LAUSR

The ability to create nanoscaffolds within living cells using DNA has the potential to become a powerful tool in synthetic biology. However, to date genetically encoded DNA nanostruc-tures are limited to simple architecture due to the lack of genetic parts that can produce multiple ssDNAs in a single bacterium. Here, we develop a system that overcomes this challenge by using a single oligo gene mimicking operons. This was achieved by converting a non-coding RNA into a long ssDNA that self-cleaves into multiple ssDNAs using R3-DNAzymes (DNAzyme-based operon). We demonstrate the ability to apply the DNAzyme-based operon to produce a four-ssDNA crosso-ver nanostructure (25 nm) that recruits split YFPs when proper-ly assembled. This system enables the formation of more com-plex DNA nanostructures in vivo and thus paves the way to further integrate the field of DNA nanotechnology into living bacteria for basic biology, bioengineering and medicine appli-cations.