Gene expression from single-stranded circular DNA – a novel component for synthetic gene circuits in vitro and in vivo

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In this proposal we will explore the use of circular single-stranded DNA molecules as switchable components for gene delivery and gene expression. In biology, circular single-stranded DNA occurs as genomic DNA or replication intermediate of a variety of viruses and bacteriophages. On the other hand, DNA nanotechnology has developed a rich methodology to create switchable molecular structures composed of DNA single strands. In particular, the well-known DNA origami technique often utilizes circular single-stranded DNA derived from bacteriophages as so-called “scaffold strands”. These scaffolds are then “folded” into specific nanoscale shapes using short complementary “staple” strands. In contrast to the origami technique, we will here study circular single-stranded DNA molecules containing gene sequences for expression in either eukaryotic or bacterial cells or cell extracts. We will investigate whether and how the intramolecular folding of such circular ssDNA molecules influences how they are processed in the biological context. Preliminary experiments indicate that circular DNA can be converted into double-stranded form in vitro, and that proteins can be expressed from the coding regions of such strands in vivo. Modulation of structure of the circular strands and incorporation into origami structures will be used to control these processes and then applied to develop a novel type of switchable gene delivery and expression platform.