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template,andthe mirror-image L-enzyme behaves similarly with D-RNAsubstrates and a D-RNA template (Fig. 2a). Furthermore, the two en-zymes can operate in a commonmixture that contains boththe L-andthe D-versionsof the substrates and template.TheD-and L-enzymes can-notinteractthrough WatsonCrick pairing anddo notappear to interactsignificantly throughcross-chiral contacts.The intermolecular reactionexhibits saturation kinetics, with a catalytic rate (kcat) of 0.019 min

1

anda Michaelisconstant (Km)of3.3 mM (ExtendedData Fig. 4).There

is no detectablereactionwhenthe templatesubstratecomplexis of thesame handedness as the enzyme, even at 50 mM concentration.The products of the ligation of twoD-RNA substrates were gel puri-

fied,then subjected to cleavage by RNaseA, whichcleaves39,59-butnot29,59-phosphodiester linkages.Cleavageat theligationjunctionwascom-plete, demonstrating that the enzyme forms the natural 39,59-linkage(Extended Data Fig. 5).

Althoughthe enzyme wasselectedon the basis of templatedligationactivity, this reaction is mechanistically similar to the templated poly-merizationof nucleoside59-triphosphates (NTPs). Otherselected ligaseshave shown at least some polymerization activity12,13, whichis the casehere too. The four L-NTPs were prepared by chemical synthesis andtested in various primer extension reactions with the D-RNA enzyme

and a separate L-RNA template. By providing a template with the se-quence 39-CCCCAGUA-59 immediately downstream from the primer-binding site, and supplying 4 mML-guanosine triphosphate (L-GTP),theD-RNA enzyme catalyses four successive GTP additions (Fig. 2b).When instead provided with D-GTP there is only a very low level ofsingle-nucleotide addition. When provided with a racemic mixture ofD,L-GTP theresults arenearlyidenticalto thereaction withL-GTPalone,with anobservedrateof 0.11 min21 inbothcases(ExtendedDataFig.3b).

Thus, thereis no chiral inhibition in theRNA-catalysed polymerizationreaction,unlikethe situation withthe non-enzymatictemplate-directedpolymerization of activated mononucleotides1.

Other templateprimer combinations were used to demonstrate theability of the D-RNAenzymeto add eachof the four L-NTPs on a com-plementary template(Fig.2b). These experiments revealed thatthe en-zyme does have sequence preferences, with addition to a 39-terminal Cor G residue being most efficient and addition to a 39-terminal A or Uresidue being poor. Addition of GTP to a 39-terminal C is especiallyefficient and mimics the ligation junction that wasused duringin vitroevolution. No attempthas yetbeenmade to select directly forNTP addi-tion or with different sequences surrounding the reaction site. None-theless, the current sequence tolerance of the enzyme is sufficient toenable the assembly of a variety of enantiomeric RNA products.

The RNA enzyme appears to be indifferent to the length of the sub-strates, so long as they are bound to a complementary template. As ademonstration of this property, a mixture ofD-mono- and oligonucle-otides wereassembled on twodifferent longD-RNAtemplates(Fig. 3a,b).Thefirst required seven ligationsand three NTP additions; the secondrequired seven ligations and two NTP additions; and both resulted inthe synthesis of full-length products. The ladder of 59-labelled materi-als demonstrates that some additions are more efficient than others,probably reflecting a mixture of sequence preference, structural con-text and competition among substrates. However, there is a clear pro-gression of successive additions, culminating in thefull-lengthproduct.The accurate assembly of the full-length materials was confirmed bysequence analysis (Extended Data Fig. 6).

As a finaltest of theability of the enzymeto synthesize enantiomericproducts, the D-RNA enzymewas used to assemble11 L-oligonucleotidesto form a mirrorcopy of itself. The tenligation junctions hadeither a Cor G residue at the 39terminus and an A, U or G residue at the 59ter-minus (Fig. 1b). The ladder of 59-labelledmaterials againdemonstratessuccessiveadditions culminating in thefull-length product (Fig.3c). Thisfull-length material was gel purified and tested for enzymatic activityinaligationreactionwithtwo D-RNA substrates anda D-RNAtemplate,confirming that it is fullyfunctional (Fig. 3d). Thisis, to ourknowledge,thefirst demonstration of an enzyme being synthesizedby itsenantiomer.

Biology is overwhelmingly homochiral, withonly sparse examplesofL-sugars and D-amino acids, such as L-arabinose in plant hemicelluloseandD-alanine in bacterial peptidoglycan. There is no known exampleof a biopolymer containing subunitsentirely of thewrong handedness.This is because the stereochemical handshake between biopolymerswould seemto demandchiraluniformity.Yet macromolecules of oppo-site handedness can interact in their own fashion, including to bringabout chemicaltransformations.Theadvantages of a cross-chiral poly-merase for RNA-based lifeare twofold:first,bothenantiomers areused,so polymerization does not deplete the supply of the correct enantio-mer;and second, the interaction between D-and L-RNA does not allowconsecutive WatsonCrick pairs that can contribute to sequence bias.

The question remains as to how a chirally pure RNA enzyme wouldarise in thefirstplace, andmoreover howthere might be both D-and L-

versions of such an enzyme. One possibility is that RNA-based life waspreceded by a genetic system based on an achiral polymer 2,14, whichthen evolved the ability to synthesize RNA polymers. An achiral cata-lyst would generate both D-and L-RNA, but could distinguish betweenthehomo-and heterochiral addition of monomers to thegrowingchain.A second possibility is that life began with the non-enzymatic replica-

tion of eitherD- orL-RNA15,16, and subsequently evolved the ability to

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Fig. 1b, but with the enzyme detached from the primer. The reactions used10mM enzyme, 0.5mM fluorescently labelled upstream substrate, 4mMdownstream substrate, 2 mM template, 250 mM MgCl2and 250 mM NaCl,which were incubatedat pH 8.5and 23uC for 0.5,2 or8 h.The markerlane(M)contains the D- and L-upstreamsubstrates alone,labelledwith either fluorescein(green) or boron-dipyrromethene (red), respectively.b, Template-directedpolymerization ofL-NTPs catalysed by a D-RNA enzyme. The L-primer wastethered to the D-enzyme as shown in Fig. 1b and the L-template was providedseparately. All templates had the primer-binding sequence shown in Fig. 1b,followed by 39-CCCCAGUA-59for GTP addition, 39-UUUUAGUA-59foradenosine triphosphate (ATP) addition, 39-GGGGAGUA-59for cytidinetriphosphate (CTP) addition, or 39-AAAAAGUA-59for uridine triphosphate(UTP) addition. The reactions used 0.5 mM enzymeprimer complex, 1 mMtemplate, and 4 mM of the appropriate NTP, under the same conditionsas described earlier, except at 17 uC for 24 h. The reaction products werephotocleaved to detach the extended primer before analysis by polyacrylamide

gel electrophoresis (PAGE).

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catalysethe cross-chiralpolymerizationof RNA. Theproducts of cross-chiral polymerization could do so similarly, ultimately displacing thechemical replication process.

The cross-chiral polymerase is still a young enzyme, only 16 roundsof selectiveamplification awayfrom random sequence.However, it hasauspicious properties that can probably be improved through further

in vitroevolution. It will be especially important to increase the cata-lytic rate of the enzyme and to enhance its ability to extend 39terminithatend ineitheran A orU residue.The ultimateaimis toachievecross-chiral RNA replication, which would require the enzyme to generate

bothstrands of an RNAduplex, that is, both the enantiomeric enzymeand its complement. Cross-chiral replication does not require the D-and L-enzymes to have the same sequence, and even if initiated withenzymes of thesame sequence,the twowould probablysoon drift apart.If early life did entail the cross-chiral polymerization of RNA, thenthere would have been an era when both sides of the mirror were in-dispensable. Subsequently, however, a keyevolutionary innovation mayhave arisen on one side of the mirror, for example, the invention of

instructed L-polypeptide synthesis byD-RNA. Then the other side ofthe mirror could go dark, leaving biology to followa homochiral path.

Online ContentMethods, along with any additional Extended Data display itemsandSourceData,are available in theonline versionof thepaper; references uniqueto these sections appear only in the online paper.

Received 16 July; accepted 16 September 2014.

Published online 29 October 2014.

1. Joyce,G. F. et al.Chiral selection in poly(C)-directed synthesis of oligo(G). Nature310, 602604 (1984).

2. Joyce, G. F., Schwartz, A. W., Miller, S. L. & Orgel, L. E. The casefor an ancestralgeneticsystem involving simple analoguesof thenucleotides. Proc. NatlAcad.Sci.USA84,43984402 (1987).

3. Klussmann, M. et al.Thermodynamic control of asymmetric amplification inamino acid catalysis. Nature441,621623 (2006).

4. Hein,J. E., Tse,E. & Blackmond,D. G. A route to enantiopure RNAprecursorsfromnearly racemic starting materials. Nat. Chem.3, 704706 (2011).5. Ashley, G. W. Modeling, synthesis, and hybridization properties of L-ribonucleic

acid.J. Am. Chem. Soc.114,97319736 (1992).6. Garbesi, A.et al. L-DNAs as potent antimessenger oligonucleotides: a

reassessment. Nucleic Acids Res.21,41594165 (1993).7. Sczepanski, J.T. & Joyce, G.F. Bindingof a structured D-RNAmoleculeby an L-RNA

aptamer. J. Am. Chem. Soc.135,1329013293 (2013).8. Johnston, W. K., Unrau, P. J., Lawrence, M. S., Glasner, M. E. & Bartel, D. P.

RNA-catalyzed RNA polymerization: accurate and generalRNA-templated primerextension.Science292,13191325 (2001).

9. Wochner, A., Attwater, J., Coulson, A. & Holliger, P. Ribozyme-catalyzedtranscription of an active ribozyme.Science332,209212 (2011).

10. Zaher,H. S.& Unrau,P. J.Selectionof an improvedRNApolymeraseribozymewithsuperior extension and fidelity. RNA13,10171026 (2007).

11. Rohatgi, R., Bartel, D. P. & Szostak, J. W. Kinetic and mechanistic analysis ofnonenzymatic, template-directed oligoribonucleotide ligation.J. Am. Chem. Soc.118, 33323339 (1996).

12. Ekland, E. H. & Bartel, D. P. RNA-catalysed RNA polymerization using nucleosidetriphosphates. Nature382,373376 (1996).

13. McGinness, K. E. & Joyce, G. F. RNA-catalyzed RNA ligation on an external RNAtemplate. Chem. Biol.9,297307 (2002).

14. Bohler, C., Nielsen, P. E. & Orgel, L. E. Template switching between PNA and RNAoligonucleotides. Nature376,578581 (1995).

15. Inoue, T. & Orgel, L. E. A nonenzymatic RNA polymerase model.Science219,859862 (1983).

16. Adamala, K. & Szostak, J. W. Nonenzymatic template-directed RNA synthesisinside model protocells.Science342, 10981100 (2013).

Supplementary Informationis available in theonline version of the paper.

AcknowledgementsThis workwas supported by grant NNX10AQ91Gfrom NASAandby grant 287624 from the Simons Foundation. J.T.S. was supported by RuthL. KirschsteinNational ResearchService Award No. F32GM101741 fromthe NationalInstitutes of Health.

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

Author InformationReprints and permissions information is available atwww.nature.com/reprints. The authors declare no competing financial interests.Readersare welcome to commenton theonline version of thepaper. Correspondenceand requests for materials should be addressed to G.F.J. ([email protected]).

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Figure 3| Cross-chiral assemblyof long RNAs. a, Assembly of 50-nucleotideand 49-nucleotide D-RNAs on complementaryD-RNA templates throughmultiple ligation and polymerization events, catalysed by the L-RNA enzyme.The reaction mixtures were sampled at 0, 1, 2 and 3 days and the 59-labelledproducts were analysed by PAGE in comparison with authentic full-lengthmaterial (M). Numbers on the right indicate the nucleotide length ofsuccessively assembled components. Dots indicate intermediate-lengthmaterials resulting from degradation of longer products. See Methods forreaction conditions.b, Sequences of substrates and templates used to assemblethetwo RNAs shown in a. Dotsindicate the junctionsfor assembly. c, Assemblyof the 83-nucleotide L-RNA enzyme on a complementaryL-RNA template,

catalysed by the D-RNA enzyme of the same sequence. The reaction mixturewas sampled at 0, 1, 3 and 5 days and the products were analysed as above.Red dots in Fig. 1c indicate the junctions for assembly, with sequencemodifications at positions13, 14,31 and32, as shown in Extended Data Fig. 2g.d, Catalytic activity of the L-RNA enzyme that had been assembled by theD-RNA enzyme. The reaction conditions are as in Fig. 2a, but with 0.5 mMenzyme, 0.2mM upstream substrate, 1 mM downstream substrate, and 0.5mMtemplate.Freact, fraction reacted.

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carriedout ina mixturecontaining 2mM ligatedRNA,variousamounts of RNase A,and 50mM Tris(pH 7.6), which wasincubated at 23uC for 1 min,thenquenchedby adding 20 mgml21 tRNA and immediately analysed by PAGE.Analysis of ligation activity.Forcis-ligation reactions, the D-RNA enzyme firstwasjoinedto Sub3in a reactionmixturecontaining 4 Uml21 T4RNAligase,10mMRNAenzyme,7 mMSub3,10mMMgCl2,1mMDTT,50mMTris(pH7.5),and10%DMSO, which wasincubated at 16uC overnight. The ligatedproductswere ethanolprecipitated, purified by PAGE, concentrated using an Amicon YM-10 spin filter,and desalted by ethanolprecipitation. The components for cross-chiral ligation were

assembledin a mixture containing 0.1mM enzyme-Sub3,2 mMSub1,1mM Tem2,250 mMNaCl and 50mM Tris(pH8.5),whichwasheatedat 70uCfor3min,thenslowly cooled to 23 uC. Ligation was initiated by adding an equal volume of a so-lution containing 500 mM MgCl2, 250 mM NaCl, 50 mM Tris (pH 8.5), and 0.1%TWEEN-20, incubating at 23 uC for various times, then quenching with EDTA.Thereactionproducts were photolysed (350 nm)at 4 uC for10 min, thenanalysedby PAGE.

For trans-ligationreactions,20 mM D- or L-16.12txenzymewas mixed with1 mM59-labelled upstreamsubstrate (L-Sub4or D-Sub5, respectively),8 mM downstreamsubstrate(L-Sub1 or D-Sub2, respectively), 4mM template( L-Tem2or D-Tem3, res-pectively), 250mM NaCl,and 50mM Tris (pH8.5), heatedat 70uC for 3 min,thenslowly cooled to 23 uC. Ligation was carried out as described earlier and the pro-ducts were analysed by PAGE.Analysisof polymerizationactivity. The D-16.12t enzyme first wasjoined to Sub3,as described earlier. The components for cross-chiralpolymerizationwere assem-bled in a mixture containing 1 mM enzyme-Sub3, 2mM various L-RNA templates

(Tem58),8 mMof theappropriate NTP, 250mM NaCl and50 mMTris(pH 8.5),which was heated at 70 uC for 3 min, then slowly cooled to 23uC. Polymerizationwas initiated by addingan equal volumeof a solution containing 500 mM MgCl2,250mM NaCl,50 mMTris (pH8.5)and 0.1% TWEEN-20,incubatingat 17uCfor

various times, then quenchingwith EDTA. The reaction products were photolysedand analysed by PAGE, as described earlier.Cross-chiral synthesis of long RNAs.The reaction components were assembledina mixture containing 80mM L-16.12tx, 10mM RNAtemplate (Tem9 or Tem10),4mM [59-32P]-labelledSub7, 20mM eachhexanucleotidesubstrate (Sub810),50 mMeach trinucleotide substrate (Sub1214), 20mM Sub11, 10 mMD-GTP, 250 mMNaCl and 50 mM Tris (pH 8.5), which was heated at 70uC for 3 min, then slowlycooledto 23 uC. Cross-chiral synthesiswas initiatedby addingan equal volume ofa solution containing 500mM MgCl2, 250 mM NaCl, 50mM Tris (pH 8.5) and

0.1%TWEEN-20, incubatingat 17 uC forvarioustimes,thenquenching with EDTA.The biotinylated template was removed by adding 0.5 mg magnetic beads,shakingat23 uCfor1h,washingwith23 0.5ml BufferA, eluting theligatedproductswith23 200ml 25 mM NaOH, and immediately neutralizing with 1 M Tris (pH 7.6).The products were ethanol precipitated and analysed by PAGE in comparison toauthentic full-length materials (Std1 for the 50-nucleotide RNA; Std2 for the 49-nucleotide RNA).

The full-length RNA was excised from the gel, eluted overnight, concentratedusing an AmiconYM-10spin filter,and reversetranscribed as described earlier.The

resultingcDNA was purifiedby PAGE, PCRamplifiedusingprimersFwd3 andRev4,and cloned and sequenced as described earlier, confirming the accurate synthesisof full-length RNA (Extended Data Fig. 6).

Forcross-chiralsynthesis of the L-16.12ts enzyme, an 83-nucleotide L-RNAtem-plate was prepared by cross-chiral ligation of Tem11 and Tem12, using S11 as asplint and D-16.12ts as the catalyst, under the same conditions used to prepareL-16.12tx. The componentsfor synthesis ofL-16.12ts were assembledin a mixturecontaining 80mMD-16.12ts, 10mM template, 50mM each trinucleotide substrate(Sub15and Sub16), 20mM eachof theothersubstrates (Sub1725), 250mM NaCland 50mM Tris(pH8.5), which was heatedat 70uC for 3 min,thenslowly cooledto23 uC. Cross-chiral synthesis wasinitiated by addingan equal volumeof a solu-tion containing 500 mM MgCl2, 250 mM NaCl, 50mM Tris (pH 7.6) and 0.1%TWEEN-20, incubatingat 17 uC for5 days, then quenching with EDTA. Thefull-lengthproductswere purified by PAGE, then testedfor activity in a trans-ligationreaction, as described earlier.

17. Paul, N., Springsteen, G. & Joyce, G. F. Conversion of a ribozyme to adeoxyribozyme through in vitroevolution. Chem. Biol.13,329338(2006).

18. Pluthero, F. G. Rapid purification of high-activityTaq DNA polymerase.NucleicAcids Res.21,48504851 (1993).

19. Wang, L.K., Ho,C. K.,Pei,Y. & Shuman,S. Mutationalanalysis ofbacteriophageT4RNAligase1: different functional groupsare requiredfor thenucleotidyl transferand phosphodiester bond formation steps of the ligation reaction.J. Biol. Chem.278, 2945429462 (2003).

20. Caton-Williams,J.,Hoxhaj,R.,Fiaz, B.& Huang, Z.Use ofa novel59-regioselectivephosphitylating reagent for one-pot synthesis of nucleoside 59-triphosphatesfrom unprotected nucleosides.Curr. Protoc. Nucleic Acid Chem. 52,Chapter 1,Unit 1.30 (2013).

21. Cadwell, R. C. & Joyce, G. F. Randomization of genes by PCR mutagenesis.PCRMethods Appl. 2,2833 (1992).

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Extended Data Figure 1| Sequences of individual clones isolated aftereach phase of the in vitroevolution process. a, Twenty-three clones isolatedafter round 10.b, Ten clones isolated after round 16, with the underlinedsequence derived from the 30 random-sequence nucleotides that were inserted

following round 10. Clone numbers are shown on the left, with numbers inparentheses indicating duplicate sequences among the clones that wereanalysed. Nucleotides within the fixed primer-binding sites are not shown.

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Extended Data Figure 2| Sequence and secondary structure of evolvedand engineered variants of the cross-chiral RNA enzyme. a, Clone 10.2,isolated after round 10. Nucleotides within the primer-binding sites are shownin 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 afterround 16.d, Truncated version of clone 16.12, replacing the primer-bindingsites with two GC pairs (dashed box).e, The 16.12t enzyme, removing the

extended portion of the P3 stem (blue box) and replacing a GU pair withinthe P3 stem by a GC pair (dashed box). f, Variant 16.12tx, replacing twobase pairs compared with 16.12t (dashed box) to facilitate assembly by RNA-catalysed ligation of two shorter RNAs. g, Variant 16.12ts, replacing twoadditionalbase pairscomparedwith 16.12tx (dashed box) to facilitate assemblycatalysed by the enantiomeric enzyme.

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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 thereaction format shown in Fig. 1b. Reaction conditions: 0.05 mM enzymeprimer, 1mM downstream substrate, 0.5 mM template, 250 mM MgCl2,250 mM NaCl, pH 8.5, 23 uC.b, Polymerization ofL-GTP by extension of anL-oligonucleotide primer on a complementaryL-RNA template (see Fig. 2b).Reaction conditions were as described earlier, but with no downstreamsubstrate and with either 4 mML-GTP (solid circles) or 4 mM eachD- andL-GTP (open circles).

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Extended Data Figure 4| Kinetic analysis of the reaction of the D-16.12tenzyme witha separate L-template/primer/substrate complex.The reactionswere carried out as described in Methods, except that the concentration ofenzyme was varied, always in at least tenfold excess over the concentration oftemplate/primer/substrate complex. Values forkobs(the observed rate ofreaction) were obtained for each concentration of enzyme ([E]) based onthe initial rate of reaction, then fit to the MichaelisMenten equation:kobs5 kcat [E]/(Km1 [E]). This gave values forkcatof 0.0196 0.001 min

1 andforKmof 3.360.3mM. Reaction conditions: 0.550 mM enzyme, 0.1 mMtemplate/primer (Tem13), 0.2mM downstream substrate (Sub1), 250 mMMgCl2, 250 mM NaCl, pH 8.5, 23 uC.

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Extended Data Figure 5| Analysis of the regiospecificity of ligation.a, D-RNA substrates and template for ligation, catalysed by the L-16.12txenzyme. Dot indicates the ligation junction, which is also the site for RNase Acleavage that is closest to the 39end of the ligated product. The downstreamsubstrate is labelled at the 39end with fluorescein (circled F).b, RNase Adigestion of the ligated products (LP) in comparison to authentic all-39,59-linked RNA of the same sequence (S10). Reaction conditions: 0100 mgml21

RNase A, 2mM RNA, 50 mM Tris (pH 7.6), 23 uC, 1 min.

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Extended Data Figure 6| Sequence analysis of long RNAs obtained bycross-chiral synthesis. a, Full-length50-nucleotide D-RNA assembledthroughseven ligations and three NTP additions.b, Full-length 49-nucleotide D-RNA

assembled through seven ligations and two NTP additions. See Fig. 3a, b forsubstratesequences and reaction products. See Methods for reaction conditionsand sequencing procedure.

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Extended Data Figure 7| Chemical structure of linkers used to preparevarious enzyme and enzymeprimer molecules. See Supplementary Table 1for sequences of all linker-containing oligonucleotides. Structure 1, CpCdinucleotide tethered via a photocleavable linker to the 59end of Tem1,enabling joining by T4 RNA ligase to the pool of RNAs in rounds 110, asshown in Fig. 1a.2, CpC dinucleotide tethered via a photocleavable,fluorescein-labelled linker to the 59end of Sub2, enabling joining by T4 RNA

ligase to the pool of RNAs in rounds 1116, as shown in Fig. 1b.3, CpCdinucleotide tethered via a photocleavable, boron-dipyrromethene-labelledlinker to the 59end of Sub4, used in the D-RNA-catalysed ligation ofL-RNA(Fig. 2a, red).4, CpC dinucleotide tethered via a photocleavable, fluorescein-labelled linker to the 59end of Sub5, used in the L-RNA-catalysed ligation ofD-RNA (Fig. 2a, green).

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Extended Data Figure 8| High-performance liquid chromatographyanalysis of synthetic L-NTPs. a, Elution of the four L-NTPs.b, Elution ofthe four authentic D-NTPs. High-performance liquid chromatographyconditions: C18 column, linear gradient of 010% acetonitrile in 20 mMtriethylammonium acetate (pH 7.0), ultraviolet detection at 254 nm. AU,absorbance units.

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