For the first time, researchers have combined an artificial DNA replication scheme with a reconstructed gene expression system using cell-free materials, in the hope of developing more complex artificial cells that can be used to produce useful substances found in living organisms.
The ability to proliferate and evolve is one of the defining characteristics of all living organisms. Genome evolution is the process by which a genome changes in sequence or size over time. Studying genome evolution involves structural analysis of the genome, comparative genomics, and the examination of genomic parasites, ancient genome duplications and polyploidy. The genome evolution field is constantly shifting due to the steadily growing number of sequenced genomes being made readily available.
Comparing genomes of both close relatives and distance ancestors can demonstrate evolution and, subsequently, highlight the differences and similarities between species and how they began to emerge. Genomic DNA undergoes evolution through many continuous generations of replication. This, coupled with gene expression and the resultant evolution, are fundamental functions of living things. However, these processes have not previously been reconstructed in cell-free systems or by using artificial materials, until now.
Adding artificial genomic DNA to a molecular system
In order to develop an artificial molecular system that can multiply and evolve, genetic information must be translated into RNA. In addition, proteins need to be expressed and the DNA replication cycle with these proteins must be sustained over a long period of time. Therefore, historically, it has been impossible to create a reaction system in which the genes necessary for DNA replication are expressed, while those genes simultaneously carry out their functions.
For the first time, researchers at the University of Tokyo combined an artificial DNA replication scheme with a reconstructed gene expression system and micro-compartmentalisation using cell-free materials alone. Specifically, circular DNA was replicated through rolling-circle replication followed by homologous recombination catalysed by phi29 DNA polymerase and Cre recombinase expressed from the DNA. The system was encapsulated in microscale water-in-oil droplets and underwent multiple serial dilution cycles.
It was found that after thirty rounds of serial dilution, the isolated circular DNAs had accumulated several common mutations and the isolated DNA clones exhibited higher replication abilities compared to the original DNA. This was largely due to the isolated DNA clones’ improved ability as a replication template, increased polymerase activity and the reduced inhibitory effect of polymerisation by the recombinase. Effectively, the researchers successfully translated genes into proteins and replicated the original circular DNA with the translated proteins. Moreover, when the artificial DNA replication cycle was continued for about 60 days, it was found that the replication efficiency increased by 10-fold compared to the original DNA.
Prospects of artificial genomic DNA evolution
Overall, the artificial genomic DNA in this study, which continuously replicated using self-encoded proteins and autonomously improved its sequence, provides a useful starting point for the development of more complex artificial cells. By adding the necessary genes for transcription and translation to artificial genomic DNA, in the future it may be possible to develop artificial cells that can grow autonomously by feeding them low-molecular-weighted compounds, such as amino acids and nucleotides. Producing useful substances using living organisms, including for drug development and food productions, would become more stable and much easier to control in these artificial cells.
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