In-ice evolution of RNA polymerase ribozyme activity

Abstract

Mechanisms of molecular self-replication have the potential to shed light on the origins of life. In particular, self-replication through RNA-catalysed templated RNA synthesis is thought to have supported a primordial 'RNA world'. However, existing polymerase ribozymes lack the capacity to synthesize RNAs approaching their own size. Here, we report the in vitro evolution of such catalysts directly in the RNA-stabilizing medium of water ice, which yielded RNA polymerase ribozymes specifically adapted to sub-zero temperatures and able to synthesize RNA in ices at temperatures as low as -19 °C. The combination of cold-adaptive mutations with a previously described 5' extension operating at ambient temperatures enabled the design of a first polymerase ribozyme capable of catalysing the accurate synthesis of an RNA sequence longer than itself (adding up to 206 nucleotides), an important stepping stone towards RNA self-replication.

Figure 1. In-ice selection for polymerase ribozyme activity

a, Short scheme of in-ice compartmentalised bead-tagging (CBT) selection (full scheme in Supplementary Fig. S1a). Solutes and microbeads are concentrated into the channels of the liquid eutectic phase (pale red) surrounding ice crystals (pale blue). Inset: scanning electron micrograph of ice selection (diameter, 0.15 mm). Ice-active ribozymes (red) extend primers linked to the same bead as themselves and their encoding gene, enabling recovery by flow cytometry. b, Secondary structures of the wild-type R18 ribozyme construct (residues not mutagenised in the starting library are shown in grey) and ribozyme Y, with mutations derived from the in-ice selected ribozyme C8 in blue. c, Polymerase activities of R18 and the evolved ribozyme Y (denaturing PAGE of extension time-courses (primer A /template I)) at 17°C and in ice at −7°C. The average number of nucleotides added per primer is indicated below each lane.

Figure 2. Cold adaptation of ribozyme activity

a, Influence of temperature upon RNA synthesis by ribozymes Y and R18: quantification of average extension (primer A / template I, 7 days) in aqueous (left panel) and frozen (right panel) reactions (error bars represent s.d. of 3 repeats, except for −7°C frozen (6 repeats)). b, Primer extension by Y in ices at different temperatures (primer A / template I; 40 days; * = −25°C, no NTPs).

Figure 3. Basis of cold adaptation

a, Representation of the relative positions of primer (orange), template (lilac) and catalytic core of Y (black), modelled on shared regions in the crystal structure of the progenitor class I ligase ribozyme. The incoming NTP (red) is positioned at the 3′ end of the primer, and the locations of the four mutations comprising Y are highlighted. The indicated single-stranded downstream template and the linker region leading to the processivity domain (not shown) are likely flexible in the polymerase. b, Backmutation analysis of Y to uncover contributions to cold adaptation. Denaturing PAGE of extension (3 days, primer A / template I) at 17°C (top panel) and at −7°C in ice (lower panel), by R18, Y, and four reversion mutants of Y as indicated. These extensions were quantified (average nucleotides added/primer), allowing calculation of the ratio of observed extension in ice versus 17°C for each ribozyme, varying ~10-fold between Y and R18 (bottom panel). c, Influence of template length (indicated) and sequence on extension by R18 and Y (denaturing PAGE of extensions of primer A upon different templates at 17°C and at −7°C in ice (7 days)).

Figure 4. Long range RNA synthesis by ribozyme tC9Y

a, Denaturing PAGE showing the tC9Y-ctalysed extension of primer BioFITC-A upon the (I-n series of templates (17°C, 7 days). Size of synthesised products (nucleotides added to primer) is indicated. b, Secondary structure of the tC9Y ribozyme, shown hybridised to the 5′ end of the template (I-n, where n is the number of central 11-nucleotide repeats) via the 5′ ss_C19 sequence (purple). c, The full spectrum of error rates within all sequenced products synthesised by tC9Y upon template (I-10 at 17°C. The total fidelity (97.4%) is calculated using a geometric mean of the total error rates opposite A, C, G and U. d, The errors shown in (c) were subdivided into rates of each error type in the final two bases of all sequenced extension products versus the rates in all preceding internal bases up to the final two. Note that deletions of a penultimate base would appear as and are assigned as substitutions, as further extension would be required to manifest their identity; thus, the displayed substitution rates in the final two bases represent overestimates. The geometric mean between the total substitution rates at A, C, G, and U is 7.1% in the final two bases of each extension product compared to 0.8% in the preceding bases.

Figure 5. Template selection

a, Sequences of five in-ice evolved templates (round 4) alongside template I-5. b, Extension of primer A by ribozyme tC9Y on ice-selected templates compared to favourable template I-5. (17°C, 7 days). c, Base composition of the central 36-nt evolved region of sequences from template pools over the course of selection (Positions sequenced: Starting pool = 271, Round 1 = 232, Round 2 = 582, Round 3 = 739, Round 4 = 3086). d, Ratio of the observed dinucleotide frequencies (O_i) to those expected (E_i) in the pool of round 4 sequenced templates (based on nucleotide composition). Those overrepresented are in blue, those underrepresented in red.