Design and Development of Nanosized DNA Assemblies in Polypod-like Structures as Efficient Vehicles for Immunostimulatory CpG Motifs to Immune Cells

Abstract

The immunostimulatory activity of phosphodiester DNA containing unmethylated cytosine-phosphate-guanine (CpG) dinucleotides, or CpG motifs, was significantly increased by the formation of Y-, X-, or dendrimer-like multibranched shape. These results suggest the possibility that the activity of CpG DNA is a function of the structural properties of branched DNA assemblies. To elucidate the relationship between them, we have designed and developed nanosized DNA assemblies in polypod-like structures (polypod-like structured DNA, or polypodna for short) using oligodeoxynucleotides (ODNs) containing CpG motifs and investigated their structural and immunological properties. Those assemblies consisting of three (tripodna) to eight (octapodna) ODNs were successfully obtained, but one consisting of 12 ODNs was not when 36-mer ODNs were annealed under physiological sodium chloride concentration. High-speed atomic force microscopy revealed that these assemblies were in polypod-like structures. The apparent size of the products was about 10 nm in diameter, and there was an increasing trend with an increase in ODN length or with the pod number. Circular dichroism spectral data showed that DNA in polypodna preparations were in the B-form. The melting temperature of polypodna decreased with increasing pod number. Each polypodna induced the secretion of tumor necrosis factor-α and interleukin-6 from macrophage-like RAW264.7 cells, with the greatest induction by those with hexa- and octapodna. Increasing the pod number increased the uptake by RAW264.7 cells but reduced the stability in serum. These results indicate that CpG DNA-containing polypodna preparations with six or more pods are a promising nanosized device with biodegradability and high immunostimulatory activity.

DNA Nanostructures as Smart Drug-Delivery Vehicles and Molecular Devices

“…Later on, Sellner et al [28] performed analogous and successful experiments with CpG-coated origamis in vivo. In addition, Li et al [40] and Mohri et al [41,42] have reported similar results when using RAW264.7 cells and tetrahedral DNA cages (Li et al), polypod-like structured DNA ( Figure 1D, Mohri et al) or DNA dendrimers (Mohri et al) as carriers for immunostimulatory CpG motifs.These aforementioned results show that DNA-based designs have tremendous potential in fully tunable and triggered celltargeting tasks and they could open up entirely new opportunities e.g. in cancer therapy.…”

“…(C) DNA structures for small interfering RNA (siRNA) delivery: a cage with cell-targeting ligands (folate or peptide) at the end of the siRNA motifs [37], and a PEGylated folatemodified nanotube [36]. (D) DNA structures for CpG-triggered immunostimulation: a DNA origami tube [39] and a polypod-like structure [41]. (E) A smart logic-gated DNA origami nanorobot for targeting cells and subsequently displaying the molecular payload [43].…”

CpG oligodeoxynucleotide nanomedicines for the prophylaxis or treatment of cancers, infectious diseases, and allergies

“…Mohri et al 79 synthesized polypod-like structures through self-assembly of 3-8 ODN molecules each of which contains CpG motifs ( Figure 1C), and investigated the relationship between the branched CpG ODN assembly properties and the immunostimulation effects. All the polypod-like CpG ODNs that were synthesized through self-assembly of natural phosphodiester ODNs of 30-90 nt were ~10 nm.…”

Drug delivery systems based on nucleic acid nanostructures

“…increased by a factor of 3 (G2) and 5 (G3). Polypods [66] With increasing branching of the pods an enhanced stimulation was found for both TNF-α and IL-6. For the prior cytokine a 5-fold to 400-fold increase was observed compared to ds DNA.…”

“…In addition to the trigonal Y-shaped unit, polypods with a higher degree of branching were evaluated regarding their immunostimulatory potential [66]. For that purpose a tri-, tetra-, hexa-and octapod consisting of three, four, six and eight ODNs, respectively, were assembled (Fig.…”