DNA nanoengineering, in particular, DNA origami has potential applications in a variety of areas including, for example, nanoelectronics, biomedical diagnostics, and therapeutics. To fully realize the potential of DNA self-assembly in these and other areas, methods must be available for economical, scalable, and reliable production of single-stranded DNA (ssDNA) scaffolds from virtually any source. In this review, we will describe the virtues and liabilities of four strategies for generating ssDNA, including Rolling Circle Amplification (RCA), strand-specific exonuclease digestion, chemical denaturation, and asymmetric PCR (aPCR), with suggestions for approaches to optimize the use of each method.

Nucleic acid nanoparticles are playing an increasingly important role in biomolecular diagnostics and therapeutics as well as a variety of other areas. The unique attributes of self-assembling DNA nanoparticles provide a potentially valuable addition or alternative to the lipid-based nanoparticles that are currently used to ferry nucleic acids in living systems. To explore this possibility, we have assessed the ability of self-assembling DNA nanoparticles to be constructed from complete gene cassettes that are capable of gene expression in vitro. In the current report, we describe the somewhat counter-intuitive result that despite extensive crossovers (the stereochemical analogs of Holliday junctions) and variations in architecture, these DNA nanoparticles are amenable to gene expression as evidenced by T7 RNA polymerase-driven transcription of a reporter gene in vitro. These findings, coupled with the vastly malleable architecture and chemistry of self-assembling DNA nanoparticles, warrant further investigation of their utility in biomedical genetics.

Nucleic acid nanoparticles are playing an increasingly important role in biomolecular diagnostics and therapeutics as well as a variety of other areas. The unique attributes of self-assembling DNA nanoparticles provide a potentially valuable addition or alternative to the lipid-based nanoparticles that are currently used to ferry nucleic acids in living systems. To explore this possibility, we have assessed the ability of self-assembling DNA nanoparticles to be constructed from complete gene cassettes that are capable of gene expression in vitro. In the current report we describe the somewhat counter-intuitive result that despite extensive crossovers (the stereochemical analogs of Holliday junctions) and variations in architecture, these DNA nanoparticles are amenable to gene expression as evidenced by T7 RNA polymerase-driven transcription of a reporter gene in vitro. These findings, coupled with the vastly malleable architecture and chemistry of self-assembling DNA nanoparticles, warrant further investigation of their utility in biomedical genetics.