M13mp18-RF Phage Vector

The fabrication of DNA origami nanostructures with highly ordered functional motifs is of great significance for biomedical applications. Here, researchers present a robust strategy to produce custom scaffolds with integrated aptamer sequences, enabling the direct construction of functional DNA origami structures. Specifically, two different thrombin aptamer sequences were simultaneously inserted into the M13mp18 phage genome. Functional DNA origami structures assembled from this aptamer-integrated scaffold exhibit improved binding efficiency to thrombin and are more than 10-fold more resistant to exonuclease degradation than those produced using traditional peg extension methods. In addition, a scaffold integrated with platelet-derived growth factor aptamers was also produced, and the assembled DNA origami structure showed significant inhibitory effects on breast cancer cells MDA-MB-231. This scalable approach to creating specifically designed scaffolds opens up a new way to build more stable and functional DNA origami structures, providing an important foundation for their broader applications.

In this study, to obtain custom-designed scaffolds containing different numbers and types of aptamers, aptamer sequences were inserted into user-defined sites in the M13mp18 phagemid as a proof of concept (Figure 1). Specifically, the researchers designed three aptamer-integrated scaffolds. The aptamer-integrated scaffold is then annealed to the corresponding backbone and folded into the designed DNA origami nanostructures. The aptamer sequence was inserted into the M13mp18 RF DNA vector at specific locations by PCR and Gibson assembly. The recombinant phagemid containing the correct assembly sequence was verified by sequencing and transformed into E. coli for amplification. The recombinant phagemid is replicated as a single strand and packaged into new phage. Single-stranded DNA is then extracted from the phage particles.

Figure 1. Schematic representation of the preparation of aptamer-functionalized synthetic scaffolds and assembly of functional DNA origami structures. (A) A custom-designed ssDNA fragment containing the aptamer sequence was inserted into the M13mp18 RF DNA vector. The recombinant phagemids are then transformed into E. coli cells and produce large amounts of in vivo packaged phage particles. The corresponding ssDNA was obtained by harvesting and purifying these phages. (B) Sequence-tailored ssDNA is used as a scaffold to assemble functional DNA origami nanostructures. (Chen X, et al., 2021)