In vivo cloning of artificial DNA nanostructures

Abstract

Mimicking nature is both a key goal and a difficult challenge for the scientific enterprise. DNA, well known as the genetic-information carrier in nature, can be replicated efficiently in living cells. Today, despite the dramatic evolution of DNA nanotechnology, a versatile method that replicates artificial DNA nanostructures with complex secondary structures remains an appealing target. Previous success in replicating DNA nanostructures enzymatically in vitro suggests that a possible solution could be cloning these nanostructures by using viruses. Here, we report a system where a single-stranded DNA nanostructure (Holliday junction or paranemic cross-over DNA) is inserted into a phagemid, transformed into XL1-Blue cells and amplified in vivo in the presence of helper phages. High copy numbers of cloned nanostructures can be obtained readily by using standard molecular biology techniques. Correct replication is verified by a number of assays including nondenaturing PAGE, Ferguson analysis, endonuclease VII digestion, and hydroxyl radical autofootprinting. The simplicity, efficiency, and fidelity of nature are fully reflected in this system. UV-induced psoralen cross-linking is used to probe the secondary structure of the inserted junction in infected cells. Our data suggest the possible formation of the immobile four-arm junction in vivo.DNA nanotechnology ͉ immobile DNA junction ͉ self-replication ͉ synthetic biology T he notion that DNA is merely the gene encoder of living systems has been eclipsed by the successful development of DNA nanotechnology (1-2). Using branched DNA as the main building block (also known as a ''tile'') and cohesive singlestranded DNA (ssDNA) ends to designate the pairing strategy for tile-tile recognition, one can rationally design and assemble complicated nanoarchitectures from specifically designed DNA oligonucleotides. Objects in both two and three dimensions with a large variety of geometries and topologies have been built from DNA with excellent yield (3-9); this development enables the construction of DNA-based nanodevices (10-11) and DNA template directed organization of other molecular species (12)(13)(14)(15)(16)(17)(18)(19). The construction of such nanoscale objects constitutes the basis of DNA nanotechnology. However, the synthetic scale of oligonucleotides limits the potential applications of DNA nanotechnology, especially when the nanostructure is designed to be made from long ssDNA (Ͼ100 bases). Replicable DNA nanostructures are thus a highly desirable goal to help overcome this barrier.To this end, several possible solutions have been explored. For example, a three-point star motif has been replicated by chemical methods in which the parental nanomotif serves as the template to direct the chemical ligation of nonidentical DNA strands to form the next generation of the nanomotif (20). Another instance involves a 1.7-kb ssDNA that was used to assemble a DNA octahedron (together with five short strands); this molecule was cloned in a plasmid in bacteria (9). A nicking end...

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