Elucidation of the Mechanism of Increased Activity of Immunostimulatory DNA by the Formation of Polypod-like Structure.

PURPOSE: We previously demonstrated that the immunostimulatory activity of CpG DNA is increased by the formation of polypod-like structures. The present study was designed to elucidate the mechanism underlying this increase.

METHODS: Tripodna (three pods) and hexapodna (six pods) were prepared. The cellular uptake of Alexa Fluor 488-labeled DNA samples was examined in several cell lines by measuring the MFI of cells. TNF-α release from RAW264.7 cells was measured after addition of polypodna containing CpG motifs. Dissociation of double stranded DNA was evaluated using FRET.

RESULTS: Tripodna and hexapodna were efficiently taken up by macrophage-like RAW264.7 cells and dendritic DC2.4 cells, but not by fibroblast or endothelial cell lines. The uptake by RAW264.7 cells was highest for hexapodna, followed by tripodna, dsDNA, and ssDNA. The release of TNF-α from RAW264.7 cells was also highest for hexapodna. The ratio of TNF-α release to cellular uptake was highest for ssDNA, and lowest for dsDNA. Tripodna and hexapodna were more easily dissociated into single strands after cellular uptake than was dsDNA.

CONCLUSIONS: The efficient cellular uptake and prompt dissociation into single strands can be directly related to the high immunostimulatory activity of polypod-like structured DNAs containing CpG motifs.