3556 Nucleic Acids Research, 2005, Vol. 33, No. 11 Scheme 3. Tentative mechanism of the thermolytic cleavage of 2-(N-formyl-N- methyl)aminoethyl thiophosphate protecting groups from CpG ODN fma1555. (i) NH3 gas ( 10 bar), 12 h, 25 C; (ii) 0.1 M TEAA (pH 7.0) or 1· PBS (pH 7.2), 90 C, 3 h. mechanism presumably proceeds through a cyclodeesteriﬁca- tion mechanism (Scheme 3) that is typically observed with many of the thermosensitive phosphate/thiophosphate protect- ing groups investigated earlier (2,4,6,7). PAGE analysis of puriﬁed and fully deprotected CpG ODN fma1555, ODN fma1556 and CpG ODN 1555 showed that each oligonucleotide migrated on the gel as a homogeneous band. A photograph of the stained gel is shown in Figure 2. Although deprotected CpG ODN 1555 and CpG ODN fma1555 exhibited an identical electrophoretic mobility indic- ative of an identical DNA sequence, deprotected ODN 1556 displayed a slightly different mobility. It should be understood that the electrophoretic mobility of a synthetic oligonucleotide depends more on its base composition than its length (42). Since ODN 1556 has a different DNA sequence than that of CpG ODN 1555, its electrophoretic mobility was expected to differ from that of CpG ODN 1555 in spite of an identical chain length. The immunostimulatory properties of CpG ODN fma1555 were evaluated in vivo in mice and compared with those of CpG ODN 1555, which were described earlier (43). Prior studies had shown that treatment with CpG ODNs induced an increase in the number of cytokine secreting cells (44). Given that the thermolytic deprotection of CpG ODN fma1555 proceeds with a half-time of 73 h at 37 C, expression of its immunoprotective properties should be delayed s1400 e t y c1200 o n e l p1000 s 6 0 1 800 / 6 - IL 600 g n ite 400 r c e s 200 s l l e 0 C s500 e t y c o en400 l p s 6 10300 / 2 1 - IL200 g n i t e rc100 e s s l l eC 0 Saline CpG ODN 1555 CpG ODN fma 1555 IL- 6 * * * 0 24 72 120 Time post administration (hr) * IL-12 0 24 72 120 Time post administration (hr) Figure 3. Kinetics of cytokine secretion in mice treated with CpG ODN fma1555. Female Balb/c mice (6–12 weeks of age, 3–6/group) were adminis- tered CpG ODN 1555 or CpG ODN fma1555 (100 mg/mouse) intraperitoneally and then euthanized 24, 72 or 120 h later. Shown in this graph are the number of cells secreting IL-6 and IL-12 (p40/p70) ex vivo, as determined by ELIspot for each time point. relative to that of CpG ODN 1555. As shown in Figure 3, splenocytes from Balb/c mice treated with CpG ODN fma1555 had a delayed increase in IL-6 and IL-12 secreting cells when compared to mice that received CpG ODN 1555. Importantly, the number of IL-12 secreting cells was still increased 120 h after treatment, as compared to mice receiving CpG ODN 1555. To assess whether the number of cytokine secreting cells was dose dependent, mice were treated intraperitoneally with CpG ODN 1555 or fma1555 (30, 100 or 300 mg/mouse, 3 mice/group) and euthanized 72 h after treatment. As shown in Figure 4, there is a direct correlation between the dose