Influence of backbone chemistry on immune activation by synthetic oligonucleotides

Depending on base sequence, DNA displays immunological activities relevant to the design of novel therapeutic agents. To determine the influence of ba ckbone structure on these activities, we tested a series of synthetic phosp hodiester and phosphorothioate oligonucleotides in in vitro cultures of mur ine spleen cells. These compounds were 30 bases long and consisted of eithe r a single base or an immunostimulatory sequence (AACGTT) flanked on 5' and 3' ends by 12 nucleotides of each base. Cell activation was assessed by bo th thymidine incorporation and expression of cell surface CD69; production of interleukin-6 and interleukin-12 was used as a measure of cytokine stimu lation. In these assays, phosphorothioate oligonucleotides induced much hig her levels of proliferation CD69 expression, and cytokine production than t he comparable phosphodiester compounds and had activity at lower concentrat ions. The sequence for optimal stimulation by phosphorothioates varied amon g responses, however. For example, whereas compounds containing an immunost imulatory sequence all induced similar levels of proliferation and CD69 exp ression, cytokine production was greatest with compounds with dA and dT fla nks. Furthermore, while single base dG oligonucleotides stimulated prolifer ation as both phosphodiesters and phosphorothioates, they failed to stimula te cytokine production. Together, these findings indicate that base sequenc e as well as backbone chemistry influence immune activation by synthetic ol igonucleotides, with the effects varying among responses. While suggesting differences in the structure-function relationships of nucleic acids in the ir immune activities, these findings also raise the possibility of the desi gn of agents with specific patterns of immune modulation. (C) 1999 Elsevier Science Inc.