Construction of Monomeric and Dimeric G-Quadruplex-Structured CpG Oligodeoxynucleotides for Enhanced Uptake and Activation in TLR9-Positive Macrophages.

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'-G ₅ )], 3'-end [1668(3'-G ₅ )], or within the sequence [1668(mid-G ₅ )]. 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'-G ₅ ) 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'-G ₅ ). GQ structuration increased CpG ODN uptake by RAW264.7 cells, and 1668(3'-G ₅ ) decomposed more slowly in serum than 1668(5'-G ₅ ). 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.