Extended Data Fig. 4: Primer-free synthesis pathways of triplet polymerase ribozyme fragments.
Above, Potential synthesis pathways to a 24 nt RNA from triplet substrates. Each node represents a synthesis intermediate, of increasing length towards the centre of the map (full length product). The intermediate at each node can be synthesized from the intermediates or triplets at each end of a line passing through the node. Larger intermediates can potentially be generated via multiple routes. Below, Maps of products detected during primer-less synthesis of three fragments (β+ (top), γ+ (middle) and δ+ (bottom)) of the t5 ribozyme4, and their inferred synthesis pathways. The maps show the distribution of polymerised triplets: the area of each node is proportional to the abundance of that intermediate (from sequencing scheme in Supplementary Fig. 4) multiplied by its length. Node sizes are uniformly scaled in each map such that the sum of intermediate node areas is constant; the triplet substrate nodes (not sequenced) are sized arbitrarily. Putative synthesis pathways are marked on each map and displayed alongside in a 5’ to 3’ direction with intervening symbols indicating how triplets are joined together into RNA products – indicating sites of initiation (triplet:triplet ligation), polymerisation (triplet addition to the 3’ of an oligonucleotide) or instances of ligation (of oligomer substrates larger than triplets). The β fragment exhibits two sites of initiation, followed by polymerisation from each with their concomitant ligation on the path to the full-length fragment. The γ fragment synthesis is characterized by multiple sites of initiation and independent ligation of the resulting oligomers, and proceeded effectively, perhaps reflecting the availability of multiple initiation sites. The δ primerless synthesis was inefficient, with negligible ligation at two junctions, and as a result no full-length product was detected. The observed products suggested a pattern of two initiation sites, followed by polymerisation and ligation. The triplet:triplet ligation observed across the three templates is evidence of widespread triplet substrate coating of single-stranded templates.
