Rolling circle enzymatic replication of a complex multi-crossover DNA nanostructure

Nature has evolved replicable biological molecules, such as DNA, as genetic information carriers. The replication process is tightly controlled by complicated cellular machinery. It is interesting to ask if artificial DNA nano-objects with a complex secondary structure can be replicated in the same way as simple DNA double helices. Here we demonstrate that paranemic crossover DNA, a structurally complicated multi-crossover DNA molecule, can be replicated successfully using Rolling Circle Amplification (RCA). The amplification efficiency is moderate with high fidelity, confirmed by native PAGE, thermal transition study, and Ferguson analysis. The structural details of the DNA structure after the full replication circle are verified by hydroxyl radical autofootprinting. We conclude that RCA can serve as a reliable method to replicate complex DNA structures. We also discuss the possibility of using viruses and bacteria to clone artificial DNA nano-objects. The findings that single stranded paranemic crossover DNA molecules can be replicated by DNA polymerase will not only be useful in nanotechnology but also may have implications for the possible existence of such complicated DNA structures in nature.