Layer-by-Layer modified DNA Origami Nanostructures as a Smart Tool for Drug Delivery

• Applicant: Privatdozentin Dr. Uta Reibetanz
• Subject Area: Pharmacy
• Term: since 2020
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 437539561

DNA origami nanostructures are based on the connection of a long single-stranded scaffold DNA with short staple oligonucleotides to build up 2d or 3d structures. We here focus on a box or cage structure consisting of an open 40 nm × 25 nm × 25 nm tube completed with two 30 nm × 25 nm × 25 nm lids. Such a structure can be easily filled with cargo molecules: a short single DNA strand (target) is attached to the inner side of the tube, whereas the cargo is bound to a complementary incumbent strand and subsequently connected with the target. After closing the tube with lids, a small DNA invader strand penetrates the cage walls to detach the target-incumbent connection via toehold-mediated strand displacement reaction. However, it turned out that the now freely movable cargo (streptavidin or bovine serum albumin) is also able to escape the cage structure, thus a balanced design is needed in the first place to both allow the small invader strand to penetrate and prevent the larger cargo to escape the closed structure. Defined modifications of the design, such as exchange of staples and changing the design of the tube-lid interface composition as well as the increase of the captured cargo molecule already lead to a sufficient enhancement of the retention capability. However, for cellular applications overall cage retention / leakage capability, stability under cellular conditions (37°C, physiological salt concentration, activity of enzymes, proteases and nucleases) and a mechanism for controlled release have to be considered and a balanced mixture of functionalization has to be assembled to realize those requirements. Thus, in this project we will concentrate on two approaches: On the one hand, the enhancement of the stability and retention capability under cellular conditions will be initiated by the application of several surface modification, such as by the assembly of polymer or lipid layers or the cross-linking of the DNA wall structure. In parallel, the integration of a switchable “hole structure” will be investigated, in particular regarding its controlled release ability under such coating conditions. On the other hand, the leaky appearance of the already designed origami nanostructure will be used and applied as a carrier with low, but constant release profile of the transported agents within the cell, assuming the cage structure provide a significant stability under cellular conditions. To monitor the properties of the origami cage structure, therapeutically active agents (e.g., doxorubicin, coupled to human serum albumin) will be used as a cargo and the cage status followed in model environment as well as in application to culture cells.

• DFG Programme: Research Grants
• Co-Investigator: Professor Dr. Ralf Seidel