Idera Pharmaceuticals, Inc., Cambridge, Massachusetts 02139, USA.
Bioconjug Chem. 2010 Jan;21(1):39-45. doi: 10.1021/bc900425s.
Bacterial and synthetic DNA containing unmethylated CpG motifs act as ligands of Toll-like receptor 9 (TLR9). Our earlier studies showed that 5'-accessibility of synthetic oligodeoxynucleotides containing CpG motif (ODN) is required for TLR9-mediated immune stimulatory activity. Blocking the 5'-end of ODN through conjugation to a variety of moieties reduces immune stimulatory activity (Bioconjugate Chem. 2002, 13, 966-974). In the present study, we conjugated a model peptide, a 28-amino-acid-long beta-amyloid peptide, to either the 5'- or the 3'-end of an ODN via C3 and C6 alkyl linkers. We compared the immune stimulatory activity of the resulting conjugates with that of a parent ODN without conjugation in TLR9-transfected cells, mouse spleen cell cultures, and in vivo in mice. ODN with the peptide conjugated at the 3'-end via C3 and C6 linkers had immune stimulatory activity similar to that of the parent ODN in both in vitro and in vivo in mice. On the contrary, conjugation of peptide at the 5'-end of the ODN significantly abrogated immune stimulatory activity. In conclusion, the results presented here demonstrate that peptide/protein conjugation to ODN is optimal at the 3'-end with either C3 or C6 linker and conjugation at the 5'-end leads to significant loss of TLR9-mediated immune stimulation.
细菌和含有未甲基化 CpG 基序的合成 DNA 可作为 Toll 样受体 9（TLR9）的配体。我们之前的研究表明，含有 CpG 基序的合成寡脱氧核苷酸（ODN）的 5'可及性是 TLR9 介导的免疫刺激活性所必需的。通过与各种基团缀合来阻断 ODN 的 5'端会降低免疫刺激活性（Bioconjugate Chem. 2002, 13, 966-974）。在本研究中，我们通过 C3 和 C6 烷基接头将模型肽（一种 28 个氨基酸长的β-淀粉样肽）缀接到 ODN 的 5'或 3'末端。我们比较了所得缀合物与未缀合的亲本 ODN 在 TLR9 转染细胞、小鼠脾细胞培养物和小鼠体内的免疫刺激活性。通过 C3 和 C6 接头在 3'末端将肽缀接到 ODN 上的 ODN 在体外和体内均具有与亲本 ODN 相似的免疫刺激活性。相反，将肽缀接到 ODN 的 5'末端会显著削弱免疫刺激活性。总之，这里呈现的结果表明，肽/蛋白质与 ODN 的缀合在 3'末端使用 C3 或 C6 接头是最佳的，而在 5'末端的缀合会导致 TLR9 介导的免疫刺激显著丧失。
