Extended sequence preferences for oligodeoxyribonucleotide activity.

Synthetic type B phosphorothioate oligodeoxyribonucleotides (ODN) activate mouse B cells via Toll-like receptor 9 (TLR9). Starting with closely related 15-mer prototype ODN, the sequence requirements for stimulatory (ST-) and inhibitory (IN-) activity were contrasted, by measuring apoptosis protection, G(1) entry and interleukin-6 secretion. ST-ODN and IN-ODN differ in that (1) ST-ODN require a 5' T, (2) the central CG is obligatory, (3) CG must be flanked 3' specifically by TT at the position where IN-ODN have GG, and (4) IN-ODN tolerate truncation of the 3' end better than ST-ODN. Features shared by ST-ODN and IN-ODN include (1) requiring CC adjacent to the 5' end, and (2) avoiding CC immediately 5' to the CG. This pattern is used to create a model of how ST-ODN binding might function to aggregate TLR9 so as to initiate the signal, and how the 5' ends of ST-ODN and IN-ODN compete for binding. Further justification for considering TLR9 to be the ODN receptor was provided by a demonstration that in HEK293 cells transfected with TLR9, the potency of a panel of ODN for activating NF-kappaB roughly parallels that seen in the biological assays in mouse B cells.