Protein-coated dsDNA nanostars with high structural rigidity and high enzymatic and thermal stability

DNA nanotechnology creates precise shape-specific nanostructures through the self-assembly of short ssDNA oligonucleotides. One such shape, which has relevant biomedical applications due to its multivalency, is the star. However, building star-like nanostructures with a large size (>100 nm) using ssDNA is complex and challenging. This study presents a novel strategy to prepare stiff and large dsDNA nanostars by assembling duplex DNA fragments into star-shapes that are subsequently coated with a virus-inspired protein. The protein binds dsDNA and overcomes the high structural flexibility of naked dsDNA. The nanostar-like dsDNA templates with up to six arms were prepared by self-assembly of PCR-produced dsDNA fragments (211 to 722 bp) with a central DNA junction. Through gel electrophoresis and Atomic Force Microscopy it is demonstrated that single dsDNA nanostars are self-assembled and coated with the protein, and this has a large stiffening effect on the nanostar. Furthermore, the coating significantly enhances stability at high temperatures and protects nanostars against nuclease degradation for at least 10 hours. This study shows that DNA-binding proteins can be harnessed as structural "rigidifiers" of flexible branched dsDNA templates. This strategy opens a way to prepare structurally defined hybrid protein-dsDNA nanostructures that could be exploited as building blocks for novel DNA nanomaterials.