Artificial DNA achieves self-replication outside living cells

A research group including Doctoral Student Yuta Yamagishi and Professor Masayuki Su'etsugu of the Department of Life Science at the Graduate School of Science at Rikkyo University designed and constructed a 53-kb long artificial genomic DNA encoding all 26 protein genes required for Escherichia coli-type DNA replication (Replication-Cycle Reaction: RCR). By adding this long artificial genomic DNA to a cell-free gene expression system capable of producing proteins from DNA without using cells, they produced a group of replication proteins encoded in the DNA within a test tube. The study was published online in Nucleic Acids Research.

Creating artificial cells requires putting many genes into DNA and establishing a self-replication mechanism in which that DNA increases itself through gene expression. However, longer DNA is more difficult to replicate, making the construction of a self-replicating system a major challenge. To express and functionalize multiple genes on a single DNA molecule, the arrangement of genes and the balance of expression are crucial. If even a portion of the required proteins is deficient, the entire replication reaction stalls.

Therefore, the research group first investigated which proteins were prone to deficiency. Based on those results, they improved the design of the long artificial genomic DNA and revised the reaction conditions, thereby establishing conditions to appropriately express the group of replication proteins required for self-replication.

Through this improvement, they confirmed that the expressed replication protein group acted on the original long artificial genomic DNA, causing the DNA itself to replicate. Furthermore, they transferred the long artificial genomic DNA amplified in a single reaction into a fresh reaction mixture and performed serial passage reactions to allow self-replication again, confirming that self-replication could be maintained repeatedly.

These results demonstrate that long artificial genomic DNA can continue to self-replicate while being passed on to subsequent reactions, serving as a foundation for constructing artificial cells with numerous proteins using artificial genomic DNA that encodes a large number of genes.

The current long artificial genomic DNA carries a gene set required for E. coli-type DNA replication. Moving forward, by adding gene sets responsible for transcription, translation, metabolism, and other functions to this DNA, it may become possible to step-by-step assemble the functions required for artificial cells.

Journal Information
Publication: Nucleic Acids Research
Title: A self-replicating artificial module-genome that generates bacterial chromosome replication system in vitro
DOI: 10.1093/nar/gkag663