Extended Data Fig. 8: γ-fragment replication reactions, sequencing and analysis.
(a) Iterative replication of exogenous or endogenous γ fragment by the TPR. Replication reactions were set up as in Fig. 4b (with 0.6 mM KCl), and either seeded with γD, no exogeneous template (TPR) or partly degraded TPR (by prior heating with Mg2+ = ‘deg. TPR’). Post-freezing (eutectic phase) concentrations of template, TPR and products are shown. Concentrations of γ+ (red) and γ– (blue) products reaching full-length were inferred from gel densitometry of triplicate reactions. Concentrations of product derived from replication of the template seed were calculated by proportional subtraction of products derived from intact ribozyme in unseeded reactions. This showed low single-cycle γ– synthesis efficiency using TPR as a template, but higher efficiency upon partial TPR degradation. Replication efficiency from γD is higher than in Fig. 4c, likely due to the 10-fold lower starting concentration of γD duplex used here. (b) Yields of full-length fragment replication products classed by γ-fragment homology. The sequence product distribution in Supplementary Fig. 7c for each sample was scaled by the modelled yield of all γ+ products in the corresponding cycle (Supplementary Fig. 7d). (c) The six most common individual sequences in the 5-cycle γD replication sample (occurrence shown out of 10,880 total sequences). Interestingly, the two of these sequences with very low γ+ identity show complementarity (underlined) to parts of the type 1 ribozyme (see Extended Data Fig. 1), a further instance of TPR acting as a template.
