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A cross-chiral RNA polymerase ribozyme

Abstract

Thirty years ago it was shown that the non-enzymatic, template-directed polymerization of activated mononucleotides proceeds readily in a homochiral system, but is severely inhibited by the presence of the opposing enantiomer1. This finding poses a severe challenge for the spontaneous emergence of RNA-based life, and has led to the suggestion that either RNA was preceded by some other genetic polymer that is not subject to chiral inhibition2 or chiral symmetry was broken through chemical processes before the origin of RNA-based life3,4. Once an RNA enzyme arose that could catalyse the polymerization of RNA, it would have been possible to distinguish among the two enantiomers, enabling RNA replication and RNA-based evolution to occur. It is commonly thought that the earliest RNA polymerase and its substrates would have been of the same handedness, but this is not necessarily the case. Replicating d- and l-RNA molecules may have emerged together, based on the ability of structured RNAs of one handedness to catalyse the templated polymerization of activated mononucleotides of the opposite handedness. Here we develop such a cross-chiral RNA polymerase, using in vitro evolution starting from a population of random-sequence RNAs. The d-RNA enzyme, consisting of 83 nucleotides, catalyses the joining of l-mono- or oligonucleotide substrates on a complementary l-RNA template, and similar behaviour occurs for the l-enzyme with d-substrates and a d-template. Chiral inhibition is avoided because the 106-fold rate acceleration of the enzyme only pertains to cross-chiral substrates. The enzyme’s activity is sufficient to generate full-length copies of its enantiomer through the templated joining of 11 component oligonucleotides.

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Figure 1: Evolution of a cross-chiral RNA ligase.
Figure 2: Cross-chiral ligation and polymerization.
Figure 3: Cross-chiral assembly of long RNAs.

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Acknowledgements

This work was supported by grant NNX10AQ91G from NASA and by grant 287624 from the Simons Foundation. J.T.S. was supported by Ruth L. Kirschstein National Research Service Award No. F32 GM101741 from the National Institutes of Health.

Author information

Authors and Affiliations

Authors

Contributions

J.T.S. and G.F.J. conceived the project, designed the experiments, and wrote the paper. J.T.S. carried out the experiments.

Corresponding author

Correspondence to Gerald F. Joyce.

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Competing interests

The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Sequences of individual clones isolated after each phase of the in vitro evolution process.

a, Twenty-three clones isolated after round 10. b, Ten clones isolated after round 16, with the underlined sequence derived from the 30 random-sequence nucleotides that were inserted following round 10. Clone numbers are shown on the left, with numbers in parentheses indicating duplicate sequences among the clones that were analysed. Nucleotides within the fixed primer-binding sites are not shown.

Extended Data Figure 2 Sequence and secondary structure of evolved and engineered variants of the cross-chiral RNA enzyme.

a, Clone 10.2, isolated after round 10. Nucleotides within the primer-binding sites are shown in red. b, Lib2, based on clone 10.2, with 30 random-sequence nucleotides (N30, green) inserted between the P1 and P3 stems. c, Clone 16.12, isolated after round 16. d, Truncated version of clone 16.12, replacing the primer-binding sites with two G–C pairs (dashed box). e, The 16.12t enzyme, removing the extended portion of the P3 stem (blue box) and replacing a G–U pair within the P3 stem by a G–C pair (dashed box). f, Variant 16.12tx, replacing two base pairs compared with 16.12t (dashed box) to facilitate assembly by RNA-catalysed ligation of two shorter RNAs. g, Variant 16.12ts, replacing two additional base pairs compared with 16.12tx (dashed box) to facilitate assembly catalysed by the enantiomeric enzyme.

Extended Data Figure 3 Catalytic activity of the d-16.12t enzyme.

a, Ligation of two l-oligonucleotides on an l-RNA template, according to the reaction format shown in Fig. 1b. Reaction conditions: 0.05 µM enzyme–primer, 1 µM downstream substrate, 0.5 µM template, 250 mM MgCl2, 250 mM NaCl, pH 8.5, 23 °C. b, Polymerization of l-GTP by extension of an l-oligonucleotide primer on a complementary l-RNA template (see Fig. 2b). Reaction conditions were as described earlier, but with no downstream substrate and with either 4 mM l-GTP (solid circles) or 4 mM each d- and l-GTP (open circles).

Source data

Extended Data Figure 4 Kinetic analysis of the reaction of the d-16.12t enzyme with a separate l-template/primer/substrate complex.

The reactions were carried out as described in Methods, except that the concentration of enzyme was varied, always in at least tenfold excess over the concentration of template/primer/substrate complex. Values for kobs (the observed rate of reaction) were obtained for each concentration of enzyme ([E]) based on the initial rate of reaction, then fit to the Michaelis–Menten equation: kobs = kcat [E]/(Km + [E]). This gave values for kcat of 0.019 ± 0.001 min–1 and for Km of 3.3 ± 0.3 µM. Reaction conditions: 0.5–50 µM enzyme, 0.1 µM template/primer (Tem13), 0.2 µM downstream substrate (Sub1), 250 mM MgCl2, 250 mM NaCl, pH 8.5, 23 °C.

Source data

Extended Data Figure 5 Analysis of the regiospecificity of ligation.

a, d-RNA substrates and template for ligation, catalysed by the l-16.12tx enzyme. Dot indicates the ligation junction, which is also the site for RNase A cleavage that is closest to the 3′ end of the ligated product. The downstream substrate is labelled at the 3′ end with fluorescein (circled F). b, RNase A digestion of the ligated products (LP) in comparison to authentic all-3′,5′-linked RNA of the same sequence (S10). Reaction conditions: 0–100 µg µl−1 RNase A, 2 μM RNA, 50 mM Tris (pH 7.6), 23 °C, 1 min.

Extended Data Figure 6 Sequence analysis of long RNAs obtained by cross-chiral synthesis.

a, Full-length 50-nucleotide d-RNA assembled through seven ligations and three NTP additions. b, Full-length 49-nucleotide d-RNA assembled through seven ligations and two NTP additions. See Fig. 3a, b for substrate sequences and reaction products. See Methods for reaction conditions and sequencing procedure.

Extended Data Figure 7 Chemical structure of linkers used to prepare various enzyme and enzyme–primer molecules.

See Supplementary Table 1 for sequences of all linker-containing oligonucleotides. Structure 1, CpC dinucleotide tethered via a photocleavable linker to the 5′ end of Tem1, enabling joining by T4 RNA ligase to the pool of RNAs in rounds 1–10, as shown in Fig. 1a. 2, CpC dinucleotide tethered via a photocleavable, fluorescein-labelled linker to the 5′ end of Sub2, enabling joining by T4 RNA ligase to the pool of RNAs in rounds 11–16, as shown in Fig. 1b. 3, CpC dinucleotide tethered via a photocleavable, boron-dipyrromethene-labelled linker to the 5′ end of Sub4, used in the d-RNA-catalysed ligation of l-RNA (Fig. 2a, red). 4, CpC dinucleotide tethered via a photocleavable, fluorescein-labelled linker to the 5′ end of Sub5, used in the l-RNA-catalysed ligation of d-RNA (Fig. 2a, green).

Extended Data Figure 8 High-performance liquid chromatography analysis of synthetic l-NTPs.

a, Elution of the four l-NTPs. b, Elution of the four authentic d-NTPs. High-performance liquid chromatography conditions: C18 column, linear gradient of 0–10% acetonitrile in 20 mM triethylammonium acetate (pH 7.0), ultraviolet detection at 254 nm. AU, absorbance units.

Supplementary information

Supplementary Table 1

This file contains sequences of oligonucleotides used in the study. (PDF 83 kb)

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Sczepanski, J., Joyce, G. A cross-chiral RNA polymerase ribozyme. Nature 515, 440–442 (2014). https://doi.org/10.1038/nature13900

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