Subscriber access provided by EMP BRAS PESQ AGROP EMBRAPA

Communication

Expanding the Genetic Code of an Animal

Sebastian Greiss, and Jason W Chin
J. Am. Chem. Soc., Just Accepted Manuscript DOI: 10.1021/ja2054034 Publication Date (Web): 08 August 2011 Downloaded from http://pubs.acs.org on August 12, 2011

Just Accepted
Just Accepted manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides Just Accepted as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. Just Accepted manuscripts appear in full in PDF format accompanied by an HTML abstract. Just Accepted manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI). Just Accepted is an optional service offered to authors. Therefore, the Just Accepted Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the Just Accepted Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these Just Accepted manuscripts.

Journal of the American Chemical Society is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 6

Journal of the American Chemical Society

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Expanding the Genetic Code of an Animal

SebastianGreiss1andJasonW.Chin1,*
1MedicalResearchCouncilLaboratoryofMolecularBiology,HillsRd,Cambridge,CB20QH,UK.

chin@mrclmb.cam.ac.uk SupportingInformationPlaceholder
ABSTRACT: Genetic code expansion, for the sitespecific incorporation of unnatural amino acids into proteins, is currently limited to cultured cells and unicellular organisms. Here we expand the genetic code of a multicellularanimal,thenematodeCaenorhabditiselegans (C.elegans). mutagenesiscouldprovidemuchneedednewinsightcan onlybestudiedinanimals.Herewereportthefirstgenetic code expansion in an animal the nematode worm C. elegans. C. elegans is an attractive target for a multicellular genetic code expansion. The genome of C. elegans is sequenced31 and the lineage of every cell during embryogenesisandpostembryonicdevelopmenthasbeen mapped in this organism32,33, which is invaluable in understanding mutant phenotypes at the cellular level. Theorganismhasaround1000somaticcellsthatmakeup a variety of tissues including muscles, nerves and intestines. The entire organism is transparent at every stage of life, making it possible to visualize expression in individual cells using fluorescent proteins. This will facilitate light mediated intervention in biological processes using genetically encoded photoresponsive amino acids, including photocrosslinkers and photocaged amino acids, as well as unnatural amino acid based imaging methods. Many biochemical and signalling pathways involved in disease are conserved between C. elegansandhumans,whichmakesC.elegansanimportant organism for identifying the molecular mechanisms of disease34. Moreover, C. elegans is the only multicellular organism where amber suppressors have been isolated and introduced into the germ line by classical genetics approaches3538, and suppression efficiencies exceeding 30% have been reported39. These observations suggest that amber suppression is not problematic for the organismthroughitsdevelopmentandreproduction. The sitespecific incorporation of unnatural amino acidsintotargetproteinsposesanumberofchallenges:we require an orthogonal amber suppressor tRNA, that is correctlytranscribed,processed,modifiedandexportedto the cytoplasm of the cell, an orthogonal aaRS that can uniquely aminoacylate the orthogonal tRNA in the cytoplasm, and an mRNA encoding a gene of interest bearing an amber codon that directs amino acid incorporation2,40. In addition, we need to combat any effects of nonsensemediated decay41,42 that may destroy transcripts bearing amber codons and limit expression of proteins bearing unnatural amino acids. The sitespecific incorporationofunnaturalaminoacidsinananimalposes additional challenges, since each of the translational components must be present in the same cell or cells within the organism to effect genetic code expansion, and we need to ensure that the unnatural amino acids are taken up by the animal and are available, within the cytoplasmofitscells,forproteintranslation.

Genetic code expansion, utilizing orthogonal aminoacyltRNA synthetase (aaRS)/tRNACUA pairs, has facilitated the sitespecific incorporation of unnatural amino acids into proteins in E. coli, yeast and cultured mammalian cells16. The application of unnatural amino acid mutagenesis to the production of recombinant proteins allows access to modified proteins, including proteins bearing defined posttranslational modifications, for structural biology, enzymology, and single molecule studies613. The genetically encoded incorporation of photocaged amino acids in living cells allows the photo control of protein interactions, protein localization, enzymatic activity and cellular signaling3,14,15, while the incorporationofphotocrosslinkingaminoacidsallowsthe mapping of weak or transient protein interactions, including those in membranes, that are challenging to define by noncovalent approaches1620, and the incorporation of bioorthogonal chemical handles and biophysical probes are facilitating emerging approaches forproteinimagingandspectroscopy2,11,2129.

Figure1.ExpandingthegeneticcodeofC.elegans.(A)DNA constructs used for genetic code expansion in C. elegans. (B) AminoacidsubstratesforPylRSusedinthisstudy.

The genetically encoded sitespecific incorporation of unnatural amino acids into proteins in multicellular organismswouldfacilitatetheextensionofmoleculartools for dissecting and controlling biological processes inside cells3,14,15,30 to the study of complex processes in whole organisms. This is important because many fundamental biological processes, including those involved in neural processinganddevelopmentwhereunnaturalaminoacid

ACS Paragon Plus Environment

Journal of the American Chemical Society

We created a reporter for amber suppression (Prps 0::mGFPTAGmcherryHANLS) in which a 5 mGFP is separated from a 3 mCherry gene by a linker region containing an amber stop codon (Figure 1). A ribosomal protein promoter (Prps0) in this construct drives expression in most cells in the worm43, the HA tag allows detectionofexpressionbyantiHAantibodies,thenuclear localization sequence (NLS) concentrates fluorescence in the nucleus and the unc54 3 untranslated region (UTR) stabilizes the mRNA throughout the worm. We injected thisreporterintoC.elegansusingaconstructcarryingwild type lin15B as a selection marker in a lin15B(n765) geneticbackground44.Weobservedthatthetransmission frequency of the transgenic extrachromosomal arrays to offspring was low (2030%). This resulted in C. elegans populationswhereamajorityofanimalsdidnotcarrythe transgenes.MoreoverweobservedthattheGFPsignalin worms carrying the reporter was much weaker than the GFPsignalproducedfromasimpleGFPgene. We reasoned that the low GFP expression was likely due to the degradation of reporter mRNA through nonsensemediated decay (NMD), a surveillance mechanism present in eukaryotes that is responsible for detecting and destroying transcripts with premature stop codons41,42. When we crossed worms expressing the reporter with smg2(e2008) worms that are deficient in NMD41,45, but otherwise healthy, we observed a striking increase of GFP signal (Figure 2 & Supporting Information (SI) Figure 1). While we see a strong GFP signalinwormstransformedwiththereporterwedonot observeanymCherryfluorescence,demonstratingthatthe reporter is functional and that the worms do not contain endogenous amber suppressors. Based on these observations we constructed all subsequent transgenic linesusingthesmg2(e2008)worms.

Page 2 of 6

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 2. Each of the components required for genetic code expansion in C. elegans can each be expressed in the animal (A) The effect of nonsensemediated decay (NMD) on GFP expression levels from worms containing the reporter construct (Prps0::mGFPTAGmCherryHANLS). Wildtype C. elegans: the GFP fluorescence of a representative wild type animal. NMD, shows the GFP fluorescence of a transgenic wormcreatedbycrossingthereporterconstructintothesmg 2(e2008)mutantbackground.(B)FLAGMmPylRS(toppanel) and MmtRNACUA (lower panel) are expressed from animals containing Prps0::FLAGMmPylRS and PCeN741::MmPylT respectively.FLAGMmPylRSwasdetectedbywesternblotin worm lysates using an antiFLAG antibody. MmtRNACUA was detected by northern blot from total RNA isolated from worms.Allexperimentsusedamixedstagepopulation.

To address the problem of low transmission levels we tested transformation markers that use a gene conferring resistance to specific antibiotics. Recent reports use puromycin46 and G41847 resistance genes, respectively, for antibiotic based selection in worms. However,

puromycin efficiently kills wild type animals only in the presence of the permeabilizing detergent, Triton X100, and G418 does not kill all wild type worms in a population.Wethereforeinvestigatedafurtherantibiotic, hygromycin B48, which is used for selection in eukaryotic cell culture, but has not been used as a selectable marker in C. elegans. We found that hygromycin B (0.5 mg/ml) kills 100% of wild type worms without the addition of Triton X100 (data not shown). When the hygromycin B phosphotransferasegene(hpt)fusedtotherps0promoter (Prps0::hpt) was injected into worms it conferred resistance to the antibiotic. Using the hpt transformation marker we were able to isolate transgenic lines that appeartohavetransmissionratesof100%inthepresence of hygromycin B (data not shown). In all subsequent experimentsweusedhygromycinBresistanceasamarker forintroducingDNAconstructsintoC.elegans. Three aminoacyltRNA synthetase/tRNACUA pairs (Ec tyrosyltRNA synthetase/tRNACUA, Ec leucyltRNA synthetase/tRNACUA and pyrrolysyltRNA synthetase (PylRS)/ tRNACUA from Methanosarcina species) are orthogonal in eukaryotic cells, and have been used to incorporate unnatural amino acids2,3,5,14,49,50. We and others have demonstrated that the PylRS/tRNACUA pairs from Methanosarcina species including M. barkeri (Mb) and M. mazei (Mm), which naturally uses pyrrolysine, can be used to incorporate a range of unnatural amino acids, includingN(tbutyloxycarbonyl)Llysine(1)andN6[(2 propynyloxy)carbonyl]Llysine (2)36,11,26,27,5154. The PylRS/tRNACUApair,unliketheotherpairsthatcanbeused in eukaryotes, can be evolved in E. coli to recognize new aminoacidsandnumerousunnaturalaminoacidscannow be incorporated using this pair and its evolved variants. Moreover, the pyrrolysyltRNA synthetases evolved in E. colicanbetransplantedintoeukaryoticcells3,5,14,50,55.The abilitytoevolvesynthetasesinE.coliandtransplantthem to other organisms makes it especially attractive to develop the PylRS/tRNACUA system for incorporating unnaturalaminoacidsinanimals. To express MmPylRS from an RNA Polymerase II (Pol II) promoter we created Prps0::FLAGMmPylRS, in which Prps0directsexpressionthroughouttheanimal,theFLAG tag allows the expression of PylRS to be detected by western blot. Western blots demonstrate that the synthetase is expressed in the worm (Figure 2 and SI Figure1). MmtRNACUArequiresRNApolymeraseIIItranscription. TranscriptionofeukaryotictRNAsbyRNAPIIIisdirected by A and B box sequences that are internal to the tRNA gene. Theses sequences are not present in the orthogonal MmtRNACUA gene and it is challenging to introduce them without disrupting the three dimensional structure and functionality of the mature tRNA4. We therefore investigatedextragenicRNApolymeraseIIIpromotersfor the transcription of MmtRNACUA. To direct the transcription of MmtRNACUA we created PCeN74 1::MmPylT::sup73,inwhichtheselectedPolIIIpromoter, derived from the stembulge noncoding RNA (ncRNA) CeN741isfusedtothe5endoftheMmtRNACUAgeneand transcription of the tRNA is terminated by the region found immediately 3 of the sup7 C. elegans tryptophanyl tRNA gene. We chose the CeN741 promoter, since it

ACS Paragon Plus Environment

Page 3 of 6

Journal of the American Chemical Society

silencing of at least one essential genetic component (synthetaseortRNAorreporter). To further demonstrate that the unnatural amino acid isincorporatedinresponsetotheambercodon,leadingto theproductionofthefulllengthGFPmCherryHANLS,we lysed worms from each line grown in the presence and absence of 1 for western blotting. AntiHA, and antiGFP western blots confirmed the unnatural amino acid dependent production of GFPmCherryHANLS in worms (Figure3B,andSIFigure2).

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

shows a high level of expression in adult animals, and some expression in larval stages56,57; we reasoned that these properties would enable us to more efficiently screen for cells or animals expressing a functional tRNA, sincewormsareintheadultstageforuptoseveralweeks but are only in the larval stages for a short period. Northern blots, using a probe specific for MmtRNACUA4, demonstrate that the tRNA is efficiently produced from thispromoterinC.elegans(Figure2&SIFigure1). Weconstructedlinescontainingallgeneticcomponents by biolistic bombardment58 of smg2(e2008) worms with plasmids encoding the reporter, synthetase, tRNA and hygromycin B phosphotransferase gene (Prps0::mGFP TAGmcherryHANLS, Prps0::FLAGMmPylRS, PCeN74 1::MmPylT,Prps0::hpt).Thetransformantsweregrownon platessupplementedwithhygromycinBfor2weekstokill off all nontransgenic worms, resulting in populations where all worms contained the extrachromosomal transgenic array Ex1[Prps0::mGFPTAGmcherryHANLS; Prps0::FLAGMmPylRS; PCeN741::MmPylT; Prps0::hpt]. Survivingwormsweregrownon5mM1andinspectedby fluorescence microscopy for the presence of mCherry in thenucleusofcellswithintheworm.Thisstepallowedus to select for animals expressing the reporter as well as functionalMmPylRSandMmtRNACUA. Weexaminedseveralthousandwormsandobserveda few(1to5)mCherrypositivewormsperhundredworms examined.IndividualwormsshowedmCherryexpression in different tissues, including intestinal cells, pharyngeal cells, neurons and body wall muscle. The mosaicism of expression from these extrachromosomal arrays is well documented and may result from either loss of the array duringmitosisorpartialorcompletesilencingofthearray. We singled out 13 mCherry positive worms and grew them in the absence of 1 and the presence of hygromycin B, to select for inheritance of the array in the resulting lines. We examined these lines for mCherry fluorescence inthepresenceandabsenceof 1.Whilealllinesselected showed amino acid dependent mCherry fluorescence, we focusedinsubsequentexperimentsontwolines(1.3.1and 1.8.1). These lines were singled out from distinct plates, and showed the strongest mCherry fluorescence in the presenceofaminoacid1.Intheabsenceofaminoacid1 we did not find any worms expressing mCherry, in the several thousand animals we screened by fluorescence microscopy.Incontrast,whenaminoacid1wasaddedto the lines we saw strong mCherry fluorescence that was easily detectable under a dissection microscope by eye (Figure3AandSIMovies14),inafractionoftheworms (approximately 5%). The mCherry fluorescence was nuclear,consistentwiththemCherryGFPfusionbearinga nuclear localization. In contrast GFP fluorescence was diffuse, as free GFP resulting from termination at the amber codon does not bear a nuclear localization signal and is found throughout the cell. Between animals in a single line we observed a large variation in both the number and identity of cells displaying mCherry fluorescence. The most likely explanations for this large variation within a line are loss of the extrachromosomal array during developmental mitosis and/or partial silencing of the extrachromosomal array, leading to

Figure 3. The orthogonal MmPylRS/MmtRNACUA pair incorporates 1 or 2 in response to the amber codon in C. elegans. (A) Representative fluorescence images of worms containing Ex1[Prps0::mGFPTAGmcherryHANLS; Prps0::FLAG MmPylRS; PCeN741::MmPylT; Prps0::hpt] in the absence (top panels) and in the presence (bottom panels) of1, see also SI Movies14).BothGFPandtheGFP::mCherryfusionprotein show mosaic expression throughout the animal. GFP mosaicism is not visible here because its fluorescence is saturatingundertheimagingconditions,butGFPmosaicismis clear in Figure 2A. mCherry is nuclear localized, making its mosaicism more obvious. (B) Lanes 16: western blot of raw lysates from mixed populations of worms grown in the absence or presence of 1. Lanes 7,8, 9: western blot of GFP::mCherry fusion protein affinity purified using an antibodyagainstmCherryfromwormsgrownintheabsence or presence of 1 or 2. GFP::mCherry was detected using an antibody against the Cterminal HA tag. Western blots using antiGFP were performed as loading controls (lanes 16) and input controls (lanes 7 and 8). Two independent lines were assayed.Moreproteinwasloadedinthenoaddedaminoacid lanes (lanes 1 and 4). (C) Genetically encoded 2 can be specifically labeled with an azidoderivative of biotin, 3. ImmunoprecipitatedGFP::mCherryfusionfromBwaslabeled with 3. The blot was probed with antiHA to check protein loading, stripped and the reaction products detected using streptavidinhorseradishperoxidase.

To demonstrate the generality of our approach for incorporating unnatural amino acids using the

ACS Paragon Plus Environment

Journal of the American Chemical Society

MmPylRS/MmtRNACUA pair in C. elegans we performed experiments using an alkyne 2, a known substrate for PylRS that we have previously sitespecifically incorporatedintoproteinsinE.coliandeukaryoticcells4,27. Worms from line 1.8.1 displayed mCherry fluorescence in thepresence,butnotabsence,of2(SIFigure3).Whenan antimCherry antibody was used to immunoprecipitate proteins from line 1.8.1 the production of fulllength protein, as detected with an antibody against the C terminalHAtag,wasdependentonthepresenceofeither 1 or 2. To further demonstrate that worms grown in the presenceof2specificallyincorporatethealkyneintoGFP mCherryHANLS we labeled the immunoprecipitated proteinswithabiotinazide,3(SIFigure3B),viaacopper (I) catalyzed cycloaddition59. The protein purified from wormsgrowninthepresenceof2wasselectivelylabeled with biotin, confirming the incorporation of the alkyne (Figure3C). Taken together the fluorescence imaging and western blotdatademonstratethattheMmPylRS/MmtRNACUApair directstheincorporationofunnaturalaminoacids,1or2, in response to an amber stop codon in C. elegans. It formally remains possible that 1 is posttranslationally modified in C. elegans. Although we have demonstrated the specificity of the PylRS/tRNACUA pair for the incorporation of 1 in prokaryotic and eukaryotic unicellular systems4,27 where overexpression of proteins bearing the unnatural amino acid in sufficient quantities for mass spectrometry is less challenging, we cannot rule out the possibility that an aspect of worm metabolismthat is not conserved in either other eukaryotes or bacterialeads to the selective post-translational modification of this amino acid in the worm. The incorporation of 2 in C.elegans was directly confirmed by its specific and selective labeling withthebiotinazide,3. In the absence of added unnatural amino acid no full lengthproteinisproduced,demonstratingthatMmtRNACUA is not appreciably aminoacylated by endogenous synthetases, and is orthogonal in worms. The orthogonality of MmPylRS in C.elegans is confirmed by experimentsdemonstratingthattheMmPylRS/MmtRNACUA does not appreciably incorporate 2 in response to sense codons60(SIFigure4). In conclusion we have demonstrated the first genetically encoded incorporation of unnatural amino acids in a multicellular organism. Since we see mCherry expressionthroughouttheorganismourdatasuggestthat the MmPylRS/MmtRNACUA pair can function in diverse tissues to incorporate unnatural amino acids. The PylRS/tRNACUA pairs and their derivatives that have been evolvedinE.colicanbeusedtodirecttheincorporationof a range of unnatural amino acids; extensions of the approach reported here should allow the introduction of posttranslational modifications, photocaged amino acids, bioorthgonalchemicalhandles,andphotocrosslinkersinto proteins in C. elegans. The approach we have developed may provide tools to dissect the molecular basis of complexbiologicalphenomenainwholeanimals.

Page 4 of 6

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Supplementary Figures and 4 Supplementary Movies. This material is available free of charge via the internet at http://pubs.acs.org.

AUTHOR INFORMATION
Corresponding Author
*chin@mrclmb.cam.ac.uk

Funding Sources
ThisresearchwassupportedbytheMRCandtheLouisJeantet Foundation.

ACKNOWLEDGMENT
We are extremely grateful to M. deBono & his group for extensivehelp&toW.Schmiedfornorthernblots.

REFERENCES
(1)Xie,J.;Schultz,P.G.NatRevMolCellBiol2006,7,775. (2)Chin,J.W.;Cropp,T.A.;Anderson,J.C.;Mukherji,M.;Zhang, Z.;Schultz,P.G.Science2003,301,964. (3)Gautier,A.;Nguyen,D.P.;Lusic,H.;An,W.;Deiters,A.;Chin, J.W.JAmChemSoc2010,132,4086. (4)Hancock,S.M.;Uprety,R.;Deiters,A.;Chin,J.W.JAmChem Soc2010,132,14819. (5)Mukai,T.;Kobayashi,T.;Hino,N.;Yanagisawa,T.;Sakamoto, K.;Yokoyama,S.BiochemBiophysResCommun2008,371,818. (6) Neumann, H.; PeakChew, S. Y.; Chin, J. W. Nat Chem Biol 2008,4,232. (7) Lammers, M.; Neumann, H.; Chin, J. W.; James, L. C. Nat ChemBiol2010,6,331. (8)Liu,C.C.;Schultz,P.G.NatBiotechnol2006,24,1436. (9) Neumann, H.; Hancock, S. M.; Buning, R.; Routh, A.; Chapman, L.; Somers, J.; OwenHughes, T.; van Noort, J.; Rhodes, D.;Chin,J.W.MolCell2009,36,153. (10)Neumann,H.;Hazen,J.L.;Weinstein,J.;Mehl,R.A.;Chin,J. W.JAmChemSoc2008,130,4028. (11)Nguyen,D.P.;GarciaAlai,M.M.;Kapadnis,P.B.;Neumann, H.;Chin,J.W.JAmChemSoc2009,131,14194. (12) Nguyen, D. P.; Garcia Alai, M. M.; Virdee, S.; Chin, J. W. ChemBiol2010,17,1072. (13) Virdee, S.; Ye, Y.; Nguyen, D. P.; Komander, D.; Chin, J. W. NatChemBiol2010,6,750. (14) Gautier, A.; Deiters, A.; Chin, J. W. J Am Chem Soc 2011, 133,2124. (15)Lemke,E.A.;Summerer,D.;Geierstanger,B.H.;Brittain,S. M.;Schultz,P.G.NatChemBiol2007,3,769. (16) Chou, C.; Uprety, R.; Davis, L.; Chin, J. W.; Deiters, A. ChemicalScience2011,2,480. (17)Carvalho,P.;Stanley,A.M.;Rapoport,T.A.Cell2010,143, 579. (18)Chin,J.W.;Martin,A.B.;King,D.S.;Wang,L.;Schultz,P.G. ProcNatlAcadSciUSA2002,99,11020. (19)Hino,N.;Okazaki,Y.;Kobayashi,T.;Hayashi,A.;Sakamoto, K.;Yokoyama,S.NatMethods2005,2,201. (20)Mori,H.;Ito,K.ProcNatlAcadSciUSA2006,103,16159. (21) Jackson, J. C.; Hammill, J. T.; Mehl, R. A. J Am Chem Soc 2007,129,1160. (22)Chin,J.W.;Santoro,S.W.;Martin,A.B.;King,D.S.;Wang, L.;Schultz,P.G.JAmChemSoc2002,124,9026. (23) Deiters, A.; Cropp, T. A.; Mukherji, M.; Chin, J. W.; Anderson,J.C.;Schultz,P.G.JAmChemSoc2003,125,11782. (24)Wang,L.;Zhang,Z.;Brock,A.;Schultz,P.G.ProcNatlAcad SciUSA2003,100,56. (25) Carrico, Z. M.; Romanini, D. W.; Mehl, R. A.; Francis, M. B. ChemCommun(Camb)2008,1205. (26)Fekner,T.;Li,X.;Lee,M.M.;Chan,M.K.AngewChemIntEd Engl2009,48,1633.

ASSOCIATED CONTENT
Supporting Information. Supplementary Reference Information, Supplementary Materials and Methods, 2

ACS Paragon Plus Environment

Page 5 of 6

Journal of the American Chemical Society

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(27) Nguyen, D. P.; Lusic, H.; Neumann, H.; Kapadnis, P. B.; Deiters,A.;Chin,J.W.JAmChemSoc2009,131,8720. (28)Wang,J.;Zhang,W.;Song,W.;Wang,Y.;Yu,Z.;Li,J.;Wu,M.; Wang,L.;Zang,J.;Lin,Q.JAmChemSoc2010,132,14812. (29)Ye,S.;Zaitseva,E.;Caltabiano,G.;Schertler,G.F.;Sakmar, T.P.;Deupi,X.;Vogel,R.Nature2010,464,1386. (30) Chou, C.; Young, D. D.; Deiters, A. Chembiochem 2010, 11,972. (31) C. elegans Sequencing Consortium, Science 1998, 282, 2012. (32)Sulston,J.E.;Horvitz,H.R.DevBiol1977,56,110. (33)Kimble,J.;Hirsh,D.DevBiol1979,70,396. (34) Kaletta, T.; Hengartner, M. O. Nat Rev Drug Discov 2006, 5,387. (35)Hodgkin,J.Genetics1985,111,287. (36)Li,L.;Linning,R.M.;Kondo,K.;Honda,B.M.MolCellBiol 1998,18,703. (37)Kondo,K.;Hodgkin,J.;Waterston,R.H.MolCellBiol1988, 8,3627. (38)Kondo,K.;Makovec,B.;Waterston,R.H.;Hodgkin,J.JMol Biol1990,215,7. (39)Waterston,R.H.Genetics1981,97,307. (40) Liu, D. R.; Magliery, T. J.; Pastrnak, M.; Schultz, P. G. Proc NatlAcadSciUSA1997,94,10092. (41) Hodgkin, J.; Papp, A.; Pulak, R.; Ambros, V.; Anderson, P. Genetics1989,123,301. (42)Longman,D.;Plasterk,R.H.A.;Johnstone,I.L.;Cceres,J. F.Genes&Development2007,21,1075. (43)HuntNewbury,R.;etal.PLoSBiol2007,5,e237. (44)Huang,L.S.;Tzou,P.;Sternberg,P.W.MolBiolCell1994, 5,395. (45)Page,M.F.;Carr,B.;Anders,K.R.;Grimson,A.;Anderson, P.MolCellBiol1999,19,5943. (46) Semple, J. I.; GarciaVerdugo, R.; Lehner, B. Nat Methods 2010. (47) GiordanoSantini, R.; Milstein, S.; Svrzikapa, N.; Tu, D.; Johnsen, R.; Baillie, D.; Vidal, M.; Dupuy, D. Nat Methods 2010, 7, 721. (48)Gritz,L.;Davies,J.Gene1983,25,179. (49)Wu,N.;Deiters,A.;Cropp,T.A.;King,D.;Schultz,P.G.JAm ChemSoc2004,126,14306. (50) Chen, P. R.; Groff, D.; Guo, J.; Ou, W.; Cellitti, S.; Geierstanger, B. H.; Schultz, P. G. Angew Chem Int Ed Engl 2009, 48,4052. (51)Li,X.;Fekner,T.;Ottesen,J.J.;Chan,M.K.AngewChemInt EdEngl2009,48,9184. (52)Polycarpo,C.R.;Herring,S.;Berube,A.;Wood,J.L.;Soll,D.; Ambrogelly,A.FEBSLett2006,580,6695. (53)Nguyen,D.P.;Elliott,T.;Holt,M.;Muir,T.W.;Chin,J.W.J AmChemSoc2011,133,11418. (54)Virdee,S.;Kapadnis,P.B.;Elliott,T.;Lang,K.;Madrzak,J.; Nguyen,D.P.;Riechmann,L.;Chin,J.W.JAmChemSoc2011,133, 10708. (55)Chin,J.W.EMBOJ2011.30,2312 (56)Deng,W.;etal.GenomeRes2006,16,20. (57) Li, T.; He, H.; Wang, Y.; Zheng, H.; Skogerb, G.; Chen, R. BMCMolBiol2008,9,71. (58) Praitis, V.; Casey, E.; Collar, D.; Austin, J. Genetics 2001, 157,1217. (59)Rostovtsev,V.V.;Green,L.G.;Fokin,V.V.;Sharpless,K.B. AngewChem2001,114,14. (60) The orthogonality of the synthetase and tRNA are best addressedinasinglelineofworms,ratherthaninseparatelines bearing only the synthetase or tRNA which may have different geneticbackgrounds.

ACS Paragon Plus Environment

Journal of the American Chemical Society

Page 6 of 6

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Paragon Plus Environment