Chiral nucleic acid adjuvant
View Patent ↗Problem The purpose of the present invention is to provide: a stereo isomer of a novel CpG oligonucleotide, which has excellent stability; and a CpG oligonucleotide which has a capability of producing interferon-α (IFNα). Solution The present invention relates to an oligonucleotide which contains two to four sequences each represented by the formula 5′-X 1 X 2 CpGX 3 X 4 -3′ (formula (I)) and has a length of 14 to 32 nucleotides. In formula (I), CpG represents a non-methylated CpG residue having a phosphate skeleton modification, X 1 X 2 represents any one of AA, AT, GA and GT, and X 3 X 4 represents any one of TT, AT, AC and CG. The oligonucleotide has at least one phosphate skeleton modification at an S-form stereoisomer located at a site other than the CpG.
1. An oligonucleotide which comprises two to four sequences each represented by 5′-X 1 X 2 CpGX 3 X 4 -3′ and has a length of 14 to 32 nucleotides,
wherein the CpG is non-methylated CpG without a modified phosphate backbone,
wherein X 1 X 2 is AA, AT, GA, or GT, and contains no modified phosphate backbones,
wherein X 3 X 4 is TT, AT, AC, TC, or CG, and contains no modified phosphate backbones, and
the oligonucleotide comprises at least one modified phosphate backbone at a site other than the parts represented by 5′-X 1 X 2 CpGX 3 X 4 -3′.
2. An oligonucleotide which comprises two to four sequences each represented by 5′-X 1 X 2 CpGX 3 X 4 -3′ and has a length of 14 to 32 nucleotides,
wherein the CpG is non-methylated CpG without a modified phosphate backbone,
wherein X 1 X 2 is AA, AT, GA, or GT, and may comprise a modified phosphate backbone,
wherein X 3 X 4 is TT, AT, AC, TA, TC, or CG, and may comprise a modified phosphate backbone, and
wherein the oligonucleotide comprises at least one Sp modified phosphate backbone at a site other than the parts represented by 5′-X 1 X 2 CpGX 3 X 4 -3′.
3. An oligonucleotide which comprises two to four sequences each represented by 5′-X 1 X 2 CpGX 3 X 4 -3′ and has a length of 14 to 32 nucleotides,
wherein the CpG is non-methylated CpG without a modified phosphate backbone,
wherein X 1 X 2 is AA, AT, GA, or GT, and may comprise a modified phosphate backbone,
wherein X 3 X 4 is TT, AT, AC, TA, TC, or CG, and may comprise a modified phosphate backbone, and
the oligonucleotide comprises at least one modified phosphate backbone at a site other than the parts represented by 5′-X 1 X 2 CpGX 3 X 4 -3′; and
wherein the oligonucleotide comprises one of the following sequences:
SEQ No.
Sequence
1
T*C*GTCGTT*T*T*GTCGTT*T*T*GTCGGG
2
G*G*GTCGTT*T*T*GTCGTT*T*T*GTCGGG
3
T*C*AACGTT*T*C*AACGTT*T*T
4
T*C*AACGTT*T*C*AACGTT*T*T*GG
5
T*C*AACGTT*T*C*AACGTT*G*G
6
T*C*AACGTT*T*C*AACGTT*G*G*G*G
7
T*C*AACGTT*T*T*AACGTT*T*T*AACGGG
8
T*C*AACGTT*T*A*ACGTT*T*T
9
T*C*AACGT*TAACGTT*T*T
10
T*C*AACGTT*T*A*AACGTT*T*A*AACGGG
11
T*C*AACGTTAACGTTAACGGG
12
T*C*GACGTT*T*T*GACGTT*T*T*GACGGG
15
G*G*GACGT*T*T*TGACGT*T*T*TGACGGGGG
16
T*C*GACGT*T*T*TGACGT*T*T*TGACGT*T*T*TGACGGG
17
T*C*GACGT*T*GACGT*T*GACGGG
20
T*C*GACGT*T*GACGT*T*GACGT*T*GACGGG
21
T*C*GACGTT*T*A*AACGTT*T*A*AACGTT*T*A*AACGGG
22
T*C*GACGTT*T*A*AACGTT*T*A*GACGTT*T*A*AACGGG
23
T*C*GACGTTAACGTTAACGTTAACGGG
24
GGGACGTT*T*A*AACGTCTAGACGGG
25
T*C*GACGT*ACGT*ACGT*ACGGG
26
T*C*GACGTT*T*T*GACGTT*T*T*G*A*C*G*G*G
29
T*C*GACGTT*T*T*GACGTT*T*T*GACGG*G*G
30
T*C*GACGTT*T*T*GACGTT*T*T*GACGT*G*G
31
T*C*GACGTT*T*T*GACGTT*T*T*GACGTG*G*G
32
T*C*GACGTT*T*T*GACGTT*T*T*G*A*C*G*G*G
33
T*C*GACGTT*T*T*GACGTT*T*T*GACGG*G*G*G*G
36
T*C*GACGTT*T*T*GACGTT*T*T*GACG*G*G*G*G
37
T*C*GACGT*T*GACGT*T*GACGTG*G*G
38
G*G*T*G*C*ATCGAT*G*C*A*G*G*G*G*G*G
39
T*C*ATCGAT*T*T*ATCGAT*T*T*ATCGGG
40
G*G*T*G*C*GACGAT*G*C*A*G*G*G*G*G*G
41
G*G*G*G*GACGATCGTCGGG*G*G*G
42
G*G*GACGATATCGTCG*G*G*G*G*G
43
G*G*GACGACGTCGTCG*G*G*G*G*G
46
G*G*GGGACGATCGTCG*G*G*G*G*G
47
G*G*GACGCGCGTCG*G*G*G*G*G*G*G
48
G*G*G*G*TCGTTCG*G*G*G
49
T*C*ATCGAT*T*T*ATCGAT*T*T*A*A*C*G*G*G
50
T*C*GACGTTTTGACGTT*T*T*G*A*C*G*G*G
51
T*C*GACGTTTTGACGTTTT*G*A*C*G*G*G
52
T*C*GACGT*T*GACGT*T*GACGG*G
53
TC*GACGT*T*GACGT*T*GACG*G*G
54
T*C*GACGT*T*GACGT*T*GACGG*G*G
55
T*C*GACGT*T*GACGT*T*GACT*G
56
T*C*GACGT*T*GACGT*T*G*A*C*G*G*G
57
T*C*GACGTTGACGT*T*G*A*C*G*G*G
58
T*C*ATCGATATCGA*T*G*A*C*G*G*G
59
T*C*GACGT*T*GACGT*T*GACG*G*G*G
60
T*C*GACGTT*T*T*GACGTT*T*T*G*G*G*G*G
61
T*C*GACGTT*T*T*GACGTT*T*T*G*A*G*G*G*G
62
T*C*GACGTT*T*T*GACGTT*T*T*G*T*G*G*G*G
63
T*C*G*ACGTT*G*ACGTT*G*A*C*G*G*G
64
C*C*GACGTT*T*T*GACGTT*T*T*GACG*G*G
65
T*C*GACGTT*T*A*GACGTT*T*A*GACG*G*G
66
T*C*GACGTT*T*T*GACGTT*T*T*GACG*A*A
67
T*C*GACGTT*T*T*GACGTT*T*T*GACG*T*T
68
T*C*AACGTT*T*T*AACGTT*T*T*GACG*G*G
69
T*C*GACGTT*T*T*GACGTT*T*T*GGG
70
T*C*GACGTT*T*T*GACGTT*T*T*GACGTTGG
71
T*C*GACGTT*GACGTT*G*G*G
72
T*C*GACGTT*T*T*T*ACGTT*T*T*G*ACG*G*G
73
T*C*G*A*CGTT*T*T*ACGTTTTGACGGG
74
T*C*G*ACGTT*T*T*G*ACGTT*T*T*G*ACG*G*G
75
T*C*GACGTA*GACGTA*GACG*G*G
76
T*A*GACGAT*T*C*GTCGTC*T*A*GACG*G*G
77
T*A*GACGA*C*GTCGT*A*GACC*G*G
78
T*C*G*ACGTTT*T*G*ACGTT*T*T*G*A*C*G*G*G
79
T*C*G*ACGTT*T*T*T*A*ACGAC*T*T*G*A*C*G*G*G
80
T*C*G*ACGTTT*T*AACGAC*T*T*G*A*C*G*G*G
81
T*C*ATCGAT*T*T*ATCGAT*T*T*GACG*G*G
82
T*C*ATCGAT*T*T*ATCGAT*T*T*ATCGA*T*G*G*G
83
T*C*ATCGAT*T*T*ATCGAT*T*T*AT*C*G*G*G
84
T*C*ATCGAT*T*T*ATCGAT*T*T*ATCGAT*T*T*ATCG*
G*G
85
T*C*ATCGAT*T*T*ATCGAT*T*T*ATCGAT*T*T*A*T*C*
G*G*G
86
T*C*ATCGAT*T*T*ATCGAT*T*T*ATCGAT*A*T*C*G*
G*G
87
T*T*ATCGAT*T*T*ATCGAT*T*T*G*A*C*G*G*G
88
T*C*ATCGATATCGAT*T*T*G*A*C*G*G*G
89
TCATCGAT*T*T*ATCGAT*T*T*A*T*C*G*G*G
90
T*C*ATCGAT*T*T*ATCGAT*T*T*G*A*C*G*A*T
91
T*C*GACGT*T*GACGT*T*GACGT*T*G*G*G
92
T*C*G*ACGT*T*G*ACGT*T*G*A*C*G*G*G
93
T*C*A*TCGAT*T*T*A*TCGAT*T*T*G*A*C*G*G*G
94
T*C*A*TCGAT*A*TCGAT*G*ACGT*T*T*G*G*G
95
T*C*GACGTTTGACGTTT*G*A*C*G*G*G
96
T*C*ATCGAT*T*T*ATCGAT*T*T*A*T*C*G*G*G
97
G*G*GACGATATCGTCG*G*G*G*G*G
98
G*G*GACGAC*G*TCGTCG*G*G*G*G*G
99
G*G*GACGACGTCGTCG*G*G*G*G
100
T*C*GACGACGTCGTCG*G*G*G*G*G
101
T*C*GACGACGTCGTCT*T*T*G*G*G
102
T*A*GACGACGTCGTCT*T*T*G*G*G
103
T*T*GACGACGTCGTCA*A*A*G*G*G
104
T*C*GACGTAGACGTCT*T*T*G*G*G
105
T*C*GACGTAGACGTTT*A*G*G*G*G
106
T*C*ATCGATATCGATT*T*T*G*G*G
107
T*T*ATCGATATCGATA*A*A*G*G*G
108
T*C*GACGTAGACGATCGA*T*G*G*G
109
T*C*GACGAC*T*T*GACGAC*T*T*G*A*C*G*G*G
110
T*C*GACGAC*T*T*GTCGTC*T*T*G*A*C*G*G*G
111
T*T*ATCGATATCGATA*T*C*G*A*T*G*G*G
112
T*T*ATCGATATCGATT*T*A*A*A*G*G*G
113
T*C*ATCGAT*T*T*ATCGAT*T*T*G*A*C*G*T*T
114
T*C*ATCGA*T*ATCGA*T*G*A*C*G*G*G*G
115
T*C*ATCGAT*ATCGA*T*G*G*G
116
T*C*GTCGTTGTCGT*T*G*A*C*G*G*G
117
T*C*G*TCGTT*T*T*G*TCGTT*T*T*G*A*C*G*G*G
118
T*C*GTCGTTGTCGTTG*A*C*G*A*C*G*G*G
wherein in the above formula, * refers to a modified phosphate backbone and at least one * in each formula is Sp, and
in the above formula, CG at a site corresponding to 5′-X 1 X 2 CpGX 3 X 4 -3′ means non-methylated CpG without a modified phosphate backbone.
4. An oligonucleotide which comprises two to four sequences each represented by 5′-X 1 X 2 CpGX 3 X 4 -3′ and has a length of 14 to 32 nucleotides,
wherein the CpG is non-methylated CpG without a modified phosphate backbone,
wherein X 1 X 2 is AA, AT, GA, or GT, and may comprise a modified phosphate backbone,
wherein X 3 X 4 is TT, AT, AC, TA, TC, or CG, and may comprise a modified phosphate backbone,
wherein the oligonucleotide comprises at least one modified phosphate backbone at a site other than the parts represented by 5′-X 1 X 2 CpGX 3 X 4 -3′, and
wherein the oligonucleotide comprises a sequence represented as -(G) m -, wherein m is an integer of 1 to 6, at 3′ end side of a CpG motif, the CpG motif being 5′-X 1 X 2 CpGX 3 X 4 -3′.
5. An oligonucleotide which comprises two to four sequences each represented by 5′-X 1 X 2 CpGX 3 X 4 -3′ and has a length of 14 to 32 nucleotides,
wherein the CpG is non-methylated CpG without a modified phosphate backbone,
wherein X 1 X 2 is AA, AT, GA, or GT, and may comprise a modified phosphate backbone,
wherein X 3 X 4 is TT, AT, AC, TA, TC, or CG, and may comprise a modified phosphate backbone,
wherein the oligonucleotide comprises at least one modified phosphate backbone at a site other than the parts represented by 5′-X 1 X 2 CpGX 3 X 4 -3′, and
wherein the oligonucleotide comprises a sequence represented as TC, TA, TG, CC or CC at 5′ end side of a CpG motif, the CpG motif being the sequence of 5′-X 1 X 2 CpGX 3 X 4 -3′.
6. An oligonucleotide which comprises two to four sequences each represented by 5′-X 1 X 2 CpGX 3 X 4 -3′ and has a length of 14 to 32 nucleotides,
wherein the CpG is non-methylated CpG without a modified phosphate backbone,
wherein X 1 X 2 is AA, AT, GA, or GT, and may comprise a modified phosphate backbone,
wherein X 3 X 4 is TT, AT, AC, TA, TC, or CG, and may comprise a modified phosphate backbone,
wherein the oligonucleotide comprises at least one modified phosphate backbone at a site other than the parts represented by 5′-X 1 X 2 CpGX 3 X 4 -3′, and
wherein the oligonucleotide comprises at least a first CpG motif and a second CpG motif, the first and the second CpG motifs being 5′-X 1 X 2 CpGX 3 X 4 -3′,
wherein the first CpG motif and the second CpG motif are bonded directly with no sequence between them, or
wherein the oligonucleotide comprises a sequence represented as -(T) n -, wherein n is an integer of 1 to 3, TA or TC between the first CpG motif and the second CpG motif.
7. The oligonucleotide as claimed in claim 3 , wherein the oligonucleotide comprises a sequence selected from Sequence Nos. 12, 17, 78, 43, 60, 67, 102, 103, 112 and 116.