ssDNA Modification on Lipid Carriers Utilizing ssDNA-Conjugated Fusion Protein with Cholesterol-Binding Ability

Lipid carriers, such as liposomes and exosomes, are nanoscale vesicular structures composed of phospholipids, glycolipids, membrane proteins, and cholesterol, which have been widely investigated as drug- and nucleic-acid-delivery systems. Functionalization of lipid carrier surfaces with ligand molecules is an effective approach for the targeted delivery of lipid carriers to specific tissues and cells. In this regard, the use of DNA modification represents a rational and promising approach as DNA aptamers exhibit high affinity and specificity for target molecules and offer several advantages over conventional ligands. The current strategies for modifying lipid carriers with DNA remain suboptimal, leaving room for further improvement. In this study, we aimed to modify lipid carriers with single-stranded (ss)DNA-conjugated fusion proteins that have cholesterol-binding abilities. The fusion protein (Rep-ALOD4) was composed of a replication initiator protein derived from porcine circovirus type 2 (Rep), which acted as an ssDNA-conjugating domain, and domain 4 of anthrolysin O (ALOD4), which acted as a cholesterol-binding domain. As Rep-ALOD4 can bind to cholesterol in lipid membranes under mild conditions, ssDNA-conjugated Rep-ALOD4 achieved ssDNA modification on liposome carriers containing more than 30 wt % cholesterol. Furthermore, cell-selective targeting using DNA aptamer- and ssDNA-modified liposomes and exosomes was successful because of the functional applicability of ssDNA-displaying Rep-ALOD4. Thus, the DNA modification platform of Rep-ALOD4 has the potential to substantially advance targeted drug delivery using lipid carriers.