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Discovery and evolution of RNA and XNA reverse transcriptase function and fidelity


The ability of reverse transcriptases (RTs) to synthesize a complementary DNA from natural RNA and a range of unnatural xeno nucleic acid (XNA) template chemistries, underpins key methods in molecular and synthetic genetics. However, RTs have proven challenging to discover and engineer, in particular for the more divergent XNA chemistries. Here we describe a general strategy for the directed evolution of RT function for any template chemistry called compartmentalized bead labelling and demonstrate it by the directed evolution of efficient RTs for 2′-O-methyl RNA and hexitol nucleic acids and the discovery of RTs for the orphan XNA chemistries d-altritol nucleic acid and 2′-methoxyethyl RNA, for which previously no RTs existed. Finally, we describe the engineering of XNA RTs with active exonucleolytic proofreading as well as the directed evolution of RNA RTs with very high complementary DNA synthesis fidelities, even in the absence of proofreading.

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Fig. 1: CBL selection scheme.
Fig. 2: XNA reverse transcriptases.
Fig. 3: Characterization of 2′-O-Me-RNA reverse transcriptase C8.
Fig. 4: A reverse transcriptase for PS 2′-MOE RNA.
Fig. 5: Proofreading XNA RTs.
Fig. 6: Selection of high-fidelity RTs.

Data availability

The authors declare that the data supporting the findings of this study are available within the article and its Supplementary Information files.

Code availability

Custom scripts used for RT fidelity analysis in this study can be found at GitHub:


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This work was supported by the Medical Research Council (MRC) grant programme no. MC_U105178804 (P.H., G.H. and A.I.T.), the UK Biotechnology and Biological Sciences Research Council grant no. 09-EuroSYNBIO-OP-013) (B.T.P.) and a research collaboration between AstraZeneca UK and the Medical Research Council, MRC-AstraZeneca Blue Sky Grant (S.A.-F. and NS).

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Authors and Affiliations



G.H,. S.A.-F. and P.H. conceived and designed experiments. G.H. performed all experiments except structural models (with S.A.-F. and B.T.P.), deep sequencing and data analysis (with B.T.P.) and RT characterization (with A.I.T. and N.S.). All authors analysed data, discussed results and co-wrote the manuscript.

Corresponding author

Correspondence to Philipp Holliger.

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The authors declare no competing interests.

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Extended data

Extended Data Fig. 1 CBL model selections, signal amplification and plasmid capture.

a, Cells expressing RT521K were spiked into an excess of cells expressing inactive RT (RT-ΔDTD) at 1:100 (top) and 1:1000 (bottom) ratios and encapsulated in w/o emulsion with beads coated with DNA primer bbFd and template TnotTest7 (Supplementary Table 7). After in-emulsion cell lysis and primer extension, beads were recovered and sorted for fluorescence using FACS (middle panel, dashed box). Plasmids recovered from sorted beads were amplified by qPCR and the amount of RT521K and RT-ΔDTD plasmids post-CBL selection quantified. Enrichment was calculated by determining the ratio of RT521K: RT-ΔDTD plasmids in the input (before CBL selection) (left panels) and comparing it with the output ratio (after CBL selection)(right panels) yielding enrichment factors of ~500-fold in both cases. b, HCR signal amplification. Cells expressing RT521K were encapsulated with beads coated with primer bbTest7 annealed to RNA template tnog_full (Supplementary Table 7) and RNA reverse transcription was performed in emulsion. After recovering the beads from emulsion, the sample was split in half and beads were treated with either a nucleic acid probe (top bead) or underwent HCR (bottom bead). Flow cytometry analysis of the beads labelled with a direct probe (top plot) or HCR (bottom plot) shows approximately an order of magnitude increase in signal and a greater percentage of fluorescently labelled beads in HCR conditions. c, Plasmid capture on microbeads. Plasmids bound to beads during CBL were quantified by qPCR. Cells expressing RT521K were combined with beads in bulk (in solution) or were encapsulated in w/o emulsions and underwent reverse transcription. Beads in w/o emulsion were extracted and bulk and emulsion treated beads were sorted for fluorescence by HCR. The number of plasmids per bead was quantified before and after FACS. Purified plasmid was bound to beads without capture oligo (untreated beads) and with capture oligo as controls showing approximately 10 plasmids captured per bead in emulsion and stably bound (surviving post-sort).

Extended Data Fig. 2 RT mutation screen.

a, Space filling surface model of the ternary structure of KOD polymerase (PDB ID 5OMF) with primer (nascent) strand (red) and template strand (green) shown. The position of mutations selected for screening are shown in blue. b, ELONA (enzyme-linked oligonucleotide assay)-based RT activity assay scheme: (from left to right) RT reactions are performed with a biotinylated primer bbTest7 (Supplementary Table 7), bound to wells in a streptavidin plate and hybridized to 2′-O-Me RNA template (cyan) TFRst 2′-O-Me (Supplementary Table 7). RT synthesized cDNA (red), which remains bound to the plate after template removal. The presence of the (correct) cDNA can be detected by a specific oligonucleotide probe FitcFd (Supplementary Table 7) labelled with FITC (green), which in turn is detected by an anti-FITC antibody (blue) conjugated to horse-radish peroxidase (yellow star). c, RT mutation activity screen: only mutations S383K, N735K and I114T show an improved signal and when combined (RT-TKK) show more than double the signal of wt (RT521K) (NTC, no template negative control, n = 3).

Extended Data Fig. 3 RT-TKK: mutations and electrostatic surface.

a, Sequence alignment of RT521K and RT-TKK with mutations shown in blue. b, Space filling model of the ternary structure of KOD polymerase (PDB ID 5OMF) with RT-TKK mutations I114T, S383K and N735K (blue), primer strand (red), template (green) and incoming deoxynucleotide triphosphate (orange). c, Zoom in of the uracil binding pocket (UBP) with uracil base bound (PDB ID 2VWJ) and V93Q mutation (orange). (present in both RT-521K and RT-TKK) and I114T mutation (blue spheres) (RT-TKK). Note how V93Q narrows the UBP (compared to wild-type UBP shown in Extended Data Fig. 1a) and sterically excludes uracil from the binding pocket. The mechanistic basis of the I114T mutation improvement of cDNA synthesis on 2′-O-Me RNA is currently unclear. The main chain NH of I114 hydrogen bonds with uracil in the wild-type UBP. Mutation to I114T may alter main chain conformation to disrupt this interaction and this may further improve uracil exclusion. d, Electrostatic potential of the primer/template binding surface of KOD (left) and its change upon N735K and S383K mutations. Note the increase of positively charged surface potential in proximity to the template strand (green), which is likely to enhance template binding.

Extended Data Fig. 4 A reverse transcriptase for 2′-MOE RNA.

a, Chemical structure of 2′-MOE RNA. b, denaturing urea-PAGE of cDNA synthesis on 2′-MOE RNA template (sequence: 10–27 Spinraza (Supplementary Table 7)) and c, RT-PCR of cDNA synthesis. Only RT-C8 can reverse transcribe 2′-MOE RNA (as well as PS 2′-MOE RNA (Fig. 4)).

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Supplementary Methods, Figs. 1–8, Tables 1–11 and References.

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Houlihan, G., Arangundy-Franklin, S., Porebski, B.T. et al. Discovery and evolution of RNA and XNA reverse transcriptase function and fidelity. Nat. Chem. 12, 683–690 (2020).

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