Towards the design and construction of self-replicating RNA nanostructures

Abstract

The transition from an RNA-based world to DNA as the primary genetic material is a pivotal topic in origin-of-life research. This study aims to prototype a hybrid genome system combining DNA and RNA, capable of self-replication and evolution, initially in vitro and potentially in vivo. Using Qß replicase, segmented RNA genomes were synthesized, incorporating the Phi29 phage pRNA for self-assembly into nanorings. The replication efficacy of these replicons was evaluated both independently and in coupled assembly-replication reactions, with Qß chosen for its high amplification efficiency and template versatility. To mitigate the challenge of parasite RNA formation, which depletes essential replication components, a water-emulsion system was employed. The modular assembly of replicons into nanorings was further investigated, with a focus on system reproducibility and the effect of Mg2+ concentrations on nanoring stability. The final goal is to integrate self-replication with self-assembly, thereby creating a segmented, modular RNA genome for in vitro genetic information storage. This work addresses key challenges and advancements in developing synthetic self-replication systems for eventual genomic transplantation in E. coli.