Figure 1. Monomer polymerisation and triplet polymerisation.

(A) Scheme outlining initial derivation of a triplet polymerase activity from a mononucleotide polymerase ribozyme via directed evolution. Z RPR truncation effects are shown in Figure 1—figure supplement 1, the selection cycle is outlined in Figure 1—figure supplement 2, and the selection conditions of rounds 1–7 are listed in Figure 1—source data 1. Below, modes of action and secondary structures of the mononucleotide polymerase ribozyme (Z RPR) and a triplet polymerase ribozyme (0core), both depicted surrounding primer (tan)/template (grey) duplexes with a mononucleoside triphosphate (NTP) or trinucleotide triphosphate (triplet) substrate present (red). Here, the templates are hybridised to the ribozyme upstream of the primer binding site, flexibly tethered to enhance local concentration and activity (via L repeats of an AACA sequence, for example L = 5 in templates SR1-4 below). Z RPR residues comprising its catalytic core (Zcore) are black; mutations in 0core arising from directed evolution of Zcore are in teal. (B) Primer extension by the Z RPR using monomers (1 mM NTPs) or by 0core using triplets (5 μM ^pppAUA and ^pppCGC), on a series of 6-nucleotide repeat templates (SR1-4, examples below) with escalating secondary structure potential that quenches Z RPR activity beyond the shortest template SR1 (−7˚C ice 17 days, 0.5 μM/RNA). Extension by the triplet polymerase ribozyme 0core can overcome these structure tendencies up to the longest template SR4. (C) Triplet concentration dependence of extension using templates SR1 (grey circles) and SR3 (black triangles) by 0core (^pppAUA and ^pppCGC, 0.1 μM of primer A10, template and ribozyme, −7˚C supercooled 15 days, ± s.d., n = 3); shown below is a model of cooperative triplet-mediated unfolding of template SR3 structure to explain the sigmoidal triplet concentration dependence (red curve) of extension upon it. Numerical values are supplied in Figure 1—source data 2.

Figure 1—figure supplement 1. Templated ligase activity from a mononucleotide polymerase.

Extension of primer A11 on template HTI by the Z RPR (Wochner et al., 2011) and a 3’ truncation of it (Zcore, Figure 1a) at −7˚C in ice for 8 days (0.5 μM of each RNA), adding as substrates either a long oligonucleotide (‘11 nt’, ^pppGCGAAGCGUGU at 0.5 μM), a triplet (‘3 nt’, ^pppGCG at 5 μM), or NTPs (‘1 nt’, at 1 mM each). Gel densitometry was used to calculate the percentage of primer extended (shown below each lane). Z RPR has only a very limited template ligation activity (slightly exceeding background nonenzymatic ligation [Rohatgi et al., 1996]). In contrast, 3’ truncation (which abolished mononucleotide polymerase activity) accommodated and enhanced templated ligation.

Figure 1—figure supplement 2. Selection scheme for in vitro evolution of triplet polymerase activity.

Illustration of the in vitro evolution strategy to enrich iterative templated ligase activity from libraries of selection constructs. Centre, an example selection construct is shown comprising Zcore with a 3’ 30 nt random domain followed by a fixed hairpin-forming reverse transcription site (1). This construct is flexibly tethered at its 5’ in cis (Tagami et al., 2017; Attwater et al., 2010) (via L repeats (3-8) of a flexible AACA linker) to a template region (grey) bound to 5’-dual biotin modified primer (tan). This construct requires successive polymerisation of two ^pppUCG triplet substrates (red) in −7˚C ice followed by self-ligation to its triphosphorylated 5’ terminus to attach itself to the biotinylated primer (2). Active triplet polymerases are then identified and size-purified by denaturing PAGE to deplete unreacted ribozymes and primer (3), then captured on streptavidin beads and subjected to a denaturing wash to ensure covalent linkage to primer. Remaining dual-purified bead-linked ribozymes were then reverse transcribed (4). cDNAs were eluted with NaOH and neutralised, before acting as templates for ‘+’ strand DNA synthesis from 5’ biotinylated ‘rescue’ oligonucleotides that only prime correctly on the complete synthesised sequence (5), ensuring that constructs that deviated from the specified templated triplet polymerization fail to prime on cDNA preventing their ‘+’ strand synthesis. ‘+’ strands were then captured on fresh streptavidin beads, stripped of cDNAs, reamplified and diversified to generate selection construct for a new round of selection (6). See Materials and Methods for protocol details and Figure 1—source data 1, Figure 2—source data 1, Figure 4—source data 1 and Supplementary file 3 for oligonucleotides used in selections.