxyamide hydroxyamideL-proline (compound 20) (640mg, 1.54mmol) was mixed with anhydrous pyridine (1mL) and azeotropically dried at room temperature. To the resulting residue were added 4, 4' -dimethoxytrityl chloride (657mg, 1.85mmol), DMAP (2mg) and anhydrous pyridine (5mL), and after stirring at room temperature for 4 hours, methanol (1mL) 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 bicarbonate. The organic layer was collected, dried over sodium sulfate, and the organic layer was filtered. With respect to the obtained filtrate, the solvent was distilled off under reduced pressure. To the resulting residue were added anhydrous acetonitrile (5mL) and a tetrahydrofuran solution containing 1mol/L tetrabutylammonium fluoride (1.42mL, 1.42mmol of tetrabutylammonium fluoride), and the mixture was stirred at room temperature overnight. After completion of the reaction, ethyl acetate (100mL) was added to the above mixture, which was washed with water and then with saturated brine. The organic layer was collected, dried over sodium sulfate, and the organic layer was filtered. With respect to the obtained filtrate, the solvent was distilled off under reduced pressure. The obtained residue was subjected to silica gel column chromatography (developing solvent CH)₂Cl₂：CH₃OH = 95: 5. containing 0.05% pyridine) to give compound 21(680mg, yield 73%) as a colorless syrup. The results of NMR of the above compound are shown below.

¹H-NMR(CDCl₃)：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，6.8Hz，1H，CH)、3.79(s，6H，OCH₃)、3.61(dd，2H，J＝11Hz，5.4Hz，CH₂)、3.50-3.54(m，1H，CH)、3.36-3.43(m，1H，CH)，3.20-3.26(m，2H，CH₂)，3.05(t，J＝6.4Hz，2H，CH₂)、2.38-2.45(m，1H，CH)、2.30(t，J＝7.8Hz，2H，CH₂)、2.05-2.25(m，1H，CH)、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，CH₂)；

Ms(FAB+)：m/z602(M⁺)、303(DMTr⁺)。

(5) DMTr-diamide-L-proline amiditesB type (Compound 22)

The obtained DMTr-hydroxydiamide-L-proline form B (compound 21) (637mg, 1.06mmol) was mixed with anhydrous acetonitrile and azeotropically dried at room temperature. To the obtained residue, tetrazolediisopropylamine (201mg, 1.16mmol) was added, and the mixture was degassed under reduced pressure and then purged with argon. To the mixture was added anhydrous acetonitrile (1mL), and further added a solution of 2-cyanoethoxy-N, N, N ', N' -tetraisopropylphosphoramidite (350mg, 1.16mmol) in anhydrous acetonitrile (1 mL). The mixture was stirred at room temperature for 4 hours under an argon atmosphere. The mixture was diluted with dichloromethane and washed with saturated aqueous sodium bicarbonate and saturated brine. The organic layer was collected, dried over sodium sulfate, and the organic layer was filtered. With respect to the obtained filtrate, the solvent was distilled off under reduced pressure. The obtained residue was subjected to column chromatography using an amino silica gel as a packing material (development solvent hexane: acetone = 7: 3), to give compound 22(680mg, purity 95%, yield 76%) as a colorless syrup. The results of NMR of the above compound are shown below.

¹H-NMR(CDCl₃)：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＝7.8Hz，1H，CH)、3.75-3.93(m，3H)、3.79(s，6H，OCH₃)、3.46-3.68(m，5H)、3.34-3.41(m，1H，CH)、3.10-3.31(m，1H，CH)、3.05(t，J＝6.3Hz，2H，CH₂)、2.62(t，J＝6.3Hz，2H，CH₂)、2.39-2.46(m，1H，CH)、2.29(t，7.3Hz，2H，CH₂)、2.03-2.19(m，1H，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，CH₂)、1.18(d，J＝6.4Hz，6H，CH₃)、1.16(d，J＝6.4Hz，6H，CH₃)；

P-NMR(CH₃CN)146.90；

Ms(FAB+)：m/z803(M⁺+1)、303(DMTr⁺)。

(example B5)

To generate the nucleic acid molecule of the present invention comprising a linker having a proline backbone, DMTr-amidoethyleneoxyethylamino-L-proline amidite (hereinafter, referred to as PEG spacer arm type) was synthesized by scheme 5 below.

[ chemical formula 20]

Scheme 5

(1) DMTr-amide hydroxyethoxyethylamino-L-proline (Compound 23)

DMTr-amide-L-proline (compound 6) (1.00g, 2.05mmol), 2- (2-hydroxyethoxy) ethyl 4-toluenesulfonate (3.10g, 12.30mmol) and a solution of potassium carbonate (0.85g, 6.15mmol) in anhydrous dimethylformamide (10mL) were mixed and stirred at room temperature under an argon atmosphere for 4 days. For the above mixture, after the solvent was distilled off at room temperature under reduced pressure, methylene chloride (20mL) was added and filtered. The filtrate was concentrated, and the obtained residue was subjected to silica gel column chromatography. As the solvent to be added for the above silica gel column chromatography, ethyl acetate containing 0.05% pyridine was used first, and CH containing 0.05% pyridine was used thereafter₂Cl₂And CH₃Mixed solution of OH (CH)₂Cl₂：CH₃OH = 9: 1). As a result, compound 23(1.15g, yield 97%) was obtained as a colorless syrup. The results of NMR of the above compound are shown below.

¹H-NMR(CDCl₃)：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，OCH₃)、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-amide hydroxyethoxyethylamino-L-proline (compound 23) (0.63g, 1.00mmol) was mixed with anhydrous pyridine, and azeotropically dried at room temperature. To the resulting residue, tetrazolediisopropylamine (206mg, 1.20mmol) was added, and the mixture was degassed under reduced pressure and purged with argon. To the mixture was added anhydrous acetonitrile (1mL), and further added a solution of 2-cyanoethoxy-N, N, N ', N' -tetraisopropylphosphoramidite (282mg, 1.12mmol) in anhydrous acetonitrile (1 mL). The mixture was stirred at room temperature for 4 hours under an argon atmosphere. Then, the above mixture was diluted with dichloromethane, washed with a saturated aqueous sodium bicarbonate solution and saturated brine. The organic layer was collected, dried over sodium sulfate, and the organic layer was filtered. The solvent was distilled off under reduced pressure from the obtained filtrate. The obtained residue was subjected to column chromatography using an amino silica gel as a packing material (developing solvent hexane: acetone = 7: 3, containing 0.05% pyridine) to obtain compound 24(0.74g, purity 100%, yield 87%) as a colorless syrup. The results of NMR of the above compound are shown below.

¹H-NMR(CD₃CN)：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，CH₂)、3.79(s，6H，OCH₃)、3.47-3.64(m，7H)、3.15-3.23(m，1H)、3.11(t，J＝6.4Hz，2H，CH₂)、3.01(t，J＝5.9Hz，2H，CH₂)、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，CH₃)；

P-NMR(CD₃CN)：148.00；

Ms(FAB+)：m/z776(M⁺)、303(DMTr⁺)201(C₈H₁₉N₂OP⁺)。

(example B6)

1. Synthesis of protected prolinol

Prolaninol (compound 3) protected with dimethoxytrityl group was synthesized according to scheme 6 shown below.

[ chemical formula 21]

Scheme 6

(1) trifluoroacetyl-L-prolinol (Compound 1)

L-prolinol (2.0g, 20mmol) was dissolved in THF20 mL. On the other hand, ethyl trifluoroacetate (3.0g, 21mmol) was dissolved in THF20 mL. Then, the latter THF solution was added dropwise to the former THF solution containing L-prolinol, and stirred for 12 hours. The reaction mixture was concentrated under reduced pressure to obtain compound 1(3.7g, yield 97%). The results of NMR of the above compound are shown below.

¹H-NMR(CDCl₃)：4.28-4，23(1.0H，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) (3.7g, 19mmol) was dissolved in pyridine, and azeotropically dried at room temperature for 3 times. The obtained residue was dissolved in 15mL of pyridine, and 4, 4' -dimethoxytrityl chloride (DMTr-Cl) (8.1g, 24mmol) was added under argon gas with stirring in an ice bath, followed by reaction at room temperature for 4 hours. Then, in order to quench the excess DMTr-Cl, 10mL of methanol was further added to the reaction solution, and the mixture was stirred for 10 minutes. Thereafter, methylene chloride was added to the reaction solution, and the reaction solution was washed with a saturated aqueous sodium bicarbonate solution and saturated brine. The recovered organic layer after washing was dried over 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)₂Cl₂：CH₃OH = 95: 5. contains 0.1% of pyridinePyridine) to obtain purified compound 2(8.5g, yield 89%). The results of NMR of the above compound are shown below.

¹H-NMR(CDCl₃)：7.39-7.18(9H，m，Ar-H)，6.82(4H，d，J＝8.6Hz，Ar-H)，3.78(6H，s，OCH₃)，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 resulting trifluoroacetyl-DMTr-L-prolinol (compound 2) (5g, 10mmol) was dissolved in THF100 mL. To the THF solution was added 100mL of a 5% aqueous solution of sodium hydroxide and the mixture was stirred. To the solution was added 5mL of a 1M tetra-n-butylammonium fluoride (TBAF) solution, and the mixture was stirred at room temperature for 12 hours. The reaction solution was washed with a saturated aqueous sodium bicarbonate solution and saturated brine. The recovered organic layer after washing was dried over sodium sulfate. The organic layer was filtered, and the obtained filtrate was concentrated under reduced pressure to obtain compound 3(3.6g, yield 90%). The results of NMR of the above compound are shown below.

Synthesis of amidites derivatives

Using the protected prolinol (compound 3) synthesized in the above "1.", amidite derivatives with prolinol, different in binding form, were synthesized by the following scheme 7.

[ chemical formula 22]

Scheme 7

(1) DMTr-carbamate-L-prolinol (Compound 4)

1, 8-octanediol (9.0g, 62mmol) was dissolved in THF90mL and placed under argon. On the other hand, carbonyldiimidazole (2.0g, 12mmol) was dissolved in THF10 mL. The latter THF solution was added to the former THF solution, and stirred at room temperature for 1 hour. The reaction solution was washed with water until the TLC point of 1, 8-octanediol disappeared. The organic layer collected after washing was washed with saturated brine, and the collected organic layer was dried over anhydrous sodium sulfate. The organic layer was filtered, and the obtained filtrate was concentrated under reduced pressure. Subjecting the residue to silica gel column chromatography (developing solvent CH)₂Cl₂：CH₃OH = 95: 5) to obtain a purified compound. This compound was a compound in which one end of 1, 8-octanediol was activated with carbonyldiimidazole (2.3g, yield 77%).

0.9g of the above compound was dissolved in 10mL of acetonitrile, and the mixture was placed under argon. On the other hand, DMTr-L-prolinol (compound 3) (1.9g, 4.8mmol) was dissolved in acetonitrile 20 mL. The latter acetonitrile solution was added to the former acetonitrile solution, and stirred at room temperature for 24 hours. Then, the reaction solution was washed with a saturated aqueous sodium hydrogencarbonate solution and saturated brine, and the recovered organic layer 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 silica gel column chromatography (development solvent dichloromethane: acetone = 9: 1, containing 0.1% pyridine) to obtain purified compound 4 (prolinol carbamate amidite) (1.5g, yield 65%). The results of NMR of the above compound are shown below.

¹H-NMR(CDCl₃)：7.40-7.14(9H，m，Ar-H)，6.82(4H，d，J＝8.6Hz，Ar-H)，4.24-3.94(2H，m，COOCH₂)，3.78(s，6H，OCH₃)，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)

Triphosgene (2.0g, 6.7mmol) was dissolved in THF10mL under argon and stirred at 0 ℃. On the other hand, DMTr-L-prolinol (Compound 3) (1.3g, 3.2mmol) and N, N-diisopropylethylamine (16g, 124mmol) were dissolved in THF10mL, and added dropwise to the above-mentioned triphosgene in THF. The reaction solution was stirred at 0 ℃ for 1 hour, and then at room temperature for 2 hours. Then, 8-amino-1-octanol (2.3g, 16mmol) and N, N-diisopropylethylamine (5.0g, 38mmol) were dissolved in THF30 mL. The stirred reaction mixture was added dropwise to the THF solution, and the mixture was stirred at 0 ℃ for 1 hour and then at room temperature for 48 hours. The reaction mixture was concentrated under reduced pressure, and the residue was dissolved in methylene chloride. The solution was washed with a saturated aqueous sodium hydrogencarbonate solution and saturated brine, and the recovered 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. In this case, a mixed solvent of acetone and water containing 0.1% of pyridine is used as the developing solvent, and the mixing ratio of acetone and water is stepwise, specifically, acetone: the molar ratio of water is in a range of 2: 8. 3: 7. 4: 6 and 5: 5 are changed. The fraction containing the objective 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 ureidoamide) (0.9g, yield 49%). The results of NMR of the above compound are shown below.

¹H-NMR(CDCl₃)：7.40-7.14(9H，m，Ar-H)，6.82(4H，m，Ar-H)，3.78(s，6H，OCH₃)，3.68-3.25(9H，m，CH₂NH，CH₂OH，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 with prolinol (Compounds 6 and 7)

The compound 4(0.80g, 1.4mmol) obtained as a modified prolinol was dissolved in acetonitrile and azeotropically dried at room temperature for 3 times. The resulting residue was dissolved in 1mL of acetonitrile and placed under argon. To the acetonitrile solution was added tetrazolediisopropylamine (0.24g, 1.4mmol) as a reaction solution. On the other hand, 2-cyanoethyl-N, N, N ', N' -tetraisopropylphosphorodiamidite (0.50g, 1.7mmol) was dissolved in acetonitrile 1 mL. This was added to the reaction solution, and stirred at room temperature for 4 hours. Methylene chloride 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 column chromatography on silica gel amide (development solvent hexane: acetone = 10: 1, containing 0.1% pyridine) to obtain purified compound 6 (DMTr-carbamate-L-prolinol amidite) (0.90g, yield 83%). The results of NMR of the above compound are shown below.

¹H-NMR(CDCl₃)：7.40-7.14(9H，m，Ar-H)，6.82(4H，d，J＝8.6Hz，Ar-H)，4.24-3.94(2H，m，COOCH₂)，3.78(s，6H，OCH₃)，3.72-2.96(11H，m，CH₂O，POCH₂，CHCH₃，H-2，H-5，H-6)，2.58(2H，m，CH₂CN)，2.10-1.46(16H，m，alkyl，H-3，H-4)，1.34-1.10(12H，m，CHCH₃)。31P-NMR(CD₃CN)146.82。

FAB-MS：776[M+H]⁺。

The modified prolinol was treated in the same manner as above except that the compound 5 was used in place of the compound 4 to obtain the purified compound 7 (DMTr-ureido-L-prolinol amidite) (0.80g, yield 74%). The results of NMR of the above compound are shown below.

¹H-NMR(CDCl₃)：7.40-7.14(9H，m，Ar-H)，6.82(4H，m，Ar-H)，3.78(s，6H，OCH₃)，3.65-3.25(13H，m，CH₂O，POCH₂，CHCH₃，H-2，CH₂NH，CH₂OH，H-2，H-5，H-6)，2.73(2H，m，CH₂CN)，2.10-1.48(16H，m，alkyl，H-3，H-4)，1.35-1.10(12H，m，CHCH₃)。

31P-NMR(CD₃CN)146.83。

FAB-MS：775[M+H]⁺。

The present invention has been described above with reference to the embodiments, but the present invention is not limited to the above embodiments. Various modifications that can be understood by those skilled in the art can be made to the structure and details of the invention of the present application within the scope of the invention of the present application.

This application claims priority based on Japanese application No. 2010-156122 filed on 7/8/2010, Japanese application No. 2010-230808 filed on 10/13/2010, Japanese application No. 2010-269824 filed on 12/2/2010, Japanese application No. 2010-174915 filed on 8/3/2010, Japanese application No. 2010-230806 filed on 10/13/2010, and Japanese application No. 2010-269823 filed on 12/2/2010, the disclosures of which are incorporated herein in their entirety.

Industrial applicability

The single-stranded nucleic acid molecule of the present invention can suppress gene expression, can be easily synthesized because it is not circular, and can be efficiently produced because it is single-stranded and does not have a double-strand annealing step. As described above, the ssNc molecule of the present invention can inhibit the expression of a target gene, and thus is useful as a research tool for pharmaceuticals, diagnostic agents, agricultural chemicals, medicine, life sciences, and the like.

Claims

1. A single-stranded nucleic acid molecule comprising an expression-suppressing sequence that suppresses expression of a target gene,

a5 '-side region Xc, an inner region Z and a 3' -side region Yc in this order from the 5 '-side to the 3' -side,

the inner region Z is formed by connecting an inner 5 'side region X and an inner 3' side region Y,

the 5 '-side region Xc is complementary to the inner 5' -side region X,

the 3 '-side region Yc is complementary to the inner 3' -side region Y,

at least one of the inner region Z, the 5 '-side region Xc and the 3' -side region Yc comprises the expression-suppressing sequence,

wherein Xc is 1-7 bases or Yc is 1-9 bases,

a connecting sub-region Lx is provided between the 5 '-side region Xc and the inner 5' -side region X, the 5 '-side region Xc and the inner 5' -side region X are connected via the connecting sub-region Lx,

a linker region Ly is provided between the 3 '-side region Yc and the inner 3' -side region Y, and the 3 '-side region Yc and the inner 3' -side region Y are connected via the linker region Ly

The number of bases of the linker region Lx and the linker region Ly is 3 to 50,

wherein the number of bases Z of the inner region Z, the number of bases X of the inner 5 '-side region X, the number of bases Y of the inner 3' -side region Y, the number of bases Xc of the 5 '-side region Xc, and the number of bases Yc of the 3' -side region Yc satisfy the conditions of the following equations (1) and (2)

Z＝X+Y···(1)

Z≥Xc+Yc···(2)，

Wherein the number of bases Z in the inner region Z is 19 to 30 bases.

2. The single-stranded nucleic acid molecule according to claim 1, wherein 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 number of bases Yc in the 3' -side region Yc satisfy any one of the following conditions (a) to (d),

(a) satisfying the following conditions (3) and (4),

X＞Xc···(3)

Y＝Yc···(4)

(b) satisfying the following conditions (5) and (6),

X＝Xc···(5)

Y＞Yc···(6)

(c) satisfying the following conditions (7) and (8),

X＞Xc···(7)

Y＞Yc···(8)

(d) satisfying the following conditions of formulas (9) and (10),

X＝Xc···(9)

Y＝Yc···(10)。

3. the single-stranded nucleic acid molecule according to claim 2, wherein in the (a) to (d), a difference between the number of bases X of the inner 5 '-side region X and the number of bases Xc of the 5' -side region Xc, and a difference between the number of bases Y of the inner 3 '-side region Y and the number of bases Yc of the 3' -side region Yc satisfy the following condition,

(a) satisfying the following conditions (11) and (12),

X-Xc. 1,2 or 3. cndot. (11)

Y-Yc＝0···(12)

(b) Satisfying the following conditions (13) and (14),

X-Xc＝0···(13)

Y-Yc 1,2 or 3 (14)

(c) Satisfying the following conditions (15) and (16),

X-Xc. 1,2 or 3. cndot. (15)

Y-Yc 1,2 or 3 (16)

(d) Satisfying the following conditions (17) and (18),

X-Xc＝0···(17)

Y-Yc＝0···(18)。

4. the single-stranded nucleic acid molecule according to claim 1 or 2, wherein the number of bases Xc in the 5' -side region Xc is 1 to 7 bases.

5. The single-stranded nucleic acid molecule according to claim 1 or 2, wherein the number of bases Xc in the 5' -side region Xc is 1 to 3 bases.

6. The single-stranded nucleic acid molecule according to claim 1 or 2, wherein the number of bases Yc in the 3' -side region Yc is 1 to 7 bases.

7. The single-stranded nucleic acid molecule according to claim 1 or 2, wherein the number of bases Yc in the 3' -side region Yc is 1 to 3 bases.

8. The single stranded nucleic acid molecule of claim 1 or 2 comprising at least 1 modified residue.

9. The single-stranded nucleic acid molecule of claim 1 or 2, comprising a labeling substance.

10. The single stranded nucleic acid molecule of claim 1 or 2 comprising a stable isotope.

11. The single-stranded nucleic acid molecule of claim 1 or 2, which is an RNA molecule.

12. The single-stranded nucleic acid molecule according to claim 1 or 2, wherein the linker region Lx and/or the linker region Ly is composed of nucleotide residues.

13. The single stranded nucleic acid molecule of claim 12 wherein the nucleotide residue is an unmodified nucleotide residue and/or a modified nucleotide residue.

14. The single-stranded nucleic acid molecule according to claim 1 or 2, wherein the total number of bases in the single-stranded nucleic acid molecule is 50 bases or more.

15. The single stranded nucleic acid molecule of claim 1 or 2 wherein the inhibition of expression of the gene is RNA interference based inhibition of expression.

16. The single-stranded nucleic acid molecule according to claim 1 or 2, wherein the base sequence of the single-stranded nucleic acid molecule is any one of the base sequences of SEQ ID Nos. 2, 7,8, 13, 14, 29 to 35, 37, 43, 44, 47, 48 and 51 to 80.

17. A composition for suppressing expression, which is a composition for suppressing expression of a target gene,

comprising a single-stranded nucleic acid molecule according to any one of claims 1 to 16.

18. A pharmaceutical composition comprising a single-stranded nucleic acid molecule according to any one of claims 1 to 16.

19. The pharmaceutical composition of claim 18, which is for the treatment of inflammation.

20. Use of the single stranded nucleic acid molecule of any one of claims 1 to 16 in the preparation of a medicament for inhibiting expression of a target gene.

21. The use of claim 20, comprising a procedure for administering the single-stranded nucleic acid molecule to a cell, tissue, or organ.

22. The use of claim 21, wherein the single stranded nucleic acid molecule is administered in vivo or in vitro.

23. The use according to any one of claims 20 to 22, wherein the inhibition of expression of the gene is inhibition of expression based on RNA interference.

24. Use of the single stranded nucleic acid molecule of any one of claims 1 to 16 in the preparation of a medicament for inducing RNA interference that inhibits expression of a target gene.

25. A nucleic acid molecule for use in the treatment of a disease,

the nucleic acid molecule is the single-stranded nucleic acid molecule according to any one of claims 1 to 16,

the single-stranded nucleic acid molecule has a sequence that suppresses expression of a gene that causes the disease as the expression suppression sequence.