The canonical genetic code includes 64codons encoding 20 amino acids andthree stop signals. It is preserved in

three kingdoms of life. The origin of the ge-netic code, whether a “frozen accident” or anexpansion from a primordial code with feweramino acids, remains an enigma (1, 2).Although proteins carry out most of the com-plex processes of life, there is clearly a needfor additional building blocks: Some func-tions are dependent on posttranslationalmodification or cofactors, and many impor-tant peptides containing unusual amino acidsare synthesized nonribosomally (3). Why on-ly 20, and why were these 20 amino acids inparticular chosen for the code? Nature en-codes two additional amino acids, selenocys-teine (Sec) and pyrrolysine (Pyl), in limitedproteins but in a distinctive way: The Sec-tRNASec is converted from a preloaded Ser-tRNASec. Special mRNA elements and elon-gation factors are also required for Sec incor-poration into proteins (4). Pyl is likely incor-porated similarly (5). Why does nature notsimply employ the standard mechanism ofdirectly loading amino acids onto their cog-nate tRNA to incorporate Sec and Pyl?

In my graduate research, I exploredwhether the genetic code can be expandedto accommodate additional amino acids, us-ing a strategy that mimics the way that thecommon amino acids are encoded. To dothis, a novel tRNA-codon pair and anaminoacyl–tRNA synthetase (aaRS) need tobe generated that uniquely incorporate anunnatural amino acid. The new componentsshould be orthogonal to the endogenousones to avoid crosstalk and should functionefficiently with the translational apparatus(see the figure) (6).

Design of a new codon-tRNA-aaRS setfrom scratch would be nearly impossible,considering their delicate interactionsevolved to ensure translational accuracy.My approach was therefore borrowing andengineering. Escherichia coli was chosenas the host organism, and the amber non-sense codon (UAG) was hijacked to en-code an unnatural amino acid. I first gen-erated an orthogonal amber suppressortRNATyr

CUA/TyrRS pair in E. coli by import-

ing a tRNATyr/TyrRS pairfrom the archaebacteriumMethanococcus jannaschii(Mj), after testing varioustRNA/aaRS pairs fromdifferent organisms (7).To optimize this pair, Ithendeveloped a generalstrategy consisting of neg-ative and positive selec-tions of a mutant suppres-sor tRNA library (8).Eleven nucleotides of Mj-tRNATyr

CUA were randomlymutated, and from the re-sulting library a mutant(mutRNATyr

CUA) was identi-fied that has almost noaffinity for E. coli syn-thetases and is stillcharged efficiently by theorthogonal Mj-TyrRS with tyrosine. The nextstep was to alter the amino acid specificity ofthe Mj-TyrRS so that it aminoacylated themutRNATyr

CUA with an unnatural amino acidonly. A combinatorial approach was pursued,in which a pool of mutant synthetases wasgenerated from the framework of the wild-type synthetase and then mutants were select-ed based on their specificity for an unnaturalamino acid relative to the common 20.

Five active-site residues of Mj-TyrRS wererandomly mutated to generate the synthetase

library. After two rounds of selection, a syn-thetase was evolved that, when coexpressedwith the mutRNATyr

CUA, incorporates O-methyl-L-tyrosine into proteins in response tothe amber codon with translational fidelityand yield rivaling those of natural amino acids(9). Thus, the genetic code of E. coli was ex-

panded for the first time. Isubsequently evolved asecond mutant synthetasethat is capable of selective-ly inserting L-3-(2-naph-thyl)-alanine, an aminoacid structurally distinctfrom tyrosine, suggestingthat this methodologyshould be generalizable tovarious unnatural aminoacids (10).

These results showthat the genetic code canindeed be expanded fur-ther using nature’s tech-nique—loading aminoacid to cognate tRNA viasynthetase—although na-ture did not repeat thismethod for a 21st amino

acid. This expansion may recapitulate howsome of the common amino acids wereadded to the genetic code, suggesting that anincremental expansion was involved in thecode’s origin. The fact that no toxic side ef-fects were observed in E. coli cells with UAGencoding an unnatural amino acid supportsthe codon reassignment hypothesis (2). To in-vestigate the evolutionary consequences ofadding novel amino acids to the geneticrepertoire, a completely autonomous21–amino acid bacterium was generated,

which biosynthesizes p-amino-L-phenylalanine from basic car-bon sources and incorporates itin response to the UAG codon(11). Directed evolution of suchorganisms under selective pres-sure is under way and may shedlight on whether additionalamino acids give an evolutionaryadvantage.

Genetically encoding newamino acids makes it possible totailor changes in proteins in livecells, and therefore protein struc-ture and function can be studieddirectly in vivo in addition to invitro. Using the same methodand system, we subsequently en-coded more than 13 unnaturalamino acids with novel function-alities in E. coli (12). For in-stance, the versatile keto group

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Expanding the Genetic CodeLei Wang

ESSAY

The author is in the Department of Pharmacology,University of California, San Diego, La Jolla, CA92093, USA. E-mail: lewang@ucsd.edu

Natural amino acid

EndogenoustRNA

AMP+PPi

Translation

Protein

Unique codon

mRNA

ATP

Endogenoussynthetase

Unnatural amino acid

OrthogonaltRNA

AMP+PPi

ATP

Orthogonalsynthetase

New building blocks. A general method for genetically en-

coding unnatural amino acids into proteins.

24 OCTOBER 2003 VOL 302 SCIENCE www.sciencemag.org

Amersham Biosciences and Scienceare pleased to present the prize-win-

ning essay by Lei Wang, a regional

winner from North America, who is

the 2003 Grand Prize winner of the

Amersham Biosciences and SciencePrize for young scientists.

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was genetically encoded in the form of p-acetyl-L-phenylalanine (13). It served as aunique chemical handle, through which pro-teins were selectively labeled with fluo-rophores for imaging, with biotin for detec-tion, and with carbohydrates for generationof homogeneous glycoprotein mimetics(14). Other agents such as spin labels, metalchelators, cross-linking agents, polyethers,fatty acids, and toxins can be attached simi-larly. Two heavy atom–containing aminoacids (p-bromo and p-iodo-L-phenylala-nine) were site-specifically incorporated in-to proteins, providing a reliable method forpreparing isomorphous heavy-atom deriva-tives of proteins for crystallography (12).

The availability of novel building blocksmay lead to protein properties that never ex-isted before. In an initial test, Tyr66 of thegreen fluorescent protein (GFP) was substi-

tuted with several tyrosine analogs, result-ing in mutant GFPs with emissions rangingfrom blue to cyan to green, as well as othernew spectral properties (15). In vivo unnat-ural amino acid mutagenesis by rational de-sign or directed protein evolution shouldgreatly expand the scope and power of pro-tein engineering.

In summary, my thesis research demon-strated that the genetic code can be expandedto include new amino acids. The methodolo-gy is generalizable to different amino acids aswell as cell types (16). It provides a newmeans for evolutionary study of the geneticcode, and powerful tools for molecular andcellular biologists to dissect protein and cel-lular function both in vitro and in vivo. Withadditional building blocks genetically encod-ed, proteins and even organisms with en-hanced or novel properties may be evolved.

References

1. F. H. Crick, J. Mol. Biol. 38, 367 (1968).2. R. D. Knight, S. J. Freeland, L. F. Landweber, Nature Rev.

Genet. 2, 49 (2001).3. C. T. Walsh et al., Curr. Opin. Chem. Biol. 5, 525

(2001).4. A. Bock et al., Mol. Microbiol. 5, 515 (1991).5. G. Srinivasan, C. M. James, J. A. Krzycki, Science 296,

1459 (2002).6. L. Wang, P. G. Schultz, Chem. Commun. 1 (2002).7. L. Wang, T. J. Magliery, D. R. Liu, P. G. Schultz, J. Am.

Chem. Soc. 122, 5010 (2000).8. L. Wang, P. G. Schultz, Chem. Biol. 8, 883 (2001).9. L. Wang, A. Brock, B. Herberich, P. G. Schultz, Science

292, 498 (2001).10. L.Wang, A. Brock, P. G. Schultz, J. Am. Chem. Soc. 124,

1836 (2002).11. R. A. Mehl et al., J. Am. Chem. Soc. 125, 935 (2003).12. L. Wang, thesis, University of California at Berkeley

(2002).13. L. Wang, Z. Zhang, A. Brock, P. G. Schultz, Proc. Natl.

Acad. Sci. U.S.A. 100, 56 (2003).14. H. Liu, L. Wang, A. Brock, C. H. Wong, P. G. Schultz, J.

Am. Chem. Soc. 125, 1702 (2003).15. L. Wang, J. Xie, A. A. Deniz, P. G. Schultz, J. Org. Chem.

68, 174 (2003).16. K. Sakamoto et al., Nucleic Acids Res. 30, 4692 (2002).

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2003 Grand Prize Winner

Lei Wang was born in Tonggu, China. He attended PekingUniversity and received his bachelor’s degree in organic chem-istry in 1994 and his master’s degree in physical chemistry in

1997. At Peking University, working in Dr. Zhongfan Liu’s labora-tory, he used scanning probe microscopy to investi-gate the properties of nanoparticles. He left Chinafor the United States to pursue graduate studies atthe University of California at Berkeley. Under theguidance of Dr. Peter G. Schultz, Dr. Wang devel-oped a general method for genetically encoding un-natural amino acids into proteins in live cells. Afterreceiving his Ph.D. in 2002, Dr. Wang joined Dr.Roger Y. Tsien’s group at the University of

California, San Diego, for postdoctoral training as a Merck Fellowof the Damon Runyon Cancer Research Foundation.

Regional WinnersNorth America: Jeff Levsky, for his essay “Simple Single Cells,”

based on his Ph.D. research in the laboratory of Dr. Robert Singerat the Albert Einstein College of Medicine, New York. Dr. Levskywas born in Washington, DC, in 1978. After receiving a bachelor’sdegree from Northwestern University, Illinois, in 1998 he joined theMedical Scientist Training Program at Albert Einstein University.In Dr. Singer’s group, he studied the development of single-cellgene expression profiling technology. Dr. Levsky is currently com-pleting his clinical training and is searching for a residency positionto pursue a physician-scientist career in imaging and computing.

Europe: Rut Carbadillo-Lopez, for her essay “ShapingBacteria: The Actin-Like Prokaryotic Cytoskeleton,” based on re-search performed under the guidance of Prof. Jeff Errington at theSir William Dunn School of Pathology, University of Oxford, UK.Dr. Carbadillo-Lopez left her home town of Barcelona, Spain, toattend the National Institute of Applied Sciences (INSA) in Lyon,France, and graduated in 1996 with an engineering degree in bio-chemistry. She spent 17 months working in industry, and then ob-tained a master’s degree in general microbiology from the PasteurInstitute of Paris before joining the University of Oxford as agraduate student. After receiving her Ph.D. in 2002, she wasawarded a Long-Term Fellowship from the Human FrontiersScience Program Organization for postdoctoral training.

Ravi Kamath, for his essay “Functional Genomics in C. ele-gans Using RNAi,” based on his Ph.D. research performed in thelaboratory of Dr. Julie Ahringer at the Wellcome Trust/CancerResearch UK Institute of Cancer and Developmental Biology. Dr.Kamath was born in Ohio. After completing an undergraduatedegree at Harvard University, he entered Harvard MedicalSchool in 1997, but took a break to pursue graduate studies at theUniversity of Cambridge as a Howard Hughes Medical InstitutePredoctoral Fellow. He completed his Ph.D. degree in 2002 andhas since returned to complete his medical degree at Harvard,where he is currently in his final year.

All Other Countries: Qing Chen, for her essay “Induction ofbgl Operon Expression in E. coli: Novel Insights into SensorStimulation and Signaling,” based on research in the laboratory ofProf. Orna Amster-Choder at the Hebrew University MedicalSchool, Israel. Dr. Chen was born in 1964 in Jinan, China. She re-ceived her M.D. degree in 1986, and a master’s degree in bio-chemistry in 1991 from the Medical School of ShandongUniversity. She worked as a lecturer at the Medical School ofBeijing University for 2 years before going to Jerusalem to pursuePh.D. studies. She was awarded her Ph.D. degree in 2002. In 1998,she left Israel for a research associate position in the laboratory ofProf. Robert Kadner at the University of Virginia. She is current-ly a research scientist in the laboratory of Dr. Malabi Venkatensanat the Walter Reed Army Institute of Research, Maryland.

David Lando, for his essay “The Huff and Puff of HIFRegulation,” based on his Ph.D. research carried out in the labo-ratory of Dr. Murray Whitelaw in the Department of MolecularBiosciences at the University of Adelaide, Australia. Dr. Landowas born in 1969 in Mildura, a small Australian country town.He obtained his B.Sc. degree in 1990 from Flinders University ofSouth Australia. After spending 6 years working for a localbiotechnology company, Dr. Lando returned to university to worktoward a doctoral degree. After completing his Ph.D., he joinedthe laboratory of Dr. Tony Kouzarides at the Wellcome TrustCancer Research UK Institute in Cambridge, where he currentlyholds a C. J. Martin Fellowship from the the National Health andMedical Research Council of Australia.

For the full text of essays by the regional winners and for infor-mation about applying for next year’s awards, see Science Online athttp://www.sciencemag.org/feature/data/pharmacia/prize/apbprize.shl.

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