RAES：a rapid and high-fidelity method for artificial extrachromosomal circular DNA synthesis and its application as a bacterial DNA-free vector for robust microRNA overexpression

Abstract

Extrachromosomal circular DNA (eccDNA) has recently gained increasing attention due to its significant role in cancer and other pathological states. The majority of circular DNAs detected by Circle-seq are small-size eccDNAs with enigmatic functions. One major technical hurdle is to synthesize eccDNA for functional assessment. Here, we describe RAES (RCA-product dependent Artificial EccDNA Synthesis), a rapid and economic method for artificial eccDNA synthesis. Eight eccDNAs carrying different microRNA genes (eccMIR) found in gastric cancer tissues, ranging from 329 bp to 2 189 bp in size, were created utilizing the RAES method. Exonuclease V and single restriction-endonuclease digestion identified the circular structure of synthetic eccDNAs. The DNA circularization efficiency afforded by RAES ranged from 15.6% to 31.1%, which was negatively correlated with the eccDNA length. In addition, we demonstrated that RAES-synthesized eccMIRs can express both miRNA-3p and -5p molecules efficiently in different cell types, independent of a canonical promoter. Further assays proved that these eccMIRs were functional as they were able to repress the luciferase gene containing a miRNA-target sequence in the 3’UTR as well as the endogenous mRNA targets. Together, RAES is an economic and efficient method to synthesize artificial eccDNAs, which also provides a promising way to produce bacterial DNA-free vectors for safe and robust miRNA overexpression in either fundamental research or clinical therapeutics.