Orthogonal Q-Ribosomes
Field of the Invention
The invention relates to ribosomes for translation of quadruplet codons.
5 Background to the Invention
10
15
20
Since each of the 64 triplet codons are used to encode natural amino acids or
polypeptide termination. new blank codons are required for cellular genetic code
expansion. In principle quadruplet codons might provide 256 blank codons.
Stoichiometrically aminoacylated extended anticodon tRNAs have been used to
incorporate unnatural amino acids in response to 4-base codons with very low
efficiency in in vitro systems 11- 13 and in limited in vivo systems. via import of previously
aminoacylated tRNA 14 15. This is a problem in the art.
In one case a 4-base suppressor and amber codon have been used. in a nongeneralizable
approach. to encode two unremarkable amino acids with low efficiency
16. Indeed. the inefficiency with which natural ribosomes decode quadruplet codons
severely limits their utility for genetic code expansion. which is a problem in the art.
The present invention seeks to overcome problem(s) associated with the prior art.
Summary of the Invention
25 The inventors have mutated certain ribosomal components to produce a ribosome with
a new technical capability of translating quadruplet codons. The mutations have
focussed on the 16S rRNA. The ribosomes produced according to the present invention
are sometimes referred to as quadruplet-ribosomes or Q-Ribosomes (RiboQ).
30 In one aspect. the invention relates to a 16S rRNA comprising a mutation at A 1196.
In one aspect. the invention relates to a 16S rRNA comprising a mutation at A 1196 and
at least one further mutation selected from C 1195T. A 1197G. C 1195A.
35 In another aspect. the invention relates to a 16S rRNA as described above further
comprising a mutation at C 1195 and/or A 1197.
5
wo 2011/077075 PCT/GB2010/002296
In another aspect. the invention relates to a 16S rRNA as described above which
comprises
(i) C1195A and A1196G; or
(ii)
(iii)
C1195T. A1196G and A1197G; or
A1196G and A1197G.
In another aspect, the invention relates to a ribosome capable of translating a
quadruplet codon. said ribosome comprising a 16S rRNA as described above.
10 In another aspect. the invention relates to use of a 16S rRNA as described above in the
translation of a mRNA comprising at least one quadruplet codon.
15
20
Detailed Descrlpflon of the lnvenflon
In one aspect the invention relates to a 16S rRNA comprising a mutation at A 1196.
Suitably said mutation is A 1196G.
In another aspect. the invention relates to a 16S rRNA as described above further
comprising a mutation at C1195 and/or A 1197.
In another aspect, the invention relates to a 16S rRNA as described above which
comprises
(i) C1195A and A 1196G; or
(ii)
25 (iii)
C1195T, A1196G and A1197G; or
A1196G and A1197G.
In another aspect. the invention relates to a 16S rRNA as described above which further
comprises A531 G and U534A.
30 In another aspect, the invention relates to a ribosome capable of translating a
quadruplet codon, said ribosome comprising a 16S rRNA as described above.
35
In another aspect. the invention relates to use of a 16S rRNA as described above in the
translation of a mRNA comprising at least one quadruplet codon.
Suitably the l6S rRNA of the invention comprising a mutation at A 1196 comprises
A 1196G. This specific mutation is common to each of the preferred 16S rRNAs
2
wo 2011/077075 PCT/GB2010/002296
exemplified herein such as Q l, Q2. Q3 and Q4, which all possess A 1196G (i.e. G at
position 1196).
Suitably the 16S rRNA of the invention further comprises a mutation at A 1197. Suitably
5 the 16S rRNA of the invention comprising a mutation at A 1197 comprises A 1197G. This
specific mutation is common to 75% of the preferred 165 rRNAs exemplified herein such
as Ql. Q2 and Q3. which all possess A1197G (i.e. Gat position 1197).
Suitably the 16S rRNA of the invention comprises a mutation at A 1196 and a mutation at
10 A1197. Most suitably the 16S rRNA of the invention comprises A1196G and A1197G.
Each of Q 1. Q2 and Q3 comprise this combination of mutations.
Suitably the 16S rRNA of the invention may comprise a mutation at C 1195. This mutation
may be C1195T or C1195A. Suitably the 16S rRNA of the invention which comprises a
15 C1195 mutation also comprises a A1196 mutation such as A1196G. Suitably when the
16S rRNA of the invention comprises A1197G. it also comprises C1195T. Suitably when
the 16S rRNA of the invention comprises A1196G and A1197G. it also comprises C1195T.
Suitably when the 16S rRNA of the invention comprises A 1196G and is wild type at
A 1197 (i.e. A at position 1197). it also comprises C 1195A.
20
Further mutations may be present or may not be present.
Rlbo-X and Rlbo-Q
25 The Ribo-Q 1 6S rRNA sequences herein have been prepared from Ribo-X as a starting
16S rRNA sequence. Ribo-X is a published 16S rRNA sequence well known to the person
skilled in the art. More specifically, Ribo-X refers to a 165 rRNA sequence which has two
substitutions compared to wild type. namely A531 G and U534A. Therefore suitably
each Ribo-Q 16S rRNA sequence described herein also possesses A531 G and U534A in
30 addition to each further mutation or substitution discussed herein. It should be
assumed that the 1 6S rRNAs of the invention each possess A531 G and U534A in addition
to any other mutations discussed, unless the context indicates otherwise. Thus. suitably
each 16S rRNA of the invention comprises at least 3 mutations compared to wild type.
namely A 1196, A531 G and U534A. most suitably A 1196G, A531 G and U534A.
35
In case any more detail is needed, Ribo-X is discussed in depth in PCT /GB2007/004562
(published as W02008/065398). This document is specifically incorporated herein by
reference expressly for the detail of the Ribo-X 16S rRNA sequence which is the
3
wo 2011/077075 PCT/GB2010/002296
'background' or parent sequence from which the Ribo-Q 16S rRNAs of the invention are
derived and/or produced.
Suitably the 16S rRNA of the invention comprises A 1196G and A 1197G (Ribo-Q 1, Ribo-
5 Q2, Ribo-Q3).
Suitably the 16S rRNA of the invention comprises C1195T and All96G and Al197G
(Ribo-Q3).
10 Suitably the 16S rRNA of the invention comprises Cl195T and A 1196G (Ribo-Q4).
In one embodiment the 16S rRNA of the invention consists of wild type 16S rRNA
sequence and A531G and U534A and A1196G and Al197G (Ribo-Q1).
15 In one embodiment the 16S rRNA of the invention consists of wild type 16S rRNA
sequence and A531 G and U534A and A 1196G and A 1197G and up to 8 further
mutations/substitutions (Ribo-Q2).
In one embodiment the 16S rRNA of the invention consists of wild type 16S rRNA
20 sequence and A531 G and U534A and C1195T and A 1196G and A 1197G (Ribo-Q3).
In one embodiment the 16S rRNA of the invention consists of wild type 16S rRNA
sequence and A531G and U534A and Cll95T and All96G (Ribo-Q4).
25 The invention relates to encoding multiple unnatural amino acids via evolution of a
quadruplet decoding ribosome.
Definitions
As the term "orthogonal" is used herein, it refers to a nucleic acid, for example rRNA or
30 mRNA, which differs from natural, endogenous nucleic acid in its ability to cooperate
with other nucleic acids. Orthogonal mRNA. rRNA and tRNA are provided in matched
groups (cognate groups) which cooperate efficiently. For example, orthogonal rRNA,
when part of a ribosome, will efficiently translate matched cognate orthogonal mRNA,
but not natural, endogenous mRNA. For simplicity, a ribosome comprising an
35 orthogonal rRNA is referred to herein as an "orthogonal ribosome," and an orthogonal
ribosome will efficiently translate a cognate orthogonal mRNA.
4
5
wo 2011/077075 PCT/GB2010/002296
An orthogonal codon or orthogonal mRNA codon is a codon in orthogonal mRNA
which is only translated by a cognate orthogonal ribosome, or translated more
efficiently, or differently, by a cognate orthogonal ribosome than by a naturaL
endogenous ribosome. Orthogonal is abbreviated to 0 (as in 0-mRNA).
Thus, by way of example, orthogonal ribosome (0-ribosome) •orthogonal mRNA (0-
mRNA) pairs are composed of: an mRNA containing a ribosome binding site that does
not direct translation by the endogenous ribosome, and an orthogonal ribosome that
efficiently and specifically translates the orthogonal mRNA, but does not appreciably
1 0 translate cellular mRNAs.
"Evolved", as applied herein for example in the expression "evolved orthogonal
ribosome", refers to the development of a function of a molecule through
diversification and selection. For example, a library of rRNA molecules diversified at
15 desired positions can be subjected to selection according to the procedures described
herein. An evolved rRNA is obtained by the selection process.
As used herein, the term "mRNA" when used in the context of an 0-mRNA 0-ribosome
pair refers to an mRNA that comprises an orthogonal codon which is efficiently
20 translated by a cognate 0-ribosome, but not by a naturaL wild-type ribosome. In
addition, it may comprise an mutant ribosome binding site (particularly the sequence
from the AUG initiation codon upstream to -13 relative to the AUG) that efficiently
mediates the initiation of translation by the 0-ribosome, but not by a wild-type
ribosome. The remainder of the mRNA can vary, such that placing the coding
25 sequence for any protein downstream of that ribosome binding site will result in an
mRNA that is translated efficiently by the orthogonal ribosome, but not by an
endogenous ribosome.
As used herein, the term "rRNA" when used in the context of an 0-mRNA 0-ribosome
30 pair refers to a rRNA mutated such that the rRNA is an orthogonal rRNA and a ribosome
containing it is an orthogonal ribosome, i.e., it efficiently translates only a cognate
orthogonal mRNA. The primary, secondary and tertiary structures of wild-type ribosomal
rRNAs are very well known, as are the functions of the various conserved structures
(stems-loops, hairpins, hinges, etc.). 0-rRNA typically comprises a mutation in 16S rRNA
35 which is responsible for binding of tRNA during the translation process. It may also
comprise mutations in the 3' regions of the small rRNA subunit which are responsible for
the initiation of translation and interaction with the ribosome binding site of mRNA.
5
wo 2011/077075 PCT/GB2010/002296
The expression of an "0-rRNA" in a cell, as the term is used herein, is not toxic to the cell.
Toxicity is measured by cell death, or alternatively, by a slowing in the growth rate by
80% or more relative to a cell that does not express the "0-mRNA." Expression of an 0-
rRNA will preferably slow growth by less than 50%, preferably less than 25%, more
5 preferably less than 1 0%, and more preferably still. not at aiL relative to the growth of
similar cells lacking the 0-rRNA.
As used herein, the terms "more efficiently translates" and "more efficiently mediates
translation" mean that a given 0-mRNA is translated by a cognate 0-ribosome at least
10 25% more efficiently, and preferably at least 2, 3, 4 or 8 or more times as efficiently as an
0-mRNA is translated by a wild-type ribosome or a non-cognate 0-ribosome in the
same cell or cell type. As a gauge, for example, one may evaluate translation
efficiency relative to the translation of an 0-mRNA encoding chloramphenicol acetyl
transferase using at least one orthogonal codon by a natural or non-cognate
15 orthogonal ribosome.
As used herein, the term "corresponding to" when used in reference to nucleotide
sequence means that a given sequence in one molecule, e.g., in a 16S rRNA, is in the
same position in another molecule, e.g., a 16$ rRNA from another species. By "in the
20 same position" is meant that the "corresponding" sequences are aligned with each
other when aligned using the BLAST sequence alignment algorithm "BLAST 2
Sequences" described by Tatusova and Madden ( 1999, "Blast 2 sequences - a new tool
for comparing protein and nucleotide sequences", FEMS Microbiol. Lett. 17 4:247-250)
and available from the U.S. National Center for Biotechnology Information (NCBI). To
25 avoid any doubt, the BLAST version 2.2.11 (available for use on the NCBI website or,
alternatively, available for download from that site) is used, with default parameters as
follows: program, blastn; reward for a match, 1; penalty for a mismatch, -2; open gap
arid extend gap penalties 5 and 2, respectively; gap x dropoff, 50; expect 1 0.0; word
size 11; and filter on.
30
As used herein, the term "selectable marker" refers to a gene sequence that permits
selection for cells in a population that encode and express that gene sequence by the
addition of a corresponding selection agent.
35 As used herein, the term "region comprising sequence that interacts with mRNA at the
ribosome binding site" refers to a region of sequence comprising the nucleotides near
the 3' terminus of 16S rRNA that physically interact. e.g., by base pairing or other
interaction, with mRNA during the initiation of translation. The "region" includes
6
wo 2011/077075 PCT/GB2010/002296
nucleotides that base pair or otherwise physically interact with nucleotides in mRNA at
the ribosome binding site, and nucleotides within five nucleotides 5' or 3' of such
nucleotides. Also included in this "region" are bases corresponding to nucleotides 722
and 723 of the E. coli 16S rRNA, which form a bulge proximal to the minor groove of the
5 Shine-Delgarno helix formed between the ribosome and mRNA.
As used herein, the term "diversified" means that individual members of a library will
vary in sequence at a given site. Methods of introducing diversity are well known to
those skilled in the art, and can introduce random or less than fully random diversity at a
10 given site. By "fully random" is meant that a given nucleotide can be any of G, A, T, or
C (or in RNA, any of G, A, U and C). By "less than fully random" is meant that a given
site can be occupied by more than one different nucleotide, but not all of G, A, T (U in
RNA) or C. for example where diversity permits either G or A, but not U or C, or permits
G, A, or U but not Cat a given site.
15
20
As used herein, the term "ribosome binding site" refers to the region of an mRNA that is
bound by the ribosome at the initiation of translation. As defined herein, the "ribosome
binding site" of prokaryotic mRNAs includes the Shine-Delgarno consensus sequence
and nucleotides -13 to + 1 relative to the AUG initiation codon.
As used herein, the term "unnatural amino acid" refers to an amino acid other than the
20 amino acids that occur naturally in protein. Non-limiting examples include: a pacetyi-
L-phenylalanine, a p-iodo-L-phenylalanine, an 0-methyi-L-tyrosine, a ppropargyloxyphenylalanine,
a p-propargyl-phenylalanine, an L-3-(2-naphthyl)alanine, a
25 3-methyl-phenylalanine, an 0-4-allyi-L-tyrosine, a 4-propyi-L-tyrosine, a tri-0-acetyiGicNAcb-
serine, an L-Dopa, a fluorinated phenylalanine, an isopropyi-L-phenylalanine,
a p-azido-L-phenylalanine, a p-acyi-L-phenylalanine, a p-benzoyi-L-phenylalanine, an
L-phosphoserine, a phosphonoserine, a phosphonotyrosine, a p-bromophenylalanine, a
p-amino-L-phenylalanine, an isopropyi-L-phenylalanine, an unnatural analogue of a
30 tyrosine amino acid; an unnatural analogue of a glutamine amino acid; an unnatural
analogue of a phenylalanine amino acid; an unnatural analogue of a serine amino
acid; an unnatural analogue of a threonine amino acid; an alkyl. aryl, acyl, azide,
cyano, halo, hydrazine, hydrazide, hydroxyl, alkenyl, alkynl, ether, thiol, sulfonyL selena,
ester, thioacid, borate, boronate, phospho, phosphono, phosphine, heterocyclic,
35 enone, imine, aldehyde, hydroxylamine, keto, or amino substituted amino acid, or a
combination thereof; an amino acid with a photoactivatable cross-linker; a spinlabeled
amino acid; a fluorescent amino acid; a metal binding amino acid; a metalcontaining
amino acid; a radioactive amino acid; a photocaged and/or
7
wo 2011/077075 PCT/GB2010/002296
photoisomerizable amino acid; a biotin or biotin-analogue containing amino acid; a
keto containing amino acid; an amino acid comprising polyethylene glycol or
polyether; a heavy atom substituted amino acid; a chemically cleavable or
photocleavable amino acid; an amino acid with an elongated side chain; an amino
5 acid containing a toxic group; a sugar substituted amino acid; a carbon-linked sugarcontaining
amino acid; a redox-active amino acid; an a-hydroxy containing acid; an
amino thio acid; an a, a disubstituted amino acid; a b-amino acid; a cyclic amino acid
other than proline or histidine, and an aromatic amino acid other than phenylalanine,
tyrosine or tryptophan.
10
International patent application PCT/GB2006/002637 describes the generation of
orthogonal ribosome/mRNA pairs in which the ribosome binding site in the 0-mRNA
binds specifically to the 0-ribosome.
15 Briefly, the bacterial ribosome is a 2.5 MDa complex of rRNA and protein responsible for
translation of mRNA into protein (The Ribosome, Vol. LXVI. (Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, New York; 2001 ). The interaction between the
mRNA and the 30S subunit of the ribosome is an early event in translation (Laursen, B.S.,
Sorensen, H.P., Mortensen, K.K. & Sperling-Petersen, H.U., Microbial Mol Bioi Rev 69, 101-
20 123 (2005)), and several features of the mRNA are known to control the expression of a
gene, including the first codon (Wikstrom, P.M., Lind, L.K., Berg, D.E. & Bjork, G.R., J Mol
Biol224, 949-966 (1992)), the ribosome-binding sequence (including the Shine Delgarno
(SD) sequence (Shine, J. & Delgarno, L., Biochem J 141, 609-615 (1974), Steitz, J.A. &
Jakes, K., Proc Natl Acad Sci US A 72, 4734-4738 (1975), Yusupova, G.Z., Yusupov, M.M.,
25 Cote, J.H. & Noller, H.F., Cell 106, 233-241 (2001)), and the spacing between these
sequences (Chen, H., Bjerknes, M., Kumar, R. & Jay, E., Nucleic Acids Res 22, 4953-4957
(1994)). In certain cases mRNA structure (Gottesman, S. et al. in The Ribosome, Vol. LXVI
(Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York; 2001), Looman,
A.C., Bodlaender, J., de Gruyter, M., Vogelaar, A. & van Knippenberg, P.H., Nucleic
30 Acids Res 14,5481-5497 (1986)), Liebhaber, S.A., Cash, F. & Eshleman, S.S., J Mol Bio1226,
609-621 (1992), or metabolite binding (Winkler, W., Nahvi, A. & Breaker, R.R., Nature 419,
952-956 (2002)), influences translation initiation, and in rare cases mRNAs can be
translated without a SD sequence, though translation of these sequences is inefficient
(Laursen, B.S., Sorensen, H.P., Mortensen, K.K. & Sperling-Petersen, H.U., Microbial Mol
35 Bioi Rev 69, 101-123 (2005)), and operates through an alternate initiation pathway,
Laursen, B.S., Sorensen, H.P., Mortensen, K.K. & Sperling-Petersen, H.U. Initiation of
protein synthesis in bacteria. Microbiol Mol Bioi Rev 69, 101-123 (2005). For the vast
majority of bacterial genes the SD region of the mRNA is a major determinant of
8
wo 2011/077075 PCT/GB2010/002296
translational efficiency. The classic SD sequence GGAGG interacts through RNA-RNA
base-pairing with a region at the 3' end of the 16S rRNA containing the sequence
CCUCC, known as the Anti Shine Delgarno (ASD). In E. coli there are an estimated 4,122
translational starts (Shultzaberger, R.K., Bucheimer, R.E., Rudd, K.E. & Schneider, T.D., J
5 Mol Bioi 313, 215-228 (200 1)), and these differ in the spacing between the SD-Iike
sequence and the AUG start codon, the degree of complementarity between the SOlike
sequence and the ribosome, and the exact region of sequence at the 3' end of
the 16S rRNA with which the mRNA interacts. The ribosome therefore drives translation
from a more complex set of sequences than just the classic Shine Delgarno (SD)
lO sequence. For clarity, mRNA sequences believed to bind the 3' end of 16S rRNA are
referred to as SD sequences and to the specific sequence GGAGG is referred to as the
classic SD sequence.
Mutations in the SD sequence often lead to rapid cell lysis and death (Lee, K., Holland-
15 Staley, C.A. & Cunningham, P.R., RNA 2, 1270-1285 ( 1996), Wood, T.K. & Peretti, S.W.,
Biotechnol. Bioeng 38, 891-906 ( 1991)). Such mutant ribosomes mis-regulate cellular
translation and are not orthogonal. The sensitivity of cell survival to mutations in the ASD
region is underscored by the observation that even a single change in the ASD can
lead to cell death through catastrophic and global mis-regulation of proteome
20 synthesis (Jacob, W.F., Santer, M. & Dahlberg, A.E., Proc Natl Acad Sci U S A 84, 4757-
4761 (1987). Other mutations in the rRNA can lead to inadequacies in processing or
assembly of functional ribosomes.
PCT/GB2006/02637 describes methods for tailoring the molecular specificity of
25 duplicated E. coli ribosome mRNA pairs with respect to the wild-type ribosome and
mRNAs to produce multiple orthogonal ribosome orthogonal mRNA pairs. In these pairs
the ribosome efficiently translates only the orthogonal mRNA and the orthogonal mRNA
is not an efficient substrate for cellular ribosomes. Orthogonal ribosomes as described
therein that do not translate endoge~ous mRNAs permit specific translation of desired
30 cognate mRNAs without interfering with cellular gene expression. The network of
interactions between these orthogonal pairs is predicted and measured, and it is shown
that orthogonal ribosome mRNA pairs can be used to post-transcriptionally program
the cell with Boolean logic.
35 PCT/GB2006/02637 describes a mechanism for positive and negative selection for
evolution of orthogonal translational machinery. The selection methods are applied to
evolving multiple orthogonal ribosome mRNA pairs (0-ribosome 0-mRNA). Also
9
wo 2011/077075 PCT/GB2010/002296
described is the successful prediction of the network of interactions between cognate
and non-cognate 0-ribosomes and 0-mRNAs.
Here we provide new, further modified orthogonal ribosomes and . methods for
5 producing such 0-ribosomes which expand the molecular decoding properties of the
ribosome. Specifically, we evolve orthogonal ribosomes that more efficiently decode
quadruplet codons.
We disclose evolved orthogonal ribosomes which enhance the efficiency of synthetic
10 genetic code expansion. We provide cellular modules composed of an orthogonal
ribosome and an orthogonal mRNA. These pairs function in parallel with, but
independent of. the natural ribosome-mRNA pair in Escherichia coli. Orthogonal
ribosomes do not synthesize the proteome and may be diverged to· operate using
different tRNA decoding rules from natural ribosomes. Here we demonstrate the
15 evolution of orthogonal ribosomes (ribo-Q's) for the efficient, high fidelity decoding of
codons such as quadruplet codons placed within the context of an orthogonal mRNA
in living cells. We combine ribo-Q, orthogonal mRNAs and orthogonal aminoacyl-tRNA
synthetase/tRNA pairs to substantially increase the efficiency of site-specific unnatural
amino acid incorporation in E. coli. This advantageously allows the efficient synthesis of
20 proteins incorporating unnatural amino acids at multiple sites, and/or minimizes the
functional and/or phenotypic effects of truncated proteins for example in experiments
that use unnatural amino acid incorporation to probe protein function in vivo.
ORTHOGONAL CODONS
25 We describe an evolved ribosome which is capable of translating an orthogonal mRNA
codon, which means that the ribosome interprets mRNA information according to a
code which is not the universal genetic code, but an orthogonal genetic code. This
introduces a number of possibilities, including the possibility of having two separate
genetic systems present in the cell, wherein cross-talk is eliminated by virtue of the
30 difference in code; or of a mRNA molecule encoding different polypeptides according
to which code is used to translate it.
An orthogonal codon, from which orthogonal genetic codes can be assembled, is a
code which is other than the universal triplet code. Table 1 below represents the
35 universal genetic code:
10
wo 2011/077075 PCT/GB2010/002296
Table 1 Second nucleotide
u c A G
UGU Cysteine
UUU Phenylalanine (Phe) UCU Serine (Ser) UAU Tyrosine (Tyr) u
(Cys}
UUC Phe UCC Ser UAC Tyr UGC Cys c
u
UUA Leucine (Leu) UCA Ser UAA STOP UGA STOP A
UGG Tryptophan
UUG Leu UCG Ser UAG STOP G
(Trp)
ccu Proline CGU Arginine
CUU Leucine (Leu) CAU Histidine (His) u
(Pro) (Arg)
cue Leu CCC Pro
c
CAC His CGC Arg c
CAA Glutamine
CUA Leu CCA Pro CGA Arg A
(Gin)
CUG Leu CCG Pro CAG Gin CGG Arg G
ACU Threonine AAU Asparagine
AUU Isoleucine (lie) AGU Serine (Ser) u
(Thr) (Asn)
AUC lie ACC Thr AAC Asn AGC Ser c
A AGA Arginine
AUA lie ACA Thr AAA Lysine (Lys) A
(Arg)
AUG Methionine (Met)
ACG Thr AAG Lys AGG Arg G
or START
GCU Alanine GAU Aspartic acid GGU Glycine
GUU Valine Val u
(Ala) (Asp) (Giy)
GUC (Val) GCC Ala GAC Asp. GGC Gly c
G
GAA Glutamic acid
GUA Val GCAAia GGAGiy A
(Giu)
GUG Val GCG Ala GAGGiu GGGGiy G
Certain variations in this code occur naturally; for example, mitochondria use UGA to
encode tryptophan (Trp) rather than as a chain terminator. In addition,
5 most animal mitochondria use AUA for methionine not isoleucine and
all vertebrate. mitochondria use AGA and AGG as chain terminators.
11
5
wo 2011/077075 PCT/GB2010/002296
Yeast mitochondria assign all codons beginning with CU to threonine instead of leucine
(which is still encoded by UUA and UUG as it is in cytosolic mRNA).
Plant mitochondria use the universal code, and this has permitted angiosperms to
transfer mitochondrial genes to their nucleus with great ease.
Violations of the universal code are far rarer for nuclear genes. A few unicellular
eukaryotes have been found that use one or two (of their three) STOP codons for amino
acids instead.
1 0 The vast majority of proteins are assembled from the 20 amino acids listed above even
though some of these may be chemically altered, e.g. by phosphorylation, at a later
time.
However, two cases have been found in nature where an amino acid that is not one of
15 the standard 20 is inserted by a tRNA into the growing polypeptide.
Selenocysteine. This amino acid is encoded by UGA. UGA is still used as a chain
terminator. but the translation machinery is able to discriminate when a UGA codon
should be used for selenocysteine rather than STOP. This codon usage has been found
20 in certain Archaea, eubacteria, and animals (humans synthesize 25 different proteins
containing selenium).
Pyrrolysine. In one gene found in a member of the Archaea, this amino acid is encoded
by UAG. How the translation machinery knows when it encounters UAG whether to
25 insert a tRNA with pyrrolysine or to stop translation is not yet known.
All of the above are. for the purposes of the present invention. considered to be part of
the universal genetic code.
30 The present invention enables novel codes, not previously known in nature. to be
developed and used in the context of orthogonal mRNA/rRNA pairs.
SELECTION FOR ORTHOGONAL RIBOSOMES
35 A selection approach for the identification of orthogonal ribosome orthogonal mRNA
pairs. or other pairs of orthogonal molecules, requires selection for translation of
orthogonal codons in 0-mRNA. The selection is advantageously positive selection, such
12
wo 2011/077075 PCT/GB2010/002296
that cells which express 0-mRNA are selected over those that do not or do so less
efficiently.
A number of different positive selection agents can be used. The most common
5 selection strategies involve conditional survival on antibiotics. Of these positive
selections, the chloramphenicol acetyl-transferase gene in combination with the
antibiotic chloramphenicol has proved one of the most useful. Others as known in the
art, such as ampicillin, kanamycin, tetracycline or streptomycin resistance, among
others, can also be used.
10
0-mRNA/0-rRNA pairs can be used to produce an orthogonal transcript in a host ceiL
for example CAT, that can only be translated by the cognate orthogonal ribosome,
thereby permitting extremely sensitive control of the expression of a polypeptide
encoded by the transcript. The pairs can thus be used to produce a polypeptide of
15 interest by, for example, introducing nucleic acid encoding such a pair to a cell, where
the orthogonal mRNA encodes the polypeptide of interest. The translation of the
orthogonal mRNA by the orthogonal ribosome results in production of the polypeptide
of interest. It is contemplated that polypeptides produced in cells encoding orthogonal
mRNA ·orthogonal ribosome pairs can include unnatural amino acids.
20
The methods described herein are applicable to the selection of orthogonal mRNA
orthogonal rRNA pairs in species in which the 0-mRNA comprises orthogonal codons
which are translated by the 0-rRNA. Thus, the methods are broadly applicable across
prokaryotic and eukaryotic species. in which this mechanism is conserved. The
25 sequence of 16$ rRNA is known for a large number of bacterial species and has itself
been used to generate phylogenetic trees defining the evolutionary relationships
between the bacterial species (reviewed, for example, by Ludwig & Schleifer, 1994,
FEMS Microbial. Rev. 15: 155-73; see also Bergey's Manual of Systematic Bacteriology
Volumes 1 and 2, Springer, George M. Garrity, ed.). The Ribosomal Database Project II
30 (Cole JR, Chai B, Farris RJ, Wang Q, Kulam SA, McGarrell DM, Garrity GM, Tiedje JM,
Nucleic Acids Res, (2005) 33(Database lssue):D294-D296. doi: 10.1 093/nar/gki038)
provides, in release 9.28 (6/17/05), 155,708 aligned and annotated 16S rRNA sequences,
along with online analysis tools.
35 Phylogenetic trees are constructed using, for example, 16S rRNA sequences and the
neighbour joining method in the ClustaiW sequence alignment algorithm. Using a
phylogenetic tree, one can approximate the likelihood that a given set of mutations
(on 16S rRNA and a codon in mRNA) that render the set orthogonal with respect to
13
wo 2011/077075 PCT/GB2010/002296
each other in one species will have a similar effect in another species. Thus, the
mutations rendering mRNA/16S rRNA pairs orthogonal with respect to each other in one
member of. for example, the Enterobacteriaceae Family (e.g., E. coli) would be more
likely to result in orthogonal mRNA/ orthogonal ribosome pairs in another member of the
5 same Family (e.g., Salmonella) than in a member of a different Family on the
phylogenetic tree.
In some instances, where bacterial species are very closely related, it may be possible
to introduce corresponding 16S rRNA and mRNA mutations that result in orthogonal
1 0 molecules in one species into the closely related species to generate an orthogonal
mRNA orthogonal rRNA pair in the related species. Also where bacterial species very
are closely related (e.g., for E. coli and Salmonella species). it may be possible to
introduce orthogonal 16S rRNA and orthogonal mRNA from one species directly to the
closely related species to obtain a functional orthogonal mRNA orthogonal ribosome
15 pair in the related species.
Alternatively, where the species in which one wishes to identify orthogonal mRNA
orthogonal ribosome pairs is not closely related (e.g., where they are not in the same
phylogenetic Family) to a species in which a set of pairs has already been selected.
20 one can use selection methods as described herein to generate orthogonal mRNA
orthogonal ribosome pairs in the desired species. Briefly, one can prepare a library of
mutated orthogonal 16S rRNA molecules. The library can then be introduced to the
. chosen species. One or more 0-mRNA sequences can be generated which comprise
a sequence encoding a selection polypeptide as described herein using one or more
25 orthogonal codons (the bacterial species must be sensitive to the activity of the
selection agents, a matter easily determined by one of skill in the art). The 0-mRNA
library can then be introduced to cells comprising the 0-rRNA library, followed by
positive selection for those cells expressing the positive selectable marker in order to
identify orthogonal ribosomes that pair with the 0-mRNA.
30
The methods described herein are applicable to the identification of molecules useful
to direct translation or other processes in a wide range of bacteria, including bacteria
of industrial and agricultural importance as well as pathogenic bacteria. Pathogenic
bacteria are well known to those of skill in the art, and sequence information. including
35 not only 16S rRNA sequence. but also numerous mRNA coding sequences. are
available in public databases, such as GenBank. Common, but non-limiting examples
include, e.g., Salmonella species, Clostridium species, e.g., Clostridium botulinum and
Clostridium perfringens, Staphylococcus sp., e.g, Staphylococcus aureus;
14
wo 2011/077075 PCT/GB2010/002296
Campylobacter species, e.g., Campylobacter jejuni, Yersinia species, e.g., Yersinia
pestis, Yersinia enterocolitica and Yersinia pseudotuberculosis, Listeria species, e.g.,
Listeria monocytogenes, Vibrio species, e.g., Vibrio cholerae, Vibrio parahaemolyticus
and Vibrio vulnificus, Bacillus cereus, Aeromonas species, e.g., Aeromonas hydrophile,
5 Shigella species, Streptococcus species, e.g., Streptococcus pyogenes, Streptococcus
faecalis, Streptococcus faecium, Streptococcus pneumoniae, Streptococcus durans,
and Streptococcus avium, Mycobacterium tuberculosis, Klebsiella species,
Enterobacter species, Proteus species, Citrobacter species, Aerobacter species,
Providencia species, Neisseria species, e.g., Neisseria gonorrhea and Neisseria
10 meningitidis, Heamophilus species, e.g., Haemophilus influenzae, Helicobacter species,
e.g., Helicobacter pylori, Bordetella species, e.g., Bordetella pertussis, Serratia species,
and pathogenic species of E. coli, e.g., Enterotoxigenic E. coli (ETEC),
enteropathogenic E. coli (EPEC) and enterohemorrhagic E. coli 0 157:H7 (EHEC).
15 RELEASE FACTOR 1 I AMBER CO DONS
20
25
Advantageously, to maximize the efficiency of full-length protein synthesis with respect
to truncated protein, the effects of release factor 1 (RF-1 )-mediated chain termination
would be minimized for the expression of a gene of interest.
Unlike the natural ribosome the orthogonal ribosome is not responsible for synthesizing
the proteome, and is therefore tolerant to mutations in the highly conserved rRNA that
cause lethal or dominant negative effects in the natural ribosome. Orthogonal
ribosomes may therefore be advantageously evolved towards decreased RF-1 binding.
We disclose the synthetic evolution of orthogonal ribosomes (ribo-Q's) for the efficient,
high fidelity decoding of quadruplet codons placed within the context of an
orthogonal mRNA in living cells. Ribo-Q's may preferably be combined with orthogonal
mRNAs and orthogonal aminoacyl-tRNA synthetase/tRNA pairs to advantageously
30 significantly increase the efficiency of site-specific unnatural amino acid incorporation
in E. coli. This increase in efficiency makes it possible to synthesize proteins
incorporating unnatural amino acids at multiple sites, and minimizes the functional and
phenotypic effects of truncated proteins in vivo. This has clear industrial application
and utility, for example in the manufacture of proteins incorporating unnatural amino
35 acids.
BACTERIAL TRANSFORMATION
15
wo 2011/077075 PCT/GB2010/002296
The methods described herein rely upon the introduction of foreign or exogenous
nucleic acids into bacteria. Methods for bacterial transformation with exogenous
nucleic acid, and particularly for rendering cells competent to take up exogenous
nucl~ic acid, is well known in the art. For example, Gram negative bacteria such as E.
5 coli are rendered transformation competent by treatment with multivalent cationic
agents such as calcium chloride or rubidium chloride. Gram positive bacteria can be
incubated with degradative enzymes to remove the peptidoglycan layer and thus form
protoplasts. When the protoplasts are incubated with DNA and polyethylene glycoL
one obtains cell fusion and concomitant DNA uptake. In both of these examples, if the
1 0 DNA is linear, it tends to be sensitive to nucleases so that transformation is most efficient
when it involves the use of covalently closed circular DNA. Alternatively, nucleasedeficient
cells (RecBC- strains) can be used to improve transformation.
Electroporation is also well known for the introduction of nucleic acid to bacterial cells.
15 Methods are well known, for example, for electroporation of Gram negative bacteria
such as E. coli, but are also well known for the electroporation of Gram positive
bacteria, such as Enterococcus faecalis, among others, as described, e.g., by Dunny et
al., 1991, Appl. Environ. Microbial. 57: 1194-1201.
20 The in vivo, genetically programmed incorporation of designer amino acids allows the
properties of proteins to be tailored with molecular precision 1• The Methanococcus
jannaschii tyrosyl-tRNA synthetase/ tRNAcuA (MJTyrRS/tRNAcuA)2· 3 and the
Methanosarcina barkeri pyrrolysyl-tRNA synthetase/tRNAcuA (MbPyiRS/tRNAcuA)4-6
orthogonal pairs have been evolved to incorporate a range of unnatural amino acids
25 in response to the amber codon in E. coli'· 6· 7. However, the potential of synthetic
genetic code expansion is generally limited to the low efficiency incorporation of a
single type of unnatural amino acid at a time, since every triplet codon in the universal
genetic code is used in encoding the synthesis of the proteome. In order to efficiently
encode multiple distinct unnatural amino acid into proteins we require i) blank codons
30 and ii) mutually orthogonal aminoacyl-tRNA synthetase/tRNA pairs that recognize
unnatural amino acids and decode the new codons. Here we synthetically evolve an
orthogonal ribosomes. 9 (riboQ 1) that efficiently decodes a series of quadruplet codons
and the amber codon, providing several blank codons on an orthogonal mRNA. which
it specifically translatess. By creating mutually orthogonal aminoacyl-tRNA synthetase/
35 tRNA pairs and combining these with riboQ 1 we direct the incorporation of distinct
unnatural amino acids in response to two of the new blank codons on the orthogonal
mRNA (Figure 5). Using this code, we genetically direct the formation of a specific,
redox insensitive, nanoscale protein cross-link via the bio-orthogonal cycloaddition of
16
wo 2011/077075 PCT/GB2010/002296
encoded azide and alkyne containing amino acidslO. Since the synthetase/tRNA pairs
used have been evolved to incorporate numerous unnatural amino acids I. 6. 7 it will be
possible to encode more than 200 unnatural amino acid combinations using this
approach. Since ribo-Q 1 independently decodes a series of quadruplet codons this
5 work provides foundational technologies for the encoded synthesis and synthetic
evolution of unnatural polymers in cells.
A ribosome must accommodate an extended anticodon tRNA into its decoding centre
to decode it17. 18. Natural ribosomes are very inefficient at, and unevolvable for
10 quadruplet decoding (Figure 6), which would enhance misreading of the prot eo me. In
contrast orthogonal ribosomess, which are specifically addressed to the or·thogonal
message, and are not responsible for synthesizing the proteome, may, in principle, be
evolved to efficiently decode quadruplet codons on the orthogonal message. To
discover evolved orthogonal ribosomes that enhance quadruplet decoding we first
15 created 11 saturation mutagenesis libraries in the 16S rRNA of ribo-X (an orthogonal
ribosome previously evolved for efficient amber codon decoding on an orthogonal
message9; taken together these libraries cover 127 nucleotides that are within 12 A of a
tRNA bound in the decoding centre19 (Figure 7). We used ribo-X as a starting point for
library generation because we hoped to discover evolved orthogonal ribosomes that
20 gain the ability to efficiently decode quadruplet codons while maintaining the ability to
efficiently decode amber codons on the orthogonal mRNA; thereby maximizing the
number of additional codons that can be decoded on the orthogonal ribosome.
To select orthogonal ribosomes that efficiently decode quadruplet codons using
25 extended anticodon tRNAs we combined each 0-ribosome library with a reporter
construct (0-cat (AAGA 146)/tRNASer2ucuu). The reporter contains a chloramphenicol
acetyl transferase gene that is specifically translated by O-ribosomes9, an in frame
AAGA quadruplet codon and tRNASer2ucuu (a designed variant of tRNASer2 that is
aminoacylated by E. coli seryl-tRNA synthetase and decodes the AAGA codon 9. 20).
30 The orthogonal cat gene is read in frame, and confers chloramphenicol resistance, only
if tRNASer2ucuu efficiently decodes the AAGA codon and restores the reading frame.
Clones surviving on chloramphenicol concentrations which kill cells containing ribo-X
and the cat reporter have 4 distinct sequences. Clone ribo-Q4 has double mutations at
C1195A and All96G, ribo-Q3 has the triple mutations at C1195T, A1196G and A1197G;
35 ribo-Q2 and ribo-Q 1 have the double mutation at A 1196G and A 1197G, ribo-Q2 also
has eight additional non-programmed mutations. While the entire decoding centre was
mutated, the selected mutations are spatially localized and might accommodate an
extended anticodon:codon interaction in the decoding centre (Figure la). The
17
wo 2011/077075 PCT/GB2010/002296
chloramphenicol resistance of cells containing tRNASer2ucuu and cat with two AGGA
codons is greatly enhanced when the cat gene is translated by ribo-Q ribosomes in
place of unevolved ribosomes (Figure 1b,c). Indeed the chloramphenicol resistance of
cells containing two AGGA codons read by the riboQ ribosomes approaches that of a
5 wild-type cat gene. This suggests that riboQ1 may decode quadruplet codons with an
efficiency approaching that for triplet decoding and with a much greater efficiency
than the unevolved ribosome. The enhancement in quadruplet decoding efficiency is
maintained for a variety of quadruplet codon-anticodon interactions (Figure 8).
10 Natural ribosomes decode triplet codons with high fidelity (error frequencies ranging
from 1 Q-2 to 10-4 errors per codon have been reported 21-23). To explicitly compare the
fidelity of triplet decoding and quadruplet decoding for the evolved orthogonal
ribosomes and the progenitor ribosome we used two independent methods: the
incorporation of 35$ cysteine into a protein, which contains no cysteine codons in its
15 gene 9 and variants of a dual luciferase system9. 23 (Figure 9). We find that the triplet
and quadruplet decoding translational fidelity is the same for the evolved ribosome
(ribo-Q1) and un-evolved and wild-type ribosomes, and that the 4th base of the
codon-anticodon interaction is discriminated equally well by all ribosomes (Figure 9).
20 To demonstrate that the enhanced amber decoding properties of ribo-X are
maintained in ribo-Q1 we compared the efficiency of incorporating p-benzoyi-Lphenylalanine
(Bpa. 1) into a recombinant GST-MBP fusion in response to an amber
codon on an orthogonal mRNA using orthogonal ribosomes and a previously evolved
p-benzoyi-L-phenylalanyl-tRNA synthetase/tRNAcuA pair3 (BpaRS/tRNAcuA)(Figure 2).
25 Ribo-Q 1 and ribo-X incorporate 1 with a comparable and high efficiency in response to
the amber codons in the orthogonal mRNA (compare lanes 4 & 6 and lanes 10 & 12 in
Figure 2a). Ribo-X and ribo-Q 1 are substantially more efficient than the wild type
ribosome at incorporating 1 via amber suppression (compare lanes 4 & 6 to lane 2 &
lanes 1 0 & 12 to lane 8 in Figure 2a).
30
To demonstrate the utility of ribo-Q1 for incorporating unnatural amino acids in response
to quadruplet codons we compared the efficiency of incorporating p-azido-Lphenylalanine
(AzPhe. 2) into a recombinant GST-MBP fusion in response to a
quadruplet codon using ribo-Q 1 or the wild-type ribosome. In order to direct the
35 incorporation of 2 we used the AzPheRS*/tRNAuccu pair (a variant of the pAzPheRS-
7 /tRNAcuA pair24 derived from the MJTyrRS/tRNAcuA pair for the incorporation of 2 as
described below). We find that ribo-Q 1 substantially inpeases the efficiency of
incorporation of 2 in response to a quadruplet codon. and even allows the
18
5
wo 2011/077075 PCT/GB2010/002296
incorporation of 2 in response to two quadruplet codons for the first time (compare
lanes 2 & 6 and lanes 4 & 8, Figure 2b). The site and fidelity of incorporation of 2 were
further confirmed by analysis of tandem mass spectrometry (MS/MS) fragmentation
series of the relevant tryptic pep tides (Figure 11).
To take advantage of ribo-Q1 for the incorporation of multiple distinct unnatural amino
acids in recombinant proteins, we required mutually orthogonal aminoacyl-tRNA
synthetase/tRNA pairs. We demonstrated that the MbPyiRS/tRNAcuA pair 4. 5 and
MJlyrRS/tRNAcuA pair 2, each of which have previously been evolved to incorporate a
10 range of unnatural amino acids 1· 6. 7. 25, are mutually orthogonal in their aminoacylation
specificity (Figure 12). We created the AzPheRS*/tRNAuccu pair. which is derived from
the MjTyrRS/tRNAcuA pair, by a series of generally applicable directed evolution steps
(figures 13-15). The MbPyiRS/tRNAcuA pair and AzPheRS* /tRNAuccu pair are mutually
orthogonal: they decode distinct codons, use distinct amino acids and are orthogonal
15 in their aminoacylation specificity (Figure 16).
To demonstrate the simultaneous incorporation of two useful unnatural amino acids into
a single protein we combined the MbPyiRS/MbtRNAcuA pair, the AzPheRS* tRNAuccu pair
and ribo-Q1 in E. coli. We used these components to produce full-length GST-
20 calmodulin containing 2 (AzPh~) and N6-[(2-propynyloxy)carbonyi]-L-Iysine (CAK, 4,
which we recently discovered is an efficient substrate for MbPyiRS 7) (Figure 3) in
response to an AGGA and UAG codon in an orthogonal gene. Production of the fulllength
protein required the addition of both unnatural amino acids. We further
confirmed the incorporation of 2 and 4 at the genetically programmed sites by MS/MS
25 sequencing of a single tryptic fragment containing both unnatural amino acids (Figure
3).
To begin to demonstrate that emergent properties may be programmed into proteins
via combinations of unnatural amino acids we genetically directed the formation of a
30 triazole cross-link, via a copper catalysed Husigen [2+3] cycloaddition reaction ("Click
reaction"). 10 We first encoded 2 and 4 at position 1 and 149 in calmodulin (Figure 4).
After incubation of calmodulin incorporating the azide (2) and alkyne (4) at these
positions with Cu (I) for 5 minutes we observe a more rapidly migrating protein band in
SDS-PAGE. MS/MS sequencing unambiguously confirms that the faster mobility band
35 results from the product of bio-orthogonal cycloaddition reaction between 2 and 4.
Our results demonstrate the genetically programmed proximity acceleration of a new
class of asymmetric, redox insensitive cross-link that can be used to specifically
constrain protein structure on the nanometer scale. Unlike existing protein cyclization
19
wo 2011/077075 PCT/GB2010/002296
methods for recombinant proteins 26. 27, these cross-links can be encoded at any
spatially compatible sites in a protein, not just placed at the termini. In contrast to the
chemically diverse cyclization methods that can be accessed with peptides by solidphase
peptide synthesis 2s these cross-links can be encoded into proteins of essentially
5 any size. Given the importance of disulfide bonds in natural therapeutic proteins and
hormones, the utility of peptide stapling strategies 29, the importance of peptide
cyclization 3o, and the improved stability of proteins cyclized by native chemical ligation
26 it will be interesting to investigate the enhancement of protein function that may be
accessed by combining the encoding of these cross-links with directed evolution
10 methods. By combining the numerous variant MJTyrRS/tRNAcuA and MbPyiRS/tRNAcuA
pairs reported for the incorporation of unnatural amino acids 1. 6· 7 (after appropriate
anticodon conversion using the steps reported here) with ribo-Q1 it will be possible to
encode more than 200 amino acid combinations in recombinant proteins.
15 Experimental (methods summary)
Methods for cloning, site-directed mutagenesis and library construction are described
in the Supplementary Materials. Ribosome libraries were screened for quadruplet
suppressors using a modification of the strategy to discover ribo-X 9•
E. coli genehogs or DH 1 OB were used in all protein expression experiments using LB
20 medium supplemented with appropriate antibiotics and unnatural amino acids.
Proteins were purified by affinity chromatography using published standard protocols.
Translational fidelity of evolved 0-ribosomes was measured by mis-incorporation of 35SIabelled
cysteine 9• Briefly, GST-MBP was produced by the 0-ribosome in the presence
of 35S-cysteine. The protein was purified; cleaved with thrombin, which cleaves the linker
25 between GST and MBP, and analysed by SDS-PAGE and phospho-imaging. A modified
Dual-luciferase assay was used to measure the fidelity of translation of 0-ribosomes 9•
Luminescence from a luciferase mutant containing an inactivating missense mutation
in this assay is a measure of translational inaccuracy of the ribsome. The DLR was
translated by the 0-ribosome, extracted in the cold and luciferase activity measured
30 using the Duai-Luciferase Reporter Assay System (Promega).
LC/MS/MS of proteins was performed by NextGen Science (Ann Arbor, USA). Proteins
were excised from Coomassie stained SDS-PAGE gels, digested with trypsin and
analysed by LC/MS/MS. Total protein mass was obtained by ESI-MS; purified protein was
dialysed into 10 mM ammonium bicarbonate pH 7.5, mixed 1:1 with 1% formic acid in
35 50% methanol and total mass determined in positive ion mode.
Cyclization reactions were performed for 5 minutes at room-temperature on purified
protein in 50 mM sodium phosphate pH 8.3 in the presence of 1 mM ascorbic acid, 1
20
wo 2011/077075 PCT/GB2010/002296
mM CuS04 and 2 mM bathophenathroline. Details of all methods can be found in the
Supplementary Materials.
5 Definitions
10
The term 'comprises' (comprise, comprising) should be understood to have its normal
meaning in the art. i.e. that the stated feature or group of features is included, but that
the term does not exclude any other stated feature or group of features from also
being present.
Brief DescrlpHon of the Figures
Figure 1. Selection and characterlzaHon of orthogonal quadruplet decoding ribosomes.
a. Mutations in quadruplet decoding ribosomes form a structural cluster close to the
15 space potentially occupied by an extended anticodon tRNA. Selected nucleotides are
shown in red. b. Ribo-Qs substantially enhances the tRNA decoding of quadruplet
codons. The tRNASer2uccu-dependent enhancement in decoding AGGA codons in the
0-cat (AGGA103, AGGA146) gene was measured by survival on increasing
concentrations of chloramphenicol (Cm). c. As in b, but measuring CAT enzymatic
20 activity directly by thin-layer chromatography acetylated chloramphenicol (AcCm).
ribo-X (Rx), ribo-Q1-4 (Ql-Q4) and the 0-ribosome (0)
Figure 2. Enhanced lncorporaHon of unnatural amino acids In response to amber and
quadruplet codons wHh rlbo-Q1. a. Ribo-Q 1 incorporates Bpa (p-benzoyi-L-
25 phenylalanine) as efficiently as ribo-X. The entire gel is shown in Figure 10. b. Ribo-Ql
enhances the efficiency AzPhe (p-azido-L-phenylalanine) in response to the AGGA
quadruplet codon using AzPheRS*/tRNAuccu. The gel showing the ratio of GST-MBP to
GST as well as MS/MS spectra of the single and double AzPhe incorporations are shown
in Figure 11. (UAG)n or (AGGA)n describes the number of amber or AGGA codons (n)
30 between gst and malE.
Figure 3. Encoding an azide and an alkyne In a single protein via orthogonal translaHon.
a. Expression of GST-CaM-His6 (a glutathione-S-transferase calmodulin his6 fusion)
containing two unnatural amino acids. An orthogonal gene producing a GST -CaM-His6
35 fusion that contains an AGGA codon at position 1 and an amber codon at position 40
of calmodulin (CaM) )was translated by ribo-Q 1 in the presence of AzPheRS* /tRNAuccu
and MbPyiRS/tRNAcuA. The entire gel is shown in Figure 17. b. LC/MS/MS analysis of the
21
wo 2011/077075 PCT/GB2010/002296
incorporation of two distinct unnatural amino acids into the linker region of GST-MBP. (2
is denoted as Y* and 4 as K*).
Figure 4. Genetically directed cyclization of calmodulin via a Cu(l)-catalyzed Huisgens
5 [3+2]-cycloaddition. a. Structure of calmodulin indicating the sites of incorporation of 2
and 4 and their triazole product. Image created using Pymol (www.pymol.orgl and
pdb-file 4CLN. b. GST-CaM-His6 lAzPhe 149CAK specifically cyclizes with Cu(l)-catalyst.
AzPhe is 2, Tyr is tyrosine, BocK is 3 and CAK is 4. Lanes 1 and 2 are from a separate gel
c. LC/MS/MS confirms the triazole formation. The MS/MS spectra of a doubly charged
10 peptide containing the crosslink (m/z= 1226.6092, which is within 1.8 ppm of the mass
expected for cross-linked peptide).
Figure 5. Strategy for the synthesis of an orthogonal genetic code. Combining the two
mutually orthogonal pairs (MbPyiRS/MbtRNAcuA and MjAzPheRS*/tRNAuccu) with
15 evolved orthogonal ribosomes (Ribo-Q) creates a system that is able to decode the
UAG and AGGA codons on an orthogonal mRNA (0-mRNA) to produce a protein that
contains two distinct unnatural amino acids at genetically encoded sites. UAG is
decoded as 4 (CAK) or 3 (BocLys) by MbPyiRS/MbtRNAcuA while AGGA is decoded as
2.
20
Figure 6. Evolving an orthogonal quadruplet decoding ribosome. The natural ribosome
(gray) and the progenitor orthogonal ribosome (green) utilize tRNAs with triplet
anticodon to decode triplet codons in both wt- (black) and orthogonal- (purple)
mRNAs, respectively. The decoding of quadruplet codons with extended anticodon
25 tRNAs (red) is of low efficiency (light gray arrows) on both ribosomes. Synthetic evolution
of the orthogonal ribosome leads to an evolved scenario in which a mutant (orange
patch) orthogonal ribosome more efficiently decodes quadruplet codons on
orthogonal mRNAs using extended anticodon tRNAs. Decoding of extended anticodon
tRNAs on natural mRNAs is unaffected because the orthogonal ribosome does not read
30 natural mRNAs and the natural ribosome is unaltered.
Figure 7. Comprehensive mutagenesis of the ribosome decoding centre.
A. Structure of the ribosomal small subunit with bound tRNAs and mRNAs. tRNA
anticodon stem loops are bound to A site (yellow), P site (cyan), andEsite (dark blue).
35 The mRNA is shown in purple. 16$ ribosomal RNA is shown in green and ribosomal
proteins in gray. The 118 residues in the decoding centre, targeted for mutation in the
11 libraries, are shown in orange (This figure was created using Pymol v0.99
(www .pymol.org) and PDB ID 2JOO). B. Secondary structure of the E. coli 16S ribosomal
22
wo 2011/077075 PCT/GB2010/002296
RNA (www.rna.ccbb.utexas.edu). The nucleotides targeted for mutation are shown
colored orange.
Figure 8. Ribo-Q enhances the tRNA dependent decoding of different quadruplet
5 codons. Ribo-X, Ribo-Q 1-4 and the 0-ribosome were produced from pRSF-0-rDNA
vectors. The tRNAser2UCUA-dependent enhancement in decoding UAGA codons in
the 0-cat (UAGA 103, UAGA 146), the tRNAser2AGGG-dependent enhancement in
decoding CCCU codons in the 0-cat (CCCU103, CCCU146), and the tRNAser2UCUUdependent
enhancement in decoding AAGA codons in the 0-cot (AAGA 146) was
10 measured by survival on increasing concentrations of chloramphenicol. pRSF-0-rDNA
vectors and corresponding 0-cat vectors were co-transformed into GeneHogs cells.
Transformed cells were recovered for 1 h in SOB medium containing 2% glucose and
used to inoculate 200 ml of LB-GKT (LB medium with 2% glucose, 25 ~g ml-1 kanamycin
and 12.5 ~g ml-1 tetracycline). After overnight growth (37°C, 250 r.p.m., 16 h), 2 ml of the
15 cells were pelleted by centrifugation (3,000g), and washed three times with an equal
volume of LB-KT (LB medium with 12.5 ~g ml-1 kanamycin and 6.25 ~g ml-1 tetracycline).
The resuspended pellet was used to inoculate 18 ml of LB-KT, and the resulting culture
incubated (37°C, 250 r.p.m. shaking, 90 min). To induce expression of plasmid encoded
0-rRNA, 2 ml of the culture was added to 18 ml LB-IKT (LB medium with 1.1 mM
20 isopropyl-0-thiogalactopyranoside (IPTG), 12.5 ~g ml-1 kanamycin and 6.25 IJg ml-1
tetracycline) and incubated for 4 h (37°C, 250 r.p.m.). Aliquots (250 1-11 optical density at
600 nm (00600) = 1.5) were plated on LB-IKT agar (LB agar with 1 mM IPTG, 12.5 ~g ml-1
kanamycin and 6.25 1-Jg ml-1 tetracycline) supplemented with 50 1-1g ml-1
chloramphenicol and incubated (37°C, 40 h).
25
Figure 9: The translation fidelity of evolved ribosomes is comparable to that of the
natural ribosome. A. The translational error frequency for triplet decoding as measured
by35S-cysteine misincorporation is indistinguishable for ribo-QL ribo-Q3-Q4, ribo-X, the
unevolved orthogonal ribosome and the wild-type ribosome. GST-MBP was synthesized
30 by each ribosome in the presence of 355-cysteine, purified on glutathione sepharose
and digested with thrombin. The left panel shows a Coomassie stain of the thrombin
digest. The un-annotated bands result primarily from the thrombin preparation. The right
panel shows 35S labeling of proteins in the same gel, imaged using a Storm
Phosphorimager. Lanes 1-6 show thrombin cleavage reactions of purified protein
35 derived from cells containing the indicated ribosome (with the ribosomal RNA
produced from pSC 1 01 * constructs that drive rRNA from a P 1 P2 promoter) and either
pO-gst-ma/E (for orthogonal ribosomes) or pgst-ma/E (for wild-type ribosomes). The size
markers are pre-stained standards (Bio-Rad 161-0305). The error frequency per codon
23
wo 2011/077075 PCT/GB2010/002296
translated by the ribo-Q ribosomes as measured by this method was less than 1x10-3.
Control experiments with the progenitor orthogonal ribosome, ribo-X and the wild-type
ribosome allowed us to put the same limit on their fidelity. ·This limit compares
favourably with previous measurements of error frequency using 35$ mis-incorporation
5 (4x10-3 errors per codon) 338. The translational fidelity of ribo-Q1 in triplet decoding is
comparable to that of the un-evolved ribosome, as measured by a dual-luciferase
assay. In this system a C-terminal firefly luciferase is mutated at codon K529(AAA),
which codes for an essential lysine residue. The extent to which the mutant codon is
misread by tRNNvs(UUU) is determined by comparing the firefly luciferase activity
10 resulting from the expression of the mutant gene to the wild-type firefly luciferase, and
normalizing any variability in expression using the activity of the co-translated N-terminal
F?.enil/a luciferase. Previous work has demonstrated that measured firefly luciferase
activities in this system result primarily from the synthesis of a small amount of protein
that mis-incorporates lysine in response to the mutant codon 23, rather than a low
15 activity resulting from the more abundant protein containing encoded mutations. In
experiments examining the fidelity of ribo-Q 1, lysate from cells containing pSC 1 0 1* -riboQ
1 and pO-DLR and its codon 529 variants were assayed. Control experiments used
lysates from cells containing pSC101*-0-ribosome and pO-DLR and its codon 529
variants. C. The quadruplet decoding fidelity of ribo-Q is comparable to that of un-
20 evolved ribosomes. Efficiencies were determined using a dual luciferase construct with
an N-terminal Renilla and C-terminal Firefly luciferase (Ren-FF). The reporter was
mutated to include a quadruplet AGGA codon in the linker between the two
luciferases (Ren-AGGA-FF). Ren-AGGA-FF was transformed into DH10B cells along with a
non-cognate anticodon Ser2A tRNA (UCUA or AGGG) and either ribo-Q or the 0-
25 ribosome. Readthrough efficiency for Ren-AGGA-FF was measured by taking the ratio
of Firely luminescence/Renilla luminescence. This data was divided by the same
Firefly/Renilla ratio when using the Ren-FF construct in the presence of tRNA (to
normalize for effects of the tRNA on sites outside the AGGA codon under investigation).
In order to obtain the level of decoding by these non-cognate tRNAs as a fraction of
30 decoding by cognate tRNA. these data were compared with that obtained from the
same experiment using a cognate Ser2A tRNA with the UCCU anti-codon. The data
represent the average of at least 4 trials. The error bars represent the standard
deviation. D Fourth base specificity in quadruplet decoding. E. coli DH 1 OB expressing
the indicated combination of an 0-ribosome, a chloramphenicol acetyltransferase
35 gene under the control of an orthogonal rbs with a quadruplet codon at a permissive
site and E. coli Ser2A tRNAuccu were scored for their ability to grow in the presence of
increasing amounts of chloramphenicol. The fractional activity is the maximal Cm
24
wo 2011/077075 PCT/GB2010/002296
resistance of the cells relative to the combination containing a cognate codon in the
mRNA and a particular o-ribosome.
Figure 10: Ribo-Q1 enhances the efficiency of BpaRS/tRNAcuA-dependent unnatural
5 amino acid incorporation in response to single and double UAG codons, maintaining
the enhanced amber decoding of ribo-X. In each lane an equal volume of protein
purified from glutathione sephcirose under identical conditions is loaded. Orthogonal
ribosomes are produced from pSCl01'-ribo-X, pSCl01'-ribo-Ql. Bpa, p-benzoyi-Lphenylalanine
(1). The BpaRS/tRNAcuA pair is produced from pSUPBpa that contains six
10 copies of MjtRNAcuA .. (UAG)n describes the number of amber stop codons (n) between
gst and malE in 0-gst(UAG)nmaiE or gst(UAG)nmaiE. The ratio of GST-MBP to GST reflects
the efficiency of amber suppression versus RFI mediated termination. A part of this gel
showing the band for full-length GST-MBP is shown in Figure 2 of the main text.
15 Figure 11: Ribo-Q 1 enhances the efficiency of AzPheRS* /tRNAuccu unnatural amino acid
incorporation in response to AGGA quadruplet codons. A. Ribo-Q1 is produced from
pSC101'-ribo-Q1. AzPhe, 2.5 mM 2. The AzPheRS*/tRNAuccu pair is produced from pDULE
AzPheRS*/tRNAuccu that contains a single copy of MjtRNAuccu. (AGGA)n describes the
number of quadruplet codons (n) between gst and malE in 0-gst(AGGA)nmaiE or
20 gst(AGGA)nmaiE. The ratio of GST-MBP to GST reflects the efficiency of frameshift
suppression. A part of this gel showing the bands for full-length GST-MBP is shown in
Figure 2 of the main text. B & C. MS/ MS spectra of tryptic fragments incorporating one
or two AzPhes respectively.
25 Figure 12. MbPyiRS/MbtRNAcuA and MJTyrRS/tRNAcuA pairs are mutually orthogonal in
their aminoacylation specificity. A. The decoding network of MbPyiRS/MbtRNAcuA (lime)
and MJTyrRS/tRNAcuA (grey) and its unnatural amino acid incorporating derivatives. A
unique unnatural amino acid is specifically recognized by each of the synthetases and
used to aminoacylate its cognate tRNA. We asked whether the MbPyiRS/tRNAcuA pair 4.
30 s. 34and MJTyrRS/tRNAcuA pair are mutually orthogonal in their aminoacylation specificity.
Our experiments demonstrate that there is no cross-acylation (grey arrows) between
the two aminoacyl-tRNA synthetase/tRNAcuA pairs (as shown by decoding the amber
codon in myo4TAGHis6 using the different combinations of synthetases and tRNAs, see
below). However, both tRNAs direct the incorporation of their amino acid in response to
35 the amber codon. B. E. coli DH 1 OB were transformed with pMyo4T AG-His6, a plasmid
holding the gene for sperm whale myoglobin with an amber codon at position 4 and a
C-terminal hexahistidine tag and an expression cassette for either MbtRNAcuA or
MjtRNAcuA. MbPyiRS or MJTyrRS were provided on pBKPyiS or pBKMJTyrRS, respectively.
25
5
wo 2011/077075 PCT/GB2010/002296
Cells expressing MbPyiRS received 10 mM 3 (Boclys) as a substrate for the synthetase.
Myoglobin-His6 produced by the cells was purified by Ni2+-affinity chromatography,
analysed by SDS-PAGE and detected with Coomassie stain or Western blot against the
His6-tag.
Figure 13. Genetically encoding 2 in response to a quadruplet codon. A. MjAzPheRS
aminoacylates its cognate amber suppressor tRNAcuA with 2. To differentiate the
codons that the two mutually orthogonal tRNAs decode and to create a pair for the
incorpordtion of an unnatural amino acid in response· to a quadruplet codon, we
10 altered the anticodon of MjtRNAcuA from CUA to UCCU to create MjtRNAuccu. After this,
the resulting tRNAuccu }s no longer a substrate of the parent MjAzPheRS. To create a
version of AzPheRS-7 that aminoacylates MjtRNAuccu we identified six residues (Y230,
C231, P232, F261, H283, 0286) in the parent synthetase that recognize the anticodon of
the tRNA 35 and mutated these residues to all possible combinations, creating a library
15 of 108 possible synthetase mutants. To select for AzPheRS mutants that specifically
aminoacylate MjtRNAuccu we created a chloramphenicol acetyl transferase reporter
(pREP JY(UCCU), derived from pREP YC-JYCUA 32), which contains the four base codon
AGGA at position 111 , a site permissive to the incorporation of a range of amino acids.
In the absence or presence of AzPheRS/MjtRNAuccu this reporter confers resistance to
20 chloramphenicol at low levels (30-50 IJg ml-1). We selected synthetase variants on 150
IJg ml-1 of chloramphenicol that, in combination with MjtRNAuccu, specifically direct the
incorporation of 2 in response to the AGGA codon on pREP JY(UCCU). We
characterized 24 synthetase/tRNAuccu pairs by their chloramphenicol resistance in the
presence of 2 and pREP JY(UCCU). The seven best synthetase/tRNAuccu combinations
25 confer a chloramphenicol resistance of 250-350 IJg ml-1 on cells containing 2 and pREP
JY(UCCU) (Figure 14). In the absence of the 2, we observe only background levels of
resistance (30 IJg ml-1) for several synthetases indicating that the synthetase/MjtRNAuccu
pairs specifically direct the incorporation of 2 in response to the quadruplet codon
AGGA. Sequencing these seven clones revealed similar but non-identical mutations
30 (Figure 14). B. Library design. Structure of MJ1yrRS (grey) bound to its cognate tRNA
(orange). Residues of the synthetase that recognize the anticodon and which are
mutated in the library, as well as bases of the natural anticodon (G34, U35, A36) are
shown in blue (Figure created using Pymol, www.pymol.org, and pdb-file 1 J 1 U). C. The
production of full-length myoglobin from myo4(AGGA)-hiS6 by the AzPheRS*-
35 2/MjtRNAuccu pair is dependent on the presence of 2. In the remainder of the text we
refer to MjAzPheRS*-2 as MjAzPheRS* for simplicity. MjAzPheRS*/tRNAuccu efficiently
suppress an AGGA codon placed into the myoglobin gene. E. coli DH 1 OB were
transformed with pMyo4TAG-His6 or pMyo4AGGA-His6, a plasmid holding the gene for
26
wo 2011/077075 PCT/GB2010/002296
sperm whale myoglobin with an amber or an AGGA codon at position 4, respectively,
and a C-terminal hexahistidine tag and an expression cassette for either MjtRNAcuA or
MjtRNAuccu. MjAzPheRS or MjAzPheRS* were provided on pBKMjAzPheRS or
pBKMjAzPheRS*, respectively. Cells received 2.5 mM 2 as a substrate for the synthetase.
5 Myoglobin-His6 produced by the cells was purified by Ni2+-affinity chromatography,
analysed by SDS-PAGE and detected with Coomassie stain. D. MjAzPheRS*/tRNAuccu
decodes AGGA codons specifically with 2. The incorporation of 2 into myoglobin-His6
purified from cells expressing Myo4(AGGA) and MjAzPheRS* /tRNAuccu in the presence
of 2.5 mM 2 was analysed by ESI-MS. The mass of the observed peak (18457.75 Do)
10 corresponds to the calculated mass of myoglobin containing a single 2 (18456.2 Do).
Figure 14: Amino acid dependent growth of selected MjAzPheRS* variants. E. coli DH 1 OB
were co-transf.ormed with isolates from a library built on pBK MjAzPheRS-7 and pREP
JY(UCCU) (coding for MjtRNAuccu and chloramphenicol acetyltransferase with an
15 AGGA codon at position D 111). Cells were grown in the presence or absence of 1 mM
2 for 5 hand pronged onto LB agar plates containing 25 IJg ml-1 kanamycin, 12.51-Jg ml-1
tetracycline and the indicated concentration of chloramphenicol with or without the
unnatural amino acid. Plates were photographed after 18 h at 37°C. Sequencing of
mutations for incorporating tyrosine, 2 and propargyi-L-tyrosine (Figure 15) in response to
20 the AGGA codon reveals clones with common mutations Y230K, C231 K and P232K, but
divergent mutations at positions F261 , H283 and D286. This suggests that amino acids
· 230, 231 and 232 confer affinity and specificity for the anticodon, and that 261, 283 and
286 may couple the identity of the anticodon to the amino acid identity.
25 Figure 15: Amino acid dependent growth of selected MjPrTyrRS* variants. E. coli DH10B
transformed as in Figure 14 using isolates from a library built on MjPrTyrRS and tested for
unnatural amino acid dependent growth. Mutations relative to MjPrTyrRS are given in
the table below.
30 Figure 16: The MbPyiRS/MbtRNAcuA and MjAzPheRS*/tRNAuccu pairs incorporate distinct
unnatural amino acids in response to distinct unique codons. A. The two orthogonal
pairs (MbPyiRS/MbtRNAcuA and MjAzPheRS*/tRNAuccu) decode two distinct codons in
the mRNA (UAG and AGGA) with two distinct amino acids (N6-[(tert.butyloxy)
carbonyi]-L-Iysine and 2). MbPyiRS does not aminoacylate MjtRNAuccu and
35 MbtRNAcuA is not a substrate for MjAzPheRS*. B. Suppression of a cognate codon at
position 4 in the gene of sperm whale myoglobin by different combinations of
MbPyiRS/MbtRNAcuA and MjAzPheRS*/tRNAuccu. E. coli DH10B were transformed with
pMyo4T AG-His6 or pMyo4AGGA-His6 as described in Figure 6C. Cells were provided with
27
wo 2011/077075 PCT/GB2010/002296
MbPyiRS (on pBKPyiS) or MjAzPheRS* (on pBKMjPheRS*) and 2.5 mM N6-[(tert.butyloxy)
carbonyi)-L-Iysine or 5 mM 2, respectively. Myoglobin-His6 produced by the
cells was purified by Ni2+-affinity chromatography, analysed by SDS-PAGE and detected
with Coomassie stain. We see weak incorporation in response to the UAG codon using
5 the MbPyiRS pair. This incorporation is independent of the presence of MjAzPheRS* and
results from a low level background acylation of the tRNA by E. coli synthetases in rich
media, as previously observed.
Figure 17: Encoding an azide and an alkyne in a single protein via orthogonal
10 translation. A. Expression of GST-CaM-His6 containing two unnatural amino acids. E. coli
DH10B were transformed with four plasmids: pCDF PyiST (expressing MbPyiRS and
MbtRNAcuA), pDULE AzPheRS* tRNAuccu (encoding MjAzPheRS*/tRNAuccu), pSC101* riboQ1
and p-O-gst-CaM-His6 1 AGGA 40UAG (a GST-CaM-His6 fusion translated by the
orthogonal ribosome that contains an AGGA codon at position 1 and an amber codon
15 at position 40 of calmodulin (CaM)). Cells were grown in LB medium containing
antibiotics to maintain the plasmids and 2.5 mM 4 and/or 5 mM 2 as indicated. Cells
were harvested, lysed and the protein purified on GSH-beads. Bound protein was
eluted with 10 mM GSH in PBS and analysed by SDS-PAGE. A part of this gel is shown in
Figure 3 of the main text. Full-length protein was produced by this method with yields of
20 upto 0.5 mg/L
Figure 18 shows Supplementary Table 1: Oligonucleotides used in this study.
The invention is. now described by way of example. These examples are intended to be
25 illustrative, and are not intended to limit the appended claims.
Examples
Plasmid construction
30 Previously described gst-MaiE protein expression vectors pgst-maiE and p0-gst-malf9,
are translated by wild type and orthogonal ribosomes respectively. These vectors were
used as templates to construct variants containing one or two quadruplet codons in
the linker region between the gst and malE open reading frame.
To create vectors containing a single AGGA quadruplet codon between gst and malE
35 (pgst(AGGA)maiE and pO-gst(AGGA)maiE) the Tyr codon, T AC, in the linker between
gst and malE was changed to AGGA by Quikchange mutagenesis (Stratagene), using
the primers GMx1 AGGAf and GMx1 AGGAr (all primers used in this study are listed in
Supplementary Table 1). For double AGGA mutants we additionally mutated the fourth
28
wo 2011/077075 PCT/GB2010/002296
codon in malE from GAA to AGGA by quick change PCR. with the primers GMx2AGGAf
and GMx2AGGAr to create the vectors pgst(AGGA)2ma/E and pO-gst(AGGA)2maiE.
The vector pO-gst-maiE(Y252AGGA) used for protein expression for mass spectrometry,
in which the codon for Y 17 of MBP was mutated to AGGA, was created by Quikchange
5 mutagenesis (Stratagene) using the primers MBPY17 AGGAf and MBPY17 AGGAr.
To create vectors for constitutive production of the selected 0-ribosomes the mutations
in pRSF-OrDNA that confer the quadruplet decoding capacity on the orthogonal
ribosome were transferred to pSClOl based 0-rRNA expression vectors. pSClOl'-ribo-X
was used as a template and the mutations in 16S rDNA were introduced by enzymatic
10 inverse PCR using the primers sclOlQr and scl01Qlf (for Ribo-Ql). sc101Q3f (forRiboQ3)
and sclOl Q4f (for Ribo-Q4).
pDULE AzPheRS* tRNAuccu (containing the gene for MjtRNAuccu and MjAzPheRS*, each
under the control of the lpp promoter) was created by changing the anticodon of the
MjtRNAcuA to UCCU by Quikchange and replacing the ORF of the MjBPA-RS with
15 MjAzPheRS*-2 via ligation of the MjAzPheRS*-2 gene, obtained by cutting pBK
MjAzPheRS*-2 with the restriction enzymes Ndel and Stul. into the same sites on pDULE
MjBPARS MjtRNAuccu. pCDF PyiST (a plasmid expressing MbPyiRS and MbtRNAcuA from
constitutive promoters) was created by cloning PCR products containing expression
cassettes for MbPyiRS and MbtRNAcuA into the BamHI and Sail or the Sail and Notl sites
20 of pCDF DUET-1 (Novagen). The PCR products were obtained by amplifying the
relevant regions of pBK PyiRS and pREP PyiT.
Plasmid encoding a fusion of GST and CaM were created by replacing the ORF of MBP
in p-0-gst-maiE with human CaM. The gene for CaM was amplified by PCR from pET3-
CaM (a kind gift from K. Nagai) using primers CamEcof and CamH6Hindr (adding a C-
25 terminal His6-tag) and cloned into the EcoRI and Hindlll sites of pO-gst-maiE.
Methionine-! of CaM was mutated to AGGA by a subsequent round of Quikchange
mutagenesis using primers CaM 1 aggaf and CaM 1 aggar (simultaneously removing part
of the linker between GST and CaM). In a second round of mutagenesis an amber
codon was introduced at position 149 using primers CaMK 149T AGf and CaMK 149T A Gr.
30 To create a sterically hindered control the amber codon was inserted at position 40
instead using primers CaM40tagf and CaM40tagr.
Construction of ribosome libraries and quadruplet decoding reporters.
11 different 16S rDNA libraries were constructed by enzymatic inverse PCR s, 31 using
35 pTrcRSF-0-ribo-X as a template. The resulting pRSF-0-rDNA libraries mutate between 7
and 13 nucleotides in defined regions on 16S rRNA and were constructed by multiple
rounds of by enzymatic inverse PCR using the library construction primers in
Supplementary Table 1. Each library has a diversity of greater than 109, ensuring more
29
wo 2011/077075 PCT/GB2010/002296
than 99% coverage. There is overlap in the nucleotides mutated in the 11 libraries and
overall they cover the entire surface of decoding centre in the A site of the ribosome.
To create a reporter of quadruplet decoding by orthogonal ribosomes. we used a
5 previously described 0-cat (UAGA 146) /tRNA(UAGA) vector as a template9. This vector
contains a variant of E. coli tRNASer2 on an lpp promoter and rrnC transcriptional
terminator. The tRNA has an altered anticodon and selector codons for serine 146 in
the chloramphenicol acetyl transferase (cat) gene downstream of an orthogonal
ribosome-binding site. Ser146 is an essential and conserved catalytic serine residue that
lO ensures the fidelity of incorporation. To create 0-cat (AAGA 103
AAGA 146)/tRNA(UCUU) the AAGA codon was introduced at position 146 and 103 and
the anticodon of the tRNA was converted to UCUU by Quikchange mutagenesis using
primers CAT146AGGAf. CATl46AGGAr and CATl03AGGAf. CAT103AGGAr. 0-cat
reporters containing the quadruplet codons AGGA. CCCU (using primers
15 CATl46CCCUf, CATl46CCCUr and CATl03CCCUf and CATl03CCUr) and the
corresponding tRNAs (Ser2AGGAf. Ser2AGGAr. Ser2CCCUf and Ser2CCCUr) were also
created by Quikchange mutagenesis. Reporters containing a single quadruplet
selector codon were intermediates in the vector construction process. Vectors having
the 0-cat gene but lacking the tRNA were created using 0-cat(UAGA 146). which does
20 not contain the tRNA cassette, as a template using Quik change primers CATl46AAGf.
CAT146AGGAr. CATl03AGGAf. CATl03AGGAr. CATl46CCCUf. CATl46CCCUr.
CATl03CCCUf and CATl03CCCUr that mutate the codons in 0-cat.
Selecflon of orthogonal ribosomes with enhanced quadruplet decoding.
25 To select 0-ribosomes with improved quadruplet decoding. each pRSF-0-rDNA library
was transformed by electroporation into GeneHog E. coli (Invitrogen) cells containing
0-cat (AAGA 146). Transformed cells were recovered for 1 h in SOB medium containing
2% glucose and used to inoculate 200 ml of LB-GKT (LB medium with 2% glucose. 25 IJg
ml·1 kanamycin and 12.5 IJg ml-1 tetracycline). After overnight growth (37 °C, 250 r.p.m ..
30 16 h). 2 ml of the cells were pelleted by centrifugation (3,000g), and washed three times
with an equal volume of LB-KT (LB medium with 12.5 IJg ml-1 kanamycin and 6.25 IJg ml-1
tetracycline). The resuspended pellet was used to inoculate 18 ml of LB-KT, and the
resulting culture incubated (37 °C, 250 r.p.m. shaking. 90 min). To induce expression of
plasmid encoded 0-rRNA. 2 ml of the culture was added to 18 ml LB-IKT (LB medium
35 with 1.1 mM isopropyi-D-thiogalactopyranoside (IPTG). 12.5 IJg ml-1 kanamycin and 6.25
IJg ml-1 tetracycline) and incubated for 4 h (37 oc. 250 r.p.m.). Aliquots (250 ml optical
density at 600 nm (OD6oo) = 1.5) were serial diluted and plated on LB-IKT agar (LB agar
with 1 mM IPTG. 12.5 IJg ml-1 kanamycin and 6.25 IJg ml-1 tetracycline) supplemented
30
wo 2011/077075 PCT/GB2010/002296
with chloramphenicol of different concentrations (7 5 iJg ml-1, 1 00 iJg ml-1, 150 iJg ml-1,
and 200 iJg ml-1 respectively) and incubated (37 °C, 40 h).
Characterization of evolved orthogonal ribosomes with enhanced quadruplet
5 decoding.
To separate selected pRSF-0-rDNA plasmids from the 0-cat (AAGA146)/tRNNer2(UCUU)
reporter plasmids, total plasmid DNA from selected clones was purified and digested
with Notl restriction endonuclease, and transformed into DH 1 OB E. coli. Individual
transformants were replica plated onto kanamycin agar and tetracycline agar and
1 0 plasmid separation of pRSF-0-rDNA from the reporter confirmed by restriction digest
and agarose gel analysis.
To quantify the quadruplet decoding activity of selected 16S rDNA clones, the selected
pRSF-0-rDNA plasm ids were co transformed with 0-cat (AGGA 103, AGGA 146)
/tRNNer2(UCCU). Cells were recovered (SOB, 2% glucose, 1 h) and used to inoculate 10
15 ml of LB-GKT, which was incubated ( 16 h, 37 oe, 250 r.p.m.). We used 1 ml of the
resulting culture to inoculate 9 ml of LB-KT, which was incubated (90 min, 37 °C, 250
r.p.m.). We used 1 ml of the LB-KT culture to inoculate 9 ml of LB-IKT medium, which was
incubated (37 °C, 250 r.p.m., 4 h). Individual clones were transferred to a 96-well block
and arrayed, using a 96-well pin tool, onto LB-IKT agar plates containing
20 chloramphenicol at concentrations from 0 to 500 iJg ml-1. The plates were incubated
(37°C, 16 h). We performed analogous experiments for other quadruplet codonanticodon
pairs.
To extract soluble celllysates for in vitro CAT assays, 1 ml of each induced LB-IKT culture
was pelleted by centrifugation at 3,000g. The cell pellets were washed three times with
25 500 iJI Washing Buffer (40 mM Tris-HCI, 150 mM NaCI; 1 mM EDTA, pH 7.5) and once with
500 iJI lysis buffer (250 mM Tris-HCI, pH 7.8). Cells were lysed in 200 iJI Lysis Buffer by five
cycles of flash-freezing in dry ice/ethanol, followed by rapid thawing in a 50 oc water
bath. Cell debris was removed from the lysate by centrifugation ( 12,000g, 5 min) and
the top 150 iJI of supernatant frozen at -20 °C. To assay CAT activity in the lysates, 10 iJI
30 of soluble cell extract was mixed with 2.5 iJI of FAST CAT Green (deoxy) substrate
(Invitrogen) and preincubated (37 °C, 5 min). We added 2.5 iJI of 9 mM acetyi-CoA
(Sigma), and incubated (37 °C, 1 h). The reaction was stopped by the addition of icecold
ethyl acetate (200 I, vortex 20 s). The aqueous and organic phases were
separated by centrifugation (12,000g, 10 min) and the top 100 IJI of the ethyl acetate
35 layer collected. We spotted 1 IJI of the collected solution onto a silica gel Thin-layer
chromatography plate (Merck) for thin-layer chromatography in chloroform:methanol
(85: 15 vol/vol). The fluorescence of the spatially resolved substrate and product was
31
wo 2011/077075 PCT/GB2010/002296
visualized and quantified using a phosphorimager (Storm 860, Amersham Biosciences)
with excitation and emission wavelengths of 450 nm and 520 nm, respectively.
Small scale expression and purification of gst-maiE fusions.
5 E. coli containing the appropriate plasmid combinations were pelleted (3,000g, 10 min)
from 50 ml overnight cultures, resuspended and lysed in 800 IJI Novagen BugBuster
Protein Extraction Reagent (supplemented with 1 x protease inhibitor cocktail (Roche), 1
mM PMSF, 1 mg ml-1 lysozyme (Sigma), 1 mg ml-1 DNase I (Sigma)), and incubated (60
min, 25 °C, 1,000 r.p.m.). The lysate was clarified by centrifugation (6 min, 25,000g, 2 °C}.
1 0 GST containing proteins from the lysate were bound in batch ( 1 h, 4 oq to 50 J,JI of
glutathione sepharose beads (GE Healthcare). Beads were washed 3 times with 1 ml
PBS, before elution by heating for 1 0 min at 80 oc in 60 J,JI 1 x SDS gel-loading buffer. All
samples were analyzed on 10% Bis-Tris gels (Invitrogen).
15 Measuring the translational fidelity of orthogonal quadruplet decoding ribosomes
355-cysteine misincorporation: E. coli containing either pO-gst-ma/E and pSC 1 0 1' -0-
ribosome, pO-gst-ma/E and pSC101'-ribo-X, pO-gst-maiE and pSCl01'-riboQ, or pgstma/
E were resuspended in LB media (supplemented with 35S-cysteine (1,000 Ci mmol-1)
to a final concentration of 3 nM, 750 J,JM methionine, 25 J,Jg ml-1 ampicillin and 12.5 J,Jg
20 ml-1 kanamycin) to an 0D6oo of 0.1, and cells were incubated (3.5 h, 37°C, 250 r.p.m.).
10 ml of the resulting culture was pelleted (5,000g, 5 min), washed twice (1 ml PBS per
wash), resuspended in 1 ml lysis buffer containing 1% Triton-X, incubated (30 min, 37°C,
1 ,000 r.p.m.) and lysed on ice by pipetting up and down. The clarified cell extract was
bound to 1 00 IJI of glutathione sepharose beads ( 1 h, 4°C) and the beads were
25 pelleted (5,000g, 10 s) and washed twice in 1 ml PBS. The beads were added to 10 ml
polypropylene column (Biorad) and washed (30 ml of PBS; 10 ml 0.5 M NaCL 0.5x PBS; 30
ml PBS) before elution in 1 ml of PBS supplemented with 10 mM glutathione. Purified
GST-MBP was digested with 12.5 units of thrombin for 1 h, to yield a GST fragment and
an MalE fragment. The reaction was precipitated with 15% trichloroacetic acid and
30 loaded onto an SDS-PAGE gel to resolve the GST, MBP and thrombin, and stained with
lnstantBiue (Expedeon). The 35S activity in the GST and MBP protein bands were
quantified by densitometry, using a Storm Phosphorimager (Molecular Dynamics) and
lmageQuant (GE Healthcare). The error frequency per codon for each ribosome
examined was determined as follows: GST contains four cysteine codons, so the
35 number of counts per second (c.p.s.) resulting from GST divided by four gives A, the cps
per quantitative incorporation of cysteine. MBP contains no cysteine codons, but
misincorporation at noncysteine codons gives B c.p.s. Because GST and MBP are
present in equimolar amounts, (A/B 410, where 410 is the number of amino acids in the
32
wo 2011/077075 PCT/GB2010/002296
MBP containing thrombin cleavage fragment, gives the number of amino acids
translated for one cysteine misincorporation C. Assuming the misincorporation
frequency for all 20 amino acids is the same as that for cysteine the number of codons
translated per misincorporation is C/20, and the error frequency per codon is given by
5 (C/20)·1.
Dual luciferase assays: The previously characterized pO-DLR contains a genetic fusion
between a 5' Renilla luciferase (R-Iuc) and a 3' firefly luciferase (F-lue) on an orthogonal
ribosome binding site 9. pO-DLR, and its K529 codon variants, were transformed into E.
1 0 coli cells with pSC 1 0 1* -0-ribosome or pSC 1 0 1* -ribo-Q 1 . Where indicated an additional E.
coli Ser2A tRNA with a mutated anticodon, as specified in individual experiments, was
supplied on plasmid p 15A-tRNA-Ser2A. In this case 25 llg ml-1 tetracyclin was added to
all culture media to maintain the additional plasmid. In experiments that used a
suppressor tRNA recognizing AGGA codons a natural AGG codon, that is followed by a
15 codon starting with an A, was removed from the linker region of pO-DLR by
QuikChange using primers DLR952AAGxfand DLR953AGGxr.
Individual colonies were incubated (37°C. 250 r.p.m., 36 h) in 2 ml LB ·supplemented
with ampicillin (50 f..lg ml·1) and kanamycin (25 f..lg ml-1), pelleted (5,000g, 5 min), washed
with ice cold Millipore water and resuspended in 300 IJI (1 mg ml-1 lysozyme, 1 mg ml-1
20 DNase I, 10 mM Tris (pH 8.0), 1 mM EDTA). Cells were incubated on ice for 20 min, frozen
on dry ice, and thawed on ice. 10 IJI samples of this extract were assayed for firefly (Flue)
and Renillo (R-Iuc) luciferase activity using the Duai-Luciferase Reporter Assay
System (Promega). Each ribosome reporter combination was assayed from four
independent cultures using an Orion microplate luminometer (Berthold Detection
25 Systems) and the data analyzed as previously described. The error reported is the
standard deviation.
Mass spectrometric characterlzaHon of p-azldo·L-phenylalanlne (2) lncorporaHon by
Rlbo-Ql
30 E. coli DH10B containing p-0-gst-ma/E(Y252AGGA), pSC101*Ribo-Q1 and pDULEAzPheRS*
tRNAuccu were used to produce protein for mass spectrometry. Protein was
expressed in the presence of 2.5 mM 2 and purified on glutathione. The purified proteins
were resolved by SDS-PAGE, stained with Instant Blue (Expedeon) and the band
containing full length GST-MBP was excised for analysis by LC/MS/MS (NextGen
35 Sciences). The samples were reduced with on at 60°C and alkylated with
iodoacetamide after cooling to room temperature. The samples were then digested
with trypsin (37°C, 4 h), and the reaction was stopped by the addition of Formic acid.
The samples were analyzed by nano LC/MS/MS on a ThermoFisher LTQ Orbitrap XL. 30 IJI
33
wo 2011/077075 PCT/GB2010/002296
of hydrolysate was loaded onto a 5 mm 75 1-1m ID C12 (Jupiter Proteo, Phenomenex)
vented column at a flow-rate of 101-11 min-1. Gradient elution was over a 15 em 751Jm ID
C 12 column at 300 nl min-1 with a 1 hour gradient. The mass spectrometer was
operated in data-dependent mode, and ions were selected for MS/MS. The Orbitrap
5 MS scan was performed at 60,000 FWHM resolution. MS/MS data was searched using
Mascot (www.matrixscience.com).
Evolution of a quadruplet decoding M}AzPheRS
pBK MjAzPheRS-7 24 (a kanamycin resistant plasmid, which contains MjAzPheRS-7 on a
1 0 GlnRS promoter and terminator) was used as a template to create a library in the
region of MjAzPheRS that recognizes the anticodon. Codons for residues Y230, C231,
P232, F26l, H283 and 0286 were randomized to NNK in two rounds of enzymatic inverse
PCR, generating a library of 108 mutant clones. pREP JY(UCCU) was created by
changing the anticodon of MjtRNAcuA in pREP YC-JYCUA 32 from CUCUAAA to
15 CUUCCUAA by QuikChange mutagenesis (Stratagene) and changing the amber
codon in the chloramphenicol acetyl transferase gene to AGGA. E. coli DH 1 OB
harbouring this plasmid were transformed with the mutant library and grown in LB-KT (LB
medium supplemented with 25 1-1g ml-1 kanamycin and 12.5 IJg ml-1 tetracycline)
supplemented with 1 mM 2. 1 09 cells were plated on LB-KT plates containing 1 mM 2
20 and concentrations of chloramphenicol ranging from 50 to 250 IJg ml-1. After incubation
(36 h, 37°C) individual clones were tested for 2 dependent growth on LB-KT plates with
0-250 IJg ml-1 chloramphenicol with and without 1 mM 2. The plasmid DNA from clones
showing amino acid dependent growth was isolated and digested with Hindlll to
eliminate pREP JY(UCCU). After transformation and reisolation of the kanamycin
25 resistant plasmid the DNA was sequenced.
To select quadruplet decoding pairs that incorporate other amino acids, the procedure
above was repeated using the relevant starting template and unnatural amino acid.
Investigating the mutual orthogonality of MbPyiRS/MbtRNAcuA and MJlyrRS/MjtRNAcuA
30 To test the ability of MbPyiRS to aminoacylate MjtRNAcuA E. coli DH10B were
transformed with a pBK MbPyiRS encoding MbPyiRS under the control of a GlnRS
promoter and terminator and pMyo4T AG-His6, expressing sperm whale myoglobin with
an amber codon at position 4 and MjtRNAcuA. The cells were grown overnight at 37°C in
LB-KT. Fresh LB-KT (50 ml) supplemented with 10 mM N6-[(tert.-butyloxy)carbonyi]-L-Iysine
35 (Boclys, 3) was inoculated 1 :50 with overnight culture. After 3 h at 37°C protein
expression was induced by addition of 0.2% arabinose. After a further 3 h cells were
harvested and washed with PBS. Proteins were extracted by shaking at 25°C in 1 ml Niwash
butter ( 10 mM Tris/CI, 20 mM imidiazole, 200 mM NaCI pH 8.0) supplemented with
34
wo 2011/077075 PCT/GB2010/002296
protease inhibitor cocktail (Roche}, 1 mM PMSF, and approx. 1 mg ml-1 lysozyme and
0.1 mg ml-1 DNAse I. The extract was clarified by centrifugation (5 min, 25000 g, 4°C},
supplemented 50 IJI Ni2+-NTA beads and incubated with agitation for 1 hat 4°C. Beads
were washed in batch three times with 1 ml Ni-wash buffer and eluted in 100 1-JI sample
5 buffer supplemented with 200 mM imidazole. To test the aminoacylation activity
between the cognate pairs or between MJTyrRS and MbtRNAcuA analogous
experiments were carried out as above using the relevant plasmids (pBK MJTyrRS or pBK
MbPyiRS and pMyo4TAG-His6 or pMyo4TAG-His6-PyiT) and unnatural amino acids (3 or
none}. Proteins were analysed by 4-12% SDS-PAGE and stained with Instant Blue.
10
15
Characterization of the quadruplet suppressing AzPheRS*
Expression and purification of myoglobin from pMyo4TAG-His6 or pMyo4AGGA-His6 was
carried out as above using the relevant pBK plasmids and 2.5 mM 2. Proteins were
analysed by 4-12% SDS-PAGE.
Characterization of Myo4AzPhe produced with AzPheRS* from pMyo4AGGA-His6 by ESI
mass spectrometry
Myogl~bin was expressed in E. coli DH 1 OB using plasm ids pBK AzPheRS* and
pMyo4AGGA-His6 essentially as described above but at 1 I scale. The protein was
20 extracted by shaking at 25°C in 30 ml Ni-wash buffer supplemented with protease
inhibitor cocktail (Roche}, 1 mM PMSF, 1 mg ml-1 lysozyme and 0.1 mg ml-1 DNAse I. The
extract was clarified by centrifugation (15 min, 38000 g, 4°C}, supplemented 0.3 ml Ni2+NT
A beads and incubated with agitation for 1 h at 4°C. Beads were poured into a
column and washed with 40 ml of Ni-wash buffer. Bound protein was eluted in 0.5 ml
25 fractions of the same buffer containing 200 mM imidazole and immediately rebuffered
to 10 mM ammonium carbonate pH 7.5 by dialysis. 50 1-11 of the sample was mixed 1:1
with 1% formic acid in 50% methanol and total mass determined on an LCT time-of-flight
mass spectrometer with electrospray ionization (Micromass}. The sample was injected
at 10 IJI min-1 and calibration performed in positive ion mode using horse heart
30 myoglobin. 50 scans were averaged and molecular masses obtained by deconvoluting
multiply charged protein mass spectra using Mass lynx version 4.1 (Micromass). The
theoretical mass of the wild-type myoglobin was calculated using Protparam
( http://us.expasy.org(tools/protparam.html}, and the theoretical mass for 2 adjusted
manually.
35
MS/MS analysis of GST -MBP 234AzPhe 239CAK
E. coli DH lOB were transformed with pDULE AzPheRS*/tRNAuccu and pCDF PyiST and
grown to logarithmic phase in LB-ST (25 IJQ ml-1 spectinomycin and 12.5 IJQ ml-1
35
wo 2011/077075 PCT/GB2010/002296
tetracycline). Electrocompetent cells were prepared and transformed with a plasmid
for the constitutive expression of an orthogonal ribosome (pSC 1 01 * Ribo-Q) and p-Ogst(
234AGGA 239TAG)ma/E. The recovery of the transformation was used to inoculate
LB-AKST (LB medium containing 50 IJg ml-1 ampicillin, 12.5 IJg ml-1 kanamycin, 25 IJg ml-1
5 spectinomycin and 12.5 IJg ml-1 tetracycline). The culture was grown to saturation at
37°C and used to inoculate the main culture 1 :50. Cells were grown overnight at 37°C,
harvested by centrifugation and stored at -20°C. The GST-MBP protein was expressed at
a scale of 100 ml using 2.5 mM of each AzPhe (2) and CAK {4). Proteins were extracted
and purified as above. After washing the beads with PBS the protein was eluted by
1 0 heating in 1 00 IJI 1 x sample buffer containing 50 mM [3-mercaptoethanol to 80°C for 5
min. The protein sample was analysed by 4-12% SDS-PAGE and stained with Instant Blue.
The band containing full-length GST-MBP was excised and submitted for LC/MS/MS
analysis (by NextGen Sciences).
15 Cycllzatlon of GST·CaM·His, lAzPhe 149CAK
E. coli DH 1 OB were transformed sequentially with four plasm ids as described above
using expression plasmids p-O-gst-CaM-His6 1 AGGA 149UAG or p-O-gst-CaM-His6
1 AGGA 40UAG. The protein was expressed at 0.5 L scale as described above using 5
mM 2 and 2.5 mM 4. The cells were extracted and GST-CaM-His6 purified as described
20 for myoglobin-His6 and dialysed against 50 mM Na2HP04 pH 8.3. To perform the
cyclization reaction, 160 IJI of protein sample was mixed with 40 IJI of a fresh solution of 5
mM ascorbic acid, 5 mM CuS04 and 10 mM bathophenanthroline. The reaction was
incubated at 4°C and analysed by 4-12% SDS-PAGE.
To analyze the cyclization product by mass spectrometry we introduced additional
25 tryptic cleavage sites around the incorporation sites of unnatural amino acids to
facilitate subsequent analysis. Therefore, the point mutations Q4K and M 146K
{numbering relative to the AGGA codon in p-O-gst-CaM-His6 1 AGGA 149UAG) and a
G3K linker directly following the TAG codon were ir:~troduced by QuikChange. The
protein was expressed, purified and cyclized as above with very similar yields. The
30 cyclized protein was subsequently excised from an SDS-PAGE gel and submitted for
mass spectrometric analysis {NextGen Sciences, Ann Arbor, USA).
36
5
10
15
20
25
30
35
40
45
50
55
wo 2011/077075 PCT/GB2010/002296
References:
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
Xie. J. & Schultz. P. G. A chemical toolkit for proteins--an expanded genetic
code. Nat Rev Mol Cell Bioi 7, 775-82 (2006).
Steer, B. A. & Schimmel. P. Major anticodon-binding region missing from an
archaebacterial tRNA synthetase. J Bioi them 274. 35601-6 { 1999).
Chin. J. W., Martin, A. B .. King, D. S., Wang, L. & Schultz. P. G. Addition of a
photocrosslinking amino acid to the genetic code of Escherichiacoli. Proc Natl
A cad Sci U S A 99. 11 020-4 {2002).
Srinivasan. G., James, C. M. & Krzycki. J. A. Pyrrolysine encoded by UAG in
Archaea: charging of a UAG-decoding specialized tRNA. Science 296, 1459-62
{2002).
Polycarpo, C. et al. An aminoacyl-tRNA synthetase that specifically activates
pyrrolysine. Proc Natl Acad Sci US A 101. 12450-4 {2004).
Neumann, H., Peak-Chew, S. Y. & Chin, J. W. Genetically encoding N{epsilon)acetyllysine
in recombinant proteins. Nat Chern Bioi 4, 232-4 {2008).
Nguyen, D. P. et al. Genetic encoding and labeling of aliphatic azides and
alkynes in recombinant proteins via a pyrrolysyl-tRNA Synthetase/tRNA{CUA)
pair and click chemistry. JAm Chem Soc 131,8720-1 {2009).
Rockham. 0. & Chin, J. W. A network of orthogonal ribosome x mRNA pairs. Nat
Chem Bioi 1, 159-66 {2005).
Wang. K., Neumann, H., Peak-Chew. S. Y. & Chin. J. W. Evolved orthogonal
ribosomes enhan'ce the efficiency of synthetic genetic code expansion. Nat
Biotechnol 25. 770-7 {2007).
Rostovtsev. V. V .. Green, L. G .. Fokin. V. V. & Sharpless, K. B. A stepwise huisgen
cycloaddition process: copper(l)-catalyzed regioselective "ligation" of azides
and terminal alkynes. Angew Chem lnt Ed Engl41, 2596-9 {2002).
Hohsaka, T. & Sisido, M. Incorporation of non-natural amino acids into proteins.
Curr Opin Chem Bioi 6. 809-15 (2002).
Ohtsuki. T .. Manabe. T. & Sisido, M. Multiple incorporation of non-natural amino
acids into a single protein using tRNAs with non-standard structures. FEBS Lett
579, 6769-74 (2005).
Murakami. H .. Hohsaka, T.. Ashizuka, Y. & Sisido, M. Site-directed incorporation of
p-nitrophenylalanine into streptavidin and site-to-site photinduced electron
transfer from a pyrenyl group to a nitrophenyl group on the protein framework.
Journal of the American Chemical Society 120. 7520-7529 ( 1998).
Rodriguez. E. A., Lester. H. A. & Dougherty, D. A. In vivo incorporation of multiple
unnatural amino acids through nonsense and frameshift suppression. Proc Natl
Acad Sci US A 103,8650-5 {2006).
Monahan, S. L.. Lester. H. A. & Dougherty, D. A. Site-specific incorporation of
unnatural amino acids into receptors expressed in mammalian cells. Chemistry
and Biology 10,573-580 (2003).
Anderson. J. C. et al. An expanded genetic code with a functional quadruplet
codon. Proc Natl Acad Sci US A 101. 7566-71 {2004).
Atkins, J. F. & Bjork, G. R. A gripping tale of ribosomal frameshifting: extragenic
suppressors of frameshift mutations spotlight P-site realignment. Microbial Mol
Bioi Rev 73. 178-210 {2009).
Stahl, G., McCarty, G. P. & Farabaugh, P. J. Ribosome structure: revisiting the
connection between translational accuracy and unconventional decoding.
Trends Biochem Sci 27. 178-83 {2002).
Selmer, M. et al. Structure of the 70S ribosome complexed with mRNA and tRNA.
Science 313, 1935-42 (2006).
Magliery, T. J., Anderson, J. C. & Schultz, P. G. Expanding the genetic code:
selection of efficient suppressors of four-base codons and identification of
37
5
10
15
20
25
wo 2011/077075 PCT/GB2010/002296
21.
22.
23.
24.
25.
26.
27.
28.
29.
30.
"shifty" four-base codons with a library approach in Escherichia coli. J Mol Bioi
307, 755-69 (2001 ).
Khazaie, K., Buchanan, J. H. & Rosenberger, R. F. The accuracy of Q beta RNA
translation. 1. Errors during the synthesis of Q beta proteins by intact Escherichia
coli cells. Eur J Biochem 144,485-9 (1984).
Laughrea, M., Latulippe, J., Filion, A. M. & Boulet, L. Mistranslation in twelve
Escherichia coli ribosomal proteins. Cysteine misincorporation at neutral amino
acid residues other than tryptophan. Eur J Biochem 169, 59-64 ( 1987).
Kramer, E. B. & Farabaugh, P. J. The frequency of translational misreading errors
in E. coli is largely determined by tRNA competition. Rna 13, 87-96 (2007).
Chin, J. W. et al. Addition of p-azido-L-phenylalanine to the genetic code o.f
Escherichia coli. JAm Chern Soc 124,9026-7 (2002).
Mukai, T. et al. Adding 1-lysine derivatives to the genetic code of mammalian
cells with engineered pyrrolysyl-tRNA synthetases. Biochem Biophys Res
Commun 371,818-22 (2008).
Camarero, J. A., Pavel, J. & Muir, T. W. Chemical Synthesis of a Circular Protein
Domain: Evidence for Folding-Assisted Cyclization. Angewandte Chemie -
International Edition 37, 347-349 (1998).
Scott, C. P., Abei-Santos, E., Wall, M., Wahnon, D. C. & Benkovic, S. J. Production
of cyclic peptides and proteins in vivo. Proc Natl Acad Sci U S A 96, 13638-43
(1999).
Li, P. & Roller, P. P. Cyclization strategies in peptide derived drug design. Curr
Top Med Chem 2, 325-4 1 (2002).
Walensky, L. D. et al. Activation of apoptosis in vivo by a hydrocarbon-stapled
BH3 helix. Science 305, 1466-70 (2004).
Trauger, J. W., Kohli, R. M., Mootz, H·. D., Marahiel, M. A. & Walsh, C. T. Peptide
cyclization catalysed by the thioesterase domain of tyrocidine synthetase.
Nature 407,215-8 (2000).
30 31. Stemmer, W. P. & Morris, S. K. Enzymatic inverse PCR: a restriction site
independent, single-fragment method for high-efficiency, site-directed
mutagenesis. Biotechniques 13,214-20 (1992).
32.
35
33.
34.
Santoro, S. W., Wang, L., Herberich, B., King, D. S. & Schultz, P. G. An efficient
system for the evolution of aminoacyl-tRNA synthetase specificity. Nat
Biotechnol20, 1044-8 (2002).
Rice, J. B., Libby, R. T. & Reeve, J. N. Mistranslation of the mRNA encoding
bacteriophage T7 0.3 protein. J Bioi Chern 259, 6505-10 ( 1984).
Hao, B. et al. A new UAG-encoded residue in the structure of a methanogen
methyltransferase. Science 296, 1462-6 (2002).
40 35. Kobayashi, T. et al. Structural basis for orthogonal tRNA specificities of tyrosyltRNA
synthetases for genetic code expansion. Nat Struct Bioi 10, 425-32 (2003).
All publications mentioned in the above specification are herein incorporated by
reference. Various modifications and variations of the described aspects and
45 embodiments of the present invention will be apparent to those skilled in the art without
departing from the scope of the present invention. Although the present invention has
been described in connection with specific preferred embodiments, it should be
understood that the invention as claimed should not be unduly limited to such specific
embodiments. Indeed, various modifications of the described modes for carrying out
50 the invention which are apparent to those skilled in the art are intended to be within
the scope of the following claims.
38
wo 2011/077075 PCT/GB2010/002296

CLAIMS
1. A 16S rRNA comprising a mutation at A 1196.
5 2. A 16S rRNA according to claim 1 wherein said mutation is A 1196G.
3. A 16S rRNA according to claim 1 or claim 2 further comprising a mutation at
Cll95 and/or Al197.
10 4. A 16S rRNA according to any preceding claim which comprises
(I) C1195A and A1196G; or
(ii) Cll95T, A 1196G and Al197G; or
(iii) All96G and A1197G.
15 5. A l6S rRNA according to any preceding claim which further comprises A531 G
and U534A.
6. A ribosome capable of translating a quadruplet codon, said ribosome
comprising a 16S rRNA according to any preceding claim.
20
7. Use of a 16S rRNA according to any preceding claim in the translation of a
mRNA comprising at least one quadruplet codon.
8. A 16S rRNA, ribosome, cell or method substantially as described herein.
25
9. A 16S rRNA, ribosome, cell or method according to claim 8 with reference to the
accompanying figures.