Sequence requirements for oligodeoxyribonucleotide inhibitory activity

Abstract

Inhibitory oligonucleotides (IN-ODN) differing from stimulatory CpG ODN (ST-ODN) by as few as two bases can block ST-ODN-induced proliferation, apoptosis protection and IL-6 secretion in B lymphocytes and the production of IL-12p40 by non-B cells. The main objective of this study was to determine the ODN sequence requirements for inhibition in mice. Starting with a strongly inhibitory 15-mer prototype phosphorothioate sequence, we tested the 60 sequences that differed from the prototype by one base, revealing the three areas that are critical for activity. Between these areas were the spacer sequences where base composition mattered little, but the number of bases was important. Truncation of three bases at the 3' end of the 15-mer and one at the 5' end was tolerated with minimal loss of activity. This approach yielded an 'optimal' sequence of 5' CC x notC notC xxGGGx or CC x notC notC xGGGxx 3', where x is any base. The sequence requirements for optimal inhibition of B cell responses to Type B (K) ODN and mixed splenocyte IL-12p40 responses to Type A (D) ODN were strikingly similar. Inhibition of ST-ODN by IN-ODN was competitive. A hypothetical model of the ODN-binding site is proposed. Synthetic IN-ODN with the sequence characteristics defined here should provide antidotes for excessive innate reactions to DNA.

Structure-Dependent Modulation of Alpha Interferon Production by Porcine Circovirus 2 Oligodeoxyribonucleotide and CpG DNAs in Porcine Peripheral Blood Mononuclear Cells

“…Recent reports have suggested possible competition between inhibitory and stimulatory ODNs. Specifically, it has been postulated that binding of inhibitory ODNs to TLR9 causes a conformational change of the MyD88 binding sites which interrupts further signaling through TLR9 (4,28). However, these studies were conducted using synthetic phosphorothioate ODNs, and ODNs with this backbone conformation have been demonstrated to exhibit unspecific binding to several proteins including TLRs (49).…”

TLR9 triggering in Burkitt's lymphoma cell lines suppresses the EBV BZLF1 transcription via histone modification

“…IRS869 competes with CpG2006 for binding to TLR9 (Ashman et al, 2005;Duramad et al, 2005), whereas chloroquine blocks the acidification and thus the maturation of endosomes (Yi et al, 1999). Indeed, both inhibitors reversed the suppressive effect of CpG2006 on BCR triggering-induced Data are represented as mean ± s.e.m.…”

Microbial nucleic acids pay a Toll in kidney disease

“…However, the immunostimulatory effects of CpG-DNA can be neutralized with synthetic ODN of certain inhibitory sequence motifs (7). We injected such G-rich inhibitory ODN into MRL lpr/lpr mice from week 11 to 24 of age.…”