Design, synthesis, and immunostimulatory properties of CpG DNAs containing alkyl-linker substitutions: role of nucleosides in the flanking sequences.
Bacterial and synthetic DNA containing unmethylated CpG dinucleotides activate the innate immune system and promote Th1-like immune responses. Recently, a receptor, TLR9, has been shown to recognize CpG DNA and activate immune cascade. But there have been no reports on the molecular mechanisms of recognition between CpG DNA and the receptor(s). Our earlier studies described a number of the chemical and structural characteristics of CpG dinucleotide and the sequences flanking the CpG dinucleotide that are critical for immunostimulatory activity. In the present study, we examined the effect of the presence and absence of a nucleoside in the flanking sequences by replacing one or two natural deoxyribonucleosides at various positions with one or more alkyl- (C2-C12), branched alkyl- (glyceryl or aminobutyryl-propanediol), or ethyleneglycol- (tri or hexa) linkers. The results suggest that a linker substitution at the first two nucleoside positions adjacent to the CpG dinucleotide on the 5'- or the 3'-side neutralizes the immunostimulatory activity, as determined by in vitro mouse spleen cell proliferation, cytokine secretion, and in vivo mouse spleen enlargement. The same substitutions placed about three to six nucleotides away from the CpG dinucleotide either did not affect or potentiated immunostimulatory activity compared with parent CpG-DNA without modifications. Substitution of deoxyribonucleosides with a C3 or C4 alkyl-linker was found to be optimal for potentiating immunostimulatory activity.
Yu
Dong
D
Hybridon, Inc., 345 Vassar Street, Cambridge, Massachusetts 02139,USA.
Kandimalla
Ekambar R
ER
Cong
Yanping
Y
Tang
Jimmy
J
Tang
Jin-Yan
JY
Zhao
Qiuyan
Q
Agrawal
Sudhir
S
D000276:Adjuvants, Immunologic / Q000138:chemical synthesis* / Q000737:chemistry / Q000494:pharmacology; D000818:Animals; D002455:Cell Division / Q000187:drug effects; D002478:Cells, Cultured; D018899:CpG Islands; D004247:DNA / Q000737:chemistry*; D003853:Deoxyribonucleosides / Q000737:chemistry*; D019855:Ethylene Glycol / Q000737:chemistry; D007378:Interleukins / Q000378:metabolism; D008214:Lymphocytes / Q000166:cytology / Q000187:drug effects; D051379:Mice; D008807:Mice, Inbred BALB C; D009841:Oligonucleotides / Q000138:chemical synthesis* / Q000737:chemistry / Q000494:pharmacology; D009929:Organ Size / Q000187:drug effects; D013154:Spleen / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D013163:Splenomegaly / Q000139:chemically induced; D013329:Structure-Activity Relationship
D000276:Adjuvants, Immunologic; D003853:Deoxyribonucleosides; D007378:Interleukins; D009841:Oligonucleotides; D004247:DNA; D019855:Ethylene Glycol