Development of Hydrophobic Interaction-based DNA Supramolecules as Efficient Delivery Carriers of CpG DNA to Immune cells

Unmethylated cytosine-phosphate-guanine (CpG) DNA stimulates mammalian immune cells through recognition by Toll-like receptor 9 (TLR9). Therefore, CpG DNA is expected to be an effective adjuvant for the treatment of immune and allergic diseases. However, challenges, such as low stability against DNase and low delivery efficiency for immune cells, still need to be resolved for the application of CpG DNA. To overcome these challenges, we developed DNA supramolecules consisting of long single-stranded DNA (lss-DNA) synthesized using rolling circle amplification (RCA) and cholesterol-modified DNA (chol-DNA). Lss-DNAs containing multiple CpG motifs were annealed with complementary chol-DNAs to form DNA supramolecules through hydrophobic interactions. Transmission electron microscopy revealed that lss-DNA mixed with chol-DNA formed micrometer-sized DNA supramolecules. The formation of DNA supramolecules increased their stability against DNase compared to lss DNA, which was evaluated using FBS. Furthermore, DNA supramolecules induced three-times higher TNF-α release from RAW264.7 cells than lss-DNA alone. These results demonstrate that DNA supramolecules are efficient delivery carriers of CpG DNA to immune cells.