Effect of chemical modifications of cytosine and guanine in a CpG-Motif ofoligonucleotides: Structure-immunostimulatory activity relationships

Oligodeoxynucleotides containing unmethylated CpG-motifs stimulate the inna te immune system, including inducing B-cell proliferation and cytokine prod uction. However, the mechanism of immunostimulation by CpG-oligonucleotides and the precise structural requirements and specific functional groups of cytosine and guanine necessary for recognition of and interaction with prot ein/receptor factors that are responsible for immune stimulation have not b een elucidated. We sought to understand the critical role of each functiona l group of the cytosine and guanine moieties in a CpG-motif in inducing imm unostimulatory activity. To this end, we examined structure-immunostimulato ry activity relationships of phosphorothio ate oligodeoxynucleotides (PS-ol igos) containing YpG- and CpR-motifs (Y and R stand for pyrimidine and puri ne analogues, respectively). The PS-oligos containing a YpG- motif in which the natural deoxycytidine was replaced with deoxy-5-hydroxycytidine or deo xy-N4-ethylcytidine showed immunostimulatory activity. Substitution of deox ycytidine with a deoxy-5-methylisocytidine, deoxyuridine, or deoxy-P-base-n ucleoside in the YpG-motif completely abolished the immune stimulatory acti vity, similar to the results observed with deoxy-5-methylcytidine. In the c ase of PS-oligos containing a CpR-motif, 7-deazaguanine substitution for na tural guanine showed immunostimulatory activity similar to that of a parent PS-oligo. These studies suggest that the 2-keto, 3-imino and 4-amino group s of cytosine, and the 1-imino, 2-amino and 6-keto groups of guanine in a C pG-motif are important for the immunostimulatory activity of CpG-PS-oligos. The absence of N7 on guanine of the CpG-motif does not affect immunostimul atory activity significantly. These studies suggest that it is possible to develop YpG- and CpR-motifs as an alternative to CpG-motifs in PS-oligos fo r immunostimulatory studies. (C) 2001 Published by Elsevier Science Ltd. Al l rights reserved.