The G-quadruplex (GQ) structure has potential applications in nucleic acid drug delivery because of its superior stability. In this study, we added one G-tract (five guanines) to an unmethylated phosphodiester-linked cytosine-phosphate-guanine oligodeoxynucleotide (CpG ODN), a potential immune adjuvant, to construct a GQ-structured CpG ODN with precise structural properties, increased biological stability, and efficient delivery to Toll-like receptor 9 (TLR9)-positive immune cells. A G-tract was added to phosphodiester-backboned CpG1668 at the 5′-end [1668(5′-G₅)], 3′-end [1668(3′-G₅)], or within the sequence [1668(mid-G₅)]. Circular dichroism analysis showed that all CpG ODNs with a G-tract formed parallel GQ structures, irrespective of its position. Electrophoresis showed that 1668(5′-G₅) formed a GQ dimer, whereas others remained GQ monomers. GQ-structured CpG ODNs induced greater tumor necrosis factor-α and interleukin-6 secretion from TLR9-positive mouse macrophage-like RAW264.7 cells than single-stranded CpG ODNs, with the highest for 1668(3′-G₅). GQ structuration increased CpG ODN uptake by RAW264.7 cells, and 1668(3′-G₅) decomposed more slowly in serum than 1668(5′-G₅). Thus, GQ formation with one G-tract is a simple and efficient strategy for CpG ODN delivery to TLR9-positive cells, and addition of a G-tract to the 3′-end is effective in obtaining monomeric GQ-structured CpG ODN with high biological stability and immunostimulatory activity.

中文翻译：

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单体和二聚体 G-四链体结构的胞嘧啶-磷酸-鸟嘌呤寡脱氧核苷酸的构建，用于增强 Toll 样受体 9 阳性巨噬细胞的摄取和激活。

G-四链体（GQ）结构由于其优越的稳定性而在核酸药物递送中具有潜在的应用。在这项研究中，我们在未甲基化的磷酸二酯连接的胞嘧啶-磷酸-鸟嘌呤寡脱氧核苷酸 (CpG ODN)（一种潜在的免疫佐剂）中添加了一个 G 束（五个鸟嘌呤），以构建具有精确结构特性的 GQ 结构的 CpG ODN，增加生物稳定性，并有效递送至 Toll 样受体 9 (TLR9​​) 阳性免疫细胞。在 5'-末端 [1668(5'-G ₅ )]、3'-末端 [1668(3'-G ₅ )] 或序列 [1668(中 G ₅)]。圆二色性分析表明，所有具有 G 束的 CpG ODN 形成平行的 GQ 结构，无论其位置如何。电泳显示1668(5'-G ₅ )形成GQ二聚体，而其他保持GQ单体。GQ 结构的 CpG ODN 比单链 CpG ODN 从 TLR9 阳性小鼠巨噬细胞样 RAW264.7 细胞中诱导更大的肿瘤坏死因子-α 和白细胞介素-6 分泌，最高的是 1668(3'-G ₅ )。GQ 结构增加了 RAW264.7 细胞对 CpG ODN 的摄取，1668(3'-G ₅ ) 在血清中的分解速度比 1668(5'-G ₅）。因此，用一个 G-tract 形成 GQ 是将 CpG ODN 传递给 TLR9 阳性细胞的一种简单有效的策略，并且在 3'-末端添加 G-tract 可有效获得具有高的单体 GQ 结构的 CpG ODN。生物稳定性和免疫刺激活性。