| Popis: |
The virtual circular genome (VCG) model was proposed as a means of going beyond template copying to indefinite cycles of nonenzymatic RNA replication during the origin of life. In the VCG model the protocellular genome is a collection of short oligonucleotides that map to both strands of a virtual circular sequence. Replication is driven by templated nonenzymatic primer extension on a subset of kinetically trapped partially base-paired configurations, followed by shuffling of these configurations to enable continued oligonucleotide elongation. Here we describe initial experimental studies of the feasibility of the VCG model for replication. We designed a small 12-nucleotide model VCG and synthesized all 247 oligonucleotides of length 2 to 12 corresponding to this genome. We experimentally monitored the fate of individual labeled primers in the pool of VCG oligonucleotides following the addition of activated nucleotides, and investigated factors such as oligonucleotide length, concentration, composition, and temperature on the extent of primer extension. We observe a surprisingly prolonged equilibration process in the VCG system that enables a considerable extent of reaction. We find that environmental fluctuations would be essential for continuous templated extension of the entire VCG system, since the shortest oligonucleotides can only bind to templates at low temperatures, while the longest oligonucleotides require high temperature spikes to escape from inactive configurations. Finally, we demonstrate that primer extension is significantly enhanced when the mix of VCG oligonucleotides is pre-activated. We discuss the necessity of ongoingin-situactivation chemistry for continuous and accurate VCG replication. |
