CpG oligonucleotides with modified termini and nicked dumbbell structure show enhanced immunostimulatory activity.
A series of 21 phosphodiester oligodeoxyribonucleotides containing the core sequence 5'-GACGTT-3' or related control sequences were prepared and tested for their immunostimulatory effect on murine macrophages. The range of structural modifications tested included substituents at 3'- or 5'-termini, N3-methylation of thymidine residues, and hexaethylene glycol linkers favoring nicked or cyclic dumbbell duplexes. Lipophilic and cationic substituents at the termini failed to increase the release of TNF-alpha and nitric oxide, but two new types of modification were found that enhance the stimulation of RAW264.7 macrophages. One is the substitution of the 5'-terminal hydroxyl group with an amino group, and the other is the introduction of linkers favoring nicked duplexes. Even for sequences without linkers, UV-melting analysis and two-dimensional NMR showed that the core sequence 5'-GACGTT-3' readily forms a duplex. The cyclic derivative of the most active nicked dumbbell sequence is inactive, however. Together these results suggest a recognition of both the 5'-terminus and the core of the CpG oligonucleotides by the putative receptor(s) and provide an entry into a class of modified oligonucleotides whose activity rivals that of phosphorothioates, but consists of synthetic compounds that are single stereoisomers.
Narayanan
Sukunath
S
Institute for Organic Chemistry, University of Karlsruhe (TH), Fritz-Haber-Weg 6, D-76131 Karlsruhe, Germany.
Dalpke
Alexander H
AH
Siegmund
Karsten
K
Heeg
Klaus
K
Richert
Clemens
C
D000276:Adjuvants, Immunologic / Q000138:chemical synthesis* / Q000737:chemistry / Q000494:pharmacology; D000818:Animals; D001854:Bone Marrow Cells / Q000187:drug effects / Q000378:metabolism; D002460:Cell Line; D018899:CpG Islands; D003713:Dendritic Cells / Q000187:drug effects / Q000378:metabolism; D018664:Interleukin-12 / Q000378:metabolism; D053762:Interleukin-12 Subunit p40; D015850:Interleukin-6 / Q000378:metabolism; D008264:Macrophages / Q000187:drug effects / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D051379:Mice; D008807:Mice, Inbred BALB C; D009569:Nitric Oxide / Q000378:metabolism; D009841:Oligonucleotides / Q000138:chemical synthesis* / Q000737:chemistry / Q000494:pharmacology; D010783:Photometry; D021122:Protein Subunits / Q000378:metabolism; D012996:Solutions; D013237:Stereoisomerism; D013329:Structure-Activity Relationship; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism; D014466:Ultraviolet Rays
D000276:Adjuvants, Immunologic; D053762:Interleukin-12 Subunit p40; D015850:Interleukin-6; D009841:Oligonucleotides; D021122:Protein Subunits; D012996:Solutions; D014409:Tumor Necrosis Factor-alpha; D018664:Interleukin-12; D009569:Nitric Oxide