Generation of DNAzyme in Bacterial Cells by a Bacterial Retron System

Abstract

DNAzymes are catalytically active single-stranded DNAs in which DNAzyme 10−23 (Dz 10−23) consists of a catalytic core and a substrate-binding arm that reduces gene expression through sequencespecific mRNA cleavage. However, the in vivo application of Dz 10−23 depends on exogenous delivery, which leads to its inability to be synthesized and stabilized in vivo, thus limiting its application. As a unique reverse transcription system, the bacterial retron system can synthesize single-stranded DNA in vivo using ncRNA msr/msd as a template. The objective of this work is to reduce target gene expression using Dz 10−23 generated in vivo by the retron system. In this regard, we successfully generated Dz 10−23 by cloning the Dz 10−23 coding sequence into the retron msd gene and tested its ability to reduce specific gene expression by examining the mRNA levels of cf p encoding cyan fluorescence protein and other functional genes such as mreB and f tsZ. We found that Dz had different repressive effects when targeting different mRNA regions, and in general, the repressive effect was stronger when targeting downstream of mRNAs. Our results also suggested that the reduction effect was due to cleavage of the substrate mRNA by Dz 10−23 rather than the antisense effect of the substrate-binding arm. Therefore, this study not only provided a retron-based method for the intracellular generation of Dz 10−23 but also demonstrated that Dz 10−23 could reduce gene expression by cleaving target mRNAs in cells. We believe that this new strategy would have great potential in the regulation of gene expression.

Intracellular Expression of a Fluorogenic DNA Aptamer Using Retron Eco2

“…Expression of ssDNA using retrons is typically achieved by encoding a cargo gene in the msd region of the ncRNA. This approach has been successfully demonstrated for recombineering donors 32 , protein-binding DNA sequences 38 and DNAzymes 36 . However, it was not clear as to what extent the folding of the cargo aptamer sequence would be affected due to the extensive secondary structure of the Eco2 msd - msr RNA-DNA hybrid.…”

“…For example, Lopez et al tested modifications to the ncRNA in case of retron Eco1 in order to boost RT-DNA abundance 32 . More recently, Liu et al also used retron Eco1 to express 10-23 DNAzyme in vivo 36 . However, it is poorly understood how the encoding of a functional ssDNA sequence in retrons will affect its activity.…”