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

在这个提议中，我们将探索使用环状单链DNA分子作为基因递送和基因表达的可切换组件。在生物学中，环状单链DNA作为基因组DNA或各种病毒和噬菌体的复制中间体出现。另一方面，DNA纳米技术已经开发了丰富的方法来创建由DNA单链组成的可切换分子结构。特别地，众所周知的DNA折纸技术通常利用源自噬菌体的环状单链DNA作为所谓的“支架链”。然后使用短的互补“钉”链将这些支架“折叠”成特定的纳米级形状。与折纸技术相反，我们将在这里研究含有在真核或细菌细胞或细胞提取物中表达的基因序列的环状单链DNA分子。我们将研究这种环状ssDNA分子的分子内折叠是否以及如何影响它们在生物学背景下的加工。初步实验表明，环状DNA可以在体外转化为双链形式，并且蛋白质可以在体内从这种链的编码区表达。调节环状链的结构并掺入折纸结构将用于控制这些过程，然后应用于开发新型的可切换基因递送和表达平台。

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.