Engineering a DNAzyme-Based Operon System for the Production of DNA Nanoscaffolds in Living Bacteria

Abstract

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 nanostructures 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 noncoding 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 crossover nanostructure (25 nm) that recruits split YFPs when properly assembled. This system enables the formation of more complex 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 applications.

Genetically Encoding Ultra-Stable Virus-like Particles Encapsulating Functional DNA Nanostructures in Living Bacteria

“…VLPs/mL (see methods). These yields are more than 1000-fold higher than the purification yields of unencapsulated ssDNA for 1 L of bacteria 36 .…”

Application of Nucleic Acid Frameworks in the Construction of Nanostructures and Cascade Biocatalysts: Recent Progress and Perspective

“…After the RNA regions were degraded by RNases, the ssDNA products self-assembled into nanostructures. In another work of them, a long ssDNA was produced with the same strategy, and then self-cleaved by DNAzymes harbored in its sequence to produce ssDNA components for the assembly of DNA nanoscaffolds in the cells (Alon et al, 2020).…”

Zn2+‐Dependent DNAzymes: From Solution Chemistry to Analytical, Materials and Therapeutic Applications

“…Recently, Elbaz and co-workers converted a noncoding RNA into a singlestranded DNA containing the IÀ R3 DNAzyme in E. coli, allowing self-cleavage into multiple DNA strands, and this can be considered as a DNAzyme-based operon. [99] An extra of 0.5 mM ZnSO 4 was supplied to assist the cleavage. The authors further demonstrated the production of a DNA crossover nanostructure assembled by such DNA that can recruit split yellow fluorescent proteins.…”

“…Using this method, hundreds of mg of assembled DNA origami structures were prepared in a cost‐efficient manner. Recently, Elbaz and co‐workers converted a noncoding RNA into a single‐stranded DNA containing the I−R3 DNAzyme in E. coli , allowing self‐cleavage into multiple DNA strands, and this can be considered as a DNAzyme‐based operon [99] . An extra of 0.5 mM ZnSO 4 was supplied to assist the cleavage.…”