Patent Publication Number: US-9885036-B2

Title: Double-stranded polynucleotide

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
     This patent application is a National Stage application filed under 35 U.S.C. 371 of International Patent Application No. PCT/JP2009/061998, filed Jun. 30, 2009, entitled “Double-Stranded Polynucleotide,” which claims priority to Japanese Application No. 2008-172174 filed Jul. 1, 2008, and to Japanese Application No. 2009-122742 filed May 21, 2009, the contents of all of which are hereby incorporated by reference in their entirety. 
     SEQUENCE LISTING 
     A Sequence Listing in computer readable form (CRF) is submitted with this application. The CRF file is named 218147US-seqlist.txt, was created Dec. 21, 2010, and contains 104 kilobytes. The entire content of the CRF file is incorporated herein by reference. 
     TECHNICAL FIELD 
     The present invention relates to a novel double-stranded polynucleotide that is resistant to RNase and has an RNA interference effect, use of the double-stranded polynucleotide, a method for inhibiting gene expression using the double-stranded polynucleotide, a pharmaceutical composition comprising the double-stranded polynucleotide, etc. 
     BACKGROUND ART 
     A method for inhibiting the expression of a target gene in cells, tissues, or individuals includes an approach by which double-stranded RNA is introduced into the cells, tissues, or individuals. By this introduction of double-stranded RNA, mRNA having homology to the sequence is degraded such that the expression of the target gene is inhibited. This effect is called “RNA interference” or “RNAi”. RNA interference was originally reported in  C. elegans  (see e.g., Non Patent Reference 1) and then also reported in plants (see e.g., Non Patent Reference 2). 
     Double-stranded RNA consisting of 21-nucleotide sense and antisense strands having a 2-nucleotide overhang at the 3′-end (small interfering RNA: siRNA) has been reported to have an RNA interference effect in cultured cells of vertebrates (see e.g., Non Patent Reference 3). 
     siRNA is useful for the identification of gene functions, screening of cell strains suitable for useful substance production, regulation of genes involved in disease, etc., but, however, it is disadvantageously degraded easily by RNase (see e.g., Non Patent Reference 4). RNA synthesis is more difficult than DNA synthesis and therefore reportedly entails a 5 to 10-times higher cost (see e.g., Non Patent Reference 5). Exemplary reasons for this include the need for a protective group for the 2′-hydroxy group, the need to deprotect the protective group, and poor RNA synthesis yields attributed to a reduction in condensation yield due to the steric hindrance of the protective group (see e.g., Non Patent Reference 6). 
     Thus, there has been demand for a polynucleotide to be developed which is highly resistant to RNase, can be produced at low cost, and retains RNAi activity. 
     To obtain siRNA having resistance to RNase, a method has been researched in which all or some of the RNA nucleotides constituting an siRNA are substituted with 2′-deoxyribonucleotides (DNAs) (see e.g., Patent Reference 1 and Non Patent References 7, 8, 9, 10, and 11). However, siRNA having both resistance to RNase and an RNA interference effect equivalent to that of natural siRNA has not been obtained yet. 
     An oligonucleotide having a phosphorothioate (PS) bond in which the non-bridged oxygen atom of the phosphate group in the phosphodiester bond has been substituted with a sulfur atom is known to be resistant to nuclease (see e.g., Non Patent Reference 12). siRNA having PS bonds substituting the phosphodiester bonds has been reported to exhibit RNA interference equivalent to that of unmodified siRNA (see e.g., Non Patent References 9, 13, and 14). However, the increased number of PS bonds in an oligonucleotide causes thermodynamic instability of double-stranded RNA and nonspecific binding with proteins and is therefore not thought to be preferable (see e.g., Non Patent Reference 15). 
     An attempt has also been made to obtain stable siRNA by substituting natural RNA with modified RNA. Since the 2′-OH group of RNA is essential for the RNase degradation reaction, this 2′-OH group is alkylated such that it does not serve as a substrate for RNase. A large number of such 2′-O-alkyl nucleoside derivatives have been reported. 2′-O-methyl nucleotides are naturally occurring modified nucleotides also found in tRNA and have been studied since the early stages of antisense research (see e.g., Non Patent Reference 16). 
     It has been reported that RNAi is completely lost by substituting either or both of the sense and antisense strands of siRNA with 2′-O-methyl nucleotides (see e.g., Non Patent References 7, 17, and 18) or that only weak RNAi is observed when substituting all ribonucleotides in the sense or antisense strand of siRNA with 2′-O-methyl nucleotides, and that RNAi is completely lost by substituting both the strands therewith (see e.g., Non Patent Reference 9). 
     There is a report that when all RNAs in a sense strand are substituted with 2′-O-methyl nucleotides, RNAi equivalent to that of unmodified siRNA is obtained, but this is influenced by the sequence of siRNA used in the experiment (see e.g., Non Patent Reference 19). 
     It has been reported that when four 2′-O-methyl nucleotides are introduced to the end of an siRNA, its RNAi is retained (see e.g., Non Patent Reference 14) and that when 2′-O-methyl nucleotides are alternately introduced to both the ends of the sense and antisense strands of siRNA, RNAi equivalent to that of unmodified siRNA is obtained (see e.g., Non Patent Reference 18). Moreover, it has been reported that the introduction of 3 consecutive 2′-O-methyl nucleotides does not cause any reduction in activity for a sense strand but does cause a reduction in activity for an antisense strand, and in particular, its introduction to the 5′-end of the antisense strand significantly reduces activity (see e.g., Non Patent Reference 20). 
     Moreover, siRNA comprising 2′-deoxyribonucleotides in the vicinity of the 3′ and 5′-ends of the sense strand and 2′-O-methyl nucleotides in the central portion thereof has been reported but has not been compared in RNAi activity with unmodified siRNA (see e.g., Non Patent Reference 21). 
     An oligonucleotide having an artificially synthesized modified RNA 2′-deoxy-2′-fluoronucleotide (2′-F) preferentially forms the same N-type conformation as that of ribonucleotides and has higher affinity for RNA (see e.g., Non Patent Reference 22). However, those having phosphodiester bonds have no resistance to nuclease and therefore, in order to have nuclease resistance, they must be substituted with phosphorothioate bonds therefor (see e.g., Non Patent Reference 22). 
     It has been reported that when a pyrimidine nucleotide in siRNA is substituted with 2′-F, the resulting siRNA exhibits RNAi equivalent to that of unmodified siRNA (see e.g., Non Patent References 9 and 14). The introduction of 3 consecutive 2′-F moieties to an antisense strand hardly reduces its activity (see e.g., Non Patent Reference 20). Moreover, it has been reported that when either a pyrimidine nucleotide or a purine nucleotide in sense and/or antisense strands is substituted by 2′-F and both the modified strands are combined, the resulting siRNA exhibits RNAi equivalent to that of unmodified siRNA (see e.g., Non Patent Reference 23). 
     However, among these, those exhibiting an RNA interference effect contain a ribonucleotide and are thus degraded by RNase. It has been reported that when a pyrimidine nucleotide in siRNA was substituted by 2′-F, the enhancement of RNAi or its prolonged effect was not observed in animal models (see e.g., Non Patent Reference 24). Moreover, it has been reported that nonnatural nucleosides 2′-deoxy-2′-fluorocytidine and 2′-deoxy-2′-fluorouridine, which allegedly exhibit no toxicity when administered to rats or woodchucks, serve as substrates for DNA polymerase or RNA polymerase through intracellular triphosphorylation and are incorporated into DNA, RNA, and mitochondrial DNA in various organs (see e.g., Non Patent References 25 and 26). A triphosphate form of 2′-deoxy-2′-fluoronucleoside is incorporated as a substrate for DNA polymerase α or γ into DNA, whereas a triphosphate form of 2′-O-methyl nucleoside has been confirmed in vitro not to serve as a substrate for DNA polymerase α or γ (see e.g., Non Patent Reference 27). The genetic toxicity of the 2′-deoxy-2′-fluoronucleoside is of concern (see e.g., Non Patent Reference 28). 
     It has been reported that when all nucleotides in siRNA are substituted with 2′-F, its RNAi is merely slightly lower than that of unmodified siRNA and that such siRNA is resistant to RNase (see e.g., Non Patent Reference 29). 
     It has been reported that when 2′-O-methyl nucleotides and 2′-F are alternately introduced into the sense and antisense strands of siRNA, the obtained siRNA has RNAi equivalent to or higher than that of unmodified siRNA and is relatively stably maintained in serum (see e.g., Non Patent Reference 30). However, cytotoxicity or side effects caused by the introduction of a large number of normatural nucleic acids is of concern. 
     ENAs (2′-O,4′-C-ethylene-bridged nucleic acids) are modified nucleic acids having stability to nuclease (see e.g., Non Patent References 31 and 32). It has been reported that when ENAs are introduced to replace 2 nucleotides in the 3′-terminal overhang site of either or both of the sense and antisense strands of siRNA, the RNAi activity is reduced (see e.g., Non Patent Reference 33). 
     It has been reported that the introduction of chemically synthesized siRNA into cells phosphorylates the 5′-ends of both sense and antisense strands (see e.g., Non Patent Reference 34). In human cells, RNA kinase hClp1 has been reported to be responsible for the 5′-phosphorylation of siRNA (see e.g., Non Patent Reference 35). When siRNA having a phosphorylated 5′-end and siRNA having an unphosphorylated 5′-end were separately introduced into cells and their RNAi activity compared, no difference in activity was seen therebetween, indicating that siRNA having an unphosphorylated 5′-end is easily subject to phosphorylation in cells (see e.g., Non Patent Reference 14). 
     The X-ray analysis of a complex of an antisense strand with Argonaute protein (Ago) known to participate in RNAi activity has showed that the 5′-terminal phosphate group of the antisense strand and its neighboring nucleotides are strongly bound by the PIWI domain of Ago (see e.g., Non Patent Reference 36). 
     As regards the chain length of siRNA, 21 nucleotides are routinely used with each of the sense and antisense strands having a 2-nucleotide overhang at the 3′-end. When an antisense strand is set to being 21 nucleotides in length and the chain length of a sense strand is varied from the 3′ or 5′-end, siRNA having a 21-nucleotides sense strand has been shown to have the strongest RNAi activity (see e.g., Non Patent References 7 and 37). Moreover, it has been reported that when a sense strand is 3′-terminally truncated to 17 or 18 nucleotides in chain length, the resulting siRNA exhibits RNAi activity equivalent to that of siRNA having a 21-nucleotide sense strand (see e.g., Non Patent Reference 38). 
     siRNA consisting of 21 nucleotides has been shown to have the strongest RNAi activity when the length of the 3′-terminal overhang is 2 nucleotides (see e.g., Non Patent Reference 7). It has been reported that when RNAi activity was examined using siRNA having the sequence AA, CC, GG, UU, or UG (wild-type) or TdG or TT (T and dG are 2′-deoxyribonucleotides) as the 3′-terminal overhang, all the sequences had RNAi activity (see e.g., Non Patent Reference 7). Moreover, it has been reported that siRNA having a UU sequence as the 3′-terminal overhang exhibits higher RNAi activity than that of siRNA having a TT sequence (see e.g., Non Patent Reference 10). 
     Double-stranded RNA such as polyl:polyC has been known as an interferon inducer for a long time, and TLR3 (Toll-like receptor 3) is involved in the mechanism. siRNA is also known to be recognized by TLR3 and its family members TLR7 and TLR8 are known to induce interferon or cytokines. Particularly, siRNA having a GU, UGUGU, or GUCCUUCAA sequence has been reported to tend to cause an immune response (see e.g., Non Patent References 39, 40, and 41). Moreover, the introduction of DNAs or chemically modified nucleotides such as 2′-OMeRNAs into siRNA has been shown to inhibit such immune response (see e.g., Non Patent References 41, 42, and 43). 
     The present inventors have conducted diligent studies to obtain a polynucleotide that is resistant to RNase, can be synthesized at low cost, and has an RNA interference effect, and have consequently completed the present invention by finding that a double-stranded polynucleotide comprising an oligonucleotide unit of DNAs and 2′-O-methyl RNAs alternately combined can solve the problems described above. 
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     SUMMARY OF THE INVENTION 
     Problems to be Solved by the Invention 
     An aspect of the present invention is to provide a double-stranded polynucleotide that is resistant to RNase and has an RNA interference effect. 
     A further aspect of the present invention is to provide a method for inhibiting gene expression using the double-stranded polynucleotide. 
     A further aspect of the present invention is to provide a pharmaceutical composition comprising the double-stranded polynucleotide. 
     Means for Solving the Problems 
     Accordingly, the present invention includes: 
     (1) a double-stranded polynucleotide or a salt thereof, comprising a polynucleotide represented by formula (I) and a polynucleotide represented by formula (II):
 
5′-X-(α-β) q -α p -λ m -3′  (I)
 
5′-δ s -(α-β) r -Y-υ n -3′  (II),
 
wherein
 
α and β each independently represent a DNA or a 2′-OMeRNA, wherein if α represents a DNA, then β represents a 2′-OMeRNA, and wherein if α represents a 2′-OMeRNA, then β represents a DNA, δ and λ, each independently represent a DNA or a 2′-OMeRNA, and each occurrence of a δ and each occurrence of a λ independently represents a DNA or a 2′-OMeRNA, υ represents a nucleotide selected from a DNA, an RNA, and a 2′-OMeRNA, and each occurrence of a υ independently represents a DNA, an RNA, or a 2′-OMeRNA, X and Y each independently represent an oligonucleotide, wherein each nucleotide in said oligonucleotide is independently selected from a DNA nucleotide, an RNA nucleotide, and a modified nucleic acid,
 
p represents an integer of 0 or 1, m is 0 when p is 0 and represents any integer of 0 to 5 when
 
p is 1, q represents any integer of 3 to 9, the total number of nucleotides in (α-β) q  and X is 17 or 18, s represents an integer of 0 or 1, n represents any integer of 0 to 5, r represents any integer of 3 to 9, the total number of nucleotides in (α-β) r  and Y is 17 or 18, X-(α-β) q -α p  in the polynucleotide represented by the formula (I) having a nucleotide sequence identical to a target gene; and
 
the nucleotide sequences of X-(α-β) q  in the formula (I) and (α-β) r -Y in the formula (II) are complementary to each other;
 
     (2) the double-stranded polynucleotide or a salt thereof according to (1), wherein in the polynucleotide represented by the formula (I), q is 3, and X is any one selected from the group consisting of (γ-β) 6 , (α-β)-(γ-β) 5 , (α-β) 2 -(γ-β) 4 , (α-β) 3 , (α-β) 4 -(γ-β) 2 , (α-β) 5 -(γ-β), (α-β) 6 , β 12 , (α-β)-β 10 , (α-β) 2 -β 8 -(α-β) 3 -β 6 , (α-β) 4 -β 4 , (α-β) 5 -β 2 , β-(γ-β) 5 , β-(α-β)-(γ-β) 4 , β-(α-β) 2 , (γ-β) 3 , β(α-β) 3 -(γ-β) 2 , β-(α-β) 4 -(γ-=), β 11 , β-(α-β)-β 8 , β-(α-β) 2 -β 6 , β-(α-β) 3 -β 4 , β-(α-β) 4 -β 2 , and β-(α-β) 5 , wherein γ represents an RNA; 
     (3) the double-stranded polynucleotide or a salt thereof according to (1) or (2), wherein in the polynucleotide represented by the formula (II), r is 3, and Y is any one selected from the group consisting of (γ-β) 6 , (γ-β) 5 -(α-β), (γ-β) 4 -(α-β) 2 , (γ-β 3 ) 3 -(α-β) 3 , (γ-β) 2 -(α-β) 4 , (γ-β)-(α-β) 5 , (α-β) 6 , α 6 -(α-β) 3 , α 4 -(α-β) 4 , α 2 -(α-β) 5 , (γ-β) 5 -α, (γ-β) 4 -(α-β)-α, (γ-β) 3 -(α-β) 2 -α, (γ-β) 2 -(α-β) 3 -α, (γ-β)-(α-β) 4 -α, α 6 -(α-β) 2 -α, α 4 -(α-β) 3 -α, β 2 -(α-β) 4 -α, and (α-β) 5 -α, wherein γ represents an RNA; 
     (4) the double-stranded polynucleotide or a salt thereof according to (1) or (2), wherein in the polynucleotide represented by the formula (I), q is 3, and X is any one selected from the group consisting of (γ-β) 6 , (α-β)-(γ-β) 5 , (α-β) 2 -(γ-β) 4 , (α-β) 3 -(γ-β) 3 , (α-β) 4 -(γ-β) 2 , (α-β) 5 -(γ-β), (α-β) 6 , β 12 , (α-β)-β 10 , (α-β) 2 β 8 , (α-β) 3 -β 6 , (α-β) 4 -β 4 , (α-β) 5 β 2 , β-(γ-β) 5 , β(α-β)-(γ-β) 4 , β(α-β) 2 -(γ-β) 3 , β-(α-β) 3 -(γβ) 2 , β-(α-β) 4 -(γ-β), β 11 , β-(α-β)β 8 , β-(α-β) 2 -β 6 , β-(α-β) 3 -β 4 , β-(α-β) 4 -β 2 , and β-(α-β) 5 ; and in the polynucleotide represented by the formula (II), r is 3, and Y is any one selected from the group consisting of (γ-β) 6 , (γ-β) 5 -(α-β), (γ-β) 4 -(α-β) 2 , (γ-β) 3 -(α-β) 3 , (γ-β) 2 -(α-β) 4 , (γ-β)-(α-β) 5 , (α-β) 6 , α 6 -(α-β) 3 , α 4 -(α-β) 4 , α 2 -(α-β) 5 , (γ-β) 5 , (γ-β) 5 -α, (γ-β) 4 -(α-β)-α, (γ-β) 3 -(α-β) 2 -α, (γ-β) 2 -(α-β) 3 -α, (γ-β)-(α-β) 4 -α, α 6 -(α-β) 2 -α, α 4 -(α-β) 3 -α, α 2 -(α-β) 4 -α, and (α-β) 5 -α, wherein γ represents an RNA; 
     (5) the double-stranded polynucleotide or a salt thereof according to (1), wherein in the polynucleotide represented by the formula (I), q is 4, and X is any one selected from the group consisting of (γ-β) 5 , (α-β)-(γ-β) 4 , (α-β) 2 -(γ-β) 3 , (α-β) 3 -(γ-β) 2 , (α-β) 4 -(γ-β), (α-β) 5 , β 10 , (α-β)-β 8 , (α-β) 2 -β 6 , (α-β) 3 -β 4 , (α-β) 4 -β 2 , β-(γ-β) 4 , β-(α-β)-(γ-β) 3 , β-(α-β) 2 -(γ-β) 2 , β-(α-β) 3 -(γ-β), β 9 , β-(α-β)-β 6 , β-(α-β) 2 -β 4 , β-(α-β) 3 -β 2 , and β-(α-β) 4 ′ wherein γ represents an RNA; 
     (6) the double-stranded polynucleotide or a salt thereof according to (1) or (5), wherein in the polynucleotide represented by the formula (II), r is 4, and Y is any one selected from the group consisting of (γ-β) 5 , (γ-β) 4 -(α-β), (γ-β) 3 -(α-β) 2 , (γ-β) 2 -(α-β) 3 , (γ-β)-(α-β) 4 , (α-β) 5 , β 6 -(α-β) 2 , β 4 -(α-β) 3 , β 2 -(α-β) 4 , (γ-β) 4 -α, (α-β) 4 -α, (γ-β) 3 -(α-β)-α, (γ-β) 2 -α(α-β) 2 -α, (γ-β)-(α-β) 3 -α, α 6 -(α-β)-α, α 4 -(α-β) 2 -α, α 2 -(α-β) 3 -α and (α-β) 4 -α, wherein γ represents an RNA; 
     (7) the double-stranded polynucleotide or a salt thereof according to (1), wherein in the polynucleotide represented by the formula (I), q is 4, and X is any one selected from the group consisting of (γ-β) 5 , (α-β)-(γ-β) 4 , (α-β) 2 -(γ-β) 3 , (α-β) 3 -(γ-β) 2 , (α-β) 4 -(γ-β), (α-β) 5 , β 10 , (α-β)-β 8 , (α-β) 2 -β 6 , (α-β) 3 -β 4 , (α-β) 4 -β 2 , β-(γ-β) 4 , β-(α-β)-(γ-β) 3 , β-(α-β) 2 -(γ-β) 2 , β-(α-β) 3 -(γ-β), β 9 , β-(α-β)-β 6 , β-(α-β) 2 -β 4 , β-(α-β) 3 -β 2 , and β-(α-β) 4 ; and in the polynucleotide represented by the formula (II), r is 4, and Y is any one selected from the group consisting of (γ-β) 5 , (γ-β) 4 -(α-β), (γ-β) 3 -(α-β) 2 , (γ-β) 2 -(α-β) 3 , (γ-β)-(α-β) 4 , (α-β) 5 , α 6 -(α-β) 2 , α 4 -(α-β) 3 , α 2 -(α-β) 4 , (γ-β) 4 -α, (α-β) 4 -α, (γ-β) 3 -(α-β)-α, (γ-β) 2 -(α-β) 2 -α, (γ-β)-(α-β) 3 -α, α 6 -(α-β)-α, α 4 -(α-β) 2 -α, α 2 -(α-β) 3 -α, and (α-β) 4 -α, wherein γ represents an RNA; 
     (8) the double-stranded polynucleotide or a salt thereof according to (1), wherein in the polynucleotide represented by the formula (I), q is 5, and X is any one selected from the group consisting of (γ-β) 4 , (α-β)-(γ-β) 3 , (α-β) 2 , (γ-β) 2 , (α-β) 3 -(γ-β), (α-β) 4 , β 8 , (α-β)-β 6 , (α-β) 2 -β 4 , (α-β) 3 -β 2 , β-(γ-β) 3 ,β-(α-β)-(γ-β) 2 , β-(α-β) 2 -(γ-β), β 7 , β-(α-β) 4 , β-(α-β) 2 β 2 , and β-(α-β) 3 , wherein γ represents an RNA; 
     (9) the double-stranded polynucleotide or a salt thereof according to (1) or (8), wherein in the polynucleotide represented by the formula (II), r is 5, and Y is any one selected from the group consisting of (γ-β) 4 , (γ-β) 3 -(α-β), (γ-β) 2 -(α-β) 2 , (γ-β)-(α-β) 3 , (α-β) 4 , β 6 -(α-β), β 4 -(α-β) 2 , β 2 -(α-β) 3 , (γ-β) 3 -α, (γ-β) 2 -(α-β)-α, (γ-β)-(α-β) 2 -α, α 7 , α 4 -(α-β)-α, α 2 -(α-β) 2 -α, and (α-β) 3 -α, wherein γ represents an RNA; 
     (10) the double-stranded polynucleotide or a salt thereof according to (1), wherein in the polynucleotide represented by the formula (I), q is 5, and X is any one selected from the group consisting of (γ-β) 4 , (α-β)-(γ-β) 3 , (α-β) 2 -(γ-β) 2 , (α-β) 3 -(γ-β), (α-β) 4 , (α-β)-β 6 , (α-β) 2 -β 4 , (α-β) 3 -β 2 , β-(γ-β) 3 , β-(α-β)-(γ-β) 2 , β-(α-β) 2 -(γ-β), β 7 , β-(α-β) 4 , β-(α-β) 2 -β 2 , and β-(α-β) 3 ; and in the polynucleotide represented by the formula (II), r is 5, and Y is any one selected from the group consisting of (γ-β) 4 , (γ-β) 3 -(α-β), (γ-β) 2 -(α-β) 2 , (γ-β)-(α-β) 3 , (α-β) 4 , α 6 -(α-β), α 4 -(α-β) 2 , α 2 -(α-β) 3 , (γ-β) 3 -α, (γ-β) 2 -(α-β)-α, (γ-β)-(α-β) 2 -α, α 7 , α 4 -(α-β)-α, α 2 -(α-β) 2 -α, and (α-β) 3 -α, wherein γ represents an RNA; 
     (11) a double-stranded polynucleotide or a salt thereof, comprising polynucleotides represented by formulas (I) and (III):
 
5′-X-(α-β) q -α p -λ m -3′  (I)
 
5′-δ s -(β-α) r -Y-υ n -3′  (III),
 
wherein
 
α and β each independently represent a DNA or a 2′-OMeRNA, wherein if α represents a DNA, then β represents a 2′-OMeRNA, and wherein if α represents a 2′-OMeRNA, then β represents a DNA, δ and λ each independently represent a DNA or a 2′-OMeRNA, and each occurrence of a δ and each occurrence of a λ independently represents a DNA or a T-OMeRNA, υ represents a nucleotide selected from a DNA, an RNA, and a 2′-OMeRNA, and each occurrence of a u independently represents a DNA, an RNA, or a T-OMeRNA,
 
X and Y each independently represent an oligonucleotide, wherein each nucleotide in said oligonucleotide is independently selected from a DNA nucleotide, an RNA nucleotide, and a modified nucleic acid,
 
p represents an integer of 0 or 1, m is 0 when p is 0 and represents any integer of 0 to 5 when p is 1, q represents any integer of 3 to 9, the total number of nucleotides in (α-β) q  and X is 17 or 18, s represents an integer of 0 or 1, n represents any integer of 0 to 5, r represents any integer of 3 to 9, the total number of nucleotides in (β-α) r  and Y is 17 or 18,
 
X-(α-β) q -α p  in the polynucleotide represented by the formula (I) having a nucleotide sequence identical to a target gene; and
 
the nucleotide sequences of X-(α-β) q  in the formula (I) and (β-α) r -Y in the formula (III) are complementary to each other;
 
     (12) the double-stranded polynucleotide or a salt thereof according to (11), wherein in the polynucleotide represented by the formula (I), q is 3, and X is any one selected from the group consisting of (γ-β) 6 , (α-β)-(γ-β) 5 , (α-β) 2 -(γ-β) 4 , (α-β) 3 -(γ-β) 3 , (α-β) 4 -(γ-β) 2 , (α-β) 5 -(γ-β), (α-β) 6 , β 12 , (α-β)-β 10 , (α-β) 2 β 8 , (α-β) 3 -β 6 , (α-β) 4 -β 4 , (α-β) 5 -β 2 , β-(γ-β) 5 , β-(α-β)-(γ-β) 4 , β-(α-β) 2 -(γ-β) 3 , β-(α-β) 3 -(γ-β) 2 , β-(α-β) 4 -(γ-β), β 11 , β-(α-β)β 8 , β-(α-β) 2 -β 6 , β-(α-β) 3 -β 4 , β-(α-β) 4 -β 2 , and β-(α-β) 5 , wherein γ represents an RNA; 
     (13) the double-stranded polynucleotide or a salt thereof according to (11), wherein in the polynucleotide represented by the formula (III), r is 3, and Y is any one selected from the group consisting of (β-γ) 6 , (β-γ) 5 -(β-α), (β-γ) 4 -(β-α) 2 , (β-γ) 3 -(β-α) 3 , (β-γ) 2 -(β-α) 4 , (β-γ)-(β-α) 5 , (β-α) 6 , β 6 -(β-α) 3 , β 4 -(β-α) 4 , β-(β-γ) 5 -β, (β-γ) 4 -(β-α)-β, (β-γ) 3 -(β-α) 2 -β, (β-γ) 2 -(β-α) 3 -β, (β-γ)-(β-α) 4 -β, β 6 -(β-α) 2 -β, β 4 -(β-α) 3 -β, β 2 -(β-α) 4 -β, and (β-α) 5 -β, wherein γ represents an RNA; 
     (14) the double-stranded polynucleotide or a salt thereof according to (11), wherein in the polynucleotide represented by the formula (I), q is 3, and X is any one selected from the group consisting of (γ-β) 6 , (α-β)-(γ-β) 5 , (α-β) 2 -(γ-β) 4 , (α-β) 3 -(γ-β) 3 , (α-β) 4 -(γ-β) 2 , (α-β) 5 -(γ-β), (α-β) 6 , β 12 , (α-β)-β 10 , (α-β) 2 -β 8 , (α-β) 3 -β 6 , (α-β) 4 -β 4 , (α-β) 5 -β 2 , β-(γ-β) 5 , β-(α-β)-(γ-β) 4 , β-(α-β) 2 -(γ-β) 3 , β-(α-β) 3 -(γ-β) 2 , β-(α-β) 4 -(γ-β), β 11 , β-(α-β)-β 8 , β-(α-β) 2 -β 6 , β-(α-β) 3 -β 4 , β-(α-β) 4 -β 2 , and β-(α-β) 5 ; and in the polynucleotide represented by the formula (III), r is 3, and Y is any one selected from the group consisting of (β-γ) 6 , (β-γ) 5 -(β-α), (β-γ) 4 -(β-α) 2 , (β-γ) 3 -(β-α) 3 , (β-γ) 2 -(β-α) 4 , (β-γ)-(β-α) 5 , (β-α) 6 , β 6 -(β-α) 3 , β 4 -(β-α) 4 , β 2 -(β-α) 5 , (β-γ) 5 -β, (β-γ) 4 -(β-α)-β, (β-γ) 3 -(β-α) 2 -β, (β-γ) 2 -(β-α) 3 -β, (β-γ)-(β-α) 4 -β, β 6 -(β-α) 2 -β, β 4 -(β-α) 3 -β, β 2 -(β-α) 4 -β, and (β-α) 5 -β, wherein γ represents an RNA; 
     (15) the double-stranded polynucleotide or a salt thereof according to (11), wherein in the polynucleotide represented by the formula (I), q is 4, and X is any one selected from the group consisting of (γ-β) 5 , (α-β)-(γ-β) 4 , (α-β) 2 -(γ-β) 3 , (α-β) 3 -(γ-β) 2 , (α-β) 4 -(γ-β), (α-β) 5 , β 10 , (α-β)-β 8 , (α-β) 2 -β 6 , (α-β) 3 -β 4 , (α-β) 4 -β 2 , β-(γ-β) 4 , β-(α-β)-(γ-β) 3 , β-(α-β) 2 -(γ-β) 2 , β-(α-β) 3 -(γ-β), β 9 , β-(α-β)-β 6 , β-(α-β) 2 -β 4 , β-(α-β) 3 -β 2 , and β-(α-β) 4 , wherein γ represents an RNA; 
     (16) the double-stranded polynucleotide or a salt thereof according to (11), wherein in the polynucleotide represented by the formula (III), r is 4, and Y is any one selected from the group consisting of (β-γ) 5 , (β-γ) 4 -(β-α), (β-γ) 3 -(β-α) 2 , (β-γ) 2 -(β-α) 3 , (β-γ)-(β-α) 4 , (β-α) 5 , β 6 -(β-α) 2 , β 4 -(β-α) 3 , β 2 -(β-α) 4 , (β-γ) 4 -β, (β-α) 4 -β, (γ-β) 3 -(βα)-β, (γ-β) 2 -(β-α) 2 β, (β-γ)-(β-α) 3 -β, β 6 -(β-α)-β, β 4 -(β-α) 2 -β, β 2 -(β-α 3 -β and (β-α) 4 -β, wherein γ represents an RNA; 
     (17) the double-stranded polynucleotide or a salt thereof according to (11), wherein in the polynucleotide represented by the formula (I), q is 4, and X is any one selected from the group consisting of (γ-β) 5 , (α-β)-(γ-β) 4 , (α-β) 2 -(γ-β) 3 , (α-β) 3 -(γ-β) 2 , (α-β) 4 -(γ-β), (α-β) 5 , β 10 , (α-β)-β 8 , (α-β) 2 -β 6 , (α-β) 3 -β 4 , (α-β) 4 -β 2 , β-(γ-β) 4 , β-(α-β)-(γ-β) 3 , β-(α-β) 2 -(γ-β) 2 , β-(α-β) 3 -(γ-β), β 9 , β-(α-β)-β 6 , β-(α-β) 2 -β 4 , β-(α-β) 3 -β 2 , and β-(α-β) 4 ; and in the polynucleotide represented by the formula (III), r is 4, and Y is any one selected from the group consisting of (β-γ) 5 , (β-γ) 4 -(β-α), (β-γ) 3 -(β-α) 2 , (β-γ) 2 -(β-α) 3 , (β-γ)-(β-α) 4 , (β-α) 5 , β 6 -(β-α) 2 , β 4 -(β-α) 3 , β 2 -(β-α) 4 , (β-γ) 4 -β, (β-α) 4 -β, (γ-β) 3 -(β-α)-β, (γ-β) 2 -(β-α) 2 -β, (β-γ)-(β-α) 3 -β, β 6 -(β-α)-β, β 4 -(β-α) 2 -β, β 2 -(β-α) 3 -β and (β-α) 4 -β, wherein γ represents an RNA; 
     (18) the double-stranded polynucleotide or a salt thereof according to (11), wherein in the polynucleotide represented by the formula (I), q is 5, and X is any one selected from the group consisting of (γ-β) 4 , (α-β)-(γ-β) 3 , (α-β) 2 -(γ-β) 2 , (α-β) 3 -(γ-β), (α-β) 4 , β 8 , (α-β)-β 6 , (α-β) 2 -β 4 , (α-β) 3 -β 2 , β-(γ-β) 3 , β-(α-β)-(γ-β) 2 , β-(α-β) 2 -(γ-β), β 7 , β-(α-β)-β 4 , β-(α-β) 2 -β 2 , and β-(α-β) 3 , wherein γ represents an RNA; 
     (19) the double-stranded polynucleotide or a salt thereof according to (11), wherein in the polynucleotide represented by the formula (III), r is 5, and Y is any one selected from the group consisting of (β-γ) 4 , (β-γ) 3 -(β-α), (β-γ) 2 -(β-α) 2 , (β-γ)-(β-α) 2 , (β-α) 4 , β 6 -(β-α), β 4 -(β-α) 2 , β 2 -(β-α) 3 , (γ-β) 3 -β, (β-γ) 2 -(β-α)-β, (β-γ)-(β-α) 2 -β, β 7 , β 4 -(β-α)-β, β 2 -(β-α) 2 -β, and (β-α) 3 -β, wherein γ represents an RNA; 
     (20) the double-stranded polynucleotide or a salt thereof according to (11), wherein in the polynucleotide represented by the formula (I), q is 5, and X is any one selected from the group consisting of (γ-β) 4 , (α-β)-(γ-β) 3 , (α-β) 2 -(γ-β) 2 , (α-β) 3 -(γ-β), (α-β) 4 , (α-β)-β 6 , (α-β) 2 -β 4 , (α-β) 3 -β 2 , β-(γ-β) 3 , β-(α-β)-(γ-β) 2 , β-(α-β) 2 -(γ-β), β 7 , β-(α-β)-β 4 , β-(α-β) 2 -β 2 , and β-(α-β) 3 ; and in the polynucleotide represented by the formula (III), r is 5, and Y is any one selected from the group consisting of (β-γ) 4 , (β-γ) 3 -(β-α), (β-γ) 2 -(β-α) 2 , (β-γ)-(β-α) 3 , (β-α) 4 , β 6 -(β-α), β 4 -(β-α) 2 , β 2 -(β-α) 3 , (γ-β) 3 -β, (β-γ) 2 -(β-α)-β, (β-α) 2 -β, δ 7 , β 4 -(β-α)-β, β 2 -(β-α) 2 -β, and (β-α) 3 -β, wherein γ represents an RNA; 
     (21) the double-stranded polynucleotide or a salt thereof according to (1) or (11), wherein in the polynucleotides represented by the formulas (I), (II), and (III), q is 9, the number of nucleotides in X is 0, p and m are 0, r is 9, and the number of nucleotides in Y is 0; 
     (22) a double-stranded polynucleotide or a salt thereof, comprising polynucleotides represented by formulas (IV) and (V):
 
5′-(α-β) 9 -α p -λ m -3′  (IV)
 
5′-δ s -(α-β) 9 -υ n -3′  (V),
 
wherein
 
α and β each independently represent a DNA or a 2′-OMeRNA, wherein if a represents a DNA, then β represents a 2′-OMeRNA, and wherein if α represents a 2′-OMeRNA, then β represents a DNA, δ and λ each independently represent a DNA or a 2′-OMeRNA, and each occurrence of a δ and each occurrence of a λ independently represents a DNA or a 2′-OMeRNA, υ represents a nucleotide selected from a DNA, an RNA, and a 2′-OMeRNA, and each occurrence of a υ independently represents a DNA, an RNA, or a 2′-OMeRNA,
 
p represents an integer of 0 or 1, m is 0 when p is 0 and represents any integer of 0 to 5 when p is 1, s represents an integer of 0 or 1, n represents any integer of 0 to 5,
 
(α-β) 9 -α p  in the polynucleotide represented by the formula (IV) having a nucleotide sequence identical to a target gene; and
 
the nucleotide sequences of (α-β) 9  in the formula (IV) and (α-β) 9  in the formula (V) are complementary to each other;
 
     (23) a double-stranded polynucleotide or a salt thereof, comprising polynucleotides represented by formulas (VI) and (VII):
 
5′-β-(α-β) 8 -α p λ m -3′  (VI)
 
5′-δ s -(α-β) 8 -(α-β)-υ n -3′  (VII),
 
wherein
 
α and β each independently represent a DNA or a 2′-OMeRNA, wherein if a represents a DNA, then β represents a 2′-OMeRNA, and wherein if α represents a 2′-OMeRNA, then β represents a DNA,
 
δ and λ each independently represent a DNA or a 2′-OMeRNA, and each occurrence of a δ and each occurrence of a λ, independently represents a DNA or a 2′-OMeRNA, υ represents a nucleotide selected from a DNA, an RNA, and a 2′-OMeRNA and each occurrence of a υ independently represents a DNA, an RNA, or a 2′-OMeRNA,
 
p represents an integer of 0 or 1,
 
m is 0 when p is 0 and represents any integer of 0 to 5 when p is 1,
 
s represents an integer of 0 or 1,
 
n represents any integer of 0 to 5,
 
β-(α-β) 8 -α p  in the polynucleotide represented by the formula (VI) having a nucleotide sequence identical to a target gene; and
 
the nucleotide sequences of (α-β) 8  in the formula (VI) and (α-β) 8  in the formula (VII) are complementary to each other;
 
     (24) the double-stranded polynucleotide or a salt thereof according to any one of (1) to (23), wherein α is a DNA, and β is a 2′-OMeRNA; 
     (25) the double-stranded polynucleotide or a salt thereof according to any one of (1) to (24), wherein λ and υ each independently comprise a DNA selected from a thymine base, an adenine base, and a guanine base; or a 2′-OMeRNA selected from a uracil base, an adenine base, and a guanine base; 
     (26) the double-stranded polynucleotide or a salt thereof according to any one of (1) to (25), wherein m is 0, and n is 2; 
     (27) the double-stranded polynucleotide or a salt thereof according to any one of (1) to (26), wherein p and m are 0, s is 1, and n is 2; 
     (28) the double-stranded polynucleotide or a salt thereof according to any one of (1) to (27), wherein any or all of 1 to 4 2′-OMeRNA residues are substituted by an ENA or a 2′,4′-BNA/LNA; 
     (29) the double-stranded polynucleotide or a salt thereof according to any one of (1) to (28), wherein any or all of 1 to 4 DNA residues are substituted by an RNA, an ENA or a 2′,4′-BNA/LNA; 
     (30) the double-stranded polynucleotide or a salt thereof according to any one of (1) to (29), wherein the nucleotides are bonded to each other via a phosphodiester or phosphorothioate bond; 
     (31) the double-stranded polynucleotide or a salt thereof according to any one of (1) to (30), wherein at least one of the polynucleotides has a phosphorylated 5′-end; 
     (32) the double-stranded polynucleotide or a salt thereof according to any one of (1) to (31), wherein the antisense strand has a phosphorylated 5′-end; 
     (33) a pharmaceutical composition comprising a double-stranded polynucleotide or a salt thereof according to any one of (1) to (32) as an active ingredient; and 
     (34) a method for inhibiting the expression of a target gene, comprising administering a double-stranded polynucleotide or a salt thereof selected from (1) to (32) to a mammal. 
     Advantages of Invention 
     The present invention has provided a novel double-stranded polynucleotide that is resistant to RNase and has an RNA interference effect. The present invention allows functional analysis of various genes using the polynucleotide and provides a pharmaceutical composition comprising the double-stranded polynucleotide. 
    
    
     
       BRIEF DESCRIPTION OF DRAWINGS 
         FIG. 1  is a diagram showing double-stranded polynucleotides against the human β-catenin gene (hereinafter, examples of combinations of polynucleotides as sense and antisense strands will be shown in each diagram; for symbols, the open square (□) represents an RNA, the filled circle (•) represents a DNA, the open circle (◯) represents a 2′-O-methyl RNA, the open rhombus (⋄) represents an ENA, and the filled rhombus (♦) represents a 2′,4′-BNA/LNA. In the diagram, s represents a phosphorothioate bond, p represents a phosphate group, and the arrow represents a predicted cleavage site by Argonaute2. The same holds true for the diagrams shown below). 
         FIG. 2  is a diagram showing double-stranded polynucleotides against the human β-catenin gene. 
         FIG. 3  is a diagram showing the results of western blot analysis of the intensities of inhibitory activities of various double-stranded polynucleotides on the expression of the human β-catenin gene. CTNNB1 represents the expression of human β-catenin proteins, and Actin represents the expression of β-actin proteins used as a control. The number represents the concentration of the double-stranded polynucleotide added. A lighter-colored band means stronger inhibitory activity on the expression of the human β-catenin gene. In the diagram, the notation “CT001/CT005” means “CT-001/CT-005”; thus, the “hyphen: -” in the notations of double-stranded polynucleotides may be omitted. The same holds true for the diagrams shown below. 
         FIG. 4  is a diagram showing double-stranded polynucleotides against the human β-catenin gene. 
         FIG. 5  is a diagram showing the results of western blot analysis of the intensities of inhibitory activities of various double-stranded polynucleotides on the expression of the human β-catenin gene. CTNNB1 represents the expression of human β-catenin proteins, and Actin represents the expression of β-actin proteins used as a control. The number represents the concentration of the double-stranded polynucleotide added. A lighter-colored band means stronger inhibitory activity on the expression of the human β-catenin gene. 
         FIG. 6  is a diagram showing double-stranded polynucleotides against the human β-catenin gene. 
         FIG. 7  is a diagram showing double-stranded polynucleotides against the human β-catenin gene. 
         FIG. 8  is a diagram showing double-stranded polynucleotides against the human β-catenin gene. 
         FIG. 9  is a diagram showing double-stranded polynucleotides against the human β-catenin gene. 
         FIG. 10  is a diagram showing the results of western blot analysis of the intensities of inhibitory activities of various double-stranded polynucleotides on the expression of the human β-catenin gene. CTNNB1 represents the expression of human β-catenin proteins, and Actin represents the expression of β-actin proteins used as a control. The number represents the concentration of the double-stranded polynucleotide added. A lighter-colored band means stronger inhibitory activity on the expression of the human β-catenin gene. 
         FIG. 11  is a diagram showing the results of western blot analysis of the intensities of inhibitory activities of various double-stranded polynucleotides on the expression of the human β-catenin gene. CTNNB1 represents the expression of human β-catenin proteins, and Actin represents the expression of β-actin proteins used as a control. The number represents the concentration of the double-stranded polynucleotide added. A lighter-colored band means stronger inhibitory activity on the expression of the human (β-catenin gene. 
         FIG. 12  is a diagram showing the results of western blot analysis of the intensities of inhibitory activities of various double-stranded polynucleotides on the expression of the human β-catenin gene. CTNNB1 represents the expression of human β-catenin proteins, and Actin represents the expression of β-actin proteins used as a control. The number represents the concentration of the double-stranded polynucleotide added. A lighter-colored band means stronger inhibitory activity on the expression of the human β-catenin gene. 
         FIG. 13  is a diagram showing double-stranded polynucleotides against the human β-catenin gene. 
         FIG. 14  is a diagram showing double-stranded polynucleotides against the human β-catenin gene. 
         FIG. 15  is a diagram showing the results of western blot analysis of the inhibitory activities of double-stranded polynucleotides on the expression of the human β-catenin gene. CTNNB1 represents the expression of human β-catenin proteins, and Actin represents the expression of β-actin proteins used as a control. The number represents the concentration of the double-stranded polynucleotide added. A lighter-colored band means stronger inhibitory activity on the expression of the human β-catenin gene. 
         FIG. 16  is a diagram showing double-stranded polynucleotides against the human β-catenin gene. 
         FIG. 17  is a diagram showing the results of western blot analysis of the inhibitory activities of double-stranded polynucleotides on the expression of the human β-catenin gene. CTNNB1 represents the expression of human β-catenin proteins, and Actin represents the expression of β-actin proteins used as a control. The number represents the concentration of the double-stranded polynucleotide added. A lighter-colored band means stronger inhibitory activity on the expression of the human β-catenin gene. 
         FIG. 18  is a diagram showing double-stranded polynucleotides against the human β-catenin gene used in an RNase degradation reaction. 
         FIG. 19  is a diagram showing results of polyacrylamide electrophoresis analysis of an RNase degradation reaction of double-stranded polynucleotides. The arrow represents the positions of bands of the double-stranded polynucleotides. 
         FIG. 20  is a diagram showing double-stranded polynucleotides against the human β-catenin gene. 
         FIG. 21  is a diagram showing double-stranded polynucleotides against the human β-catenin gene. 
         FIG. 22  is a diagram showing double-stranded polynucleotides against the human β-catenin gene. 
         FIG. 23  is a diagram showing the results of western blot analysis of the intensities of inhibitory activities of various double-stranded polynucleotides on the expression of the human β-catenin gene. CTNNB1 represents the expression of human β-catenin proteins, and Actin represents the expression of β-actin proteins used as a control. The number represents the concentration of the double-stranded polynucleotide added. A lighter-colored band means stronger inhibitory activity on the expression of the human β-catenin gene. 
         FIG. 24  is a diagram showing double-stranded polynucleotides against the human β-catenin gene. 
         FIG. 25  is a diagram showing the results of western blot analysis of the intensities of inhibitory activities of various double-stranded polynucleotides on the expression of the human β-catenin gene. CTNNB1 represents the expression of human β-catenin proteins, and Actin represents the expression of β-actin proteins used as a control. The number represents the concentration of the double-stranded polynucleotide added. A lighter-colored band means stronger inhibitory activity on the expression of the human β-catenin gene. 
         FIG. 26  is a diagram showing the results of western blot analysis of the intensities of inhibitory activities of various double-stranded polynucleotides on the expression of the human β-catenin gene. CTNNB1 represents the expression of human β-catenin proteins, and Actin represents the expression of β-actin proteins used as a control. The number represents the concentration of the double-stranded polynucleotide added. A lighter-colored band means stronger inhibitory activity on the expression of the human β-catenin gene. 
         FIG. 27  is a diagram showing double-stranded polynucleotides against the human β-catenin gene. 
         FIG. 28  is a diagram showing double-stranded polynucleotides against the human β-catenin gene. 
         FIG. 29  is a diagram showing the results of western blot analysis of the intensities of inhibitory activities of various double-stranded polynucleotides on the expression of the human β-catenin gene. CTNNB1 represents the expression of human β-catenin proteins, and Actin represents the expression of β-actin proteins used as a control. The number represents the concentration of the double-stranded polynucleotide added. A lighter-colored band means stronger inhibitory activity on the expression of the human β-catenin gene. 
         FIG. 30  is a diagram showing the results of western blot analysis of the intensities of inhibitory activities of various double-stranded polynucleotides on the expression of the human β-catenin gene. CTNNB1 represents the expression of human β-catenin proteins, and Actin represents the expression of β-actin proteins used as a control. The number represents the concentration of the double-stranded polynucleotide added. A lighter-colored band means stronger inhibitory activity on the expression of the human β-catenin gene. 
         FIG. 31  is a diagram showing double-stranded polynucleotides against the human β-catenin gene. 
         FIG. 32  is a diagram showing the results of western blot analysis of the intensities of inhibitory activities of various double-stranded polynucleotides on the expression of the human β-catenin gene. CTNNB1 represents the expression of human β-catenin proteins, and Actin represents the expression of β-actin proteins used as a control. The number represents the concentration of the double-stranded polynucleotide added. A lighter-colored band means stronger inhibitory activity on the expression of the human β-catenin gene. 
         FIG. 33  is a diagram showing the results of western blot analysis of the intensities of inhibitory activities of various double-stranded polynucleotides on the expression of the human β-catenin gene. CTNNB1 represents the expression of human β-catenin proteins, and Actin represents the expression of β-actin proteins used as a control. The number represents the concentration of the double-stranded polynucleotide added. A lighter-colored band means stronger inhibitory activity on the expression of the human β-catenin gene. 
         FIG. 34  is a diagram showing double-stranded polynucleotides against the human β-catenin gene. 
         FIG. 35  is a diagram showing the results of western blot analysis of the intensities of inhibitory activities of various double-stranded polynucleotides on the expression of the human β-catenin gene. CTNNB1 represents the expression of human β-catenin proteins, and Actin represents the expression of β-actin proteins used as a control. The number represents the concentration of the double-stranded polynucleotide added. A lighter-colored band means stronger inhibitory activity on the expression of the human β-catenin gene. 
         FIG. 36  is a diagram showing double-stranded polynucleotides against the human β-catenin gene. 
         FIG. 37  is a diagram showing the results of western blot analysis of the intensities of inhibitory activities of various double-stranded polynucleotides on the expression of the human β-catenin gene. CTNNB1 represents the expression of human β-catenin proteins, and Actin represents the expression of β-actin proteins used as a control. The number represents the concentration of the double-stranded polynucleotide added. A lighter-colored band means stronger inhibitory activity on the expression of the human β-catenin gene. 
         FIG. 38  is a diagram showing double-stranded polynucleotides against the human β-catenin gene. 
         FIG. 39  is a diagram showing the results of western blot analysis of the intensities of inhibitory activities of various double-stranded polynucleotides on the expression of the human β-catenin gene. CTNNB1 represents the expression of human β-catenin proteins, and Actin represents the expression of β-actin proteins used as a control. The number represents the concentration of the double-stranded polynucleotide added. A lighter-colored band means stronger inhibitory activity on the expression of the human β-catenin gene. 
         FIG. 40  is a diagram showing double-stranded polynucleotides against the human β-catenin gene. 
         FIG. 41  is a diagram showing the results of western blot analysis of the intensities of inhibitory activities of various double-stranded polynucleotides on the expression of the human β-catenin gene. CTNNB1 represents the expression of human β-catenin proteins, and Actin represents the expression of β-actin proteins used as a control. The number represents the concentration of the double-stranded polynucleotide added. A lighter-colored band means stronger inhibitory activity on the expression of the human β-catenin gene. 
         FIG. 42  is a diagram showing double-stranded polynucleotides against the human β-catenin gene. 
         FIG. 43  is a diagram showing the results of western blot analysis of the intensities of inhibitory activities of various double-stranded polynucleotides on the expression of the human β-catenin gene. CTNNB1 represents the expression of human β-catenin proteins, and Actin represents the expression of β-actin proteins used as a control. The number represents the concentration of the double-stranded polynucleotide added. A lighter-colored band means stronger inhibitory activity on the expression of the human β-catenin gene. 
         FIG. 44  is a diagram showing double-stranded polynucleotides against the human β-catenin gene. 
         FIG. 45  is a diagram showing the results of western blot analysis of the intensities of inhibitory activities of various double-stranded polynucleotides on the expression of the human β-catenin gene. CTNNB1 represents the expression of human β-catenin proteins, and Actin represents the expression of β-actin proteins used as a control. The number represents the concentration of the double-stranded polynucleotide added. A lighter-colored band means stronger inhibitory activity on the expression of the human β-catenin gene. 
         FIG. 46  is a diagram showing double-stranded polynucleotides against the human β-catenin gene. 
         FIG. 47  is a diagram showing the results of western blot analysis of the intensities of inhibitory activities of various double-stranded polynucleotides on the expression of the human β-catenin gene. CTNNB1 represents the expression of human β-catenin proteins, and Actin represents the expression of β-actin proteins used as a control. The number represents the concentration of the double-stranded polynucleotide added. A lighter-colored band means stronger inhibitory activity on the expression of the human β-catenin gene. 
         FIG. 48  is a diagram showing the change in IFN-α production caused by the administration of double-stranded polynucleotides without the use of a transfection reagent. The ordinate shows IFN-α production with the administration of a reagent known to act on Toll-like receptors (TLR) or the double-stranded polynucleotides. “Control” shows IFN-α production without the administration of a reagent known to act on Toll-like receptors (TLR) or the double-stranded polynucleotides. In the diagram, “TLR3” represents Toll-like receptor 3, “TLR7” represents Toll-like receptor 7, “TLR8” represents Toll-like receptor 8, and “TLR9” represents Toll-like receptor 9. The same holds true for  FIG. 49 . 
         FIG. 49  is a diagram showing the change in IFN-α production caused by the administration of double-stranded polynucleotides with the use of a transfection reagent. The ordinate shows IFN-α production with the administration of a reagent known to act on Toll-like receptors (TLR) or the double-stranded polynucleotides. “Medium control” shows IFN-α production without the administration of a reagent known to act on Toll-like receptors (TLR) or the double-stranded polynucleotides. “Transfection control” shows IFN-α production with the addition of only the transfection reagent. 
         FIG. 50  is a diagram showing double-stranded polynucleotides against the human β-catenin gene. 
         FIG. 51  is a diagram showing the results of western blot analysis of the intensities of inhibitory activities of various double-stranded polynucleotides on the expression of the human β-catenin gene. CTNNB1 represents the expression of human β-catenin proteins, and Actin represents the expression of β-actin proteins used as a control. The number represents the concentration of the double-stranded polynucleotide added. A lighter-colored band means stronger inhibitory activity on the expression of the human β-catenin gene. 
         FIG. 52  is a diagram showing double-stranded polynucleotides against the human β-catenin gene. 
         FIG. 53  is a diagram showing the results of western blot analysis of the intensities of inhibitory activities of various double-stranded polynucleotides on the expression of the human β-catenin gene. CTNNB1 represents the expression of human β-catenin proteins, and Actin represents the expression of β-actin proteins used as a control. The number represents the concentration of the double-stranded polynucleotide added. A lighter-colored band means stronger inhibitory activity on the expression of the human β-catenin gene. 
         FIG. 54  is a diagram showing double-stranded polynucleotides against the DDX3 (DEAD (Asp-Glu-Ala-Asp) box polypeptide 3, X-linked) gene. 
         FIG. 55  is a diagram showing double-stranded polynucleotides against the human β-catenin gene. 
         FIG. 56  is a diagram showing the intensities of inhibitory activities of DD-016/DD-017 (in the diagram, 016/017; the same holds true for the description below), DD-022/DD-017 (022/017), DD-022/DD-023 (022/023), and DD-022/DD-024 (022/024), and a natural double-stranded polynucleotide DDX3 siRNA#5 (#5) on the expression of the DDX3 gene. That derived from cells treated with only a lipofection reagent was used as a control and is indicated as “non” in the diagram. 
     
    
    
     DESCRIPTION OF EMBODIMENTS 
     Description of Terms 
     In the present specification, the “target gene” is not particularly limited as long as it can be translated to produce mRNA and/or protein in cells, tissues, or individuals to which or to whom this gene is introduced (hereinafter, they may be referred to as “recipients”). Specifically, the target gene may be endogenous to the recipients for introduction or may be exogenous and introduced thereto by an approach such as gene transfer. It may also be a gene present on the chromosome or an extrachromosomal gene. Examples of the exogenous gene include, but are not limited to, viruses, bacteria, fungi, and those derived from protozoans, which can infect the recipients. The function of a gene may be known or unknown. 
     Examples of such a target gene can include, but are not limited to, the human β-catenin gene and the DDX3 (DEAD (Asp-Glu-Ala-Asp) box polypeptide 3, X-linked) gene. 
     In the present specification, the “natural nucleoside” refers to a 2′-deoxynucleoside such as 2′-deoxyadenosine, 2′-deoxyguanosine, 2′-deoxycytidine, 2′-deoxy-5-methylcytidine, and thymidine or a ribonucleoside such as adenosine, guanosine, cytidine, 5-methylcytidine, and uridine. Moreover, the “oligonucleotide” refers to an oligonucleotide composed of a compound in which the sugar moiety of the nucleoside forms an ester with phosphoric acid. 
     In the present specification, 2′-deoxyadenosine may be referred to as A t ; 2′-deoxyguanosine may be referred to as G t ; T-deoxycytidine may be referred to as C t ; 2′-deoxy-5-methylcytidine may be referred to as 5meC t ; thymidine may be referred to as T t ; T-deoxyuridine may be referred to as U t ; adenosine may be referred to as A rt ; guanosine may be referred to as AG rt ; cytidine may be referred to as C rt ; 5-methylcytidine may be referred to as 5meC rt ; and uridine may be referred to as U rt . Moreover, in the present specification, 2′-deoxyadenosine nucleotide may be referred to as A p ; 2′-deoxyguanosine nucleotide may be referred to as G p ; 2′-deoxycytidine nucleotide may be referred to as C p ; 2′-deoxy-5-methylcytidine nucleotide may be referred to as 5meC p ; a thymidine nucleotide may be referred to as T p ; a 2′-deoxyuridine nucleotide may be referred to as U p ; an adenosine nucleotide may be referred to as A rp ; a guanosine nucleotide may be referred to as G rp ; a cytidine nucleotide may be referred to as C rp ; a 5-methylcytidine nucleotide may be referred to as 5meC rp ; and a uracil nucleotide may be referred to as U rp . 
     In the present specification, where there are phosphorothioate ester forms instead of phosphoester forms of a nucleotide, a counterpart of A p  may be referred to as A s ; a counterpart of G p  may be referred to as G s ; a counterpart of C p  may be referred to as C s ; a counterpart of 5meC p  may be referred to as 5meC s ; a counterpart of T p  may be referred to as T s ; a counterpart of U p  may be referred to as U s ; a counterpart of A rp  may be referred to as A rs ; a counterpart of G rp  may be referred to as G rs ; a counterpart of C rp  may be referred to as C rs ; a counterpart of 5meC rp  may be referred to as 5meC rs ; and a counterpart of U rp  may be referred to as U rs . 
     In the present specification, the term “nucleoside with modified sugar” refers to a nucleoside whose sugar moiety has been modified. 
     Particularly, as examples of 2′-O-methyl modification, a counterpart of A rt  may be referred to as A m1t ; a counterpart of G rt  may be referred to as G m1t ; a counterpart of C rt  may be referred to as C m1t ; a counterpart of 5meC rt  may be referred to as 5meC m1t ; a counterpart of U rt  may be referred to as U m1t ; a counterpart of A rp  may be referred to as A m1p ; a counterpart of G rp  may be referred to as G m1p ; a counterpart of C rp  may be referred to as C m1p ; a counterpart of 5meC rp  may be referred to as 5meC m1p ; a counterpart of U rp  may be referred to as U m1p ; a counterpart of A rs  may be referred to as A m1s ; a counterpart of G rs  may be referred to as G m1s ; a counterpart of C rs  may be referred to as C m1s ; a counterpart of 5meC s  may be referred to as 5meC m1s ; and a counterpart of U′ s  may be referred to as U m1s . 
     In the present specification, the 2′-O,4′-C-ethylene nucleotide unit and the “ENA unit” refer to those nucleosides and nucleotides having an ENA and also refer to nucleosides and nucleotides having an ENA unit: a counterpart of A t  may be referred to as A 2t ; a counterpart of A p  may be referred to as A c2p ; a counterpart of A s  may be referred to as A e2s ; a counterpart of G t  may be referred to as G 2t ; a counterpart of GP may be referred to as G e2P ; a counterpart of G s  may be referred to as G e2s ; a counterpart of 5meC t  may be referred to as C 2t ; a counterpart of 5meC p  may be referred to as C e2p ; a counterpart of 5meC s  may be referred to as C e2s ; a counterpart of T t  may be referred to as T 2t ; a counterpart of T p  may be referred to as T e2p ; and a counterpart of T s  may be referred to as T e2s . 
     In the present specification, the 2′-O,4′-C-methylene nucleotide unit and the “2′,4′-BNA/LNA unit” refer to those nucleosides and nucleotides having a 2′,4′-BNA/LNA and also refer to nucleosides and nucleotides having a 2′,4′-BNA/LNA unit: a counterpart of A t  may be referred to as A 1t ; a counterpart of A p  may be referred to as A e1p ; a counterpart of A s  may be referred to as A e1s ; a counterpart of G t  may be referred to as G 1t ; a counterpart of GP may be referred to as G e1p ; a counterpart of G s  may be referred to as G e1s ; a counterpart of 5meC t  may be referred to as C 1t ; a counterpart of 5meC p  may be referred to as C e1p ; a counterpart of 5meC s  may be referred to as C e1s ; a counterpart of T t  may be referred to as T 1t ; a counterpart of T p  may be referred to as T e1P ; and a counterpart of T s  may be referred to as T e1s . 
     Hereinafter, the structural formula of each nucleotide will be shown. 
     
       
         
         
             
             
         
       
       
         
         
             
             
         
       
       
         
         
             
             
         
       
       
         
         
             
             
         
       
       
         
         
             
             
         
       
       
         
         
             
             
         
       
       
         
         
             
             
         
       
       
         
         
             
             
         
       
       
         
         
             
             
         
       
       
         
         
             
             
         
       
       
         
         
             
             
         
       
       
         
         
             
             
         
       
       
         
         
             
             
         
       
       
         
         
             
             
         
       
       
         
         
             
             
         
       
       
         
         
             
             
         
       
       
         
         
             
             
         
       
       
         
         
             
             
         
       
     
     In the present specification, the term “complementary nucleotides” refers to nucleotides whose base moieties are complementary to each other and refers specifically to nucleotides complementary to each other by way of adenine and thymine, guanine and cytosine, guanine and 5-methylcytosine, and adenine and uracil as base moieties. 
     In the present specification, the “complementary nucleotide sequence” includes a nucleotide sequence consisting of nucleotides, all of which are complementary to a target nucleotide sequence, and also includes a nucleotide sequence forming base pairs with a target oligonucleotide and/or polynucleotide, albeit with one or more nucleotides that are not complementary. 
     In the present specification, the “double-stranded polynucleotide” is a polynucleotide assuming a duplex by Watson-Crick base pairs formed between complementary nucleotides, though not all the nucleotides in the polynucleotide may form Watson-Crick base pairs. 
     In the present specification, of the double-stranded polynucleotide, a strand comprising a nucleotide sequence identical to a target gene is called a passenger strand or sense strand, whereas a strand comprising a nucleotide sequence complementary to the target gene is called a guide strand or antisense strand. 
     In the present specification, the phrase “having a nucleotide sequence identical to a target gene” refers to having a sequence identical to at least a partial nucleotide sequence of the target gene. It includes a completely identical sequence and also includes a substantially identical sequence as long as the resulting double-stranded polynucleotide has an RNA interference effect. Moreover, when the target gene is known to have SNPs or the like, a sequence having these variations is also included as an identical nucleotide sequence. 
     A polynucleotide having a sequence identical or substantially identical to at least a partial nucleotide sequence of the target gene is a polynucleotide having a sequence identical or substantially identical to any 18-nucleotide or more sequence in the nucleotide sequence of the target gene. In this context, the “substantially identical sequence” refers to a sequence having 70% or higher, preferably 80% or higher, more preferably 90% or higher homology, to the nucleotide sequence of the target gene. The homology of the nucleotide sequence can be calculated using gene analysis software known in the art such as BLAST (registered trademark). 
     In the item &lt;223&gt; for each sequence in the Sequence Listing attached to the present specification, “cm” represents 2′-O-methylcytidine; “um” represents 2′-O-methyluridine; and “gm” represents 2′-O-methylguanosine. 
     Double-Stranded Polynucleotide 
     The chain length of the double-stranded polynucleotide according to the present invention may be any length from 18 nucleotides to the full length of the open reading frame (ORF) of the target gene as long as it has an RNA interference effect. The sense strand is preferably 18 to 21 nucleotides, more preferably 18 or 19 nucleotides, in chain length. The antisense strand is preferably 19 to 21 nucleotides, more preferably 21 nucleotides, in chain length. The double-stranded polynucleotide does not have to be a duplex as a whole and includes those partially overhanging at the 5′ and/or 3′-ends. The overhanging end has 1 to 5 nucleotides, preferably 1 to 3 nucleotides, more preferably 2 nucleotides. Moreover, the most preferable examples thereof include those having a structure in which the 3′-end of the polynucleotide which is the antisense strand overhangs by 2 nucleotides (overhang structure). 
     2-1. 
     An example of the double-stranded polynucleotide can include a double-stranded polynucleotide comprising a sense strand comprising a polynucleotide represented by the following formula (I) and an antisense strand comprising a polynucleotide represented by the following formula (II):
 
5′-X-(α-β) q -α p -λ m -3′  (I)
 
5′-δ s -(α-β) r -Y-υ n -3′  (II),
 
wherein
 
α and β each independently represent a DNA or a 2′-OMeRNA, wherein if α represents a DNA, then β represents a T-OMeRNA, and wherein if α represents a 2′-OMeRNA, then β represents a DNA. δ and λ each independently represent a DNA or a 2′-OMeRNA, and each occurrence of a δ and each occurrence of a λ independently represents a DNA or a 2′-OMeRNA, (e.g. where s is 2, then δ can be DNA-DNA, DNA-2′-OMeRNA, T-OMeRNA-DNA, or 2′-OMeRNA-2′-OMeRNA. Likewise, e.g. where m is 2, then λ can be DNA-DNA, DNA-2′-OMeRNA, 2′-OMeRNA-DNA, or 2′-OMeRNA-2′-OMeRNA), υ represents a nucleotide selected from a DNA, an RNA, and a 2′-OMeRNA, and each occurrence of a υ independently represents a DNA, an RNA, or a 2′-OMeRNA (e.g., for υ n , if n is 2, υ 2  can be DNA-DNA, RNA-RNA and 2′-OMeRNA-2′OMeRNA, and also can be DNA-2′-OMeRNA, DNA-RNA, RNA-2′-OMeRNA, 2′-OMeRNA-DNA, RNA-DNA and 2′-OMeRNA-RNA), X and Y each independently represent an oligonucleotide, wherein each nucleotide in said oligonucleotide is independently selected from a DNA nucleotide, an RNA nucleotide, and a modified nucleic acid,
 
p represents an integer of 0 or 1, m is 0 when p is 0 and represents any integer of 0 to 5 when p is 1, q represents any integer of 3 to 9, the total number of nucleotides in (α-β) q  and X is 17 or 18, s represents an integer of 0 or 1, n represents any integer of 0 to 5, r represents any integer of 3 to 9, the total number of nucleotides in (α-β), and Y is 17 or 18,
 
X-(α-β) q -α p  in the polynucleotide represented by the formula (I) having a nucleotide sequence identical to a target gene; and
 
the nucleotide sequences of X-(α-β) q  in the formula (I) and (α-β) r -Y in the formula (II) are complementary to each other.
 
     In this context, p, q, r, s, m, and n are numbers representing the number of nucleotides. For example, (α-β) 2  means (α-β)-(α-β). This means that when p in α p  is 0, this nucleotide is absent. α p  represents α when p is 1 and represents α-α when p is 2. 
     2-2. 
     An example of the double-stranded polynucleotide represented by the formulas (I) and (II) can include a double-stranded polynucleotide wherein both q and r are 9, and the number of nucleotides in X and Y is 0. 
     2-3. 
     Another example of the double-stranded polynucleotide can include a double-stranded polynucleotide comprising a sense strand which is a polynucleotide wherein in the polynucleotide represented by the formula (I): 
     q is 3, and X is any one selected from the group consisting of (γ-β) 6 , (α-β)-(γ-β) 5 , (α-β) 2 -(γ-β) 4 , (α-β) 3 -(γ-β) 3 , (α-β) 4 -(γ-β) 2 , (α-β) 5 -(γ-β), (α-β) 6 , β 12 , (α-β)-β 10 , (α-β) 2 -β 8 , (α-β) 3 -β 6 , (α-β) 4 -β 4 , (α-β) 5 -β 2 , β-(γ-β) 5 , β-(α-β)-(γ-β) 4 , β-(α-β) 2 -(γ-β) 3 , β-(α-β) 3 -(γ-β) 2 , β-(α-β) 4 -(γ-β), β 11 , β-(α-β)-β 8 , β-(α-β) 2 -β 6 , β-(α-β) 3 -β 4 , β-(α-β) 4 -β 2 , and β-(α-β) 5 ; q is 4, and X is any one selected from the group consisting of (γ-β) 5 , (α-β)-(γ-β) 4 , (α-β) 2 -(γ-β) 3 , (α-β) 3 -(γ-β) 2 , (α-β) 4 -(γ-β), (α-β) 5 , β 10 , (α-β)-β 8 , (α-β) 2 -β 6 , (α-β) 3 -β 4 , (α-β) 4 -β 2 , β-(γ-β) 4 , β-(α-β) 4 , β-(α-β)-(γ-β) 3 , β(α-β) 2 -(γ-β) 2 , β-(α-β) 3 -(γ-β), β 9 , β-(α-β)β 6 , β-(α-β) 2 -β 4 , β-(α-β) 3 -β 2 , and β-(α-β) 4 ; or q is 5, and X is any one selected from the group consisting of (γ-β) 4 , (α-β)-(γ-β) 3 , (α-β) 2 -(γ-β) 2 , (α-β) 3 -(γ-β), (α-β) 4 , β 8 , (α-β)-β 6 , (α-β) 2 -β 4 , (α-β) 3 -β 2 , β-(γ-β) 3 , β-(α-β)-(γ-β) 2 , β-(α-β) 2 -(γ-β), β 7 , β-(α-β)β 4 , β-(α-β) 2 -β 2 , and β-(α-β) 3 , wherein γ represents an RNA, and an antisense strand which is a polynucleotide wherein in the polynucleotide represented by the formula (II): 
     r is 3, and Y is any one selected from the group consisting of (γ-β) 6 , (γ-β) 5 -(α-β), (γ-β) 4 -(α-β) 2 , (γ-β) 3 -(α-β) 3 , (γ-β) 2 -(α-β) 4 , (γ-β)-(α-β) 5 , (α-β) 6 , α 6 -(α-β) 3 , α 4 -(α-β) 4 , α 2 -(α-β) 5 , (γ-β) 5 -α, (γ-β) 4 -(α-β)-α, (γ-β) 3 -(α-β) 2 -α, (γ-β) 2 -(α-β) 3 -α, (γ-β)-(α-β) 4 -α, α 6 -(α-β) 2 -α, α 4 -(α-β) 3 -α, β 2 -(α-β) 4 -α, and (α-β) 5 -α; r is 4, and Y is any one selected from the group consisting of (γ-β) 5 , (γ-β) 4 -(α-β), (γ-β) 3 -(α-β) 2 , (γ-β) 2 -(α-β) 3 , (γ-β)-(α-β) 4 , (α-β) 5 , β 6 -(α-β) 2 , β 4 -(α-β) 3 , β 2 -(α-β) 4 , (γ-β) 4 -α, (α-β) 4 -α, (γ-β) 3 -(α-β)-α, (γ-β) 2 -(α-β) 2 -α, (γ-β)-(α-β) 3 -α, α 6 -(α-β)-α, α 4 -(α-β) 2 -α, α 2 -(α-β) 3 -α and (α-β) 4 -α; or r is 5, and Y is any one selected from the group consisting of (γ-β) 4 , (γ-β) 3 -(α-β), (γ-β) 2 -(α-β) 2 , (γ-β)-(α-β) 3 , (α-β) 4 , β 6 -(α-β), β 4 -(α-β) 2 , β 2 -(α-β) 3 , (γ-β) 3 -α, (γ-β) 2 -(α-β)-α, (γ-β)-(α-β) 2 -α, α 7 , α 4 -(α-β)-α, α 2 -(α-β) 2 -α, and (α-β) 3 -α, wherein γ represents an RNA. 
     2-4. 
     Another example of the double-stranded polynucleotide can include a double-stranded polynucleotide comprising a sense strand comprising a polynucleotide represented by the following formula (I) and an antisense strand comprising a polynucleotide represented by the following formula (III):
 
5′-X-(α-β) q -α p -λ m -3′  (I)
 
5′-δ s -(β-α) r -Y-υ n -3′  (III),
 
wherein
 
α and β each independently represent a DNA or a 2′-OMeRNA, wherein if α represents a DNA, then β represents a 2′-OMeRNA, and wherein if α represents a 2′-OMeRNA, then β represents a DNA,
 
δ and λ each independently represent a DNA or a 2′-OMeRNA, and each occurrence of a δ and each occurrence of a λ independently represents a DNA or a 2′-OMeRNA,
 
υ represents a nucleotide selected from a DNA, an RNA, and a 2′-OMeRNA, and each occurrence of a υ independently represents a DNA, an RNA, or a 2′-OMeRNA,
 
X and Y each independently represent an oligonucleotide, wherein each nucleotide in said oligonucleotide is independently selected from a DNA nucleotide, an RNA nucleotide, and a modified nucleic acid,
 
p represents an integer of 0 or 1,
 
m is 0 when p is 0 and represents any integer of 0 to 5 when p is 1,
 
q represents any integer of 3 to 9,
 
the total number of nucleotides in (α-β) q  and X is 17 or 18,
 
s represents an integer of 0 or 1,
 
n represents any integer of 0 to 5,
 
r represents any integer of 3 to 9,
 
the total number of nucleotides in (β-α) r  and Y is 17 or 18,
 
X-(α-β) q -α p  in the polynucleotide represented by the formula (I) having a nucleotide sequence identical to a target gene; and
 
the nucleotide sequences of X-(α-β) q  in the formula (I) and (β-α) r -Y in the formula (III) are complementary to each other.
 
     2-5. 
     An example of the double-stranded polynucleotide represented by the formulas (I) and (III) can include a double-stranded polynucleotide wherein both q and r are 9, and the number of nucleotides in X and Y is 0. 
     2-6. 
     Another example of the double-stranded polynucleotide can include a double-stranded polynucleotide comprising a sense strand which is a polynucleotide wherein in the polynucleotide represented by the formula (I); 
     q is 3, and X is any one selected from the group consisting of (γ-β) 6 , (α-β)-(γ-β) 5 , (α-β) 2 -(γ-β) 4 , (α-β) 3 -(γ-β) 3 , (α-β) 4 -(γ-β) 2 , (α-β) 5 -(γ-β), (α-β) 6 , β 12 , (α-β)-β 10 , (α-β) 2 -β 8 , (α-β) 3 -β 6 , (α-β) 4 -β 4 , (α-β) 5 -β 2 , β-(γ-β) 5 , β-(α-β)-(γ-β) 4 , β-(α-β) 2 -(γ-β) 3 , β-(α-β) 3 -(γ-β) 2 , β-(α-β) 4 -(γ-β), β 11 , β-(α-β)-β 8 , β-(α-β) 2 -β 6 , β-(α-β) 3 -β 4 , β-(α-β) 4 -β 2 , and β-(α-β) 5 ; q is 4, and X is any one selected from the group consisting of (γ-β) 5 , (α-β)-(γ-β) 4 , (α-β) 2 -(γ-β) 3 , (α-β) 3 -(γ-β) 2 , (α-β) 4 -(γ-β), (α-β) 5 , β 10 , (α-β)-β 8 , (α-β) 2 -β 6 , (α-β) 3 -β 4 , (α-β) 4 -β 2 , β-(γ-β) 4 , β-(α-β)-(γ-β) 3 , β-(α-β) 2 -(γ-β) 2 , β-(α-β) 3 -(γ-β), β 9 , β-(α-β)-β 6 , β-(α-β) 2 -β 4 , β-(α-β) 3 -β 2 , and β-(α-β) 4 ; or q is 5, and X is any one selected from the group consisting of (γ-β) 4 , (α-β)-(γ-β) 3 , (α-β) 2 -(γ-β) 2 , (α-β) 3 -(γ-β), (α-β) 4 , β 8 , (α-β)-β 6 , (α-β) 2 -β 4 , (α-β) 3 -β 2 , β-(γ-β) 3 , β-(α-β)-(γ-β) 2 , β-(α-β) 2 -(γ-β), β 7 , β-(α-β)-β 4 , β-(α-β) 2 -β 2 , and β-(α-β) 3 , wherein γ represents an RNA, and an antisense strand which is a polynucleotide wherein in the polynucleotide represented by the formula (III): 
     r is 3, and Y is any one selected from the group consisting of (β-γ) 6 , (β-γ) 5 -(β-α), (β-γ) 4 -(β-α) 2 , (β-γ) 3 -(β-α) 3 , (β-γ) 2 -(β-α) 4 , (β-γ)-(β-α) 5 , (β-α) 6 , β 6 -(β-α) 3 , β 4 -(β-α) 4 , β 2 -(β-α) 5 , (β-γ) 5 -β, (β-γ) 4 -(β-α)-β, (β-γ) 3 -(β-α) 2 -β, (β-γ) 2 -(β-α) 3 -β, (β-γ)-(β-α) 4 -β, β 6 -(β-α) 2 β, β 4 -(3-α) 3 -β, β 2 -(β-α) 4 -β, and (β-α) 5 -β; r is 4, and Y is any one selected from the group consisting of (β-γ) 5 , (β-γ) 4 -(β-α), (β-γ) 3 -(β-α) 2 , (β-γ) 2 -(β-α) 3 , (β-γ)-(β-α) 4 , (β-α) 5 , β 6 -(β-α) 2 , β 4 -(β-α) 3 , β 2 -(β-α) 4 , (β-γ) 4 -β, (β-α) 4 -β, (γ-β) 3 -(β-α)-β, (γ-β) 2 -(β-α) 2 -β, (β-γ)-(β-α) 3 -β, β 6 -(β-α)-β, β 4 -(β-α) 2 -β, β 2 -(β-α) 3 -β, and (β-α) 4 -β; or r is 5, and Y is any one selected from the group consisting of (β-γ) 4 , (β-γ) 3 -(β-α), (β-γ) 2 -(β-α) 2 , (β-γ)-(β-α) 3 , (β-α) 4 , β 6 -(β-α), β 4 -(β-α) 2 , β 2 -(β-α) 3 , (γ-β) 3 -β, (β-γ) 2 -(β-α)-β, (γ-γ)-(β-α) 2 -β, β 7 , β 4 -(β-α)-β, β 2 -(β-α) 2 -β, and (β-α) 3 -β, wherein y represents an RNA. 
     2-7. 
     Another example of the double-stranded polynucleotide can include a double-stranded polynucleotide comprising a sense strand comprising a polynucleotide represented by the following formula (IV) and an antisense strand comprising a polynucleotide represented by the following formula (V):
 
5′-(α-β) 9 -α p -λ m -3′  (IV)
 
5′-δ s -(α-β) 9 -υ n -3′  (V),
 
wherein
 
α and β each independently represent a DNA or a 2′-OMeRNA, wherein if α represents a DNA, then β represents a 2′-OMeRNA, and wherein if α represents a 2′-OMeRNA, then β represents a DNA, δ and λ each independently represent a DNA or a 2′-OMeRNA, and each occurrence of a δ and each occurrence of a λ independently represents a DNA or a 2′-OMeRNA, υ represents a nucleotide selected from a DNA, an RNA, and a 2′-OMeRNA, and each occurrence of a υ independently represents a DNA, an RNA, or a 2′-OMeRNA,
 
p represents an integer of 0 or 1, m is 0 when p is 0 and represents any integer of 0 to 5 when p is 1, s represents an integer of 0 or 1, n represents any integer of 0 to 5, (α-β) 9 -α p  in the polynucleotide represented by the formula (IV) having a nucleotide sequence identical to a target gene; and
 
the nucleotide sequences of (α-β) 9  in the formula (IV) and (α-β) 9  in the formula (V) are complementary to each other.
 
     2-8. 
     Another example of the double-stranded polynucleotide can include a double-stranded polynucleotide comprising a sense strand comprising a polynucleotide represented by the following formula (VI) and an antisense strand comprising a polynucleotide represented by the following formula (VII):
 
5′-β-(α-β) 8 -α p -λ m -3′  (VI)
 
5′-δ s -(α-β) 8 -(α-β)-υ n -3′  (VII),
 
wherein
 
α and β each independently represent a DNA or a 2′-OMeRNA, wherein if α represents a DNA, then β represents a 2′-OMeRNA, and wherein if α represents a 2′-OMeRNA, then β represents a DNA, δ and λ each independently represent a DNA or a 2′-OMeRNA, and each occurrence of a δ and each occurrence of a λ independently represents a DNA or a 2′-OMeRNA, υ represents a nucleotide selected from a DNA, an RNA, and a 2′-OMeRNA and each occurrence of a υ independently represents a DNA, an RNA, or a 2′-OMeRNA,
 
p represents an integer of 0 or 1, m is 0 when p is 0 and represents any integer of 0 to 5 when p is 1, s represents an integer of 0 or 1, n represents any integer of 0 to 5, β-(α-β) 8 -α p  in the polynucleotide represented by the formula (VI) having a nucleotide sequence identical to a target gene; and
 
the nucleotide sequences of (α-β) 8  in the formula (VI) and (α-β) 8  in the formula (VII) are complementary to each other.
 
     2-9. 
     Another example of the double-stranded polynucleotide can include a double-stranded polynucleotide represented by the following formulas (VIII) and (IX):
 
5′-X-(α-β) q -α-α m -3′  (VIII)
 
5′-β-(α-β) r —Y-α n -3′  (IX),
 
wherein
 
α represents a DNA, β represents a 2′-OMeRNA, and X and Y each independently represent an oligonucleotide, wherein each nucleotide in said oligonucleotide is independently selected from a DNA nucleotide, an RNA nucleotide, and a modified nucleic acid;
 
q represents any integer from 3 to 9, the total number of nucleotides in (α-β) q  and X is 18, r represents any integer from 3 to 9, the total number of nucleotides in (α-β) r  and Y is 18, and n and m each independently represent an integer from 0 to 5;
 
X-(α-β) q -α in the polynucleotide represented by the formula (VIII) has a nucleotide sequence identical to a target gene; and
 
the nucleotide sequences of X-(α-β) q -α in the formula (VIII) and β-(α-β) r -Y in the formula (IX) are complementary to each other.
 
     In the polynucleotides represented by the formulas (VIII) and (IX), the numbers represented by n and m are preferably 2. 
     Moreover, examples of nucleotides as preferable α in α m  and α n  can include thymidine for all of them. 
     An example of the double-stranded polynucleotide represented by the formulas (VIII) and (IX) can include a double-stranded polynucleotide wherein both q and r are 9, and the number of nucleotides in X and Y is 0. 
     Another example of the double-stranded polynucleotide can include a double-stranded polynucleotide wherein in the formula (VIII), q is 3, and X is any one selected from the group consisting of (γ-β) 6 , (α-β)-(γ-β) 5 , (α-β) 2 -(γ-β) 4 , (α-β) 3 -(γ-β) 3 , (α-β) 4 -(γ-β) 2 , (α-β) 5 -(γ-β), (α-β) 3 -β 6 , (α-β) 4 -β 4 , and (α-β) 5 -β 2  wherein α represents a DNA, β represents a 2′-OMeRNA, and γ represents an RNA. 
     Another example of the double-stranded polynucleotide can include a double-stranded polynucleotide or a salt thereof, wherein in the formula (IX), r is 3, and Y is any one selected from the group consisting of (γ-β) 6 , (γ-β) 5 -(α-β), (γ-β) 4 -(α-β) 2 , (γ-β) 3 -(α-β) 3 , (γ-β) 2 -(α-β) 4 , (γ-β)-(α-β) 5 , α 6 -(α-β) 3 , α 4 -(α-β) 4 , and α 2 -(α-β) 5  wherein α represents a DNA, β represents a 2′-OMeRNA, and γ represents an RNA. 
     A further example of the double-stranded polynucleotide can include a double-stranded polynucleotide wherein in the formula (VIII), q is 3, and X is any one selected from the group consisting of (γ-β) 6 , (α-β)-(γ-β) 5 , (α-β) 2 -(γ-β) 4 , (α-β) 3 -(γ-β) 3 , (α-β) 4 -(γ-β) 2 , (α-β) 5 -(γ-β), (α-β) 3 -β 6 , (α-β) 4 -β 4 , and (α-β) 5 -β 2 ; and in the formula (IX), r is 3, and Y is any one selected from the group consisting of (γ-β) 6 , (γ-β) 5 -(α-β), (γ-β) 4 -(α-β) 2 , (γ-β) 3 -(α-β) 3 , (γ-β) 2 -(α-β) 4 , (γ-β)-(α-β) 5 , α 6 -(α-β), α 4 -(α-β) 4 , and α 2 -(α-β) 5  wherein α represents a DNA, β represents a 2′-OMeRNA, and γ represents an RNA. 
     Another example of the double-stranded polynucleotide can include a double-stranded polynucleotide wherein in the formula (VIII), q is 4, and X is any one selected from the group consisting of (γ-β) 5 , (α-β)-(γ-β) 4 , (α-β) 2 -(β-β) 3 , (α-β) 3 -(γ-β) 2 , (α-β) 4 -(γ-β) (α-β) 2 -β 6 , (α-β) 3 -β 4 , and (α-β) 4 -β 2  wherein α represents a DNA, β represents a 2′-OMeRNA, and γ represents an RNA. 
     Another example of the double-stranded polynucleotide can include a double-stranded polynucleotide wherein in the formula (IX), r is 4, and Y is any one selected from the group consisting of (γ-β) 5 , (γ-β) 4 -(α-β), (γ-β) 3 -(α-β) 2 , (γ-β) 2 -(α-β) 3 , (γ-β)-(α-β) 4 , α 6 -(α-β) 2 , α 4 -(α-β) 3 , and α 2 -(α-β) 4  wherein α represents a DNA, β represents a 2′-OMeRNA, and γ represents an RNA. 
     A further example of the double-stranded polynucleotide can include a double-stranded polynucleotide wherein in the formula (VIII), q is 4, and X is any one selected from the group consisting of (γ-β) 5 , (α-β)-(γ-β) 4 , (α-β) 2 -(γ-β) 3 , (α-β) 3 -(γ-β) 2 , (α-β) 4 -(γ-β), (α-β) 2 -β 6 , (α-β) 3 -β 4 , and (α-β) 4 -β 2 ; and in the formula (IX), r is 4, and Y is any one selected from the group consisting of (γ-β) 5 , (γ-β) 4 -(α-β), (γ-β) 3 -(α-β) 2 , (γ-β) 2 -(α-β) 3 , (γ-β)-(α-β) 4 , β 6 -(α-β) 2 , β 4 -(α-β) 3 , and β 2 -(α-β) 4  wherein α represents a DNA, β represents a 2′-OMeRNA, and γ represents an RNA. 
     Another example of the double-stranded polynucleotide can include a double-stranded polynucleotide wherein in the formula (VIII), q is 5, and X is any one selected from the group consisting of (γ-β) 4 , (α-β)-(γ-β) 3 , (α-β) 2 -(γ-β) 2 , (α-β) 3 -(γ-β), (α-β)-β 6 , (α-β) 2 -β 4 , and (α-β) 3 -β 2  wherein α represents a DNA, β represents a 2′-OMeRNA, and γ represents an RNA. 
     Another example of the double-stranded polynucleotide can include a double-stranded polynucleotide wherein in the formula (IX), r is 5, and Y is any one selected from the group consisting of (γ-β) 4 , (β-β) 3 -(α-β), (γ-β) 2 -(α-β) 2 , (γ-β)-(α-β) 3 , α 6 -(α-β) α 4 -(α-β) 2 , and α 2 -(α-β) 3  wherein α represents a DNA, β represents a 2′-OMeRNA, and γ represents an RNA. 
     A further example of the double-stranded polynucleotide can include a double-stranded polynucleotide wherein in the formula (VIII), q is 5, and X is any one selected from the group consisting of (γ-β) 4 , (α-β)-(γ-β) 3 , (α-β) 2 -(α-β) 2 , (α-β) 3 -(γ-β), (α-β)-β 6 , (α-β) 2 -β 4 , and (α-β) 3 -β 2 ; and in the formula (IX), r is 5, and Y is any one selected from the group consisting of (γ-β) 4 , (γ-β) 3 -(α-β), (γ-β) 2 -(α-β) 2 , (γ-β)-(α-β) 3 , β 6 -(α-β), β 4 -(α-β) 2 , and β 2 -(α-β) 3  wherein α represents a DNA, β represents a 2′-OMeRNA, and γ represents an RNA. 
     The nucleotide with a modified sugar encompasses all manner of sugar modification known in the technical field to which the present invention belongs. The nucleotide with a modified sugar can retain every heterocyclic base site and internucleoside bond and further includes nucleotides with a modified sugar different from the sugar modifications described above. The group of nucleotides with modified sugars includes 2′-modified nucleotides, 4′-thio-modified nucleotides, 4′-thio-2′-modified nucleotides, and bicyclic nucleotides each with a modified sugar. 
     The 2′-modified nucleotides are, for example, halo, allyl, amino, azide, O-allyl, O—C 1 -C 10  alkyl, OCF 3 , O—(CH 2 ) 2 —O—CH 3 , 2′-O(CH 2 ) 2 SCH 3 , O—(CH 2 ) 2 —O—N(R m )(R n ), and O—CH 2 —C(═O)—N(R m )(R n ), wherein R m  and R n  are each individually H, an amino protective group, or substituted or unsubstituted C 1 -C 10  alkyl. A preferable 2′-modification is —F, —OCH 3 , or —O—(CH 2 ) 2 —O—CH 3 , more preferably —OCH 3 . 
     Examples of the 4′-thio-modified nucleosides can include β-D-ribonucleosides in which the 4′-oxygen atom has been substituted by a sulfur atom (Hoshika, S. et al. FEBS Lett. 579, p. 3115-3118, (2005); Dande, P. et al. J. Med. Chem. 49, p. 1624-1634 (2006); and Hoshika, S. et al. ChemBioChem. 8, p. 2133-2138, (2007)). 
     Examples of the 4′-thio-2′-modified nucleosides can include 4′-thio-2′-modified nucleosides retaining 2′-H or 2′-O-methyl (Matsugami, et al. Nucleic Acids Res. 36, 1805 (2008)). 
     Examples of the bicyclic nucleosides with a modified sugar can include nucleosides retaining the second ring formed by bridging two atoms of the ribose ring. Examples of such nucleosides can include: 2′,4′-BNAs/LNAs (bridged nucleic acids/locked nucleic acids) in which the 2′-oxygen atom and the 4′-carbon atom are bridged by a methylene chain (Obika, S. et al. Tetrahedron Lett., 38, p. 8735-(1997); Obika, S. et al., Tetrahedron Lett., 39, p. 5401-(1998); A. A. Koshkin, A. A. et al. Tetrahedron, 54, p. 3607 (1998); and Obika, S. Bioorg. Med. Chem., 9, p. 1001 (2001); and ENAs (2′-O,4′-C-ethylene-bridged nucleic acids) bridged by an ethylene chain longer by one carbon than the methylene chain of the 2′,4′-BNA/LNA (Morita, K. et al. Bioorg. Med. Chem. Lett., 12, p. 73 (2002); and Morita, K. et al. Bioorg. Med. Chem., 11, p. 2211 (2003).). 
     When arbitrary 1 to 4 2′-OMeRNA residues in the double-stranded polynucleotide are substituted by nucleotides with a modified sugar, more preferable nucleotides with a modified sugar are each independently an ENA or a 2′,4′-BNA/LNA counterparts of the nucleotides with the modified sugar. 
     The double-stranded polynucleotide also includes a double-stranded polynucleotide in which 1 to 4 DNA residues in the polynucleotide are each independently substituted by an RNA, an ENA, or a 2′,4′-BNA/LNA. 
     The double-stranded polynucleotide also includes those comprising the antisense strand represented by the formula (II), (III), (V), (VII), or (IX) further having a phosphorylated 5′-end. 
     The double-stranded polynucleotide also includes those having phosphorothioate bonds instead of phosphoester bonds. The number of phosphorothioate bonds is preferably 0 to 5. Moreover, the phosphorothioate bonds are preferably positioned in the vicinity of the 5′ and 3′-ends of each polynucleotide. 
     The method for preparing each polynucleotide constituting the double-stranded polynucleotide is not particularly limited, and a known chemical synthesis method can be used, for example, a phosphotriester, phosphoramidite, or H-phosphonate method. For example, it can be synthesized using a commercially available nucleic acid synthesizer and commercially available reagents used in DNA/RNA synthesis. 
     The polynucleotide having a phosphorylated 5′-end can also be synthesized by a known synthesis method and can be synthesized, for example, using Phosphalink (manufactured by Applied Biosystems). 
     In the chemical synthesis, single-stranded polynucleotides having complementarity to each other can be synthesized separately and associated by an appropriate method to form a duplex. A specific example of the association method includes a method by which the synthesized single-stranded polynucleotides are mixed at a molar ratio of preferably at least 3:7, more preferably approximately 4:6, most preferably equimolar ratio (5:5), then heated to a dissociation temperature of the duplex, and then gradually cooled. The associated double-stranded polynucleotide is purified, if necessary, by a method usually used and known per se in the art. For example, a method can be used as the purification method, by which the association is confirmed using an agarose gel or the like, and residual single-stranded polynucleotides are removed, for example, by degradation with an appropriate enzyme. 
     The double-stranded polynucleotide also includes: a double-stranded polynucleotide comprising a cholesterol, lipid, or vitamin E unit introduced therein (see e.g., Lorenz, C. et al. Bioorg. Med. Chem. Lett., 14, p. 4975-4977 (2004); Soutschek, J., et al. Nature, 432, p. 173-178, (2004); Wolfrum, C. et al. Nature Biotech. 25, p. 1149-1157, (2007); Kubo, T. et al. Oligonucleotides, 17, p. 1-20, (2007); Kubo, T., et al. Biochem. Biophys. Res. Comm. 365, p. 54-61, (2008); and Nishina, K., et al., Mol. Ther. 16, p. 734-740, (2008)); and a double-stranded polynucleotide bound at the end with an aptamer, a protein-binding nucleic acid molecule. 
     The double-stranded polynucleotide also includes a double-stranded polynucleotide bound to a monoclonal antibody (or an appropriate binding site thereof) or a protein (or an appropriate oligopeptide fragment thereof) (see e.g., Song, et al. Nature Biotech. 23, p. 709-717 (2005); Xia et al. Pharm. Res. 24, p. 2309-2316 (2007); and Kumar, et al. Nature, 448, p. 39-43 (2007)). 
     Moreover, the double-stranded polynucleotide also includes a positively charged complex of a double-stranded polynucleotide supplemented with a cationic polymer (see, as successful examples achieving distribution in organs and cells, Leng et al. J. Gen. Med. 7, p. 977-986 (2005); Baigude et al. 2, p. 237-241, ACS Chem. Biol. (2007); and Yadava et al. Oligonucleotide 17, p. 213-222 (2007)). 
     The double-stranded polynucleotide includes every pharmaceutically acceptable salt or ester of the double-stranded polynucleotide, or salts of such esters. 
     Preferable examples of the pharmaceutically acceptable salt of the double-stranded polynucleotide can include: alkali metal salts such as a sodium salt, a potassium salt, and a lithium salt, alkaline earth metal salts such as a calcium salt and a magnesium salt, and metal salts such as an aluminum salt, an iron salt, a zinc salt, a copper salt, a nickel salt, and a cobalt salt; amine salts including inorganic salts such as an ammonium salt and organic salts such as a t-octylamine salt, a dibenzylamine salt, a morpholine salt, a glucosamine salt, a phenylglycine alkyl ester salt, an ethylenediamine salt, an N-methylglucamine salt, a guanidine salt, a diethylamine salt, a triethylamine salt, a dicyclohexylamine salt, an N,N′-dibenzylethylenediamine salt, a chloroprocaine salt, a procaine salt, a diethanolamine salt, an N-benzyl-phenethylamine salt, a piperazine salt, a tetramethylammonium salt, a tris(hydroxymethyl)aminomethane salt; inorganic acid salts such as a hydrohalide (e.g., a hydrofluoride, a hydrochloride, a hydrobromide, and a hydroiodide), a nitrate, a perchlorate, a sulfate, and a phosphate; organic acid salts such as lower alkanesulfonates (e.g., a methanesulfonate, a trifluoromethanesulfonate, and an ethanesulfonate), arylsulfonates (e.g., a benzenesulfonate and a p-toluenesulfonate), an acetate, a malate, a fumarate, a succinate, a citrate, a tartrate, an oxalate, and a maleate; and amino acid salts such as a glycine salt, a lysine salt, an arginine salt, an ornithine salt, a glutamate, and an aspartate. 
     A composition comprising the double-stranded polynucleotide is mixed, encapsulated, or conjugated with another molecule, molecular structure, or mixture of compounds, for example, as a liposome, a receptor-targeting molecule, an oral, rectal, or local formulation, or other formulations for assisting in uptake, distribution, and/or absorption. 
     When the double-stranded polynucleotide is used as a preventive or therapeutic drug for disease, the polynucleotide or a pharmacologically acceptable salt thereof can be administered either by itself or after mixing with an appropriate pharmacologically acceptable excipient, diluent, or the like, as an oral formulation such as tablets, capsules, granules, powders, or syrups or as a parenteral formulation such as injections, suppositories, patches, or external preparations. 
     These preparations are produced by a well-known method using additives such as excipients (examples thereof can include organic excipients including: sugar derivatives such as lactose, saccharose, glucose, mannitol, and sorbitol; starch derivatives such as corn starch, potato starch, a starch, and dextrin; cellulose derivatives such as crystalline cellulose; gum arabic; dextran; and pullulan, and inorganic excipients including: silicate derivatives such as light anhydrous silicic acid, synthetic aluminum silicate, calcium silicate, and magnesium aluminometasilicate; a phosphate such as calcium hydrogen phosphate; a carbonate such as calcium carbonate; and a sulfate such as calcium sulfate), lubricants (examples thereof can include: metal salts of stearic acid such as stearic acid, calcium stearate, and magnesium stearate; talc; colloidal silica; waxes such as beeswax and spermaceti; boric acid; adipic acid; a sulfate such as sodium sulfate; glycol; fumaric acid; sodium benzoate; DL leucine; a lauryl sulfate such as sodium lauryl sulfate and magnesium lauryl sulfate; silicic acids such as anhydrous silicic acid and silicic hydrate; and the starch derivatives described above), binders (examples thereof can include hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinylpyrrolidone, macrogol, and the same compounds as the excipients), disintegrants (examples thereof can include: cellulose derivatives such as low substituted hydroxypropylcellulose, carboxymethylcellulose, carboxymethylcellulose calcium, and internally bridged carboxymethylcellulose sodium; and chemically modified starches/celluloses such as carboxymethyl starch, carboxymethyl starch sodium, and bridged polyvinylpyrrolidone), emulsifying agents (examples thereof can include: colloidal clay such as bentonite and veegum; a metal hydroxide such as magnesium hydroxide and aluminum hydroxide; anionic surfactants such as sodium lauryl sulfate and calcium stearate; cationic surfactants such as benzalkonium chloride; and nonionic surfactants such as polyoxyethylene alkyl ether, polyoxyethylene sorbitan fatty acid ester, and sucrose fatty acid ester), stabilizers (examples thereof can include: p-oxybenzoic acid esters such as methylparaben and propylparaben; alcohols such as chlorobutanol, benzyl alcohol, and phenylethyl alcohol; benzalkonium chloride; phenols such as phenol and cresols; thimerosal; dehydroacetic acid; and sorbic acid), corrigents (examples thereof can include sweeteners, acidulants, and flavors that are usually used), and diluents. 
     Introduction of Double-Stranded Polynucleotide to Cells, Tissues, or Individuals, and Regulation of Expression of Target Gene 
     Recipients to which or to whom the double-stranded polynucleotide thus prepared is introduced are not particularly limited as long as the target gene can be intracellularly transcribed into RNA or translated into a protein therein. The recipients mean cells, tissues, or individuals. 
     The cells for which the double-stranded polynucleotide is used may be any of germline cells, somatic cells, totipotent cells, pluripotent cells, cleaved cells, non-cleaved cells, parenchymal cells, epithelial cells, immortalized cells, transformed cells, nerve cells, and immunocytes. 
     The tissues include 1-cell embryos or constitutive cells, or polyploid embryos, embryonic tissues, or the like. Moreover, examples of the above differentiated cells include adipocytes, fibroblasts, myocytes, cardiomyocytes, endothelial cells, nerve cells, glial cells, blood cells, megakaryocytes, lymphocytes, macrophages, neutrophils, eosinophils, basophiles, mast cells, leukocytes, granulocytes, keratinocytes, chondrocytes, osteoblasts, osteoclasts, hepatocytes, and endocrine or exocrine cells. For example, CHO-K1 cells (RIKEN Cell bank),  Drosophila  S2 cells (Schneider, 1. et al., J. Embryol. Exp. Morph., 27, p. 353-365 (1972)), human HeLa cells (ATCC: CCL-2), and human HEK293 cells (ATCC: CRL-1573) are preferably used as such cells. 
     Furthermore, specific examples of the individuals used as recipients of the double-stranded polynucleotide include plants, animals, protozoans, viruses, bacteria, and those belonging to the  Eumycetes . The plants may be monocots, dicots, or gymnosperms. The animals may be vertebrates or invertebrate. The vertebrates are preferably mammals including mice, rats, monkeys, dogs, and humans. 
     When the recipients are cells or tissues, a calcium phosphate method, electroporation, a lipofection method, viral infection, immersion in a double-stranded polynucleotide solution, or transformation, or the like is used as a method for introducing the double-stranded polynucleotide into the recipients. Moreover, examples of a method for introduction into embryos include microinjection, electroporation, and viral infection. When the recipients are plants, a method involving injection or perfusion into the cavities or interstitial cells or the like of the plants or spraying thereonto is used. Moreover, for animal individuals, a method involving systemic introduction through, for example, oral, local, hypodermic, intramuscular, intravenous, parenteral, transvaginal, rectal, nasal, ocular, or transmucosal administration, or electroporation, viral infection, or the like is used. A method by which the double-stranded polynucleotide is directly mixed with a diet for the organisms can also be used as an oral introduction method. 
     In addition to these approaches, a colloidal dispersion system can be used as a method for introducing the double-stranded polynucleotide into patients. 
     The colloidal dispersion system is expected to have the effect of enhancing the in-vivo stability of the compound or the effect of efficiently transporting the compound to particular organs, tissues, or cells. 
     The colloidal dispersion system used is not limited as long as it is usually applicable. Examples thereof can include polymer complexes, nanocapsules, microspheres, beads, and water-in-oil emulsifying agents, micelles, mixed micelles, and lipid-based dispersion systems including liposomes. Preferably, the colloidal dispersion system is a plurality of liposomes or artificial membrane vesicles having the effect of efficiently transporting the compound to particular organs, tissues, or cells (Mannino et al., Biotechniques, 1988, 6, p. 682-; Blume and Cevc, Biochem. et Biophys. Acta, 1990, 1029, p. 91-; Lappalainen et al., Antiviral Res., 1994, 23, p. 119-; and Chonn and Cullis, Current Op. Biotech., 1995, 6, p. 698-). 
     Unilamellar liposomes of 0.2 to 0.4 μm in size range are capable of encapsulating a considerable amount of an aqueous buffer containing macromolecules, and the contents are encapsulated in this aqueous inner membrane and transported in a biologically active form to brain cells (Fraley et al., Trends Biochem. Sci., 1981, 6, p. 77-). 
     The liposome composition is usually a complex of lipid, particularly phospholipid, specifically phospholipid having a high phase transition temperature, with one or more steroids, particularly cholesterols. 
     Examples of the lipid useful for liposome production include phosphatidyl compounds such as phosphatidyl glycerol, phosphatidyl choline, phosphatidyl serine, sphingolipid, phosphatidyl ethanolamine, cerebroside, and ganglioside. 
     Diacyl phosphatidyl glycerol is particularly useful, wherein the lipid moiety contains 14 to 18 carbon atoms and is saturated (devoid of any internal double bonds in the chain of 14 to 18 carbon atoms) and, in particular, contains 16 to 18 carbon atoms. 
     Typical phospholipids encompass phosphatidyl choline, dipalmitoyl phosphatidyl choline, and distearoyl phosphatidyl choline. 
     Targeting by the colloidal dispersion system including liposomes may be passive or active. 
     Such passive targeting is achieved by use of the fundamental tendency of liposomes to be distributed to reticuloendothelial cells in organs containing sinusoids. 
     On the other hand, examples of the active targeting can include liposome modification approaches involving binding particular ligands such as viral coat protein (Morishita et al., Proc. Natl. Acad. Sci. (U.S.A.), 1993, 90, p. 8474-), monoclonal antibodies (or appropriate binding sites thereof), sugars, glycolipids, or proteins (or appropriate oligopeptide fragments thereof) to liposomes or changing liposome composition to achieve distribution to organs and cell types other than naturally occurring sites of localization. 
     The surface of the colloidal dispersion system may be modified in various ways for targeting purposes. 
     In the liposomal targeted delivery system, a lipid group can be incorporated into the lipid bilayer of the liposome to maintain target ligands through tight association with the lipid bilayer. 
     Various linking groups may be used for linking the lipid chain to the target ligands. 
     The target ligands binding to particular cell surface molecules predominantly found on cells desired to receive the delivery of the double-stranded polynucleotide can be, for example, (1) hormones, growth factors, or appropriate oligopeptide fragments thereof, binding to particular cell receptors predominantly expressed by the cells desired to receive the delivery, or (2) polyclonal or monoclonal antibodies or appropriate fragments thereof (e.g., Fab or F(ab′)2) specifically binding to antigenic epitopes predominantly found on the target cells. 
     Two or more bioactive agents can also be compounded within the single liposome and administered. 
     A medicament for enhancing the intracellular stability of the contents and/or targeting may further be added to the colloidal dispersion system. 
     The amount of the double-stranded polynucleotide or pharmacologically acceptable salt thereof used differs depending on symptoms, ages, etc. 1 mg (preferably, 30 mg) as the lower limit to 2000 mg (preferably, 1500 mg) as the upper limit of the polynucleotide or the salt per dose for oral administration or 0.5 mg (preferably, 5 mg) as the lower limit to 500 mg (preferably, 250 mg) as the upper limit of the polynucleotide or the salt per dose for intravenous administration is preferably administered to an adult once to six times a day according to symptoms. 
     Pharmaceutical compositions and formulations for local administration include transdermal patches, ointments, lotions, creams, gels, troches, suppositories, sprays, liquids, and powders. 
     EXAMPLES 
     Hereinafter, the present invention will be described more specifically with reference to Examples, Reference Examples, and Test Examples. However, the present invention is not intended to be limited to them. In the Examples below, procedures of genetic engineering were performed by the methods described in “Molecular Cloning” [Sambrook, J., Fritsch, E. F. and Maniatis, T., published in 1989 by Cold Spring Harbor Laboratory Press] or according to the instructions of the commercially available reagents or kits used, unless otherwise specified. 
     Example 1 
     Synthesis of HO-G p -C m1p -A p -C m1p -A p -A m1p -G p -A m1p -A rp -U m1p -G rp -G m1p -A p -U m1p -C p -A m1p -C p -A m1p -A p -T p -T t -H (SEQ ID NO: 1 of the Sequence Listing) (CT-095) 
     CT-095 was synthesized according to an RNA synthesis program on the scale of 0.2 μmol using an automatic nucleic acid synthesizer (manufactured by PerkinElmer Inc., ABI model 394 DNA/RNA synthesizer). Solvents, reagents, and phosphoramidites were used in each synthesis cycle at the same concentrations as in natural oligodeoxynucleotide synthesis. 
     When deoxynucleoside phosphoramidites were used, 5′-O-dimethoxytrityl-6-N-benzoyl-2′-deoxyadenosine-3′-O-(2-cyanoethyl N,N-diisopropyl)phosphoramidites, 5′-O-dimethoxytrityl-2-N-isobutyryl-2′-deoxyguanosine-3′-O-(2-cyanoethyl N,N-diisopropyl)phosphoramidites, 5′-O-dimethoxytrityl-4-N-benzoyl-2′-deoxycytidine-3′-O-(2-cyanoethyl N,N-diisopropyl)phosphoramidites, and 5′-O-dimethoxytritylthymidine-3′-O-(2-cyanoethyl N,N-diisopropyl)phosphoramidites were purchased from Proligo and appropriately adjusted for use. 
     When 2′-O-methyl nucleoside phosphoramidites were used, 5′-O-dimethoxytrityl-6-N-benzoyl-2′-O-methyladenosine-3′-O-(2-cyanoethyl N,N-diisopropyl)phosphoramidites, 5′-O-dimethoxytrityl-2-N-isobutyryl-2′-O-methylguanosine-3′-O-(2-cyanoethyl N,N-diisopropyl)phosphoramidites, 5′-O-dimethoxytrityl-4-N-acetyl-2′-O-methylcytidine-3′-O-(2-cyanoethyl N,N-diisopropyl)phosphoramidites, and 5′-O-dimethoxytrityl-2′-O-methyluridine-3′-O-(2-cyanoethyl N,N-diisopropyl)phosphoramidites were purchased from Glen Research Corp. and appropriately adjusted for use. 
     When ribonucleoside phosphoramidites were used, 5′-O-dimethoxytrityl-6-N-benzoyl-2′-O-(tert-butyldimethylsilyl)adenosine-3′-O-(2-cyanoethyl N,N-diisopropyl)phosphoramidites, 5′-O-dimethoxytrityl-2-N-dimethylformamidine-2′-O-(tert-butyldimethylsilyl)guanosine-3′-O-(2-cyanoethyl N,N-diisopropyl)phosphoramidites, 5′-O-dimethoxytrityl-4-N-acetyl-2′-O-(tert-butyldimethylsilyl)cytidine-3′-O-(2-cyanoethyl N,N-diisopropyl)phosphoramidites, and 5′-O-dimethoxytrityl-2′-O-(tert-butyldimethylsilyl)uridine-3′-O-(2-cyanoethyl N,N-diisopropyl)phosphoramidites were purchased from Proligo and appropriately adjusted for use. 
     When 2′-O,4′-C-ethylene nucleoside phosphoramidites were used, compounds of Example 14 (5′-O-dimethoxytrityl-2′-O,4′-C-ethylene-6-N-benzoyladenosine-3′-O-(2-cyanoethyl N,N-diisopropyl)phosphoramidites), Example 27 (5′-O-dimethoxytrityl-2′-O,4′-C-ethylene-2-N-isobutyrylguanosine-3′-O-(2-cyanoethyl N,N-diisopropyl)phosphoramidites), Example 22 (5′-O-dimethoxytrityl-2′-O,4′-C-ethylene-4-N-benzoyl-5-methylcytidine-3′-O-(2-cyanoethyl N,N-diisopropyl)phosphoramidites), and Example 9 (5′-O-dimethoxytrityl-2′-O,4′-C-ethylene-5-methyluridine-3′-O-(2-cyanoethyl N,N-diisopropyl)phosphoramidites) of Japanese Patent No. 3420984 were appropriately prepared for use. 
     The phosphoramidites were appropriately supplied to the automatic nucleic acid synthesizer to synthesize a polynucleotide having the desired sequence. 0.5 μmol of CPG (controlled pore glass; manufactured by Applied Biosystems or Glen Research Corp.) bound with the desired nucleosides was used as a solid-phase carrier to synthesize the title polynucleotide. 
     The protected polynucleotide analog having the sequence of interest was treated with 2 mL of an ammonia water:ethanol solution (3:1 v/v) at 55° C. for 16 hours to excise the oligomer from the support and to remove the cyanoethyl group acting as a protective group for the phosphate group and the protective group on the base of the nucleic acid. CPG was removed by filtration. After washing with ethanol, the filtrate and the wash were combined, and the solvent was distilled off under reduced pressure. To the residue, 0.3 mL of triethylamine trihydrofluoride was added, and the mixture was left at room temperature for 19 hours. 60 μL of H 2 O and 3 mL of n-butanol were added thereto, and the mixture was left at −20° C. for 1 hour. Then, precipitates were collected by centrifugation. The obtained precipitates were dissolved in 200 μL of H 2 O and purified by 20% polyacrylamide gel electrophoresis containing 7 M urea (1×TBE, 600 V, 4 hours). After the electrophoresis, bands were visualized using a UV lamp, and the bands of interest were excised using a knife. 1 mL of a solution containing 0.2 M NaCl and 10 mM EDTA (pH 7.2) was added thereto, and the mixture was left overnight to elute the polynucleotide from the gel slice. The oligonucleotide was precipitated by the addition of ethanol and collected by centrifugation. The molecular weight of the present polynucleotide was identified by negative ion ESI mass spectrometry (calculated value: 6721.46, measured value: 6721.14). 
     The nucleotide sequence of the present polynucleotide comprises a sequence of nucleotide Nos. 3139-3157 of the human β-catenin gene (GenBank accession No. NM_001904.3). The nucleotide sequence of the human β-catenin gene is also shown in SEQ ID NO: 2 of the Sequence Listing, and its amino acid sequence is shown in SEQ ID NO: 3 of the Sequence Listing. 
     Example 2 
     Synthesis of HO-U m1p -T p -G m1p -T p -G m1p -A p -U m1p -C p -C m1p -A rp -U m1p -U rp -C m1p -T p -U m1p -G p -U m1p -G p -C m1p -T p -T t -H (SEQ ID NO: 4 of the Sequence Listing) (CT-096) 
     CT-096 was synthesized in the same way as for Example 1. 
     Molecular weight calculated value: 6632.29, measured value: 6632.17 
     Nucleotide sequence: comprising a sequence complementary to nucleotide Nos. 3139-3157 of the human β-catenin gene (GenBank accession No. NM_001904.3) 
     Example 3 
     Synthesis of HO-G p -C m1p -A p -C m1p -A p -A m1p -G rp -A m1p -A rp -U m1p -G rp -G m1p -A p -U m1p -C p -A m1p -C p -A m1p -A p -T p -T t -H (SEQ ID NO: 5 of the Sequence Listing) (CT-097) 
     CT-097 was synthesized in the same way as for Example 1, and the molecular weight was measured. 
     Molecular weight calculated value: 6737.46, measured value: 6737.38 
     Nucleotide sequence: comprising a sequence of nucleotide Nos. 3139-3157 of the human β-catenin gene (GenBank accession No. NM_001904.3) 
     Example 4 
     Synthesis of HO-U m1p -T p -G m1p -T p -G m1p -A p -U m1p -C p -C m1p -A rp -U m1p -U rp -C m1p -U rp -U m1p -G p -U m1p -G p -C m1p -T p -T t -H (SEQ ID NO: 6 of the Sequence Listing) (CT-098) 
     CT-098 was synthesized in the same way as for Example 1. 
     Molecular weight calculated value: 6634.26, measured value: 6634.90 
     Nucleotide sequence: comprising a sequence complementary to nucleotide Nos. 3139-3157 of the human β-catenin gene (GenBank accession No. NM_001904.3) 
     Example 5 
     Synthesis of HO-G rp -C m1p -A rp -C m1p -A rp -A m1p -G rp -A m1p -A rp -U m1p -G rp -G m1p -A p -U m1p -C p -A m1p -C p -A m1p -A p -T p -T t -H (SEQ ID NO: 7 of the Sequence Listing) (CT-099) 
     CT-099 was synthesized in the same way as for Example 1. 
     Molecular weight calculated value: 6785.46, measured value: 6785.12 
     Nucleotide sequence: comprising a sequence of nucleotide Nos. 3139-3157 of the human β-catenin gene (GenBank accession No. NM_001904.3) 
     Example 6 
     Synthesis of HO-U m1p -T p -G m1p -T p -G m1p -A p -U m1p -C p -C m1p -A rp -U m1p -U rp -C m1p -U rp -U m1p -G rp -U m1p -G rp -C m1p -T p -T t -H (SEQ ID NO: 8 of the Sequence Listing) (CT-100) 
     CT-100 was synthesized in the same way as for Example 1. 
     Molecular weight calculated value: 6666.26, measured value: 6665.71 
     Nucleotide sequence: comprising a sequence complementary to nucleotide Nos. 3139-3157 of the human β-catenin gene (GenBank accession No. NM_001904.3) 
     Example 7 
     Synthesis of HO-G p -C m1p -A p -C m1p -A p -A m1p -G p -A m1p -A p -T e2p -G p -G m1p -A p -U m1p -C p  A m1p -C p -A m1p -A p -T p -T t -H (SEQ ID NO: 9 of the Sequence Listing) (CT-112) 
     CT-112 was synthesized in the same way as for Example 1. However, in the final step of the automatic nucleic acid synthesizer, acid treatment was not performed (the dimethoxytrityl group was bound to the oligonucleotide). The present polynucleotide was treated with an ammonia water:ethanol solution (3:1 v/v) and then purified by reverse-phase HPLC (LC-10VP manufactured by Shimadzu Corp., column (Merck, Chromolith Performance RP-18e (4.6×100 mm)), Solution A: 5% acetonitrile, 0.1 M aqueous triethylamine acetate solution (TEAA), pH 7.0, Solution B: acetonitrile, B %: 10%→60% (10 min, linear gradient); 60° C.; 2 ml/min; 260 nm) to gather peaks of the product of interest having the dimethoxytrityl group. Water was added thereto, and TEAA was distilled off under reduced pressure. An 80% aqueous acetic acid solution (2 mL) was added thereto, and the mixture was left for 20 minutes to deprotect the dimethoxytrityl group. The solvent was distilled off, and the residue was dissolved in 500 μl of water, washed with ethyl acetate, and freeze-dried to obtain the oligonucleotide of interest. 
     Molecular weight calculated value: 6715.50, measured value: 6714.92 
     Nucleotide sequence: comprising a sequence of nucleotide Nos. 3139-3157 of the human β-catenin gene (GenBank accession No. NM_001904.3) 
     Example 8 
     Synthesis of HO-G p -C m1p -A p -C m1p -A p -A m1p -G p -A m1p -A e2p -U m1p -G p -G m1p -A p -U m1p -C p -A m1p -C p -A m1p -A p -T p -T t -H (SEQ ID NO: 10 of the Sequence Listing) (CT-113) 
     CT-113 was synthesized in the same way as for Example 7. 
     Molecular weight calculated value: 6731.50, measured value: 6732.22 
     Nucleotide sequence: comprising a sequence of nucleotide Nos. 3139-3157 of the human β-catenin gene (GenBank accession No. NM_001904.3) 
     Example 9 
     Synthesis of HO-G p -C m1p -A p -C m1p -A p -A m1p -G p -A e2p -A p -U m1p -G p -G m1p -A p -U m1p -C p -A m1p -C p -A m1p -A p -T p -T t -H A (SEQ ID NO: 11 of the Sequence Listing) (CT-114) 
     CT-114 was synthesized in the same way as for Example 7. 
     Molecular weight calculated value: 6701.47, measured value: 6701.06 
     Nucleotide sequence: comprising a sequence of nucleotide Nos. 3139-3157 of the human β-catenin gene (GenBank accession No. NM_001904.3) 
     (Example 10) 
     Synthesis of HO-G p -C m1p -A p -C e2p -A p -A m1p -G p -A e2P -A p -U m1p -G p -G m1p -A p -U m1p -C p -A m1p -C p -A m1p -A p -T p -T t -H (SEQ ID NO: 12 of the Sequence Listing) (CT-115) 
     CT-115 was synthesized in the same way as for Example 7. 
     Molecular weight calculated value: 6727.51, measured value: 6728.07 
     Nucleotide sequence: comprising a sequence of nucleotide Nos. 3139-3157 of the human β-catenin gene (GenBank accession No. NM_001904.3) 
     Example 11 
     Synthesis of HO-U m1p -T p -G m1p -T p -G m1p -A p -U m1p -C p -C m1p -A e2P -U m1p -T p -C m1p -T p -U m1p -G p -U m1p -G p -C m1p -T p -T t -H (SEQ ID NO: 13 of the Sequence Listing) (CT-116) 
     CT-116 was synthesized in the same way as for Example 7. 
     Molecular weight calculated value: 6656.35, measured value: 6655.97 
     Nucleotide sequence: comprising a sequence complementary to nucleotide Nos. 3139-3157 of the human β-catenin gene (GenBank accession No. NM_001904.3) 
     Example 12 
     Synthesis of HO-U m1p -T p -G m1p -r-G m1p -A p -U m1p -C p -C m1p -A p -T e2p -T p -C m1p -T p -U m1p -G p -U m1p -G p -C m1p -T p -T t -H (SEQ ID NO: 14 of the Sequence Listing) (CT-117) 
     CT-117 was synthesized in the same way as for Example 7. 
     Molecular weight calculated value: 6640.35, measured value: 6640.88 
     Nucleotide sequence: comprising a sequence complementary to nucleotide Nos. 3139-3157 of the human β-catenin gene (GenBank accession No. NM_001904.3) 
     Example 13 
     Synthesis of HO-U m1p -T p -G m1p -T p -G m1p -A p -U m1p -C p -C m1p -A p -T e2p -T p -C m1p -T p -T e2p -G p -U m1p -G p -C m1p -T p -T t -H (SEQ ID NO: 15 of the Sequence Listing) (CT-118) 
     CT-118 was synthesized in the same way as for Example 7. 
     Molecular weight calculated value: 6666.39, measured value: 6666.04 
     Nucleotide sequence: comprising a sequence complementary to nucleotide Nos. 3139-3157 of the human β-catenin gene (GenBank accession No. NM_001904.3) 
     Example 14 
     Synthesis of HO-G p -C m1p -A p -C m1p -A p -A m1p -G p -A m1p -A p -U m1p -G p -G m1p -A p -U m1p -C p -A m1p -C p -A m1p -A p -T p -T t -H (SEQ ID NO: 16 of the Sequence Listing) (CT-091) 
     CT-091 was synthesized in the same way as for Example 7. 
     Molecular weight calculated value: 6689.46, measured value: 6689.81 
     Nucleotide sequence: comprising a sequence of nucleotide Nos. 3139-3157 of the human β-catenin gene (GenBank accession No. NM_001904.3) 
     Example 15 
     Synthesis of HO-U m1p -T p -G m1p -T p -G m1p -A p -U m1p -C p -C m1p -A p -U m1p -T p -C m1p -T p -U m1p -G p -U m1p -G p -C m1p -T p -T t -H (SEQ ID NO: 17 of the Sequence Listing) (CT-092) 
     CT-092 was synthesized in the same way as for Example 7. 
     Molecular weight calculated value: 6614.31, measured value: 6614.80 
     Nucleotide sequence: comprising a sequence complementary to nucleotide Nos. 3139-3157 of the human β-catenin gene (GenBank accession No. NM_001904.3) 
     Example 16 
     Synthesis of HO-G p -C m1p -A p -C m1p -A p -A m1p -G p -A m1p -A m1p -U m1p -G p -G m1p -A p -U m1p -C p -A m1p -C p -A m1p -A p -T p -T t -H (SEQ ID NO: 18 of the Sequence Listing) (CT-101) 
     CT-101 was synthesized in the same way as for Example 7. 
     Molecular weight calculated value: 6719.49, measured value: 6719.67 
     Nucleotide sequence: comprising a sequence of nucleotide Nos. 3139-3157 of the human fβ-catenin gene (GenBank accession No. NM_001904.3) 
     Example 17 
     Synthesis of HO-G p -C m1p -A p -C m1p -A p -A m1p -G p -A m1p -A rp -U m1p -G p -G m1p -A p -U m1p -C p -A m1p -C p -A m1p -A p -T p -T t -H (SEQ ID NO: 19 of the Sequence Listing) (CT-102) 
     CT-102 was synthesized in the same way as for Example 1. 
     Molecular weight calculated value: 6705.46, measured value: 6705.58 
     Nucleotide sequence: comprising a sequence of nucleotide Nos. 3139-3157 of the human β-catenin gene (GenBank accession No. NM_001904.3) 
     Example 18 
     Synthesis of HO-U m1p -T p -G m1p -T p -G m1p -A p -U m1p -C p -C m1p -A m1p -U m1p -T p -C m1p -T p -U m1p -G p -U m1p -G p -C m1p -T p -T t -H (SEQ ID NO: 20 of the Sequence Listing) (CT-107) 
     CT-107 was synthesized in the same way as for Example 7. 
     Molecular weight calculated value: 6644.34, measured value: 6644.47 
     Nucleotide sequence: comprising a sequence complementary to nucleotide Nos. 3139-3157 of the human β-catenin gene (GenBank accession No. NM_001904.3) 
     Example 19 
     Synthesis of HO-U m1p -T p -G m1p -T p -G m1p -A p -U m1p -C p -C m1p -A rp -U m1p -T p -C m1p -T p -U m1p -G p -U m1p -G p -C m1p -T p -T t -H (SEQ ID NO: 21 of the Sequence Listing) (CT-108) 
     CT-108 was synthesized in the same way as for Example 1. 
     Molecular weight calculated value: 6630.31, measured value: 6630.48 
     Nucleotide sequence: comprising a sequence complementary to nucleotide Nos. 3139-3157 of the human β-catenin gene (GenBank accession No. NM_001904.3) 
     Example 20 
     Synthesis of HO-G p -C m1p -A p -C m1p -A p -A m1p -G p -A m1p -A p -U m1p -G p -G m1p -A p -U m1p -C p -A m1p -C p -A m1p -A t -H (SEQ ID NO: 22 of the Sequence Listing) (CT-103) 
     CT-103 was synthesized in the same way as for Example 7. 
     Molecular weight calculated value: 6081.07, measured value: 6081.08 
     Nucleotide sequence: comprising a sequence of nucleotide Nos. 3139-3157 of the human β-catenin gene (GenBank accession No. NM_001904.3) 
     Example 21 
     Synthesis of HO-U m1p -T p -G m1p -T p -G m1p -A p -U m1p -C p -C m1p -A p -U m1p -T p -C m1p -T p -U m1p -G p -U m1p -G p -C m1t -H (SEQ ID NO: 23 of the Sequence Listing) (CT-109) 
     CT-109 was synthesized in the same way as for Example 7. 
     Molecular weight calculated value: 6005.92, measured value: 6005.89 
     Nucleotide sequence: comprising a sequence complementary to nucleotide Nos. 3139-3157 of the human β-catenin gene (GenBank accession No. NM_001904.3) 
     Example 22 
     Synthesis of HO-G p -C m1p -A p -C m1p -A p -A m1p -G p -A m1p -A m1p -U m1p -G m1p -G m1p -A p -U m1p -C p -A m1p -C p -A m1p -A p -T p -T t -H (SEQ ID NO: 24 of the Sequence Listing) (CT-127) 
     CT-127 was synthesized in the same way as for Example 7. 
     Molecular weight calculated value: 6749.52, measured value: 6749.26 
     Nucleotide sequence: comprising a sequence of nucleotide Nos. 3139-3157 of the human β-catenin gene (GenBank accession No. NM_001904.3) 
     Example 23 
     Synthesis of HO-G p -C m1p -A p -C m1p -A p -A m1p -G m1p -A m1p -A m1p -U m1p -G m1p -G m1p -A p -U m1p -C p -A m1p -C p -A m1p -A p -T p -T t -H (SEQ ID NO: 25 of the Sequence Listing) (CT-128) 
     CT-128 was synthesized in the same way as for Example 7. 
     Molecular weight calculated value: 6779.54, measured value: 6779.31 
     Nucleotide sequence: comprising a sequence of nucleotide Nos. 3139-3157 of the human β-catenin gene (GenBank accession No. NM_001904.3) 
     Example 24 
     Synthesis of HO-G p -C m1p -A p -C m1p -A m1p -A m1p -G m1p -A m1p -A m1p -U m1p -G m1p -G m1p -A p -U m1p -C p -A m1p -C p -A m1p -A p -T p -T t -H (SEQ ID NO: 26 of the Sequence Listing) (CT-129) 
     CT-129 was synthesized in the same way as for Example 7. 
     Molecular weight calculated value: 6809.57, measured value: 6809.23 
     Nucleotide sequence: comprising a sequence of nucleotide Nos. 3139-3157 of the human β-catenin gene (GenBank accession No. NM_01904.3) 
     Example 25 
     Synthesis of HO-G p -C m1p -A p -C m1p -A p -A m1p -G m1p -A m1p -A m1p -U m1p -G p -G m1p -A p -U m1p -C p -A m1p -C p -A m1p -A p -T p -T t -H (SEQ ID NO: 27 of the Sequence Listing) (CT-130) 
     CT-130 was synthesized in the same way as for Example 7. 
     Molecular weight calculated value: 6749.52, measured value: 6749.21 
     Nucleotide sequence: comprising a sequence of nucleotide Nos. 3139-3157 of the human β-catenin gene (GenBank accession No. NM_001904.3) 
     Example 26 
     Synthesis of HO-G p -C m1p -A p -C m1p -A m1p -A m1p -G m1p -A m1p -A m1p -U m1p -G p -G m1p -A p -U m1p -C p -A m1p -C p -A m1p -A p -T p -T t -H (SEQ ID NO: 28 of the Sequence Listing) (CT-131) 
     CT-131 was synthesized in the same way as for Example 7. 
     Molecular weight calculated value: 6779.54, measured value: 6779.17 
     Nucleotide sequence: comprising a sequence of nucleotide Nos. 3139-3157 of the human β-catenin gene (GenBank accession No. NM_001904.3) 
     Example 27 
     Synthesis of HO-G p -C m1p -A p -C m1p -A p -A m1p -G p -A e2p -A p -T e2P -G p -G m1p -A p -U m1p -C p -A m1p -C p -A m1p -A p -T p -T t -H (SEQ ID NO: 29 of the Sequence Listing) (CT-132) 
     CT-132 was synthesized in the same way as for Example 7. 
     Molecular weight calculated value: 6727.51, measured value: 6728.00 
     Nucleotide sequence: comprising a sequence of nucleotide Nos. 3139-3157 of the human β-catenin gene (GenBank accession No. NM_001904.3) 
     Example 28 
     Synthesis of HO-G p -C m1p -A p -C m1p -A p -A e2p -G p -A e2p -A p -U m1p -G p -G m1p -A p -U m1p -C p -A m1p -C p -A m1p -A p -T p -T t -H (SEQ ID NO: 30 of the Sequence Listing) (CT-133) 
     CT-133 was synthesized in the same way as for Example 7. 
     Molecular weight calculated value: 6713.48, measured value: 6713.77 
     Nucleotide sequence: comprising a sequence of nucleotide Nos. 3139-3157 of the human β-catenin gene (GenBank accession No. NM_001904.3) 
     Example 29 
     Synthesis of HO-G p -C m1p -A p -C m1p -A p -A e2p -G p -A m1p -A p -T e2p -G p -G m1p -A p -U m1p -C p -A m1p -C p -A m1p -A p -T p -T t -H (SEQ ID NO: 31 of the Sequence Listing) (CT-134) 
     CT-134 was synthesized in the same way as for Example 7. 
     Molecular weight calculated value: 6727.51, measured value: 6728.04 
     Nucleotide sequence: comprising a sequence of nucleotide Nos. 3139-3157 of the human β-catenin gene (GenBank accession No. NM_001904.3) 
     Example 30 
     Synthesis of HO-G p -C m1p -A p -C m1p -A p -A e2p -G p -A e2p -A p -T e2p -G p -G m1p -A p -U m1p -C p -A m1p -C p -A m1p -A p -T p -T t -H (SEQ ID NO: 32 of the Sequence Listing) (CT-135) 
     CT-135 was synthesized in the same way as for Example 7. 
     Molecular weight calculated value: 6739.52, measured value: 6740.48 
     Nucleotide sequence: comprising a sequence of nucleotide Nos. 3139-3157 of the human β-catenin gene (GenBank accession No. NM_001904.3) 
     Example 31 
     Synthesis of HO-G p -C m1p -A p -C e2p -A p -A m1p -G p -A e2p -A p -T e2p -G p -G m1p -A p -U m1p -C p -A m1p -C p -A m1p -A p -T p -T t -H (SEQ ID NO: 33 of the Sequence Listing) (CT-137) 
     CT-137 was synthesized in the same way as for Example 7. 
     Molecular weight calculated value: 6753.55, measured value: 6754.15 
     Nucleotide sequence: comprising a sequence of nucleotide Nos. 3139-3157 of the human β-catenin gene (GenBank accession No. NM_001904.3) 
     Example 32 
     Synthesis of HO-G p -C m1p -A p -C e2p -A p -A e2p -G p -A e2p -A p -U m1p -G p -G m1p -A p -U m1p -C p -A m1p -C p -A m1p -A p -T p -T t -H (SEQ ID NO: 34 of the Sequence Listing) (CT-136) 
     CT-136 was synthesized in the same way as for Example 7. 
     Molecular weight calculated value: 6739.52, measured value: 6739.51 
     Nucleotide sequence: comprising a sequence of nucleotide Nos. 3139-3157 of the human β-catenin gene (GenBank accession No. NM_001904.3) 
     Example 33 
     Synthesis of HO-G p -C m1p -A p -C e2p -A p -A e2p -G p -A e2p -A p -T e2p -G p -G m1p -A p -U m1p -C p -A m1p -C p -A m1p -A p -T p -T t -H (SEQ ID NO: 35 of the Sequence Listing) (CT-138) 
     CT-138 was synthesized in the same way as for Example 7. 
     Molecular weight calculated value: 6765.56, measured value: 6765.76 
     Nucleotide sequence: comprising a sequence of nucleotide Nos. 3139-3157 of the human β-catenin gene (GenBank accession No. NM_001904.3) 
     Example 34 
     Synthesis of HO-G m1p -C p -A m1p -C p -A m1p -A p -G m1p -A p -A m1p -T p -G m1p -G p -A m1p -T p -C m1p -A p -C m1p (SEQ ID NO: 36 of the Sequence Listing) (CT-119) 
     CT-119 was synthesized in the same way as for Example 7. 
     Molecular weight calculated value: 6747.54, measured value: 6747.39 
     Nucleotide sequence: comprising a sequence of nucleotide Nos. 3139-3157 of the human ii-catenin gene (GenBank accession No. NM_001904.3) 
     Example 35 
     Synthesis of HO-T p -U m1p -G p -U m1p -G p -A m1p -T p -C m1p -C p -A m1p -T p -U m1p -C p -U m1p -T p -G m1p -T p -G m1p -C p -T p -T t -H (SEQ ID NO: 37 of the Sequence Listing) (CT-120) 
     CT-120 was synthesized in the same way as for Example 7. 
     Molecular weight calculated value: 6598.32, measured value: 6598.26 
     Nucleotide sequence: comprising a sequence complementary to nucleotide Nos. 3139-3157 of the human β-catenin gene (GenBank accession No. NM_001904.3) 
     Example 36) 
     Synthesis of HO-G s -C m1s -A p -C m1p -A p -A m1p -G rp -A m1p -A rp -U m1p -G rp -G m1p -A p -U m1p -C P -A m1p -C p -A m1s -A s -T s -T t -H (SEQ ID NO: 38 of the Sequence Listing) (CT-0975) 
     CT-097S is synthesized in the same way as for Example 1. However, the moiety having a phosphorothioate bond can be synthesized by treatment with a 0.2 M phenylacetyl disulfide/pyridine-acetonitrile (1:1 v/v) solution for 3 minutes instead of oxidation with an iodine/tetrahydrofuran/pyridine/H 2 O solution (Ravikumar, V. T. et al. Bioorg. Med. Chem. Lett. (2006) 16, p. 2513-2517). CT-097S is identified by negative ion mass spectrometry. 
     Nucleotide sequence: comprising a sequence of nucleotide Nos. 3139-3157 of the human β-catenin gene (GenBank accession No. NM_001904.3) 
     Example 37 
     Synthesis of HO-U m1s -T s -G m1p -T p -G m1p -A p -U m1p -C p -C m1p -A rp -U m1p -U rp -C m1p -U rp -U m1p )-G p -U m1p -G s -C m1s -T s -T t -H (SEQ ID NO: 39 of the Sequence Listing) (CT-098S) 
     CT-098S is synthesized in the same way as for Example 36. CT-098S is identified by negative ion mass spectrometry. 
     Nucleotide sequence: comprising a sequence complementary to nucleotide Nos. 3139-3157 of (GenBank accession No. NM_001904.3) 
     Example 38 
     Synthesis of HO-G s -C m1s -A p -C m1p -A p -A m1p -G p -A e2p -A p -U m1p -G p -G m1p -A p -U m1p -C p -A m1p -C p -A m1s -A s -T s -T t -H (SEQ ID NO: 40 of the Sequence Listing) (CT-139) 
     CT-139 was synthesized in the same way as for Example 7. However, the moiety having a phosphorothioate bond was treated with a 0.2 M phenylacetyl disulfide/pyridine-acetonitrile (1:1 v/v) solution for 3 minutes instead of oxidation with an iodine/tetrahydrofuran/pyridine/H 2 O solution (Ravikumar, V. T. et al. Bioorg. Med. Chem. Lett. (2006) 16, p. 2513-2517). CT-139 was identified by negative ion ESI mass spectrometry. 
     Molecular weight calculated value: 6781.78, measured value: 6781.89 
     Nucleotide sequence: comprising a sequence of nucleotide Nos. 3139-3157 of the human β-catenin gene (GenBank accession No. NM_001904.3) 
     Example 39 
     Synthesis of HO-U m1s -T s -G m1p -T p -G m1p -A p -U m1p -C p -C m1p -A p -U m1p -T p -C m1p -T p -U m1p )-G p -U m1p -G s -C m1s -T s -T t -H (SEQ ID NO: 41 of the Sequence Listing) (CT-141) 
     CT-141 was synthesized in the same way as for Example 38. 
     Molecular weight calculated value: 6829.82, measured value: 6830.13 
     Nucleotide sequence: comprising a sequence complementary to nucleotide Nos. 3139-3157 of the human β-catenin gene (GenBank accession No. NM_001904.3) 
     Example 40 
     Synthesis of HO-G s -C m1s -A p -C m1p -A p -A m1p -G m1p -A m1p -A m1p -U m1p -G p -G m1p -A p -U m1p -C p -A m1p -C p -A m1s -A s -T s -T t -H (SEQ ID NO: 42 of the Sequence Listing) (CT-140) 
     CT-140 was synthesized in the same way as for Example 38. 
     Molecular weight calculated value: 6694.62, measured value: 6694.71 
     Nucleotide sequence: comprising a sequence of nucleotide Nos. 3139-3157 of the human β-catenin gene (GenBank accession No. NM_001904.3) 
     Example 41 
     Synthesis of HO-G p -C m1p -A p -C m1p -A p -A m1p -G p -A e1p -A p -U m1p -G p -G m1p -A p -U m1p -C p -A m1p -C p -A m1p -A p -T p -T t -H (SEQ ID NO: 43 of the Sequence Listing) (CT-114L) 
     CT-114L is synthesized in the same way as for Example 7. However, the 2′,4′-BNA/LNA moiety is synthesized using 5′-O-dimethoxytrityl-2′-O,4′-C-methylene-6-N-benzoyladenosine-3′-O-(2-cyanoethyl N,N-diisopropyl)phosphoramidites as described in the literature (A. A. Koshkin, A. A. et al. Tetrahedron, 54, p. 3607-(1998)). CT-114L is identified by negative ion ESI mass spectrometry. 
     Nucleotide sequence: comprising a sequence of nucleotide Nos. 3139-3157 of the human β-catenin gene (GenBank accession No. NM_001904.3) 
     Example 42) 
     Synthesis of HO-P(═O)(OH)—O-U m1p -T p -G m1p -T p -G m1p -A p -U m1p -C p -C m1p -A p -U m1p -T p -C m1p -T p -U m1p -G p -U m1p -G p -C m1p -T p -T t -H (SEQ ID NO: 17 of the Sequence Listing) (CT-149) 
     CT-149 was synthesized in the same way as for Example 7. However, the 5′-terminal phosphate group moiety was synthesized using PHOSPHALINK (manufactured by Applied Biosystems). 
     Molecular weight: calculated value: 6694.28, measured value: 6694.55 
     Nucleotide sequence: comprising a sequence complementary to nucleotide Nos. 3139-3157 of the human β-catenin gene (GenBank accession No. NM_001904.3) 
     Example 43 
     Synthesis of HO-P(═O)(OH)—O-U m1p -T p -G m1p -T p -G m1p -A p -U m1p -C p -C m1p -A P -U m1p -T p -C m1p -T p -U m1p -G p -U m1p -G p -C m1p -U rp -T t -H (SEQ ID NO: 52 of the Sequence Listing) (CT-155) 
     CT-155 was synthesized in the same way as for Example 42. 
     Molecular weight: calculated value: 6696.27, measured value: 6696.44 
     Nucleotide sequence: comprising a sequence complementary to nucleotide Nos. 3139-3157 of the human β-catenin gene (GenBank accession No. NM_001904.3) 
     Example 44 
     Synthesis of HO-P(═O)(OH)—O-U m1p -T p -G m1p -T p -G m1p -A p -U m1p -C p -C m1p -A p -U m1p -T p  C m1p -T p -U m1p -G p -U m1p -G p -C m1p -U m1p -T t -H (SEQ ID NO: 53 of the Sequence Listing) (CT-156) 
     CT-156 was synthesized in the same way as for Example 42. 
     Molecular weight: calculated value: 6710.29, measured value: 6710.13 
     Nucleotide sequence: comprising a sequence complementary to nucleotide Nos. 3139-3157 of the human β-catenin gene (GenBank accession No. NM_001904.3) 
     Example 45) 
     Synthesis of HO-P(═O)(OH)—O-U m1p -T p -G m1p -T p -G m1p -A p -U m1p -C p -C m1p -A p -U m1p -T p -C m1p -T p -U m1p -G p -U m1p -G p -C m1p -T p -U m1t -H (SEQ ID NO: 54 of the Sequence Listing) (CT-157) 
     CT-157 was synthesized in the same way as for Example 42. 
     Molecular weight: calculated value: 6710.29, measured value: 6710.39 
     Nucleotide sequence: comprising a sequence complementary to nucleotide Nos. 3139-3157 of the human β-catenin gene (GenBank accession No. NM_001904.3) 
     Example 46 
     Synthesis of HO-P(═O)(OH)—O-U m1p -T p -G m1p -T p -G m1p -A p -U m1p -C p -C m1p -A p -U m1p -T p -C m1p -T p -U m1p -G p -U m1p -G p -C m1p -U rp -U m1t -H (SEQ ID NO: 55 of the Sequence Listing) (CT-158) 
     CT-158 was synthesized in the same way as for Example 42. 
     Molecular weight: calculated value: 6712.27, measured value: 6712.50 
     Nucleotide sequence: comprising a sequence complementary to nucleotide Nos. 3139-3157 of the human β-catenin gene (GenBank accession No. NM_001904.3) 
     Example 47 
     Synthesis of HO-P(═O)(OH)—O-U m1p -T p -G m1p -T p -G m1p -A p -U m1p -C p -C m1p -A p -U m1p -T p -C m1p -T p -U m1p -G p -U m1p -G p -C m1p -U m1p -U m1t -H (SEQ ID NO: 56 of the Sequence Listing) (CT-159) 
     CT-159 was synthesized in the same way as for Example 42. 
     Molecular weight: calculated value: 6726.29, measured value: 6726.40 
     Nucleotide sequence: comprising a sequence complementary to nucleotide Nos. 3139-3157 of the human β-catenin gene (GenBank accession No. NM_001904.3) 
     Example 48 
     Synthesis of HO-P(═O)(OH)—O-U m1p -T p -G m1p -T p -G m1p -A p -U m1p -C p -C m1p -A p -U m1p -T p -C m1p -T p -U m1p -G p -U m1p -G p -C m1p -T p -U rt -H (SEQ ID NO: 57 of the Sequence Listing) (CT-160) 
     CT-160 was synthesized in the same way as for Example 42. 
     Molecular weight: calculated value: 6696.27, measured value: 6696.26 
     Nucleotide sequence: comprising a sequence complementary to nucleotide Nos. 3139-3157 of the human β-catenin gene (GenBank accession No. NM_001904.3) 
     Example 49 
     Synthesis of HO-P(═O)(OH)—O-U m1p -T p -G m1p -T p -G m1p -A p -U m1p -C p -C m1p -A p -U m1p -T p -C m1p -T p -U m1p -G p -U m1p -G p -C m1p -U rp -U rt -H (SEQ ID NO: 58 of the Sequence Listing) (CT-161) 
     CT-161 was synthesized in the same way as for Example 42. 
     Molecular weight: calculated value: 6698.24, measured value: 6698.34 
     Nucleotide sequence: comprising a sequence complementary to nucleotide Nos. 3139-3157 of the human β-catenin gene (GenBank accession No. NM_001904.3) 
     Example 50 
     Synthesis of HO-P(═O)(OH)—O-U m1p -T p -G m1p -T p -G m1p -A p -U m1p -C p -C m1p -A p -U m1p -T p -C m1p -T p -U m1p -G p -U m1p -G p -C m1p -U m1p -U rt -H (SEQ ID NO: 59 of the Sequence Listing) (CT-162) 
     CT-162 was synthesized in the same way as for Example 42. 
     Molecular weight: calculated value: 6712.27, measured value: 6712.30 
     Nucleotide sequence: comprising a sequence complementary to nucleotide Nos. 3139-3157 of the human β-catenin gene (GenBank accession No. NM_001904.3) 
     Example 51 
     Synthesis of HO-G p -C m1p -A p -C m1p -A p -A m1p -G p -A m1p -A p -U m1p -G p -G m1p -A p -U m1p -C p -A m1p -C p -A m1t -H (SEQ ID NO: 60 of the Sequence Listing) (CT-169) 
     CT-169 was synthesized in the same way as for Example 7. 
     Molecular weight: calculated value: 5767.86, measured value: 5767.78 
     Nucleotide sequence: comprising a sequence of nucleotide Nos. 3139-3156 of the human β-catenin gene (GenBank accession No. NM_001904.3) 
     Example 52 
     Synthesis of HO-G p -C m1p -A p -C m1p -A p -A m1p -G p -A m1p -A p -U m1P -G p -G m1P -A p -U m1P -C p -A m1p -C p -H (SEQ ID NO: 61 of the Sequence Listing) (CT-170) 
     CT-170 was synthesized in the same way as for Example 7. 
     Molecular weight: calculated value: 5424.62, measured value: 5424.47 
     Nucleotide sequence: comprising a sequence of nucleotide Nos. 3139-3155 of the human β-catenin gene (GenBank accession No. NM_001904.3) 
     Example 53 
     Synthesis of HO-G p -C m1p -A p -C m1p -A p -A m1p -G p -A m1p -A p -U m1p -G p -G m1p -A P -U m1p -C p -A m1t -H(SEQ ID NO: 62 of the Sequence Listing) (CT-171) 
     CT-171 was synthesized in the same way as for Example 7. 
     Molecular weight: calculated value: 5135.44, measured value: 5134.53 
     Nucleotide sequence: comprising a sequence of nucleotide Nos. 3139-3154 of the human β-catenin gene (GenBank accession No. NM_001904.3) 
     Example 54 
     Synthesis of HO-C m1p -A p -C m1p -A p -A m1p -G p -A m1p -A p -U m1p -G p -G m1p -A p -U m1p -C p -A m1p )-C p -A m1p -A t -H (SEQ ID NO: 63 of the Sequence Listing) (CT-172) 
     CT-172 was synthesized in the same way as for Example 7. 
     Molecular weight: calculated value: 5751.86, measured value: 5751.80 
     Nucleotide sequence: comprising a sequence of nucleotide Nos. 3140-3157 of the human β-catenin gene (GenBank accession No. NM_001904.3) 
     Example 55 
     Synthesis of HO-A p -C m1p -A p -A m1p -G p -A m1p -A p -U m1p -G p -G m1p -A p -U m1p -C p -A m1p -C p -A m1p -A t -H (SEQ ID NO: 64 of the Sequence Listing) (CT-173) 
     CT-173 was synthesized in the same way as for Example 7. 
     Molecular weight: calculated value: 5432.65, measured value: 5432.62 
     Nucleotide sequence: comprising a sequence of nucleotide Nos. 3141-3157 of the human β-catenin gene (GenBank accession No. NM_001904.3) 
     Example 56 
     Synthesis of HO-C m1p -A p -A m1p -G p -A m1p -A p -U m1p -G p -G m1p -A p -U m1p -C p -A m1p -A t -H (SEQ ID NO: 65 of the Sequence Listing) (CT-174) 
     CT-174 was synthesized in the same way as for Example 7. 
     Molecular weight: calculated value: 5119.44, measured value: 5119.39 
     Nucleotide sequence: comprising a sequence of nucleotide Nos. 3142-3157 of the human (β-catenin gene (GenBank accession No. NM_001904.3) 
     Example 57 
     Synthesis of HO-C m1p -A p -C m1p -A p -A m1p -G p -A m1p -A p -U m1p -G p -G m1p -A p -U m1p -C p -A m1p -C p -A m1t -H (SEQ ID NO: 66 of the Sequence Listing) (CT-175) 
     CT-175 was synthesized in the same way as for Example 7. 
     Molecular weight: calculated value: 5438.65, measured value: 5438.55 
     Nucleotide sequence: comprising a sequence of nucleotide Nos. 3140-3156 of the human β-catenin gene (GenBank accession No. NM_001904.3) 
     Example 58 
     Synthesis of HO-C m1p -A p -C m1p -A p -A m1p -G p -A m1p -A p -U m1p -G p -G m1p -A p -U m1p -C p -A m1p -C pt -H (SEQ ID NO: 67 of the Sequence Listing) (CT-176) 
     CT-176 was synthesized in the same way as for Example 7. 
     Molecular weight: calculated value: 5095.42, measured value: 5095.25 
     Nucleotide sequence: comprising a sequence of nucleotide Nos. 3140-3155 of the human β-catenin gene (GenBank accession No. NM_0.001904.3) 
     Example 59 
     Synthesis of HO-A p -C m1p -A p -A m1p -G p -A m1p -A p -U m1p -G p -G m1p -A p -U m1p -C p -A m1p -C p -A m1t -H (SEQ ID NO: 68 of the Sequence Listing) (CT-177) 
     CT-177 was synthesized in the same way as for Example 7. 
     Molecular weight: calculated value: 5119.44, measured value: 5119.33 
     Nucleotide sequence: comprising a sequence of nucleotide Nos. 3141-3156 of the human β-catenin gene (GenBank accession No. NM_001904.3) 
     Example 60 
     Synthesis of HO-P(═O)(OH)—O-U m1p -T p -G m1p -T p -G m1p -A p -U m1p -C p -C m1p -A p -U m1p -T p -C m1p -T p -U m1p -G p -U m1p -G p -C m1p -A p -U m1t -H (SEQ ID NO: 69 of the Sequence Listing) (CT-204) 
     CT-204 was synthesized in the same way as for Example 42. 
     Molecular weight: calculated value: 6719.31, measured value: 6719.99 
     Nucleotide sequence: comprising a sequence complementary to nucleotide Nos. 3139-3157 of the human β-catenin gene (GenBank accession No. NM_001904.3) 
     Example 61 
     Synthesis of HO-P(═O)(OH)—O-U m1p -T p -G m1p -T p -G m1p -A p -U m1p -C p -C m1p -A p -U m1p -T p -C m1p -T p -U m1p -G p -U m1p -G p -C m1p -G p -U m1t -H (SEQ ID NO: 70 of the Sequence Listing) (CT-205) 
     CT-205 was synthesized in the same way as for Example 42. 
     Molecular weight: calculated value: 6735.31, measured value: 6735.79 
     Nucleotide sequence: comprising a sequence complementary to nucleotide Nos. 3139-3157 of the human β-catenin gene (GenBank accession No. NM_001904.3) 
     Example 62 
     Synthesis of HO-P(═O)(OH)—O-U m1p -T p -G m1p -T p -G m1p -A p -U m1p -C p -C m1p -A p -U m1p -T p -C m1p )-T p -U m1p -G p -U m1p -G p -C m1p -C p -U m1t -H (SEQ ID NO: 71 of the Sequence Listing) (CT-206) 
     CT-206 was synthesized in the same way as for Example 42. 
     Molecular weight: calculated value: 6695.28, measured value: 6696.00 
     Nucleotide sequence: comprising a sequence complementary to nucleotide Nos. 3139-3157 of the human β-catenin gene (GenBank accession No. NM_001904.3) 
     Example 63 
     Synthesis of HO-P(═O)(OH)—O-U m1p -T p -G m1p -T p -G m1p -A p -U m1p -C p -C m1p -A p -U m1p -T p -C m1p -T p -U m1p -G p -U m1p -G p -C m1p -T p -A m11 -14 (SEQ ID NO: 72 of the Sequence Listing) (CT-207) 
     CT-207 was synthesized in the same way as for Example 42. 
     Molecular weight: calculated value: 6733.33, measured value: 6733.98 
     Nucleotide sequence: comprising a sequence complementary to nucleotide Nos. 3139-3157 of the human β-catenin gene (GenBank accession No. NM_001904.3) 
     Example 64 
     Synthesis of HO-P(═O)(OH)—O-U m1p -T p -G m1p -T p -G m1p -A p -U m1p -C p -C m1p -A p -U m1p -T p -C m1p -T p -U m1p -G p -U m1p -G p -C 1P -T p -G m1t -H (SEQ ID NO: 73 of the Sequence Listing) (CT-208) 
     CT-208 was synthesized in the same way as for Example 42. 
     Molecular weight: calculated value: 6749.33, measured value: 6750.11 
     Nucleotide sequence: comprising a sequence complementary to nucleotide Nos. 3139-3157 of the human β-catenin gene (GenBank accession No. NM_001904.3) 
     Example 65 
     Synthesis of HO-P(═O)(OH)—O-U m1p -T p -G m1p -T p -G m1p -A p -U m1p -C p -C m1p -A p -U m1p -T p -C m1p -T p -U m1p -G p -U m1p -G p -C m1p -T p -C m1t -H (SEQ ID NO: 74 of the Sequence Listing) (CT-209) 
     CT-209 was synthesized in the same way as for Example 42. 
     Molecular weight: calculated value: 6709.31, measured value: 6709.81 
     Nucleotide sequence: comprising a sequence complementary to nucleotide Nos. 3139-3157 of the human β-catenin gene (GenBank accession No. NM_001904.3) 
     Example 66 
     Synthesis of HO-P(═O)(OH)—O-A m1p -T p -G m1p -T p -G m1p -A p -U m1p -C p -C m1p -A p -U m1p -T p -C m1p -T p -U m1p -G p -U m1p -G p -C m1p -T p -U m1t -H (SEQ ID NO: 75 of the Sequence Listing) (CT-221) 
     CT-221 was synthesized in the same way as for Example 42. 
     Molecular weight: calculated value: 6733.23, measured value: 6733.00 
     Nucleotide sequence: comprising a sequence complementary to nucleotide Nos. 3139-3156 of the human β-catenin gene (GenBank accession No. NM_001904.3) 
     Example 67 
     Synthesis of HO-P(═O)(OH)—O-G m1p -T p -G m1p -T p -G m1p -A p -U m1p -C p -C m1p -A p -U m1p -T p -C m1p -T p -U m1p -G p -U m1p -G p -C m1p -T p -U m1t -H (SEQ ID NO: 76 of the Sequence Listing) (CT-222) 
     CT-222 was synthesized in the same way as for Example 42. 
     Molecular weight: calculated value: 6749.33, measured value: 6749.06 
     Nucleotide sequence: comprising a sequence complementary to nucleotide Nos. 3139-3156 of the human β-catenin gene (GenBank accession No. NM_001904.3) 
     Example 68 
     Synthesis of HO-P(═O)(OH)—O-C m1p -T p -G m1p -T p -G m1p -A p -U m1p -C p -C m1p -A p -U m1p -T p -C m1p -T p -U m1p -G p -U m1p -G p -C m1p -T p -U m1t -H (SEQ ID NO: 77 of the Sequence Listing) (CT-223) 
     CT-223 was synthesized in the same way as for Example 42. 
     Molecular weight: calculated value: 6709.31, measured value: 6709.00 
     Nucleotide sequence: comprising a sequence complementary to nucleotide Nos. 3139-3156 of the human fβ-catenin gene (GenBank accession No. NM_001904.3) 
     Example 69) 
     Synthesis of HO-P(═O)(OH)—O-U m1p -T p -G m1p -T p -G m1p -A p -U m1p -C p -C m1p -A p -U m1p -T p -C m1p -T p -U m1p -G p -U m1p -G p -T p -U m1t -H (SEQ ID NO: 78 of the Sequence Listing) (CT-202) 
     CT-202 was synthesized in the same way as for Example 42. 
     Molecular weight: calculated value: 6391.06, measured value: 6391.70 
     Nucleotide sequence: comprising a sequence complementary to nucleotide Nos. 3140-3157 of the human β-catenin gene (GenBank accession No. NM_001904.3) 
     Example 70 
     Synthesis of HO-P(═O)(OH)—O-T p -G m1p -T p -G m1p -A p -U m1p -C p -C m1p -A p -U m1p -T p -C m1p -T p -U m1p -G p -U m1p -G p -C m1p -T p -U m1t -H (SEQ ID NO: 79 of the Sequence Listing) (CT-203) 
     CT-203 was synthesized in the same way as for Example 42. 
     Molecular weight: calculated value: 6390.10, measured value: 6390.72 
     Nucleotide sequence: comprising a sequence complementary to nucleotide Nos. 3139-3156 of the human β-catenin gene (GenBank accession No. NM_001904.3) 
     Example 71 
     Synthesis of HO-P(═O)(OH)—O-G m1p -C p -A m1p -C P -A m1p -A p -G m1p -A p -A m1p -T p -G m1p -G p -A m1p -T p -C m1p -A p -C m1p -A p -A m1p -T p -U m1t -H (SEQ ID NO: 80 of the Sequence Listing) (CT-210) 
     CT-210 was synthesized in the same way as for Example 42. 
     Molecular weight: calculated value: 6843.52, measured value: 6844.23 
     Nucleotide sequence: comprising a sequence of nucleotide Nos. 3139-3157 of the human β-catenin gene (GenBank accession No. NM_001904.3) 
     Example 72 
     Synthesis of HO-T p -U m1p -G p -U m1p -G p -A m1p -T p -C m1p -C p -A m1p -T p -U m1p -C p -U m1p -T p -G m1p -T p -G m1p -C t -H (SEQ ID NO: 81 of the Sequence Listing) (CT-211) 
     CT-211 was synthesized in the same way as for Example 42. 
     Molecular weight: calculated value: 5989.92, measured value: 5990.31 
     Nucleotide sequence: comprising a sequence complementary to nucleotide Nos. 3139-3157 of the human β-catenin gene (GenBank accession No. NM_001904.3) 
     Example 73 
     Synthesis of HO-T p -U m1p -G p -U m1p -G p -A m1p -T p -C m1p -C p -A m1p -T p -U m1p -C p -U m1p -T p -G m1p -T p -G m1t -H (SEQ ID NO: 82 the Sequence Listing) (CT-212) 
     CT-212 was synthesized in the same way as for Example 7. 
     Molecular weight: calculated value: 5700.74, measured value: 5701.15 
     Nucleotide sequence: comprising a sequence complementary to nucleotide Nos. 3140-3157 of the human β-catenin gene (GenBank accession No. NM_001904.3) 
     Example 74 
     Synthesis of HO-P(═O)(OH)—O-A p -T p -G m1p -T p -G m1p -A p -U m1p -C p -C m1p -A p -U m1p -T p -C m1p -T p -U m1p -G p -U m1p -G p -C m1p -T p -U m1t -H (SEQ ID NO: 83 of the Sequence Listing) (CT-243) 
     CT-243 was synthesized in the same way as for Example 42. 
     Molecular weight: calculated value: 6703.31, measured value: 6703.35 
     Nucleotide sequence: comprising a sequence complementary to nucleotide Nos. 3139-3156 of the human β-catenin gene (GenBank accession No. NM_001904.3) 
     Example 75 
     Synthesis of HO-P(═O)(OH)—O-G p -T p -G m1p -T p -U m1p -A p -U m1p -C p -C m1p -A p -U m1p -T p -C m1p -T p -U m1p -G p -U m1p -G p -C m1p -T p -U m1t -H (SEQ ID NO: 84 of the Sequence Listing) (CT-244) 
     CT-244 was synthesized in the same way as for Example 42. 
     Molecular weight: calculated value: 6719.13, measured value: 6719.46 
     Nucleotide sequence: comprising a sequence complementary to nucleotide Nos. 3139-3156 of the human iβ-catenin gene (GenBank accession No. NM_001904.3) 
     Example 76 
     Synthesis of HO-P(═O)(OH)—O-C p -T p -G m1p -T p -G m1p -A p -U m1p -C p -C m1p -A p -U m1p -T p -C m1p -T p -U m1p -G p -U m1p -G p -C m1p -T p -U m1t -H (SEQ ID NO: 85 of the Sequence Listing) (CT-245) 
     CT-245 was synthesized in the same way as for Example 42. 
     Molecular weight: calculated value: 6679.28, measured value: 6679.43 
     Nucleotide sequence: comprising a sequence complementary to nucleotide Nos. 3139-3156 of the human β-catenin gene (GenBank accession No. NM_001904.3) 
     Example 77 
     Synthesis of HO-P(═O)(OH)—O-T p -T p -G m1p -T p -G m1p -A p -U m1p -C p -C m1p -A p -U m1p -T p -C m1p -T p -U m1p -G p -U m1p -G p -C m1p -T p -U m1t -H (SEQ ID NO: 86 of the Sequence Listing) (CT-246) 
     CT-246 was synthesized in the same way as for Example 42. 
     Molecular weight: calculated value: 6694.29, measured value: 6694.49 
     Nucleotide sequence: comprising a sequence complementary to nucleotide Nos. 3139-3157 of the human β-catenin gene (GenBank accession No. NM_001904.3) 
     Example 78 
     Synthesis of HO-P(═O)(OH)—O-U m1p -T p -G m1p -T p -G m1p -A p -U m1p -C p -C m1p -A p -U m1p -T p -C m1p -T p -U m1p -G p -U m1p -G p -C m1p -G p -A m1t -H (SEQ ID NO: 87 of the Sequence Listing) (CT-247) 
     CT-247 was synthesized in the same way as for Example 42. 
     Molecular weight: calculated value: 6758.35, measured value: 6758.46 
     Nucleotide sequence: comprising a sequence complementary to nucleotide Nos. 3139-3157 of the human β-catenin gene (GenBank accession No. NM_001904.3) 
     Example 79) 
     Synthesis of HO-P(═O)(OH)—O-U m1p -T p -G m1p -A p -U m1p -C p -C m1p -A p -U m1p -T p -C m1p -T p -U m1p -G p -U m1p -G p -C m1p -G p -G m1t -H (SEQ ID NO: 88 of the Sequence Listing) (CT-248) 
     CT-248 was synthesized in the same way as for Example 42. 
     Molecular weight: calculated value: 6774.35, measured value: 6774.55 
     Nucleotide sequence: comprising a sequence complementary to nucleotide Nos. 3139-3157 of the human β-catenin gene (GenBank accession No. NM_001904.3) 
     Example 80 
     Synthesis of HO-P(═O)(OH)—O-U m1p -T p -G m1p -T p -G m1p -A p -U m1p -C p -C m1p -A p -U m1p -T p -C m1p -T p -U m1p -G p -U m1p -G p -C m1p -G p -C m1t -H (SEQ ID NO: 89 of the Sequence Listing) (CT-249) 
     CT-249 was synthesized in the same way as for Example 42. 
     Molecular weight: calculated value: 6734.32, measured value: 6734.35 
     Nucleotide sequence: comprising a sequence complementary to nucleotide Nos. 3139-3157 of the human β-catenin gene (GenBank accession No. NM_001904.3) 
     Example 81 
     Synthesis of HO-P(═O)(OH)—O-T p -T p -G m1p -T p -G m1p -A p -U m1p -C p -C m1p -A p -U m1p -T p -C m1p -T p -U m1p -G p -U m1p -G p -C m1p -G p -U m1t -H (SEQ ID NO: 90 of the Sequence Listing) (CT-253) 
     CT-253 was synthesized in the same way as for Example 42. 
     Molecular weight: calculated value: 6719.31, measured value: 6719.44 
     Nucleotide sequence: comprising a sequence complementary to nucleotide Nos. 3139-3157 of the human β-catenin gene (GenBank accession No. NM_001904.3) 
     Example 82 
     Synthesis of HO-P(═O)(OH)—O-T p -T p -G m1p -T p -G m1p -A p -U m1p -C p -C m1p -A p -U m1p -T p -C m1p -T p -U m1p -G p -U m1p -G p -C m1p -G p -A m1t -H (SEQ ID NO: 91 of the Sequence Listing) (CT-254) 
     CT-254 was synthesized in the same way as for Example 42. 
     Molecular weight: calculated value: 6742.35, measured value: 6742.45 
     Nucleotide sequence: comprising a sequence complementary to nucleotide Nos. 3139-3157 of the human β-catenin gene (GenBank accession No. NM_001904.3) 
     Example 83 
     Synthesis of HO-P(═O)(OH)—O-T p -T p -G m1p -T p -G m1p -A p -U m1p -C p -C m1p -A p -U m1p -T p -C m1p -T p -U m1p -G p -U m1p -G p -C m1p -G p -G m1t -H (SEQ ID NO: 92 of the Sequence Listing) (CT-255) 
     CT-255 was synthesized in the same way as for Example 42. 
     Molecular weight: calculated value: 6758.35, measured value: 6758.66 
     Nucleotide sequence: comprising a sequence complementary to nucleotide Nos. 3139-3157 of the human β-catenin gene (GenBank accession No. NM_001904.3) 
     Example 84 
     Synthesis of HO-P(═O)(OH)—O-T p -T p -G m1p -T p -G m1p -A p -U m1p -C p -C m1p -A p -U m1p -T p -C m1p -T p -U m1p -G p -U m1p -G p -C m1p -G p -C m1t -H (SEQ ID NO: 93 of the Sequence Listing) (CT-256) 
     CT-256 was synthesized in the same way as for Example 42. 
     Molecular weight: calculated value: 6718.32, measured value: 6718.59 
     Nucleotide sequence: comprising a sequence complementary to nucleotide Nos. 3139-3157 of the human β-catenin gene (GenBank accession No. NM_001904.3) 
     Example 85 
     Synthesis of HO-P(═O)(OH)—O-T p -T p -G m1p -T p -G m1p -A p -U m1p -C p -C m1p -A p -U m1p -T p -C m1p -T p -U m1p -G p -U m1p -G p -C m1p -A p -A m1t -H (SEQ ID NO: 94 of the Sequence Listing) (CT-257) 
     CT-257 was synthesized in the same way as for Example 42. 
     Molecular weight: calculated value: 6726.35, measured value: 6726.52 
     Nucleotide sequence: comprising a sequence complementary to nucleotide Nos. 3139-3157 of the human β-catenin gene (GenBank accession No. NM_001904.3) 
     Example 86 
     Synthesis of HO-P(═O)(OH)—O-T p -T p -G m1p -T p -G m1p -A p -U m1p -C p -C m1p -A p -U m1p -T p -C m1p -T p -U m1p -G p -U m1p -G p -C m1p -A p -G m1t -H (SEQ ID NO: 95 of the Sequence Listing) (CT-258) 
     CT-258 was synthesized in the same way as for Example 42. 
     Molecular weight: calculated value: 6742.35, measured value: 6742.54 
     Nucleotide sequence: comprising a sequence complementary to nucleotide Nos. 3139-3157 of the human β-catenin gene (GenBank accession No. NM_001904.3) 
     Example 87 
     Synthesis of HO-P(═O)(OH)—O-U p -T p -G m1p -T p -G m1p -A p -U m1p -C p -C m1p -A p -U m1p -T p -C m1p -T p -U m1p -G p -U m1p -G p -C m1p -T p -U m1t -H (SEQ ID NO: 96 of the Sequence Listing) (CT-264) 
     CT-264 was synthesized in the same way as for Example 42. However, the U p  moiety was synthesized using DMT-deoxyuridine-β-cyanoethyl phosphoramidite (DMT-dUridine Amidite, manufactured by Sigma-Aldrich Corp.). 
     Molecular weight: calculated value: 6680.27, measured value: 6680.29 
     Nucleotide sequence: comprising a sequence complementary to nucleotide Nos. 3139-3157 of the human β-catenin gene (GenBank accession No. NM_001904.3) 
     Example 88 
     Synthesis of HO-P(═O)(OH)—O-5meC p -T p -G m1p -T p -G m1p -A p -U m1p -C p -C m1p -A p -U m1p -T p -C m1p -T p -U m1p -G p -U m1p -G p -C m1p -T p -U m1t -H (SEQ ID NO: 97 of the Sequence Listing) (CT-265) 
     CT-265 was synthesized in the same way as for Example 42. However, the 5MeC p  moiety was synthesized using DMT-5-methyl-deoxycytidine(ac)-3-cyanoethyl phosphoramidite (5-Methyl-dC(ac) Amidite, manufactured by Sigma-Aldrich Corp.). 
     Molecular weight: calculated value: 6693.31, measured value: 6693.23 
     Nucleotide sequence: comprising a sequence complementary to nucleotide Nos. 3139-3156 of the human β-catenin gene (GenBank accession No. NM_001904.3) 
     Example 89 
     Synthesis of HO-P(═O)(OH)—O-C p -T p -G m1p -T p -G m1p -A p -U m1p -C p -C m1p -A p -U m1p -T p -C m1p -T p -U m1p -G p -U m1p -G p -C m1p -G p -G m1t -H (SEQ ID NO: 98 of the Sequence Listing) (CT-266) 
     CT-266 was synthesized in the same way as for Example 42. 
     Molecular weight: calculated value: 6743.34, measured value: 6743.78 
     Nucleotide sequence: comprising a sequence complementary to nucleotide Nos. 3139-3156 of the human β-catenin gene (GenBank accession No. NM_001904.3) 
     Example 90 
     Synthesis of HO-P(═O)(OH)—O-5meC p -T p -G m1p -T p -G m1p -A p -U m1p -C p -C m1p -A p -U m1p -T p -C m1p -T p -U m1p -G p -U m1p -G p -C m1p -G p -G m1t -H (SEQ ID NO: 99 of the Sequence Listing) (CT-267) 
     CT-267 was synthesized in the same way as for Example 88. 
     Molecular weight: calculated value: 6757.36, measured value: 6757.52 
     Nucleotide sequence: comprising a sequence complementary to nucleotide Nos. 3139-3156 of the human β-catenin gene (GenBank accession No. NM_001904.3) 
     Example 91 
     Synthesis of HO-G m1p -C p -A m1p -C p -A m1p -A p -G m1p -A p -A m1p -T p -G m1p -G p -A m1p -T p -C m1p -A p -C m1p -A t -H (SEQ ID NO: 102 of the Sequence Listing) (CT-288) 
     CT-288 was synthesized in the same way as for Example 7. 
     Molecular weight: calculated value: 5795.91, measured value: 5795.76 
     Nucleotide sequence: comprising a sequence of nucleotide Nos. 3139-3156 of the human β-catenin gene (GenBank accession No. NM_001904.3) 
     Example 92 
     Synthesis of HO-P(═O)(OH)—O-T p -U m1p -G p -U m1p -G p -A m1p -T p -C m1p -C p -A m1p -T p -U m1p -C p -U m1p -T p -G m1p -T p -G m1p -C p -T p -U m1t -H (SEQ ID NO: 103 of the Sequence Listing) (CT-289) 
     CT-289 was synthesized in the same way as for Example 42. 
     Molecular weight: calculated value: 6694.29, measured value: 6694.09 
     Nucleotide sequence: comprising a sequence complementary to nucleotide Nos. 3139-3157 of the human β-catenin gene (GenBank accession No. NM_001904.3) 
     Example 93 
     Synthesis of HO-G p -G m1p -A p -C m1p -A p -A m1p -G p -G m1p -A p -A m1p -G p -C m1p -T p -G m1p -C p -A m1p -G p -A m1t -H (SEQ ID NO: 104 of the Sequence Listing) (CT-278) 
     CT-278 was synthesized in the same way as for Example 7. 
     Molecular weight: calculated value: 5876.95, measured value: 5877.25 
     Nucleotide sequence: comprising a sequence of nucleotide Nos. 2137-2154 of the human β-catenin gene (GenBank accession No. NM_001904.3) 
     Example 94 
     Synthesis of HO-P(═O)(OH)—O-U m1p -T p -C m1p -T p -G m1p -C p -A m1p -G p -C m1p -T p -U m1p -C p -C m1p -T P -U m1p -G p -U m1p -C p -C m1p -T p -U m1t -H (SEQ ID NO: 105 of the Sequence Listing) (CT-279) 
     CT-279 was synthesized in the same way as for Example 42. 
     Molecular weight: calculated value: 6645.26, measured value: 6645.44 
     Nucleotide sequence: comprising a sequence complementary to nucleotide Nos. 2137-2155 of the human β-catenin gene (GenBank accession No. NM_001904.3) 
     Example 95 
     Synthesis of HO-P(═O)(OH)—O-T p -T p -C m1p -T p -G m1p -C p -A m1p -G p -C m1p -T p -U m1p -C p -C m1p -T p -U m1p -G p -U m1p -C p -C m1p -T p -U m1p -H (SEQ ID NO: 106 of the Sequence Listing) (CT-280) 
     CT-280 was synthesized in the same way as for Example 42. 
     Molecular weight: calculated value: 6629.26, measured value: 6629.51 
     Nucleotide sequence: comprising a sequence complementary to nucleotide Nos. 2137-2155 of the human β-catenin gene (GenBank accession No. NM_001904.3) 
     Example 96 
     Synthesis of HO-P(═O)(OH)—O-T p -T p -C m1p -T p -G m1p -C p -A m1p -G p -C m1p -T p -U m1p -C p -C m1p -T p -U m1p -G p -U m1p -C p -C m1p -A p -G m1p -H (SEQ ID NO: 107 of the Sequence Listing) (CT-281) 
     CT-281 was synthesized in the same way as for Example 42. 
     Molecular weight: calculated value: 6677.31, measured value: 6677.65 
     Nucleotide sequence: comprising a sequence complementary to nucleotide Nos. 2137-2155 of the human β-catenin gene (GenBank accession No. NM_001904.3) 
     Example 97 
     Synthesis of HO-G p -C m1p -C p -U m1p -C p -A m1p -G p -A m1p -T p -U m1p -C p -G m1p -T p -A m1p -G p -A m1p -A p -U m1p -A t -H (SEQ ID NO: 108 of the Sequence Listing) (DD-016) 
     DD-016 was synthesized in the same way as for Example 7. 
     Molecular weight: calculated value: 6040.00, measured value: 6040.44 
     Nucleotide sequence: comprising a sequence of nucleotide Nos. 1929-1947 of the DDX3 (DEAD (Asp-Glu-Ala-Asp) box polypeptide 3, X-linked) gene (GenBank accession No. NM_001356.3) 
     Example 98 
     Synthesis of HO-P(═O)(OH)—O-U m1p -A p -U m1p -T p -C m1p -T p -A m1p -C p -G m1p -A p -A m1p -T p -C m1p -T p -G m1p -A p -G m1p -G p -C m1p -T p -U m1p -H (SEQ ID NO: 109 of the Sequence Listing) (DD-017) 
     DD-017 was synthesized in the same way as for Example 42. 
     Molecular weight: calculated value: 6779.41, measured value: 6780.29 
     Nucleotide sequence: comprising a sequence complementary to nucleotide Nos. 1929-1947 of the DDX3 gene (GenBank accession No. NM_001356.3) 
     Example 99 
     Synthesis of HO-G p -C m1p -C p -U m1p -C p -A m1p -G p -A m1p -T p -U m1p -C p -G m1p -T p -A m1p -G p -A m1p -A p -U m1t -H (SEQ ID NO: 110 of the Sequence Listing) (DD-022) 
     DD-022 was synthesized in the same way as for Example 7. 
     Molecular weight: calculated value: 5726.79, measured value: 5726.89 
     Nucleotide sequence: comprising a sequence of nucleotide Nos. 1929-1946 of the DDX3 gene (GenBank accession No. NM_001356.3) 
     Example 100 
     Synthesis of HO-P(═O)(OH)—O-T p -A p -U m1p -T p -C m1p -T p -A m1p -C p -G m1p -A p -A m1p -T p -C m1p -T p -G m1p -A p -G m1p -G p -C m1p -T p -U m1p -H (SEQ ID NO: 111 of the Sequence Listing) (DD-023) 
     DD-023 was synthesized in the same way as for Example 42. 
     Molecular weight: calculated value: 6763.42, measured value: 6763.69 
     Nucleotide sequence: comprising a sequence complementary to nucleotide Nos. 1929-1947 of the DDX3 gene (GenBank accession No. NM_001356.3) 
     Example 101 
     Synthesis of HO-P(═O)(OH)—O-T p -A p -U m1p -T p -C m1p -T p -A m1p -C p -G m1p -A p -A m1p -T p -C m1p -T p -G m1p -A p -G m1p -G p -C m1p -A p -G m1t -H (SEQ ID NO: 112 of the Sequence Listing) (DD-024) 
     DD-024 was synthesized in the same way as for Example 42. 
     Molecular weight: calculated value: 6811.47, measured value: 6811.66 
     Nucleotide sequence: comprising a sequence complementary to nucleotide Nos. 1929-1947 of the DDX3 gene (GenBank accession No. NM_001356.3) 
     Example 102 
     Synthesis of HO-G s -C m1p -A p -C m1p -A p -A m1p -G p -A m1p -A p -U m1p -G p -G m1p -A p -U m1p -C s -A m1s -C s -A m1p -H (SEQ ID NO: 113 of the Sequence Listing) (CT-169S) 
     CT-169S is synthesized in the same way as for Example 36. CT-169S is identified by negative ion mass spectrometry. 
     Nucleotide sequence: comprising a sequence of nucleotide Nos. 3139-3156 of the human β-catenin gene (GenBank accession No. NM_001904.3) 
     Example 103 
     Synthesis of HO-P(═O)(OH)—O-U m1s -T s -G m1p -T p -G m1p -A p -U m1p -C p -C m1p -A p -U m1p -T p -C m1p -T p -U m1p -G p -U m1p -G s -C m1s -T s -U m1t -H (SEQ ID NO: 114 of the Sequence Listing) (CT-157S) 
     CT-157S is synthesized in the same way as for Example 42. However, the moiety having a phosphorothioate bond is treated with a 0.2 M phenylacetyl disulfide/pyridine-acetonitrile (1:1 v/v) solution for 3 minutes instead of oxidation with an iodine/tetrahydrofuran/pyridine/H 2 O solution (Ravikumar, V. T. et al. Bioorg. Med. Chem. Lett. (2006) 16, p. 2513-2517). CT-157S is identified by negative ion mass spectrometry. 
     Nucleotide sequence: comprising a sequence complementary to nucleotide Nos. 3139-3157 of the human β-catenin gene (GenBank accession No. NM_001904.3) 
     Reference Example 1 
     Synthesis of HO-C p -C m1p -A rp -C m1p -A rp -A m1p -G rp -A m1p -A rp -U m1p -G rp -G m1p -A rp -U m1p -C rp -A m1p -C rp -A m1p -A rp -T p -T t -H (SEQ ID NO: 44 of the Sequence Listing) (CT-001) 
     CT-001 was synthesized in the same way as for Example 1. 
     Molecular weight: calculated value: 6849.46, measured value: 6850.8 
     Nucleotide sequence: comprising a sequence of nucleotide Nos. 3139-3157 of the human β-catenin gene (GenBank accession No. NM_001904.3) 
     Reference Example 2 
     Synthesis of HO-U m1p -U rp -G m1p -U rp -G m1p -A rp -U m1p -C rp -C m1p -A rp -U m1p -U rp -C m1p -U rp -U m1p -G rp -U m1p -G rp -C m1p -T p -T t -H (SEQ ID NO: 45 of the Sequence Listing) (CT-005) 
     CT-005 was synthesized in the same way as for Example 1. 
     Molecular weight: calculated value: 6702.20, measured value: 6702.2 
     Nucleotide sequence: comprising a sequence complementary to nucleotide Nos. 3139-3157 of the human β-catenin gene (GenBank accession No. NM_001904.3) 
     Reference Example 3 
     Synthesis of HO-G rp -C rp -A rp -C rp -A rp -A rp -G rp -A rp -A rp -U rp -U rp -G rp -A rp -U rp -C rp -A rp -C rp -A rp -A rp -U rp -U rt -H (SEQ ID NO: 46 of the Sequence Listing) (CT-106) 
     CT-106 was synthesized in the same way as for Example 1. 
     Molecular weight: calculated value: 6727.16, measured value: 6726.73 
     Nucleotide sequence: comprising a sequence of nucleotide Nos. 3139-3157 of the human β-catenin gene (GenBank accession No. NM_001904.3) 
     Reference Example 4 
     Synthesis of HO-U rp -U rp -G rp -U rp -G rp -A rp -U rp -C rp -C rp -A rp -U rp -U rp -C rp -U rp -U rp -G rp -A rp -G rp -C rp -U rp -U rt -H (SEQ ID NO: 47 of the Sequence Listing) (CT-041) 
     CT-041 was synthesized in the same way as for Example 1. 
     Molecular weight: calculated value: 6565.88, measured value: 6565.34 
     Nucleotide sequence: comprising a sequence complementary to nucleotide Nos. 3139-3157 of the human β-catenin gene (GenBank accession No. NM_001904.3) 
     Reference Example 5) 
     Synthesis of HO-G rp -C rp -A rp -C rp -A rp -A rp -G rp -A rp -A rp -U rp -G rp -G p -A p -T p -C p -A p -C p -A p -A p -T p -T t -H (SEQ ID NO: 48 of the Sequence Listing) (CT-104) 
     CT-104 was synthesized in the same way as for Example 1. 
     Molecular weight: calculated value: 6609.25, measured value: 6608.98 
     Nucleotide sequence: comprising a sequence of nucleotide Nos. 3139-3157 of the human β-catenin gene (GenBank accession No. NM_001904.3) 
     Reference Example 6 
     Synthesis of HO-T p -T p -G p -T p -G p -A p -T p -C p -C rp -A rp -U rp -U rp -C rp -U rp -U rp -G rp -U rp -G rp -C rp -T p -T t -H (SEQ ID NO: 49 of the Sequence Listing) (CT-110) 
     CT-110 was synthesized in the same way as for Example 1. 
     Molecular weight: calculated value: 6490.05, measured value: 6489.61 
     Nucleotide sequence: comprising a sequence complementary to nucleotide Nos. 3139-3157 of the human β-catenin gene (GenBank accession No. NM_001904.3) 
     Reference Example 7 
     Synthesis of HO-G p -C p -A p -C p -A p -A p -G p -A p -A p -T p -G p -G p -A p -T p -C p -A p -C p -A p -A p -T p -T t -H (SEQ ID NO: 50 of the Sequence Listing) (CT-105) 
     CT-105 was synthesized in the same way as for Example 7. 
     Molecular weight: calculated value: 6447.28, measured value: 6447.58 
     Nucleotide sequence: comprising a sequence of nucleotide Nos. 3139-3157 of the human fβ-catenin gene (GenBank accession No. NM_001904.3) 
     Reference Example 8 
     Synthesis of HO-T p -T p -G p -T p -G p -A p -T p -C p -C p -A p -T p -T p -C p -T p -G p -T p -G p -C p -T p -T t -H (SEQ ID NO: 51 of the Sequence Listing) (CT-111) 
     CT-111 was synthesized in the same way as for Example 7. 
     Molecular weight: calculated value: 6384.19, measured value: 6384.05 
     Nucleotide sequence: comprising a sequence complementary to nucleotide Nos. 3139-3157 of the human β-catenin gene (GenBank accession No. NM_001904.3) 
     Reference Example 9 
     Synthesis of HO-G rp -C rp -A rp -C rp -A rp -A rp -G rp -A rp -A rp -U rp -C p -G rp -A 1P -U rp -C p -A rp -C p -A rp -A rt -H (SEQ ID NO: 100 of the Sequence Listing) (CT-125) 
     CT-125 was synthesized in the same way as for Example 1. 
     Molecular weight: calculated value: 6114.82, measured value: 6114.59 
     Nucleotide sequence: comprising a sequence of nucleotide Nos. 3139-3157 of the human β-catenin gene (GenBank accession No. NM_001904.3) 
     Reference Example 10 
     Synthesis of HO-U rp -U rp -G rp -U rp -G rp -A rp -U rp -C rp -C rp -A rp -U rp -U rp -C rp -U rp -U rp -G rp -U rp -G rp -C rt -H (SEQ ID NO: 101 of the Sequence Listing) (CT-126) 
     CT-126 was synthesized in the same way as for Example 1. 
     Molecular weight: calculated value: 5953.54, measured value: 5953.38). 
     Nucleotide sequence: comprising a sequence complementary to nucleotide Nos. 3139-3157 of the human β-catenin gene (GenBank accession No. NM_001904.3) 
     Reference Example 11 
     Synthesis of HO-G rp -G rp -A rp -C rp -A rp -A rp -G rp -G rp -A rp -A rp -G rp -C rp -U rp -G rp -C rp -A rp -G rp -A rp -A rp -T p -T t -H (SEQ ID NO: 115 of the Sequence Listing) (CT-165) 
     CT-165 was synthesized in the same way as for Example 1. 
     Molecular weight: calculated value: 6818.28, measured value: 6818.27 
     Nucleotide sequence: comprising a sequence of nucleotide Nos. 2137-2155 of the human β-catenin gene (GenBank accession No. NM_001904.3) 
     Reference Example 12 
     Synthesis of HO-U rp -U rp -C rp -U rp -G rp -C rp -A rp -G rp -C rp -U rp -U rp -C p -C rp -U rp -U rp -G rp -U rp -C rp -C rp -T p -T t -H (SEQ ID NO: 116 of the Sequence Listing) (CT-166) 
     CT-166 was synthesized in the same way as for Example 1. 
     Molecular weight: calculated value: 6496.90, measured value: 6496.99 
     Nucleotide sequence: comprising a sequence complementary to nucleotide Nos. 2137-2155 of the human β-catenin gene (GenBank accession No. NM_001904.3) 
     Example 104 
     Annealing for Formation of Double-Stranded Polynucleotide 
     The polynucleotides synthesized in the above Examples and Reference Examples were placed in the combinations shown in Tables 1 and 2 into one tube at concentrations of 300 pmol of each sense and antisense strand and dried under reduced pressure. 30 μL of an siRNA suspension buffer (QIAGEN) was added thereto, and the mixture was heated at 65° C. for 1 minute and then left at room temperature for 5 minutes for annealing of the sense and antisense strands to obtain a 10 μM double-stranded polynucleotide solution. 
     Reference Example 13 
     Double-stranded RNA was purchased from Ambion (Silencer Pre-designed siRNA, Gene Symbol: DDX3X, Locus ID: 1654 [Ambion] siRNA ID #145804) and used. Hereinafter, this RNA is referred to as DDX3 siRNA#5 (SEQ ID NOS: 117 and 118 of the 
     SEQUENCE LISTING 
     
       
         
           
               
               
               
               
               
             
               
                   
                 TABLE 1 
               
               
                   
                   
               
             
            
               
                   
                 CT-105 
                 CT-041 
                 CT-115 
                 CT-092 
               
               
                   
                 CT-106 
                 CT-111 
                 CT-135 
                 CT-092 
               
               
                   
                 CT-106 
                 CT-041 
                 CT-137 
                 CT-092 
               
               
                   
                 CT-091 
                 CT-092 
                 CT-119 
                 CT-120 
               
               
                   
                 CT-101 
                 CT-092 
                 CT-119 
                 CT-092 
               
               
                   
                 CT-102 
                 CT-092 
                 CT-091 
                 CT-120 
               
               
                   
                 CT-091 
                 CT-107 
                 CT-091 
                 CT-116 
               
               
                   
                 CT-091 
                 CT-108 
                 CT-091 
                 CT-117 
               
               
                   
                 CT-103 
                 CT-109 
                 CT-091 
                 CT-118 
               
               
                   
                 CT-095 
                 CT-096 
                 CT-113 
                 CT-092 
               
               
                   
                 CT-097 
                 CT-098 
                 CT-114 
                 CT-092 
               
               
                   
                 CT-099 
                 CT-100 
                 CT-115 
                 CT-092 
               
               
                   
                 CT-097 
                 CT-092 
                 CT-115 
                 CT-118 
               
               
                   
                 CT-091 
                 CT-098 
                 CT-136 
                 CT-092 
               
               
                   
                 CT-127 
                 CT-092 
                 CT-138 
                 CT-092 
               
               
                   
                 CT-128 
                 CT-092 
                 CT-001 
                 CT-092 
               
               
                   
                 CT-129 
                 CT-092 
                 CT-139 
                 CT-141 
               
               
                   
                 CT-101 
                 CT-092 
                 CT-140 
                 CT-141 
               
               
                   
                 CT-130 
                 CT-092 
                 CT-097S 
                 CT-098S 
               
               
                   
                 CT-131 
                 CT-092 
                 CT-114L 
                 CT-092 
               
               
                   
                 CT-112 
                 CT-092 
                 CT-091 
                 CT-149 
               
               
                   
                 CT-114 
                 CT-092 
               
               
                   
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 2 
               
             
            
               
                   
               
               
                 Double-stranded polynucleotide 
               
            
           
           
               
               
               
               
            
               
                 Sense strand 
                 Antisense strand 
                 Sense strand 
                 Antisense strand 
               
               
                   
               
               
                 CT-125 
                 CT-126 
                 CT-103 
                 CT-157 
               
               
                 CT-103 
                 CT-092 
                 CT-103 
                 CT-221 
               
               
                 CT-091 
                 CT-109 
                 CT-103 
                 CT-222 
               
               
                 CT-103 
                 CT-109 
                 CT-103 
                 CT-223 
               
               
                 CT-103 
                 CT-149 
                 CT-169 
                 CT-157 
               
               
                 CT-106 
                 CT-041 
                 CT-169 
                 CT-221 
               
               
                 CT-103 
                 CT-149 
                 CT-169 
                 CT-222 
               
               
                 CT-103 
                 CT-155 
                 CT-169 
                 CT-223 
               
               
                 CT-103 
                 CT-156 
                 CT-172 
                 CT-202 
               
               
                 CT-103 
                 CT-157 
                 CT-169 
                 CT-203 
               
               
                 CT-103 
                 CT-158 
                 CT-210 
                 CT-211 
               
               
                 CT-103 
                 CT-159 
                 CT-210 
                 CT-212 
               
               
                 CT-103 
                 CT-160 
                 CT-169 
                 CT-243 
               
               
                 CT-103 
                 CT-161 
                 CT-169 
                 CT-244 
               
               
                 CT-103 
                 CT-162 
                 CT-169 
                 CT-245 
               
               
                 CT-169 
                 CT-149 
                 CT-169 
                 CT-246 
               
               
                 CT-170 
                 CT-149 
                 CT-169 
                 CT-205 
               
               
                 CT-171 
                 CT-149 
                 CT-169 
                 CT-247 
               
               
                 CT-172 
                 CT-149 
                 CT-169 
                 CT-248 
               
               
                 CT-173 
                 CT-149 
                 CT-169 
                 CT-249 
               
               
                 CT-174 
                 CT-149 
                 CT-169 
                 CT-254 
               
               
                 CT-175 
                 CT-149 
                 CT-169 
                 CT-255 
               
               
                 CT-176 
                 CT-149 
                 CT-169 
                 CT-256 
               
               
                 CT-177 
                 CT-149 
                 CT-169 
                 CT-257 
               
               
                 CT-103 
                 CT-204 
                 CT-169 
                 CT-258 
               
               
                 CT-103 
                 CT-205 
                 CT-169 
                 CT-253 
               
               
                 CT-103 
                 CT-206 
                 CT-169 
                 CT-264 
               
               
                 CT-103 
                 CT-207 
                 CT-169 
                 CT-265 
               
               
                 CT-103 
                 CT-208 
                 CT-169 
                 CT-266 
               
               
                 CT-103 
                 CT-209 
                 CT-169 
                 CT-267 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 3 
               
             
            
               
                   
               
               
                 Double-stranded polynucleotide 
               
            
           
           
               
               
               
               
            
               
                 Sense strand 
                 Antisense strand 
                 Sense strand 
                 Antisense strand 
               
               
                   
               
               
                 CT-169 
                 CT-157 
                 CT-278 
                 CT-281 
               
               
                 CT-288 
                 CT-289 
                 DDX3 
                 siRNA #5 
               
               
                 CT-288 
                 CT-157 
                 DD-016 
                 DD-017 
               
               
                 CT-169 
                 CT-289 
                 DD-022 
                 DD-017 
               
               
                 CT-165 
                 CT-166 
                 DD-022 
                 DD-023 
               
               
                 CT-278 
                 CT-279 
                 DD-022 
                 DD-024 
               
               
                 CT-278 
                 CT-280 
                 CT-169S 
                 CT-157S 
               
               
                   
               
            
           
         
       
     
     Test Example 1 
     (1) Transfection 
     A human colon cancer SW480 cell strain (derived from human adenocarcinoma of the large intestine) was cultured in an RPMI1640 medium (Invitrogen Corp.) containing 10% fetal bovine serum. The culture solution of SW480 was seeded at a concentration of 100000 cells/well onto a 12-well plate and cultured overnight. Next, a lipofection reagent, HiPerFect Transfection Reagent (QIAGEN), at a final concentration of 0.5% and a double-stranded polynucleotide solution at a final concentration of 30, 3, 0.3, or 0.03 nM (or 30, 3, 1, 0.3, 0.1, or 0.03 nM) were added to each well, and the culture was further continued for 3 days. Then, the medium was removed, and the cells were washed with PBS (phosphate buffered saline) and then lysed by the direct addition of 100 pt of Laemmli Sample Buffer containing 5% 2-mercaptoethanol. The cell lysate was collected into a tube and then heated at 100° C. for 5 minutes to effect protein denaturation. The structures and nucleotide sequences of the double-stranded polynucleotides are shown in  FIGS. 1, 2, 4, 6, 7, 8, 9, 13, 14, 16, 18, 20 , and  21 . 
     (2) Western Blot Analysis 
     Each sample (1 μg in terms of protein amount) was separated by polyacrylamide gel electrophoresis (5-20% gradient gel) and electrically transferred to a nitrocellulose membrane. The membrane was blocked with a 5% skim milk solution. Then, β-catenin proteins were detected using rabbit anti-β-catenin antibodies (Cell Signaling Technology, Inc.) as primary antibodies and HRP-labeled anti-rabbit IgG antibodies (GE Healthcare Life Sciences) as secondary antibodies. β-actin proteins were detected as a negative control using anti-β-actin monoclonal antibodies (GE Healthcare Life Sciences) and HRP-labeled anti-mouse IgG antibodies (GE Healthcare Life Sciences). Each protein detection was performed by visualization based on High Performance Chemiluminescence Film (GE Healthcare Life Sciences) photosensitized with chemiluminescence generated with Western Lightning (PerkinElmer Life Sciences) as a substrate. 
     (3) Results of Western Blot Analysis 
     (a) Gene Expression Inhibitory Activities of Double-Stranded Polynucleotides Synthesized as Reference Examples 
     The experiment was carried out on a double-stranded polynucleotide consisting of the combination CT-106/CT-041 in which all nucleotides in the double-stranded polynucleotide consisted of RNAs (hereinafter, each double-stranded polynucleotide may be indicated only in the combination of sense and antisense strands, i.e., for example, the double-stranded polynucleotide consisting of the combination CT-106/CT-041 may be simply referred to as “CT-106/CT-041”), and on double-stranded polynucleotides CT-104/CT-110, CT-105/CT-111, CT-105/CT-041, and CT-106/CT-111 in which some or all of RNAs constituting the double-stranded polynucleotide were substituted by DNAs. The structures of these double-stranded polynucleotides are shown in  FIG. 1 . 
     As shown in  FIG. 3 , CT-106/CT-041 strongly inhibited the expression of the human β-catenin gene. On the other hand, CT-104/CT-110, CT-105/CT-111, CT-105/CT-041, and CT-106/CT-111 displayed little or no inhibition of the expression of the β-catenin gene. These results demonstrated that strong inhibitory effect on gene expression is lost by substituting DNAs for either or both of the sense and antisense strands of a double-stranded polynucleotide, the 3′ sequence of the sense strand, or the 5′ sequence of the antisense strand. This result was mostly consistent with the previously reported results (EMBO J., 20, p. 6877-6888 (2001); Nucleic Acids Res. 30, p. 1757-1766 (2002); and RNA, 9, p. 1034-1048, (2003)). 
     (b) Analysis of Gene Inhibitory Activities of Double-Stranded Polynucleotides-1- 
     CT-001/CT-005 comprising RNAs alternated with 2′-O-methyl RNAs, and DNAs in the overhang moiety (see  FIG. 2 ) inhibited the expression of the human β-catenin gene at a level equivalent to CT-106/CT-041 in which all nucleotides consisted of RNAs ( FIG. 3 ). In the subsequent experiments, CT-001/CT-005 was used as a control. 
     Double-stranded polynucleotides CT-091/CT-092, CT-095/CT-096, CT-097/CT-098, and CT-099/CT-100 in which RNAs in CT-001/CT-005 were partially (from the end) or wholly substituted by DNAs (for their structures, see  FIG. 4 ) were examined for their gene expression inhibitory activities. 
     As shown in  FIG. 5 , CT-091/CT-092, CT-095/CT-096, CT-097/CT-098, and CT-099/CT-100 strongly inhibited the expression of the human β-catenin gene. Particularly, CT-095/CT-096, CT-097/CT-098, and CT-099/CT-100 inhibited the gene expression at a level equivalent to CT-001/CT-005. This shows that a double-stranded polynucleotide comprising alternately located 2′ methyl RNAs or DNAs and partially having RNAs (or having no RNA in some cases) has strong gene expression inhibitory activity. 
     (c) Analysis of Gene Inhibitory Activities of Double-Stranded Polynucleotides-2- 
     Results of experiment on double-stranded polynucleotides CT-091/CT-092, CT-097/CT-092, and CT-091/CT-098 (see  FIG. 6 ) are shown in  FIG. 10 . 
     Both the double-stranded polynucleotides CT-097/CT-092 and CT-091/CT-098 strongly inhibited the expression of the β-catenin gene. Particularly CT-097/CT-092 inhibited the gene expression more strongly than the double-stranded polynucleotide CT-091/CT-098 and comparably to CT-001/CT-005. These results demonstrated that as seen in CT-097/CT-092, strong gene expression inhibitory activity can be retained even if a site other than the central portion of the sense strand of a double-stranded polynucleotide is substituted by 2′-O-methyl RNAs or DNAs. 
     (d) Analysis of Gene Inhibitory Activities of Double-Stranded Polynucleotides-3- 
     Results are shown in  FIGS. 10 and 11  as to double-stranded polynucleotides CT-127/CT-092, CT-128/CT-092, CT-129/CT-092, CT-101/CT-092, CT-130/CT-092, and CT-131/CT-092 in which a site other than the central portion of the sense strand of the double-stranded polynucleotide comprised 2′-O-methyl RNAs or DNAs and the central portion of the sense strand was substituted by 2′-O-methyl RNAs (see  FIG. 7 ). 
     The double-stranded polynucleotides CT-127/CT-092, CT-128/CT-092, CT-129/CT-092, CT-101/CT-092, CT-130/CT-092, and CT-131/CT-092 strongly inhibited the expression of the β-catenin gene. Particularly, CT-130/CT-092 exhibited gene expression inhibitory activity stronger than that of the double-stranded polynucleotide CT-091/CT-092 and comparable to that of CT-001/CT-005. 
     (e) Analysis of Gene Inhibitory Activities of Double-Stranded Polynucleotides-4- 
     Results are shown in  FIGS. 11, 12, and 17  as to double-stranded polynucleotides CT-112/CT-092, CT-114/CT-092, CT-132/CT-092, CT-133/CT-092, CT-134/CT-092, CT-115/CT-092, CT-135/CT-092, and CT-137/CT-092 (see  FIG. 8 ), CT-136/CT-092 and CT-138/CT-092 (see  FIG. 16 ) in which a site other than the central portion of the sense strand of the double-stranded polynucleotide comprised 2′-O-methyl RNAs or DNAs and ENAs were introduced in the central portion of the sense strand. 
     The double-stranded polynucleotides CT-114/CT-092, CT-132/CT-092, CT-133/CT-092, CT-134/CT-092, CT-115/CT-092, CT-135/CT-092, CT-137/CT-092, CT-136/CT-092, and CT-138/CT-092 strongly inhibited the expression of the β-catenin gene. Particularly, CT-114/CT-092 inhibited the expression of the (β-catenin gene more strongly than the double-stranded polynucleotide CT-091/CT-092 shown in  FIG. 10  and comparably to the double-stranded polynucleotide CT-001/CT-005 shown in  FIG. 10 . 
     (f) Analysis of Gene Inhibitory Activities of Double-Stranded Polynucleotides-5- 
     Double-stranded polynucleotides CT-102/CT-092, CT-091/CT-107, and CT-091/CT-108 (see  FIG. 2 ), CT-091/CT-116, CT-091/CT-117, and CT-091/CT-118 (see  FIG. 13 ), CT-113/CT-092 and CT-115/CT-118 (see  FIG. 14 ), in which a portion of the double-stranded polynucleotide CT-091/CT-092 was substituted by RNAs, ENAs, or 2′-O-methyl RNAs, were analyzed for their inhibitory activities on the expression of the human β-catenin gene, and the results are shown in  FIGS. 3 and 15 . All the double-stranded polynucleotides exhibited inhibitory activity on the expression of the human β-catenin gene equivalent to that of CT-091/CT-092. 
     (g) Comparison in Activity Based on Presence or Absence of Overhang Moiety of Double-Stranded Polynucleotide 
     The intensity of gene expression inhibitory activity was compared between a double-stranded polynucleotide CT-091/CT-092 and its overhang moiety-free double-stranded polynucleotide CT-103/CT-109 (see  FIG. 2 ). As shown in  FIG. 3 , CT-091/CT-092 having an overhang moiety inhibited the expression of the human β-catenin gene more strongly than overhang-free CT-103/CT-109. This shows that an overhang is important for designing a double-stranded polynucleotide. 
     (h) Analysis of Gene Inhibitory Activities of Double-Stranded Polynucleotides-6- 
     The inhibitory activities of CT-119/CT-120, in which the sequences of DNAs and 2′-O-methyl RNAs of the double-stranded polynucleotide CT-091/CT-092 were altered, and CT-119/CT-092 and CT-091/CT-120, in which the combinations of the sense and antisense strands of CT-091/CT-092 and CT-119/CT-120 were altered (see  FIG. 9 ), on the expression of the human β-catenin gene are shown in  FIG. 12 . CT-119/CT-120, CT-119/CT-092, and CT-091/CT-120 inhibited the expression of the β-catenin gene, whereas CT-091/CT-092 (see  FIG. 10 ) inhibited the gene expression more strongly. 
     Test Example 2 
     The intensity of gene expression inhibitory activity was compared among double-stranded polynucleotides as follows in the same way as in Test Example 1. 
     (a) Comparison in Activity Based on Presence or Absence of Overhang Moiety and 5′-Phosphate Group of Double-Stranded Polynucleotide 
     The intensity of gene expression inhibitory activity was compared in the same way as in Test Example 1 among a double-stranded polynucleotide CT-091/CT-092, a double-stranded polynucleotide CT-103/CT-109 having no overhang moiety at the 3′-ends of both the strands, CT-103/CT-092 having a 3′-terminal overhang moiety only in the antisense strand, CT-091/CT-109 having a 3′-terminal overhang moiety only in the sense strand, a double-stranded polynucleotide CT-091/CT-149 having an overhang moiety in both the strands and having a phosphate group at the 5′-end of the antisense strand, and a double-stranded polynucleotide CT-091/CT-149 having a 3′-terminal overhang moiety and a 5′-terminal phosphate group in the antisense strand (see  FIG. 22 ). 
     As shown in  FIG. 23 , CT-091/CT-092 and CT-103/CT-092 having an overhang moiety at the 3′-end of the antisense strand inhibited the expression of the human β-catenin gene more strongly than CT-103/CT-109 and CT-091/CT-109 having no overhang at the 3′-end of the antisense strand. Moreover, the double-stranded polynucleotide CT-091/CT-149 having a phosphate group at the 5′-end of the antisense strand inhibited the expression of the human β-catenin gene more strongly than the double-stranded polynucleotide CT-091/CT-092 having no 5′-terminal phosphate group. CT-103/CT-149 having a phosphate group at the 5′-end of the antisense strand inhibited the expression of the human β-catenin gene more strongly than the double-stranded polynucleotide CT-103/CT-092 having no 5′-terminal phosphate group. This shows that the 5′-terminal phosphate group and 3′-terminal overhang of an antisense strand are important for designing a double-stranded polynucleotide. 
     (b) Comparison in Activity Based on Difference in Nucleotides in 3′-Overhang Moiety of Antisense Strand of Double-Stranded Polynucleotide 
     A double-stranded polynucleotide CT-103/CT-149 having a 5′-terminal phosphate group in the antisense strand has a TT dimer sequence of DNAs in the 3′-overhang moiety of the antisense strand (CT-149). The intensity of gene expression inhibitory activity was compared among double-stranded polynucleotides CT-103/CT-155, CT-103/CT-156, CT-103/CT-157, CT-103/CT-158, CT-103/CT-159, CT-103/CT-160, CT-103/CT-161, and, CT-103/CT-162 in which the thymidine dimer of DNAs in the 3′-overhang moiety of the antisense strand (CT-149) was converted into a dimer of uridine, thymidine, and 2′-O-methyluridine in various possible combinations (see  FIG. 24 ). 
     As shown in  FIGS. 25 and 26 , CT-103/CT-155, CT-103/CT-156, CT-103/CT-157, CT-103/CT-158, CT-103/CT-159, CT-103/CT-160, CT-103/CT-161, and CT-103/CT-162 strongly inhibited the expression of the human β-catenin gene. Particularly CT-103/CT-157, CT-103/CT-158, CT-103/CT-159, CT-103/CT-160, CT-103/CT-161, and CT-103/CT-162 having 3′-terminal uridine or 2′-O-methyluridine more strongly inhibited the expression of the human β-catenin gene. 
     (c) Comparison in Activity Among Double-Stranded Polynucleotides Having 3′- or 5′-Terminally Truncated Sense Strand 
     The intensity of gene expression inhibitory activity was compared among double-stranded polynucleotides CT-169/CT-149, CT-170/CT-149, and CT-171/CT-149 in which 1 to 3 nucleotides were deleted from the 3′-end of the sense strand of the double-stranded polynucleotide CT-103/CT-149 (see  FIG. 27 ). As shown in  FIG. 29 , the double-stranded polynucleotide CT-169/CT-149 in which 1 nucleotide was deleted from the 3′-end of the sense strand strongly inhibited the expression of the human β-catenin gene, as with CT-103/CT-149. However, the double-stranded polynucleotides CT-170/CT-149 and CT-171/CT-149 in which 2 or 3 nucleotides were deleted from the 3′-end of the sense strand had reduced inhibitory activity on the expression of the human β-catenin gene, compared with CT-103/CT-149. 
     The intensity of gene expression inhibitory activity was compared among double-stranded polynucleotides CT-172/CT-149, CT-173/CT-149, and CT-174/CT-149 in which 1 to 3 nucleotides were deleted from the 5′-end of the sense strand of the double-stranded polynucleotide CT-103/CT-149 (see  FIG. 27 ). As shown in  FIGS. 29 and 30 , the double-stranded polynucleotide CT-172/CT-149 in which 1 nucleotide was deleted from the 5′-end of the sense strand strongly inhibited the expression of the human β-catenin gene, as with CT-103/CT-149. However, the double-stranded polynucleotides CT-173/CT-149 and CT-174/CT-149 in which 2 or 3 nucleotides were deleted from the 5′-end of the sense strand had reduced inhibitory activity on the expression of the human β-catenin gene, compared with CT-103/CT-149. 
     The intensity of gene expression inhibitory activity was compared among double-stranded polynucleotides CT-175/CT-149, CT-176/CT-149, and CT-177/CT-149 in which 1 or 2 nucleotides were deleted from the 3′ and 5′-ends of the sense strand of the double-stranded polynucleotide CT-103/CT-149 (see  FIG. 28 ). As shown in  FIGS. 29 and 30 , the double-stranded polynucleotide CT-175/CT-149 in which 1 nucleotide was deleted from each of the 3′ and 5′-ends of the sense strand strongly inhibited the expression of the human β-catenin gene, as with CT-103/CT-149. However, the double-stranded polynucleotides CT-176/CT-149 and CT-177/CT-149 in which 1 or 2 nucleotides were deleted from the 3′ and 5′-ends of the sense strand had reduced inhibitory activity on the expression of the human β-catenin gene, compared with CT-103/CT-149. 
     This demonstrated that even a double-stranded polynucleotide of 18 or 17 base pairs obtained by deleting 1 nucleotide from either or both of the 3′ and 5′-ends of a sense strand in its design exhibits gene expression inhibitory activity. 
     (d) Comparison in Activity Based on Difference in Nucleotide Sequence of 3′-Overhang Moiety in Antisense Strand of Double-Stranded Polynucleotide 
     A double-stranded polynucleotide CT-103/CT-157 having a 5′-terminal phosphate group in the antisense strand has a dimer sequence consisting of thymidine and 2′-O-methyluridine in the 3′-overhang moiety of the antisense strand (CT-157). The intensity of gene expression inhibitory activity was compared among double-stranded polynucleotides CT-103/CT-204, CT-103/CT-205, CT-103/CT-206, CT-103/CT-207, CT-103/CT-208, and CT-103/CT-209 in which the dimer sequence in the 3′-overhang moiety of the antisense strand (CT-157) was converted (see  FIG. 31 ). 
     As shown in  FIGS. 32 and 33 , CT-103/CT-204, CT-103/CT-205, CT-103/CT-206, CT-103/CT-207, CT-103/CT-208, and CT-103/CT-209 strongly inhibited the expression of the human β-catenin gene. 
     (e) Comparison in Activity Based on Difference in 5′-Terminal Nucleotide Sequence of Antisense Strand of Double-Stranded Polynucleotide 
     A double-stranded polynucleotide CT-103/CT-157 has 2′-O-methyluridine at the 5′-end of the antisense strand (CT-157). The intensity of gene expression inhibitory activity was compared among double-stranded polynucleotides CT-103/CT-221, CT-103/CT-222, and CT-103/CT-223 in which the base moiety of 5′-terminal 2′-O-methyluridine in the antisense strand (CT-157) was converted (see  FIG. 34 ). 
     As shown in  FIG. 35 , CT-103/CT-221, CT-103/CT-222, and CT-103/CT-223 strongly inhibited the expression of the human β-catenin gene at a level equivalent to CT-103/CT-157. 
     Furthermore, CT-169/CT-157 in which 1 nucleotide was deleted from the 3′-end of the sense strand of the double-stranded polynucleotide CT-103/CT-157 (see  FIG. 34 ) strongly inhibited the expression of the human β-catenin gene at a level equivalent to the double-stranded polynucleotide CT-103/CT-157 (see  FIG. 35 ). The intensity of gene expression inhibitory activity was compared among double-stranded polynucleotides CT-169/CT-221, CT-169/CT-222, and CT-169/CT-223 in which the base moiety of 5′-terminal 2′-O-methyluridine in the antisense strand (CT-157) of the double-stranded polynucleotide CT-169/CT-157 was converted (see  FIG. 34 ). 
     As shown in  FIG. 35 , CT-169/CT-221, CT-169/CT-222, and CT-169/CT-223 strongly inhibited the expression of the human β-catenin gene at a level equivalent to CT-169/CT-157. 
     (f) Gene Inhibitory Activities of Double-Stranded Polynucleotides Consisting of 18 Base Pairs 
     A double-stranded polynucleotide CT-103/CT-157 having a 5′-terminal phosphate group in the antisense strand has a duplex structure of 19 base pairs. The intensity of gene expression inhibitory activity was compared between a double-stranded polynucleotide CT-172/CT-202 in which 1 base pair was deleted from the 5′-end of the sense strand and the 3′-end of the antisense, and a double-stranded polynucleotide CT-169/CT-203 in which 1 base pair was deleted from the 3′-end of the sense strand and the 5′-end of the antisense strand (see  FIG. 36 ). 
     As shown in  FIG. 37 , CT-172/CT-202 and CT-169/CT-203 strongly inhibited the expression of the human β-catenin gene. 
     (g) Prevention of Off-Target of Asymmetric Double-Stranded Polynucleotide 
     Focusing on the terminal structures of a double-stranded polynucleotide CT-103/CT-157 having a 5′-terminal phosphate group in the antisense strand, the sense and antisense strands are asymmetric. Specifically, in the double-stranded polynucleotide CT-103/CT-157, the antisense strand has a 5′-terminal phosphate group and a 3′-terminal overhang structure, while the sense strand has neither 5′-terminal phosphate group nor 3′-terminal overhang structure. Thus, a double-stranded polynucleotide CT-210/CT-211, in which the sense strand was allowed to have a 5′-terminal phosphate group and a 3′-terminal overhang structure and the antisense strand was allowed to have neither 5′-terminal phosphate group nor 3′-terminal overhang structure, was synthesized (see  FIG. 38 ) to examine gene expression inhibitory activity depending on the antisense sequence having neither 5′-terminal phosphate group nor 3′-terminal overhang structure. As shown in  FIG. 39 , CT-210/CT-211 ended in slight reduction in activity compared with CT-103/CT-157. 
     In a double-stranded polynucleotide CT-169/CT-157, the antisense strand has a 5′-terminal phosphate group and a 3′-terminal overhang structure, while the sense strand has neither of them. Furthermore, it comprised 18 base pairs due to the deletion of 1 nucleotide from the 3′-end of the sense strand (CT-169). As in CT-103/CT-157, a double-stranded polynucleotide CT-210/CT-212, in which the sense strand was allowed to have a 5′-terminal phosphate group and a 3′-terminal overhang structure and the antisense strand was allowed to have neither 5′-terminal phosphate group nor 3′-terminal overhang structure, was synthesized (see  FIG. 38 ) to compare, with CT-169/CT-157, gene expression inhibitory activity depending on the antisense sequence having neither 5′-terminal phosphate group nor 3′-terminal overhang structure. 
     As shown in  FIG. 39 , CT-210/CT-212 had significantly reduced activity compared with CT-169/CT-157. This shows that, among double-stranded polynucleotides having 18 base pairs consisting of a polynucleotide strand having a 5′-terminal phosphate group and a 3′-terminal overhang structure and a polynucleotide strand which is truncated by 1 nucleotide, a polynucleotide strand having a 5′-terminal phosphate group and a 3′-terminal overhang structure has gene expression inhibitory activity but a polynucleotide strand having neither a 5′-terminal phosphate group nor a 3′-terminal overhang structure does not have gene expression inhibitory activity. 
     (h) Comparison in Activity Based on Substitution of DNA for 5′-Terminal Nucleotide in Antisense Strand of Asymmetric Double-Stranded Polynucleotide 
     An asymmetric double-stranded polynucleotide CT-169/CT-157 has 2′-O-methyluridine at the 5′-end of the antisense strand (CT-157). The intensity of gene expression inhibitory activity was compared among double-stranded polynucleotides CT-169/CT-243, CT-169/CT-244, CT-169/CT-245, and CT-169/CT-226 in which the 5′-terminal 2′-O-methyluridine in the antisense strand (CT-157) was substituted by a DNA (see  FIG. 40 ). 
     As shown in  FIG. 41 , CT-169/CT-243, CT-169/CT-244, and CT-169/CT-245 strongly inhibited the expression of the human β-catenin gene at a level equivalent to CT-169/CT-157 and CT-001/CT-005. Moreover, CT-169/CT-246 inhibited the expression of the human β-catenin gene more strongly than CT-169/CT-157 and CT-001/CT-005. 
     (i) Comparison in Activity Based on Difference in Nucleotide Sequence of 3′-Overhang Moiety in Antisense Strand of Asymmetric Double-Stranded Polynucleotide 
     An asymmetric double-stranded polynucleotide CT-169/CT-157 has a dimer sequence consisting of thymidine and 2′-O-methyluridine in the 3′-overhang moiety of the antisense strand. The intensity of gene expression inhibitory activity was compared among double-stranded polynucleotides CT-169/CT-205, CT-169/CT-247, CT-169/CT-248, and CT-169/CT-249 in which the dimer sequence of the 3′-overhang moiety in the antisense strand (CT-157) was converted (see  FIG. 42 ). 
     As shown in  FIG. 43 , CT-169/CT-205, CT-169/CT-247, and CT-169/CT-249 strongly inhibited the expression of the human β-catenin gene at a level equivalent to CT-169/CT-157 and CT-001/CT-005. Moreover, CT-169/CT-248 inhibited the expression of the human β-catenin gene more strongly than CT-169/CT-157 and CT-001/CT-005. 
     (j) Comparison in Activity Based on Substitution of DNA for 5′-Terminal Nucleotide of Antisense Strand of Asymmetric Double-Stranded Polynucleotide and Difference in Nucleotide Sequence of 3′-Overhang Moiety 
     An asymmetric double-stranded polynucleotide CT-169/CT-246 has thymidine at the 5′-end of the antisense strand (CT-246). The intensity of gene expression inhibitory activity was compared among double-stranded polynucleotides CT-169/CT-253, CT-169/CT-254, CT-169/CT-255, CT-169/CT-256, CT-169/CT-257, and CT-169/CT-258 in which the dimer sequence of the 3′-overhang moiety in the antisense strand (CT-246) was converted (see  FIG. 44 ). 
     As shown in  FIG. 45 , CT-169/CT-253, CT-169/CT-254, CT-169/CT-255, CT-169/CT-256, CT-169/CT-257, and CT-169/CT-258 strongly inhibited the expression of the human β-catenin gene at a level equivalent to or higher than CT-001/CT-005. 
     Moreover, the intensity of gene expression inhibitory activity was compared among double-stranded polynucleotides CT-169/CT-264, CT-169/CT-265, CT-169/CT-266, and CT-169/CT-267 in which the DNA as a 5′-terminal nucleotide in the antisense strand (CT-246) was substituted by 2′-deoxyuridine, 2′-deoxycytidine, or 5-methyl-2′-deoxycytidine and the dimer sequence of the 3′-overhang moiety was converted (see  FIG. 46 ). 
     As shown in  FIG. 47 , CT-169/CT-264, CT-169/CT-265, CT-169/CT-266, and CT-169/CT-267 strongly inhibited the expression of the human β-catenin gene at a level equivalent to or higher than CT-001/CT-005. 
     (Test Example 3) Test on IFN-α Production of Peripheral Mononuclear Cells Using siRNA 
     (a) Without the Use of Transfection Reagent 
     Peripheral mononuclear cells of healthy people were prepared by Ficoll-Paque density-gradient centrifugation. The prepared human peripheral mononuclear cells (2×10 5  cells/well) were cultured for 24 hours in the presence of a double-stranded polynucleotide using a 96-well plate, and IFN-α in the collected supernatant was assayed using an ELISA kit (Human IFN-α ELISA Kit, Pestka Biomedical Laboratories, Inc.). Various double-stranded polynucleotides were used as a solution containing PolyI:C (Sigma-Aldrich Corp.): 2 μg/ml, imiquimod (Invivogen): 10 −5  M, ssRNA40 (Invivogen): 2 μg/ml ssRNA41 (Invivogen): 2 μg/ml, ODN 2336 (Invivogen): 10 −5  M, ODN 2336 control (Invivogen): 10 −5  M, and one of unmodified double-stranded siRNA CT-106/041, double-stranded polynucleotide CT-103/157, or double-stranded polynucleotide CT-169/157: 10 −7 -10 −5  M. 
     As shown in  FIG. 48 , IFN-α production was observed for the unmodified siRNA CT-106/041 at 10 −5  M and however, not observed for the double-stranded polynucleotides CT-103/157 and CT-169/157. This result shows that the double-stranded polynucleotides can reduce IFN-α production and provide a polynucleotide with fewer adverse reactions. 
     (b) With the Use of Transfection Reagent 
     Peripheral mononuclear cells of healthy people were prepared by Ficoll-Paque density-gradient centrifugation. The prepared human peripheral mononuclear cells (1.1×10 6  cells/well) were cultured for 24 hours in the presence of a double-stranded polynucleotide using a 24-well plate, and IFN-α in the collected supernatant was assayed using an ELISA kit (Human IFN-α ELISA Kit, Pestka Biomedical Laboratories, Inc.). When a transfection reagent was used, the double-stranded polynucleotides (CT-103/157 and CT-169/157) and (CT-106/041) described in the Examples and Reference Examples were separately mixed in advance with Lipofectamine 2000 (Invitrogen Corporation), and each mixture was added to the cell culture solution. A solution containing PolyI:C (Sigma-Aldrich Corp.): 2 μg/ml, imiquimod (Invivogen): 10 −5  M, ssRNA40 (Invivogen): 2 μg/ml, ssRNA41 (Invivogen): 2 
     ODN 2336 (Invivogen): 10 −5  M, and ODN 2336 control (Invivogen): 10 −5  M was used as a standard compound without the use of the transfection reagent. A solution of unmodified siRNA CT-106/041, double-stranded polynucleotide CT-103/157, or double-stranded polynucleotide CT-169/157: 10 −7  M was tested using the transfection reagent. 
     As shown in  FIG. 49 , IFN-α production was observed for the unmodified siRNA CT-106/041 but not observed for the double-stranded polynucleotides CT-103/157 and CT-169/157. This shows that the polynucleotides of the present Examples can potentially reduce IFN-α production and provide a polynucleotide with fewer adverse reactions. 
     (Test Example 4) Test on Resistance to RNase 
     50 μmol of each of double-stranded polynucleotides CT-106/CT-041, CT-105/CT-111, CT-001/CT-005, CT-091/CT-092, CT-095/CT-096, CT-097/CT-098, CT-099/CT-100, CT-001/CT-092, and CT-104/CT-110 (see  FIG. 18 ) was brought to the total volume of 14.5 μL with RNase One buffer (10 mM Tris-HCl pH 7.5, 5 mM EDTA, 200 mM DTT, 200 mM sodium acetate, Promega) and treated at 37° C. by the addition of 5 U RNase One (0.5 μL, Promega). 
     A 4 μL aliquot was taken from each reaction solution after a lapse of 3 hours and 20 hours, supplemented with 1.0 μL of a loading solution (50% glycerol, 1 mM EDTA pH 8.0, 0.25% bromophenol blue, 0.25% xylene cyanol FF), and stored at −20° C. until analysis. 
     To analyze the degradation reaction of the double-stranded polynucleotide by RNase One, the sample was subjected to 20% polyacrylamide electrophoresis (1× Tris-Borate-EDTA, 200 V, 2 hours) and stained with SYBR Gold (Invitrogen Corp.). siRNA Ladder Markers manufactured by TAKARA BIO INC. were used as makers indicating the sizes of double-stranded nucleic acids. The stained gel was visualized with Molecular Imager FX Fluorescent Imager system (manufactured by Bio-Rad Laboratories Inc.). 
     CT-105/CT-111 in which all nucleotides constituting the double-stranded polynucleotide consisted of DNAs was not degraded by RNase ( FIG. 19 ). However, CT-105/CT-111 does not inhibit β-catenin expression, as shown in Test Example 1. 
     CT-106/CT-041 in which all nucleotides constituting the double-stranded polynucleotide consisted of RNAs, and CT-001/005 in which the duplex region consisted of RNAs or 2′-O-methyl RNAs exhibited the inhibition of β-catenin expression, as shown in Test Example 1, and were easily degraded by RNase (see  FIG. 19 ). 
     On the other hand, double-stranded RNA (CT-104/CT-110) having DNAs in the vicinity of the 3′-end of the sense strand and the 5′-end of the antisense strand, which was obtained by a modification method used in WO2003/044188, was also easily degraded by RNase. Furthermore, a double-stranded nucleic acid (CT-001/CT-092) having a sense strand comprising RNAs or 2′-O-methyl RNAs and an antisense strand comprising DNAs or 2′-O-methyl RNAs, which was obtained by a duplex region modification method used in WO2004/044136, was also easily degraded by RNase. 
     By contrast, CT-099/CT-100 corresponding to a form in which RNAs in CT-001/CT-005 were partially substituted by DNAs was only slightly degraded by RNase. CT-095/CT-096 and CT-097/CT-098 having further substitution by DNAs and CT-091/CT-092 having complete substitution of RNAs by DNAs were not degraded by RNase. 
     Test Example 5 
     The intensity of gene expression inhibitory activity was compared among double-stranded polynucleotides as follows in the same way as in Test Example 1. 
     (a) Comparison in Activity Based on Difference in DNA and 2′-O-Methyl RNA Modification Position in Asymmetric Double-Stranded Polynucleotide 
     The inhibitory activities of a double-stranded polynucleotide CT-169/CT-157, CT-288/CT-289 in which the sequences of DNA and 2′-O-methyl RNA modifications were altered, and CT-288/CT-157 and CT-169/CT-289 in which the combinations of the sense and antisense strands in CT-169/CT-157 and CT-288/CT-289 were altered (see  FIG. 50 ) on the expression of the human β-catenin gene are shown in  FIG. 51 . CT-169/CT-157 and CT-288/CT-157 inhibited the gene expression more strongly than CT-288/CT-289 and CT-169/CT-289. 
     (b) Gene Expression Inhibitory Activities of Double-Stranded Polynucleotides Targeting Different Sequences of Human β-Catenin Gene 
     Sequences different from the target sequence used in Test Examples 1 and 2 were selected from within the human β-catenin gene to study the inhibitory activities of double-stranded polynucleotides on the expression of the human β-catenin gene. CT-165/CT-166 having TT in the 3′-terminal overhang moiety and all the other nucleotides consisting of RNAs (see  FIG. 52 ) inhibited the expression of the human β-catenin gene, as shown in  FIG. 53 . Double-stranded polynucleotides CT-278/CT-279, CT-278/CT-280 and CT-278/CT-281 consisting of 2′-O-methyl RNAs or DNAs inhibited the expression of the human (β-catenin gene, as with CT-165/CT-166. 
     Test Example 6 
     The intensity of DDX3 gene expression inhibitory activity was compared among double-stranded polynucleotides as follows. 
     (1) Transfection 
     A human lung cancer NCI-H322 cell strain (derived from human bronchioloalveolar carcinoma) was adjusted to a concentration of 200000 cells/mL in an RPMI1640 medium (Invitrogen Corp.) containing 10% fetal bovine serum. Then, the solution was seeded at a concentration of 100 μL/well onto a 96-well flat-bottomed plate (manufactured by Corning Inc.) and cultured at 37° C. for 1 day under 5.0% CO 2  gas. A lipofection reagent Lipofectamine RNAiMAX (manufactured by Invitrogen Corp.) at a final concentration of 0.3 fit and a double-stranded polynucleotide solution at a final concentration of 5 nM were mixed in an RPMI1640 medium and left standing at room temperature for 20 minutes. The mixture was added to each well, and the culture was further continued for 3 days. 
     (2) Real-Time PCR 
     After the transfection, the culture supernatant was removed from each well, and mRNA was extracted using RNeasy Micro kit (manufactured by QIAGEN). The mRNA was quantified by real-time PCR as follows using a TaqMan probe for DDX3 (manufactured by Applied Biosystems), a TaqMan probe for β-actin (manufactured by Applied Biosystems) as an internal standard, and a real-time PCR kit (manufactured by QIAGEN) containing reagents necessary for PCR. 10 μL of QuantiTect Probe RT-PCR Master Mix, 4-8 μL at of RNase-Free Water, and 0.2 μL of QuantiTect RT Mix included in the real-time PCR kit were injected per well of a 96-well PCR plate (manufactured by Applied Biosystems). The solution was further brought to a total volume of 20 μL by the addition of 14 of the TaqMan probes and 4 μL, of the extracted mRNA solution and loaded in Mx3000P (manufactured by STRATAGENE), followed by PCR under the following conditions: 
     Reverse transcription reaction at 50° C. for 30 minutes 
     PCR initial activation at 95° C. for 15 minutes 
     PCR at 94° C. for 15 seconds 
     60° C. for 1 minute 
     This PCR cycle was repeated 48 times. A calibration curve was prepared by the UV quantification of mRNA extracted from cells treated only with the lipofection reagent. Based on the calibration curve, DDX3 and β-actin in each transfectant were quantified, and a value determined by dividing the amount of DDX3 by the amount of β-actin was plotted in a graph. Real-time PCR was conducted on N=3, and SE is shown in the graph (the structures and nucleotide sequences of the double-stranded polynucleotides are shown in  FIGS. 1, 2, 4, 6, 7, 8, 9, 13, 14, 16, 18, 20, and 21 ). 
     (3) Results of Real-Time PCR Analysis 
     A natural double-stranded polynucleotide against the human DDX3 gene (DDX3 siRNA#5) and double-stranded polynucleotides were studied by real-time PCR for their inhibitory activities on the expression of the human DDX3 gene. Double-stranded polynucleotides DD-016/DD-017, DD-022/DD-017, DD-022/DD-023, and DD-022/DD-024 comprising 2′-O-methyl RNAs or DNAs (see  FIG. 54 ) inhibited the expression of the human DDX3 gene at a level equivalent to the natural double-stranded polynucleotide (DDX3 siRNA#5), as shown in  FIG. 56 . 
     INDUSTRIAL APPLICABILITY 
     The present invention could provide a double-stranded polynucleotide that is resistant to RNase, can be synthesized at low cost, and has a genetic interference effect. 
     The double-stranded polynucleotide can be used in the functional analysis of genes, pharmaceutical compositions, etc. However, the industrial field of the present double-stranded polynucleotide is not limited as long as it can be used therein.