Putta Mallikarjuna Reddy, Zhu FuGang, Li Yukui, Bhagat Lakshmi, Cong YanPing, Kandimalla Ekambar R, Agrawal Sudhir
Idera Pharmaceuticals, Inc. 345 Vassar Street, Cambridge, MA 02139, USA.
Nucleic Acids Res. 2006 Jun 23;34(11):3231-8. doi: 10.1093/nar/gkl430. Print 2006.
Synthetic oligodeoxynucleotides containing unmethylated CpG motifs activate Toll-Like Receptor 9 (TLR9). Our previous studies have shown the role of hydrogen-bond donor and acceptor groups of cytosine and guanine in the CpG motif and identified synthetic immunostimulatory motifs. In the present study to elucidate the significance of N3-position of cytosine and N1-position of guanine in the CpG motif, we substituted C or G of a CpG dinucleotide with N3-Me-cytosine or N1-Me-guanine, respectively, in immunomodulatory oligodeoxynucleotides (IMOs). IMOs containing N-Me-cytosine or N-Me-guanine in C- or G-position, respectively, of the CpG dinucleotide showed activation of HEK293 cells expressing TLR9, but not TLR3, 7 or 8. IMOs containing N-Me-cytosine or N-Me-guanine modification showed activity in mouse spleen cell cultures, in vivo in mice, and in human cell cultures. In addition, IMOs containing N-Me-substitutions reversed antigen-induced Th2 immune responses towards a Th1-type in OVA-sensitized mouse spleen cell cultures. These studies suggest that TLR9 tolerates a methyl group at N1-position of G and a methyl group at N3-position of C may interfere with TLR9 activation to some extent. These are the first studies elucidating the role of N3-position of cytosine and N1-position of guanine in a CpG motif for TLR9 activation and immune stimulation.
含有未甲基化CpG基序的合成寡脱氧核苷酸可激活Toll样受体9（TLR9）。我们之前的研究已经表明了CpG基序中胞嘧啶和鸟嘌呤的氢键供体和受体基团的作用，并鉴定出了合成免疫刺激基序。在本研究中，为了阐明CpG基序中胞嘧啶的N3位和鸟嘌呤的N1位的重要性，我们在免疫调节寡脱氧核苷酸（IMO）中分别用N3-甲基胞嘧啶或N1-甲基鸟嘌呤取代了CpG二核苷酸中的C或G。在CpG二核苷酸的C或G位置分别含有N-甲基胞嘧啶或N-甲基鸟嘌呤的IMO可激活表达TLR9的HEK293细胞，但不能激活TLR3、7或8。含有N-甲基胞嘧啶或N-甲基鸟嘌呤修饰的IMO在小鼠脾细胞培养物、小鼠体内以及人细胞培养物中均表现出活性。此外，在卵清蛋白致敏的小鼠脾细胞培养物中，含有N-甲基取代的IMO可将抗原诱导的Th2免疫反应逆转至Th1型。这些研究表明，TLR9可耐受G的N1位上的甲基，而C的N3位上的甲基可能在一定程度上干扰TLR9的激活。这些是首次阐明CpG基序中胞嘧啶的N3位和鸟嘌呤的N1位在TLR9激活和免疫刺激中的作用的研究。
