Extended Data Fig. 5: Terminal transferase activity by a triplet polymerase ribozyme.

(a) Terminal transferase activity by the t5⁺¹ TPR⁴ (the precursor of the 5TU⁺¹ TPR²¹) when extending 12 different primer/template (P/T) pairs. These template incorporation of ^pppCCC preceding and following one of 12 different triplets (indicated). As substrates, either all 64 triplets (^pppNNN) or only the specific templated triplet and ^pppCCC were provided. With ^pppNNN, on average 29% of the full-length products were extended by a fourth triplet in a non-templated manner. Extensions were carried out as previously described (ref. ⁴, ‘Fidelity assay’ section of Methods). (b) RNA products in a mixture of the reactions that used ^pppNNN in (a) were sequenced (see ref. ⁴). The probabilities of each of the 64 triplets being added (non-templated) to a full-length product were plotted by triplet GC content. GC-rich triplets were better substrates for terminal transfer (means ± s.d. after logarithmic conversion, n = 8 (0 & 3 GCs) or 24 (1 & 2 GCs)); from ^pppNNN, 19% of all non-templated triplets added were ^pppCCC. (c) Despite the uniform presence of the best terminal transferase substrate ^pppCCC in (a), terminal transferase activity was only notable when an additional G-rich triplet was present. We propose that duplex growth proceeds through blunt-ended addition of short RNA duplexes formed by pre-associated GC-rich Watson:Crick-paired triplet dimers, whose independent existence in solution was previously inferred by the influence of complementary triplets upon TPR misincorporation frequencies⁴.

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