TY - JOUR

T1 - Circle-seq based method for eccDNA synthesis and its application as a canonical promoter independent vector for robust microRNA overexpression

AU - Yu, Jiaying

AU - Zhang, Haoran

AU - Han, Peng

AU - Jiang, Xianming

AU - Li, Jing

AU - Li, Bo

AU - Yang, Shaohua

AU - He, Chunxiao

AU - Mao, Shuang

AU - Dang, Yonghui

AU - Xiang, Xi

N1 - Publisher Copyright: © 2023 The Authors

PY - 2024

Y1 - 2024

N2 - Extrachromosomal circular DNA (eccDNA) has recently gained increasing attention due to its significant role in cancer and other pathophysiologic 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 identification. Here, we describe CAES (Circle-seq based Artificial EccDNA Synthesis), a promising and reliable method for artificial eccDNA synthesis. Eight eccDNAs carrying different microRNA genes (eccMIR) found in gastric cancer tissues, ranging from 329 bp to 2189 bp in size, were created utilizing the CAES method. Exonuclease V and single restriction-endonuclease digestion identified the circular structure of synthetic eccDNAs. The DNA circularization efficiency afforded by CAES ranged from 15.6% to 31.1%, which was negatively correlated with the eccDNA length. In addition, we demonstrated that CAES-synthesized eccMIRs can express both miRNA-3p and − 5p molecules efficiently independent of a canonical promoter in human cell lines. 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. Finally, kinetics study revealed that eccDNA exhibited a decay rate similar to the standard plasmids and linear DNA in cultured cells. Together, this study offers a rapid and convenient method for Circle-seq users to synthesize artificial eccDNAs. It also demonstrates the promising potential of eccMIR as a bacterial DNA-free vector for safe and robust miRNA overexpression in both basic research and therapeutic applications.

AB - Extrachromosomal circular DNA (eccDNA) has recently gained increasing attention due to its significant role in cancer and other pathophysiologic 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 identification. Here, we describe CAES (Circle-seq based Artificial EccDNA Synthesis), a promising and reliable method for artificial eccDNA synthesis. Eight eccDNAs carrying different microRNA genes (eccMIR) found in gastric cancer tissues, ranging from 329 bp to 2189 bp in size, were created utilizing the CAES method. Exonuclease V and single restriction-endonuclease digestion identified the circular structure of synthetic eccDNAs. The DNA circularization efficiency afforded by CAES ranged from 15.6% to 31.1%, which was negatively correlated with the eccDNA length. In addition, we demonstrated that CAES-synthesized eccMIRs can express both miRNA-3p and − 5p molecules efficiently independent of a canonical promoter in human cell lines. 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. Finally, kinetics study revealed that eccDNA exhibited a decay rate similar to the standard plasmids and linear DNA in cultured cells. Together, this study offers a rapid and convenient method for Circle-seq users to synthesize artificial eccDNAs. It also demonstrates the promising potential of eccMIR as a bacterial DNA-free vector for safe and robust miRNA overexpression in both basic research and therapeutic applications.

KW - Artificial eccDNA

KW - CAES

KW - Circle-seq

KW - DdPCR

KW - EccDNA

KW - EccDNA function

KW - MicroRNA therapeutics

U2 - 10.1016/j.csbj.2023.12.019

DO - 10.1016/j.csbj.2023.12.019

M3 - Journal article

C2 - 38223344

AN - SCOPUS:85180361869

VL - 23

SP - 358

EP - 368

JO - Computational and Structural Biotechnology Journal

JF - Computational and Structural Biotechnology Journal

SN - 2001-0370

ER -