DNA density-dependent uptake of DNA origami-based two-or three-dimensional nanostructures by immune cells

DNA nanostructures are expected to be applied for targeted drug delivery to immune cells. However, the structural properties of DNA nanostructures required for the delivery have not fully been elucidated. In this study, we focused on the DNA density that can be important for the their recognition and uptake by immune cells. To examine this, DNA nanostructures with almost identical molecular weights and structural flexibility, but with different shapes and DNA densities, were designed using DNA origami technology. We compared the following five types of DNA nanostructures, all of which consisted of ten DNA helices using an identical circular, single-stranded scaffold and staples. Rec180 had a rectangular-shaped, almost flat structure. Rec90, Rec50 and Rec0 were bent forms of Rec180 at the center by 90, 50 or 0 degrees, respectively. Rec50/50 has two bends of 50 degrees each so that the both ends stick together to form a triangular prism shape. The fluctuation, or flexibility, of these DNA nanostructures under solution conditions was estimated using CanDo software. The DNA density estimated from the average distance between any two of the ten DNA helices in the DNA nanostructures was different among them; Rec50, Rec0 and Rec50/50 had a higher density than Rec180 and Rec90. Agarose gel electrophoresis and atomic force microscopy showed that all of the nanostructures were prepared with high yield. Flow cytometry analysis revealed that the uptake of DNA nanostructures by murine macrophage-like RAW264.7 cells was higher for those with higher DNA density than those with low density. There was a positive correlation between the density and cellular uptake. These results indicate that DNA nanostructures with high DNA density are suitable for delivery to immune cells.