WO2005007870A2 - COMPOSITIONS OF ORTHOGONAL LEUCYL-tRNA AND AMINOACYL-tRNA SYNTHETASE PAIRS AND USES THEREOF - Google Patents
COMPOSITIONS OF ORTHOGONAL LEUCYL-tRNA AND AMINOACYL-tRNA SYNTHETASE PAIRS AND USES THEREOF Download PDFInfo
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/67—General methods for enhancing the expression
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Definitions
- the invention pertains to the field of translation biochemistry.
- the invention relates to methods for producing and compositions of orthogonal leucyl tRNAs, orthogonal leucyl aminoacyl-tRNA synthetases and pairs thereof.
- the invention also relates to methods of producing proteins in cells using such pairs and related compositions.
- Unnatural amino acids have been microinjected into cells.
- unnatural amino acids were introduced into the nicotinic acetylcholine receptor in Xenopus oocytes (e.g., M.W. Nowak, et al. (1998), In vivo incorporation of unnatural amino acids into ion channels in Xenopus oocyte expression system, Method Enzymol. 293:504-529) by microinjection of a chemically misacylated Tetrahymena thermophila tRNA (e.g., M.E. Saks, et al.
- new components e.g., orthogonal tRNAs, orthogonal aminoacyl-tRNA synthetases and pairs thereof, were added to the protein biosynthetic machinery of the prokaryote Escherichia coli (E. coli) (see e.g., L. Wang, et al., (2001), Science 292:498-500), which allowed genetic encoding of unnatural amino acids in vivo.
- E. coli prokaryote Escherichia coli
- a number of new amino acids with novel chemical, physical or biological properties, including photoaffinity labels and photoisomerizable amino acids, photocrosslinking amino acids see, e.g., Chin, J. W., et al. (2002) Proc. Natl. Acad.
- the invention provides compositions of and methods for producing orthogonal leucyl-tRNAs, orthogonal leucyl aminoacyl-tRNA synthetases and pairs thereof. These translational components can be used to incorporate a selected amino acid in a specific position in a growing polypeptide chain (during nucleic acid translation) in response to a selector codon.
- compositions of the invention include a composition comprising an orthogonal leucyl-tRNA (leucyl-O-tRNA), where the leucyl O-tRNA comprises an anticodon loop comprising a CU(X) n XXXAA sequence, and comprises at least about a 25% suppression activity in presence of a cognate synthetase in response to a selector codon as compared to a comparable control (e.g., in the absence of the selector codon).
- the selector codon is an amber codon
- the leucyl O-tRNA comprises a stem region comprising matched base pairs and a conserved discriminator base at position 73. This position is indicated in Figure 4, Panel A.
- the leucyl O-tRNA comprises a C:G base pair at position 3:70.
- the selector codon is a four-base codon and the leucyl
- O-tRNA comprises a first pair selected from U28:A42, G28:C42 and or C28:G42, and a second pair selected from G:49:C65 or C49:G65, where the numbering corresponds to that indicated in Figure 4, Panel A.
- the first pair is C28:G42 and the second pair is C49:G65.
- a composition comprising a leucyl O-tRNA can further include an orthogonal leucyl aminoacyl-tRNA synthetase (leucyl O-RS), where the leucyl O-RS preferentially aminoacylates the leucyl O-tRNA with a selected amino acid.
- a composition including a leucyl O-tRNA can further include a (e.g., in vitro or in vivo) translation system.
- a composition of the invention also includes a cell (e.g., a non-eukaryotic cell (e.g., an E. coli cell), or a eukaryotic cell) comprising a translation system.
- the translation system includes an orthogonal leucyl-tRNA (leucyl-O-tRNA), where the leucyl- O-tRNA comprises at least about a 25% suppression activity in presence of a cognate synthetase in response to a selector codon as compared to a control lacking the selector codon; an orthogonal aminoacyl-leucyl-tRNA synthetase (leucyl-O-RS); and, a first selected amino acid.
- a cell e.g., a non-eukaryotic cell (e.g., an E. coli cell), or a eukaryotic cell
- the translation system includes an orthogonal leucyl-tRNA (leucyl-O-t
- the leucyl O-tRNA comprises an anticodon loop comprising a CU(X) n XXXAA sequence and recognizes the first selector codon and the leucyl O-RS preferentially aminoacylates the leucyl O-tRNA with the first selected amino acid.
- the cell translation system comprises a leucyl-O-tRNA and cognate synthetase, or a conservative variant thereof, where these components are at least 50% as effective at suppressing a selector codon as a eucyl O-tRNA of S ⁇ Q ID NO: 3, 6, 7 or 12, in combination with a cognate synthetase.
- the cell can further include an additional different O-tRNA/O-RS pair and a second selected amino acid, where the O-tRNA recognizes a second selector codon and the O-RS preferentially aminoacylates the O-tRNA with the second selected amino acid.
- the cell further comprises a nucleic acid that comprises a polynucleotide that encodes a polypeptide of interest, where the polynucleotide comprises/encodes a selector codon that is recognized by the leucyl O- tRNA.
- an E. coli cell includes an orthogonal leucyl-tRNA
- leucyl-O-tRNA where the leucyl-O-tRNA comprises at least about a 25% suppression activity in presence of a cognate synthetase in response to a selector codon as compared to a control lacking the selector codon; and an orthogonal leucyl aminoacyl-tRNA synthetase (leucyl-O-RS), where the O-RS preferentially aminoacylates the O-tRNA with a selected amino acid.
- leucyl-O-RS an orthogonal leucyl aminoacyl-tRNA synthetase
- the coli cell also includes the selected amino acid, and, a nucleic acid that comprises a polynucleotide that encodes a polypeptide of interest, where the polynucleotide comprises a selector codon that is recognized by the leucyl O-tRNA.
- the leucyl O-tRNA is derived from Halobacterium sp NRC-1 and the leucyl O-RS is derived from Methanobacterium thermoaautotropicum.
- a leucyl O-tRNA of the invention comprises or is encoded by a polynucleotide sequence as set forth in any one of S ⁇ Q RO NO.: 3, 6, 7 or 12, or a complementary polynucleotide sequence thereof.
- the leucyl-O-tRNA and cognate synthetase, or a conservative variant thereof are at least 50% as effective at suppressing a selector codon as a leucyl O-tRNA of S ⁇ Q RO NO: 3, 6, 7 or 12, in combination with a cognate synthetase.
- thymine (t) is, of course, replaced by uracil (u).
- a leucyl O-RS comprises an amino acid sequence as set forth in any one of S ⁇ Q RO NO.: 15 or 16, or a conservative variation thereof.
- the leucyl O-RS or a portion thereof is encoded by a polynucleotide sequence as set forth in any one of S ⁇ Q RO NO.: 13 or 14, a conservative variant of S ⁇ Q RO NO: 13 or 14, or a complementary polynucleotide sequence thereof.
- the leucyl O-tRNA and/or the leucyl O-RS of the invention can be derived from any of a variety of organisms (e.g., both eukaryotic and non-eukaryotic organisms).
- the leucyl O-tRNA is derived from an archael tRNA (e.g., from
- Halobacterium sp NRC-1) and/or the leucyl O-RS is derived from a non-eukaryotic organism (e.g., Methanobacterium thermoaautotropicum).
- a polynucleotide of the invention includes a polynucleotide comprising a nucleotide sequence as set forth in any one of S ⁇ Q RO NO.: 1-2, 4-7, 12, and/or is complementary to or that encodes a polynucleotide sequence of the above.
- a polynucleotide of the invention also includes a nucleic acid that hybridizes to a polynucleotide described above, under highly stringent conditions over substantially the entire length of the nucleic acid.
- a polynucleotide of the invention also includes a polynucleotide that is, e.g., at least 75%, at least 80%, at least 90%, at least 95%, at least 98% or more identical to that of a naturally occurring leucyl tRNA or a consensus sequence of multiple naturally occurring leucyl tRNAs, e.g., the laucyl tRNA of SEQ RO NO: 12, and comprises an anticodon loop comprising a CU(X) n XXXAA sequence, an stem region lacking noncanonical base pairs and a conserved discriminator base at position 73.
- a polynucleotide of the invention also includes a polynucleotide that is, e.g., at least 75%, at least 80%, at least 90%, at least 95%, at least 98% or more identical to that of a naturally occurring leucyl tRNA and comprises an anticodon loop comprising a CUUCCUAA sequence, a first pair selected from T28:A42, G28:C42 and/or C28:G42, and a second pair selected from G:49:C65 or C49:G65, where the numbering corresponds to that indicated in Figure 4, Panel A.
- Polynucleotides that are, e.g., at least 80%, at least 90%, at least 95%, at least 98% or more identical to any of the above and/or a polynucleotide comprising a conservative variation of any the above or in Table 3 are also polynucleotides of the invention.
- Vectors comprising or encoding a polynucleotide of the invention are also a feature of the invention.
- a vector optionally includes any of: a plasmid, a cosmid, a phage, a virus, an expression vector, and/or the like.
- a cell comprising a vector of the invention is also a feature of the invention.
- Methods of producing an orthogonal tRNA (O-tRNA), e.g., a leucyl O- tRNA are also a feature of the invention.
- An O-tRNA, e.g., a leucyl O-tRNA, produced by the method is also a feature of the invention.
- a method includes mutating an anticodon loop on members of a pool of tRNAs (e.g., pool of leucyl tRNAs) to allow recognition of a selector codon, thereby providing a plurality of potential O-tRNAs; and analyzing secondary structure of at least one member of the plurality potential O-tRNA to identify non-canonical base pairs in the secondary structure, and, optionally, mutating the non-canonical base pairs (e.g., mutating the non-canonical base pairs to canonical base pairs).
- the non-canonical base pairs are located in stem region of the secondary structure.
- a population of cells of a first species where the cells individually comprise at least one member of the plurality of potential O-tRNAs are subjected to a negative selection, thereby eliminating cells that comprise a member of the plurality of potential O-tRNAs that is aminoacylated by an aminoacyl-tRNA synthetase (RS) that is endogenous to the cell, and providing a pool of tRNAs that are orthogonal to the cell of the first species.
- the selector codon includes an amber codon, an opal codon, a four base codon, etc.
- the method can further include adding an additional sequence (CCA) to a 3' terminus of each of the pool of tRNAs and/or measuring suppression activity.
- CCA additional sequence
- the pool of tRNAs is obtained by aligning a plurality of tRNA sequences; determining a consensus sequence; and generating a library of mutant tRNAs using the consensus sequence, where the pool of tRNAs comprise the library of mutant tRNAs.
- the subjecting step comprises a polynucleotide that encodes a negative selection marker.
- the polynucleotide that encodes the negative selection marker comprises at least one selector codon.
- a negative selection marker includes, but is not limited to, ⁇ -lactamase, ⁇ -galactosidase, and or the like.
- the negative selection marker fluoresces or catalyzes a luminescent reaction in the presence of a suitable reactant.
- a product of the negative selection marker is detected by fluorescence- activated cell sorting (FACS) or by luminescence.
- the negative selection marker includes an affinity based screening marker.
- the subjecting step comprises growing the population of cells in the presence of a selective agent (e.g., an antibiotic, such as ampicillin).
- the method further comprises subjecting to positive selection a second population of cells of the first species.
- the cells comprise a member of the pool of tRNAs that are orthogonal to the cell of the first species, a cognate aminoacyl- tRNA synthetase, and a positive selection marker.
- Cells are selected/screened for cells that comprise a member of the pool of tRNAs that is aminoacylated by the cognate aminoacyl- tRNA synthetase and that shows a desired response in the presence of the positive selection marker, thereby providing an O-tRNA.
- O-RS orthogonal aminoacyl-tRNA synthetase
- a method includes subjecting to positive selection a population of cells of a first species, where the cells each comprise: 1) a member of a plurality of aminoacyl-tRNA synthetases (RSs), where the plurality of RSs comprise mutant RSs, RSs derived from a species other than the first species or both mutant RSs and RSs derived from a species other than the first species; 2) the orthogonal tRNA (O-tRNA) (e.g., from a species other than the first species, from at least a second species, etc.); and 3) a polynucleotide that encodes a positive selection marker and comprises at least one selector codon.
- RSs aminoacyl-tRNA synthetases
- the plurality of RSs comprises leucyl RSs.
- the O-tRNA comprises a leucyl O-tRNA (e.g., where leucyl O-tRNA includes at least about a 25% suppression activity in presence of a cognate synthetase in response to a selector codon as compared to a control lacking the cognate synthetase).
- leucyl O-tRNA includes at least about a 25% suppression activity in presence of a cognate synthetase in response to a selector codon as compared to a control lacking the cognate synthetase.
- Cells are selected or screened for those that show an enhancement in suppression efficiency compared to cells lacking or having a reduced amount of the member of the plurality of RSs. These selected screened cells comprise an active RS that aminoacylates the O-tRNA.
- the level of aminoacylation (in vitro or in vivo) by the active RS of a first set of tRNAs from the first species is compared to the level of aminoacylation (in vitro or in vivo) by the active RS of a second set of tRNAs from a second species; where the level of aminoacylation is determined by a detectable substance (e.g., a labeled amino acid).
- a detectable substance e.g., a labeled amino acid
- the active RS that more efficiently aminoacylates the second set of tRNAs compared to the first set of tRNAs is selected, thereby providing the orthogonal aminoacyl-tRNA synthetase, e.g., leucyl O-RS, for use with the O-tRNA, e.g., the leucyl O-tRNA.
- orthogonal aminoacyl-tRNA synthetase identified by the method is also a feature of the invention.
- a method includes growing, in an appropriate medium, a cell, where the cell comprises a nucleic acid that comprises at least one selector codon and encodes a protein; and, providing the selected amino acid.
- the cell further comprises: an orthogonal leucyl-tRNA (leucyl-O-tRNA) that functions in the cell and recognizes the selector codon; and, an orthogonal leucyl aminoacyl-tRNA synthetase (leucyl O-RS) that preferentially aminoacylates the leucyl-O- tRNA with the selected amino acid.
- an orthogonal leucyl-tRNA leucyl-O-tRNA
- leucyl O-RS orthogonal leucyl aminoacyl-tRNA synthetase
- the leucyl-O-tRNA comprises at least about a 25% suppression activity in presence of a cognate synthetase in response to a selector codon as compared to a control lacking the cognate synthetase.
- a protein produced by this method is also a feature of the invention.
- Orthogonal leucyl-tRNA As used herein, an orthogonal leucyl-tRNA
- leucyl-O-tRNA is a tRNA that is orthogonal to a translation system of interest, where the tRNA is: (1) identical or substantially similar to a naturally occurring leucyl tRNA, (2) derived from a naturally occurring leucyl tRNA by natural or artificial mutagenesis (3) derived by any process that takes a sequence of a wild-type or mutant leucyl tRNA sequence of (1) or (2) into account, (4) homologous to a wild-type or mutant leucyl tRNA; (5) homologous to any example tRNA that is designated as a substrate for a leucyl tRNA synthetase in Table 3, or (6) a conservative variant of any example tRNA that is designated as a substrate for a leucyl tRNA synthetase in Table 3.
- the leucyl tRNA can exist charged with -parnino acid, or in an uncharged state. It is also to be understood that a "leucyl-O- tRNA" optionally is charged (aminoacylated) by a cognate synthetase with an amino acid other than leucine. Indeed, it will be appreciated that a leucyl-O-tRNA of the invention is advantageously used to insert essentially any amino acid, whether natural or artificial, into a growing polypeptide, during translation, in response to a selector codon.
- an orthogonal leucyl amino acid synthetase is an enzyme that preferentially aminoacylates the leucyl-O-tRNA with an amino acid in a translation system of interest.
- the amino acid that the leucyl O-RS loads onto the leucyl O-tRNA can be any amino acid, whether natural or artificial, and is not limited herein.
- the synthetase is optionally the same as or homologous to a naturally occurring leucyl amino acid synthetase, or the same as or
- the leucyl O-RS can be a conservative variant of a leucyl O-RS of Table 3, and/or can be at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or more identical in sequence to a leucyl O-RS of Table 3.
- Homologous Proteins and/or protein sequences are "homologous" when they are derived, naturally or artificially, from a common ancestral protein or protein sequence.
- nucleic acids and/or nucleic acid sequences are homologous when they are derived, naturally or artificially, from a common ancestral nucleic acid or nucleic acid sequence.
- any naturally occurring nucleic acid can be modified by any available mutagenesis method to include one or more selector codon.
- this mutagenized nucleic acid encodes a polypeptide comprising one or more selected amino acid, e.g. unnatural amino acid.
- the mutation process can, of course, additionally alter one or more standard codon, thereby changing one or more standard amino acid in the resulting mutant protein as well.
- Homology is generally inferred from sequence similarity between two or more nucleic acids or proteins (or sequences thereof).
- sequence similarity between sequences that is useful in establishing homology varies with the nucleic acid and protein at issue, but as little as 25% sequence similarity is routinely used to establish homology. Higher levels of sequence similarity, e.g., 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% or more, can also be used to establish homology. Methods for determining sequence similarity percentages (e.g., BLASTP and BLASTN using default parameters) are described herein and are generally available.
- Orthogonal refers to a molecule (e.g., an orthogonal tRNA (O-tRNA) and/or an orthogonal aminoacyl tRNA synthetase (O-RS)) that functions with endogenous components of a cell with reduced efficiency as compared to a corresponding molecule that is endogenous to the cell or translation system, or that fails to function with endogenous components of the cell.
- O-tRNA orthogonal tRNA
- OF-RS orthogonal aminoacyl tRNA synthetase
- orthogonal refers to an inability or reduced efficiency, e.g., less than 20 % efficiency, less than 10 % efficiency, less than 5 % efficiency, or less than 1% efficiency, of an orthogonal tRNA to function with an endogenous tRNA synthetase compared to the ability of an endogenous tRNA to function with the endogenous tRNA synthetase; or of an orthogonal aminoacyl-tRNA synthetase to function with an endogenous tRNA compared to the ability of an endogenous tRNA synthetase to function with the endogenous tRNA.
- the orthogonal molecule lacks a functionally normal endogenous complementary molecule in the cell.
- an orthogonal tRNA in a cell is aminoacylated by any endogenous RS of the cell with reduced or even undetectable efficiency, when compared to aminoacylation of an endogenous tRNA by the endogenous RS.
- an orthogonal RS aminoacylates any endogenous tRNA in a cell of interest with reduced or even undetectable efficiency, as compared to aminoacylation of the endogenous tRNA by an endogenous RS.
- a second orthogonal molecule can be introduced into the cell that functions with the first orthogonal molecule.
- an orthogonal tRNA/RS pair includes introduced complementary components that function together in the cell with an efficiency (e.g., 45 % efficiency, 50% efficiency, 60% efficiency, 70% efficiency, 75% efficiency, 80% efficiency, 90% efficiency, 95% efficiency, or 99% or more efficiency) as compared to that of a control, e.g., a corresponding tRNA RS endogenous pair, or an active orthogonal pair (e.g., a tyrosyl orthogonal tRNA/RS pair).
- an efficiency e.g., 45 % efficiency, 50% efficiency, 60% efficiency, 70% efficiency, 75% efficiency, 80% efficiency, 90% efficiency, 95% efficiency, or 99% or more efficiency
- a control e.g., a corresponding tRNA RS endogenous pair, or an active orthogonal pair (e.g., a tyrosyl orthogonal tRNA/RS pair).
- an active orthogonal pair e.g., a tyrosyl orthogonal t
- Preferentially aminoacylates refers to an efficiency, e.g., 70 % efficient, 75 % efficient, 85% efficient, 90% efficient, 95 % efficient, or 99% or more efficient, at which an O-RS aminoacylates an O-tRNA with a , selected amino acid, e.g., an unnatural amino acid, as compared to the O-RS aminoacylating a naturally occurring tRNA or a starting material used to generate the O-tRNA.
- Selector codon refers to codons recognized by the O-tRNA in the translation process and not recognized by an endogenous tRNA.
- the O- tRNA anticodon loop recognizes the selector codon on the mRNA and incorporates its amino acid, e.g., a selected amino acid, such as an unnatural amino acid, at this site in the polypeptide.
- Selector codons can include, e.g., nonsense codons, such as, stop codons, e.g., amber, ochre, and opal codons; four or more base codons; rare codons; codons derived from natural or unnatural base pairs and/or the like.
- a suppressor tRNA is a tRNA that alters the reading of a messenger RNA (mRNA) in a given translation system, e.g., by providing a mechanism for incorporating an amino acid into a polypeptide chain in response to a selector codon.
- mRNA messenger RNA
- a suppressor tRNA can read through, e.g., a stop codon, a four base codon, or a rare codon.
- Suppression activity refers, in general, to the ability of a tRNA (e.g., a suppressor tRNA) to allow translational read- through of a codon (e.g. a selector codon that is an amber codon or a 4-or-more base codon) that would otherwise result in the termination of translation or mistranslation (e.g., frame- shifting).
- Suppression activity of a suppressor tRNA can be expressed as a percentage of translational read-through observed compared to a second suppressor tRNA, or as compared to a control system, e.g., a control system lacking an O-RS.
- Percent suppression of a particular OtRNA and ORS against a selector codon (e.g., an amber codon) of interest refers to the percentage of activity of a given expressed test marker (e.g., LacZ), that includes a selector codon, in a nucleic acid encoding the expressed test marker, in a translation system of interest, where the translation system of interest includes an O-RS and an O-tRNA, as compared to a positive control construct, where the positive control lacks the O-tRNA, the O-RS and the selector codon.
- a selector codon e.g., an amber codon
- percent suppression of a test construct comprising the selector codon is the percentage of X that the test marker construct displays under essentially the same environmental conditions as the positive control marker was expressed under, except that the test marker construct is expressed in a translation system that also includes the O-tRNA and the O-RS.
- the translation system expressing the test marker also includes an amino acid that is recognized by the O-RS and O-tRNA.
- the percent suppression measurement can be refined by comparison of the test marker to a
- background or “negative” control marker construct which includes the same selector codon as the test marker, but in a system that does not include the O-tRNA, O-RS and/or relevant amino acid recognized by the O-tRNA and/or O-RS.
- This negative control is useful in normalizing percent suppression measurements to account for background signal effects from the marker in the translation system of interest.
- Suppression efficiency can be determined by any of a number of assays known in the art.
- a ?-galactosidase reporter assay can be used, e.g., a derivatized lacZ plasmid (where the construct has a selector codonpi the lacZ nucleic acid sequence) is introduced into cells from an appropriate organism (e.g., an organism where the orthogonal components can be used) along with plasmid comprising an O-fRNA of the invention.
- a cognate synthetase can also be introduced (either as a polypeptide or a polynucleotide that encodes the cognate synthetase when expressed).
- the cells are grown in media to a desired density, e.g., to an OD 600 of about 0.5, and ⁇ -galactosidase assays are performed, e.g., using the BetaFluorTM ⁇ -Galactosidase Assay Kit (Novagen). Percent suppression can be calculated as the percentage of activity for a sample relative to a comparable control, e.g., the value observed from the derivatived lacZ construct, where the construct has a corresponding sense codon at desired position rather than a selector codon.
- Translation system refers to the components that incorporate an amino acid into a growing polypeptide chain (protein).
- Components of a translation system can include, e.g., ribosomes, tRNAs, synthetases, mRNA and the like.
- the O-tRNA and/or O-RS of the invention can be added to or be a part of an in vitro or in vivo translation system, e.g., in a non-eukaryotic cell, e.g., a bacterium (such as E coli), or in a eukaryotic cell, e.g., a yeast cell, a mammalian cell, a plant cell, an algae cell, a fungus cell, an insect cell, and/or the like.
- a non-eukaryotic cell e.g., a bacterium (such as E coli)
- a eukaryotic cell e.g., a yeast cell, a mammalian cell, a plant cell, an algae cell, a fungus cell, an insect cell, and/or the like.
- Selected amino acid refers to any desired naturally occurring amino acid or unnatural amino acid.
- unnatural amino acid refers to any amino acid, modified amino acid, and/or amino acid analogue that is not one of the 20 common naturally occurring amino acids or seleno cysteine or pyrolysine.
- derived from refers to a component that is isolated from or made using a specified molecule or organism, or information from the specified molecule or organism.
- Positive selection or screening marker refers to a marker that, when present, e.g., expressed, activated, or the like, results in identification of a cell with the positive selection marker from those without the positive selection marker.
- Negative selection or screening marker refers to a marker that, when present, e.g., expressed, activated or the like, allows identification of a cell that does not possess a specified property (e.g., as compared to a cell that does possess the property).
- reporter refers to a component that can be used to identify and/or select target components of a system of interest.
- a reporter can include a protein, e.g., an enzyme, that confers antibiotic resistance or sensitivity (e.g., ⁇ -lactamase, chloramphenicol acetyltransferase (CAT), and the like), a fluorescent screening marker (e.g., green fluorescent protein (e.g., (GFP), YFP, EGFP, RFP), a luminescent marker (e.g., a firefly luciferase protein), an affinity based screening marker, or positive or negative selectable marker genes such as lacZ, ⁇ -gal/lacZ ( ⁇ - galactosidase), Adh (alcohol dehydrogenase), his3, ura3, leu2, lys2, or the like.
- a protein e.g., an enzyme, that confers antibiotic resistance or sensitivity (e.g., ⁇ -lactamase, chlor
- Eukaryote refers to organisms belonging to the phylogenetic domain Eucarya, such as animals (e.g., mammals, insects, reptiles, birds, etc.), ciliates, plants (e.g., monocots, dicots, algae, etc.), fungi, yeasts, flagellates, microsporidia, protists, etc.
- Non-eukaryote As used herein, the term “non-eukaryote” refers to non- eukaryotic organisms.
- a non-eukaryotic organism can belong to the Eubacteria (e.g., Escherichia coli, Thermus thennophilus, Bacillus stearothennophilus, etc.) phylogenetic domain, or the Archaea (e.g., Methanococcus jannaschii, Methanobacterium thennoautotrophicum, Halobacterium such as Haloferax volcanii and Halobacterium species NRC-1, Archaeoglobus fulgidus, Pyrococcus furiosus, Pyrococcus horikoshii, Aeuropyrum pernix, etc.) phylogenetic domains.
- Eubacteria e.g., Escherichia coli, Thermus thennophilus, Bacillus stearothennophilus, etc.
- Archaea e.g., Methanococcus jannaschii, Methanobacterium thennoautotrophicum, Halobacterium such as
- Conservative variant in reference to a translation component such as an O-tRNA or O-RS refers to a translation component that has a substantailly similar activity as the component on which the conservative variant is similar to, e.g., an O-tRNA or O-RS, but has variations in the sequence as compared to the base component.
- an O-RS will aminoacylate a complementary O-tRNA or a conservative variant O-tRNA with a selected amino acid, e.g., an unnatural amino acid, although the O-tRNA and the conservative variant O-tRNA do not have the same sequence.
- the conservative variant can have, e.g., one variation, two variations, three variations, four variations, or five or more variations in its sequence, as long as the conservative variant functionally interacts with a corresponding O-tRNA or O-RS in substantailly the same manner as the non- variant form.
- Selection or screening agent refers to an agent that, when present, allows for selection/screening of certain components from a population.
- a selection or screening agent can be, but is not limited to, e.g., a nutrient, an antibiotic, a wavelength of light, an antibody, an expressed polynucleotide, or the like.
- the selection agent can be varied, e.g., by concentration, intensity, etc.
- Encode refers to any process whereby the information in a polymeric macromolecule or sequence string is used to direct the production of a second molecule or sequence string that is different from the first molecule or sequence string.
- the term is used broadly, and can have a variety of applications.
- the term “encode” describes the process of semi-conservative DNA replication, where one strand of a double-stranded DNA molecule is used as a template to encode a newly synthesized complementary sister strand by a DNA-dependent DNA polymerase.
- the term "encode” refers to any process whereby the information in one molecule is used to direct the production of a second molecule that has a different chemical nature from the first molecule.
- a DNA molecule can encode an RNA molecule (e.g., by the process of transcription incorporating a DNA- dependent RNA polymerase enzyme).
- an RNA molecule can encode a polypeptide, as in the process of translation.
- the term “encode” also extends to the triplet codon that encodes an amino acid.
- an RNA molecule can encode a DNA molecule, e.g., by the process of reverse transcription incorporating an RNA-dependent DNA polymerase.
- a DNA molecule can encode a polypeptide, where it is understood that "encode” as used in that case incorporates both the processes of transcription and translation.
- FIG. 1 Panels A, B and C schematically illustrate leucyl tRNAs and synthetases, and their phylogenetic relationships.
- Panel A provides a ClustalW analysis of aminoacyl-tRNA synthetases, where Archaeal tRNA synthetases are labeled using a dashed line, prokaryotic using a solid line, and eukaryotic sequences using a dotted line. This analysis reveals the halobacterial synthetase to be unusual in its homology to prokaryotic rather than archaeal and eukaryotic synthetases.
- Panel B provides a ClustalW analysis of Halobacterial tRNAs which all share high homology to other archaeal tRNAs. Dendrograms were generated using the program PhyloDraw. Panel C provides a sequence alignment of multiple sequences of the family of archaeal leucyl tRNAs examined as potential orthogonal suppressors. Sequences examined as potential amber suppressors by changing the anticodon (boxed) to CUA are shown in bold as is the consensus sequence. The highly conserved positions G37 and A73 are indicated with underlining. [0055] Figure 2 provides a histogram showing the identification of a leucyl orthogonal pair. The suppression efficiency of seven synthetases expressed with 5 orthogonal amber suppressor reporter constructs was measured using a ⁇ -lactamase amber suppression assay.
- Panels A and B provide graphs illustrating aminoacylation in vitro by archaeal leucyl-tRNA synthetases.
- Panel A illustrates charging of crude total halobacterial tRNA determined by aminoacylation assays with [ 3 H] leucine by AfLRS ( ⁇ ), MjLRS (•), MtLRS (A), EcLRS ( ⁇ ), and no synthetase (D).
- Panel B illustrates charging of crude total E. coli tRNA.
- Panels A and B illustrates the optimization of suppressor tRNAs.
- Panel A illustrates regions (shown in boxes) of the halobacterial orthogonal tRNA subjected to mutagenesis in an effort to improve the efficiency or selectivity of TAG and AGGA suppressor tRNAs.
- Panel B illustrates that active mutant TAG suppressors identified by positive selection conserve A73. Less cross-reactive mutants identified by a double-sieve selection strategy conserve a C3:G70 base pair. The most active and selective suppressor tRNA is shown with double boxes.
- Figure 5 illustrates a consensus-derived frameshift suppressor.
- a consensus sequence was obtained by multiple sequence alignment of all known archaeal leucyl tRNAs, and the anticodon loop is changed to UCUCCUAA. The variations observed for tRNAs identified by selection are shown in boxes. The most active mutations are shown with double boxes. DETAILED DESCRIPTION
- orthogonal pairs of an aminoacyl-tRNA synthetase and a tRNA are needed that can function efficiently in the translational machinery. Desired characteristics of the orthogonal pairs include tRNA that decode or recognize only a specific new codon, e.g., a selector codon, that is not decoded by any endogenous tRNA, and aminoacyl-tRNA synthetases that preferentially aminoacylate (or charge) its cognate tRNA with only a specific selected amino acid, e.g., an unnatural amino acid.
- the O-tRNA is also not typically aminoacylated by endogenous synthetases. For example, in E.
- an orthogonal pair will include an aminoacyl-tRNA synthetase that does not significantly cross-react with any of the endogenous tRNA, which there are 40 in E. coli, and an orthogonal tRNA that is not significantly aminoacylated by any of the endogenous synthetases, e.g., of which there are 21 in E. coli.
- the O-tRNA is capable of mediating incorporation of a selected amino acid into a protein that is encoded by a polynucleotide, which comprises a selector codon that is recognized by the O-tRNA, e.g., in vivo.
- the anticodon loop of the O-tRNA recognizes the selector codon on an mRNA and incorporates its amino acid, e.g., a selected amino acid, such as an unnatural amino acid, at this site in the polypeptide. Any of a number of selector codons can be used with the invention.
- selector codons can include, e.g., nonsense codons, such as, stop codons, e.g., amber, ochre, and opal codons; four or more base codons; rare codons; codons derived from natural or unnatural base pairs and/or the like. See also the section herein entitled "Selector codon.”
- orthogonal tRNA/synthetase pairs can be developed that allow the simultaneous incorporation of multiple selected amino acids, e.g., unnatural amino acids, using these different selector codons.
- This invention provides compositions of and methods for identifying and producing additional orthogonal tRNA-aminoacyl-tRNA synthetase pairs, e.g., leucyl O-tRNA/leucyl O-RSs, using any of a number of selector codons, e.g., an amber codon, an opal codon, an extended codon (such as a four-base codon), and the like.
- selector codons e.g., an amber codon, an opal codon, an extended codon (such as a four-base codon)
- Such translation systems of the invention generally comprise cells that include an orthogonal leucyl tRNA (leucyl O-tRNA), an orthogonal leucyl aminoacyl tRNA synthetase (leucyl O-RS), and a selected amino acid, e.g., an unnatural amino acid, where the leucyl O-RS aminoacylates the leucyl O-tRNA with the selected amino acid.
- An orthogonal pair of the invention is composed of a leucyl O-fRNA, e.g., a suppressor tRNA, a frameshift tRNA, or the like, and an leucyl O-RS.
- the leucyl-O-tRNA recognize a first selector codon and has at least about a 25% suppression activity in presence of a cognate synthetase in response to a selector codon as compared to a control lacking the cognate synthetase.
- the leucyl O-tRNA also comprises an anticodon loop comprising a CU(X) n XXXAA sequence.
- the cell uses the components to incorporate the selected amino acid into a growing polypeptide chain.
- a nucleic acid that comprises a polynucleotide that encodes a polypeptide of interest can also be present, where the polynucleotide comprises a selector codon that is recognized by the leucyl O-tRNA.
- the translation system can also be an in vitro system.
- a cell of teh invention e.g., an E. coli cell
- an E. coli cell of the invention can include an orthogonal leucyl-tRNA (leucyl-O-tRNA), where the leucyl-O-tRNA comprises at least about a 25% suppression activity in presence of a cognate synthetase in response to a selector codon as compared to a control lacking the cognate synthetase; an orthogonal leucyl aminoacyl-tRNA synthetase (leucyl-O-RS); a selected amino acid; and, a nucleic acid that comprises a polynucleotide that encodes a polypeptide of interest, where the polynucleotide comprises a selector codon that is recognized by the leucyl O-tRNA.
- leucyl-O-tRNA orthogonal leucyl-tRNA
- leucyl-O-tRNA an
- the invention also features multiple O-tRNA/O-RS pairs in a cell, which allows incorporation of more than one selected amino acid.
- the cell can further include an additional different O-tRNA/O-RS pair and a second selected amino acid, where the O-tRNA recognizes a second selector codon and the O-RS preferentially aminoacylates the O-tRNA with the second selected amino acid.
- a cell can further comprise, e.g., an amber suppressor tRNA-aminoacyl tRNA synthetase pair derived from the tyrosyl-tRNA synthetase of Methanococcus jantiaschii.
- the leucyl O-tRNA and/or the leucyl O-RS can be naturally occurring or can be derived by mutation of a naturally occurring tRNA and or RS, e.g., which generates libraries of tRNAs and/or libraries of RSs, from a variety of organisms.
- one strategy of producing an orthogonal leucyl tRNA/leucyl aminoacyl-tRNA synthetase pair involves importing a heterologous tRNA/synthetase pair from, e.g., a source other than the host cell, or multiple sources, into the host cell.
- the properties of the heterologous synthetase candidate include, e.g., that it does not charge any host cell tRNA, and the properties of the heterologous tRNA candidate include, e.g., that it is not aminoacylated by any host cell synthetase.
- the heterologous tRNA is orthogonal to all host cell synthetases.
- a second strategy for generating an orthogonal pair involves generating mutant libraries from which to screen and or select a leucyl O-tRNA or leucyl O-RS. These strategies can also be combined.
- the leucyl O-tRNA and leucyl O-RS are derived from at least one organism.
- the leucyl O-tRNA is derived from a naturally occurring or mutated naturally occurring tRNA from a first organism and the leucyl O-RS is derived from naturally occurring or mutated naturally occurring RS from a second organism.
- the first and second organism is different.
- an orthogonal pair of the invention includes a leucyl-tRNA synthetase derived from Methanobacterium thennoautotrophicum, and a leucyl tRNA derived from an archael tRNA (e.g., from Halobacterium sp. NRC-1).
- the first and second organism are the same. See the section entitled “Sources and Hosts" herein for additional information.
- a leucyl O-tRNA of the invention comprises or is encoded or transcribed by or from a polynucleotide sequence as set forth in any one of SEQ ID NO.: 3, 6, 7 or 12, or a complementary polynucleotide sequence thereof.
- a leucyl O-RS comprises an amino acid sequence as set forth in any one of SEQ ID NO.: 15 or 16, or a conservative variation thereof.
- the leucyl O-RS, or a portion thereof can also be encoded or transcribed by or from a polynucleotide sequence as set forth in any one of SEQ RO NO.: 13 or 14, or a complementary polynucleotide sequence thereof. See also, the section entitled “Nucleic Acid and Polypeptide Sequence and Variants," herein.
- Orthogonal tRNA (O-tRNA) [0070]
- An orthogonal leucyl tRNA (leucyl O-tRNA) mediates incorporation of a selected amino acid into a protein that is encoded by a polynucleotide that comprises a selector codon that is recognized by the leucyl O-tRNA, e.g., in vivo.
- a leucyl O-tRNA of the invention comprises an anticodon loop comprising a CU(X) n XXXAA sequence.
- the CU(X) n XXXAA sequence is found in the anticodon loop, where X refers to any nucleotide, and (X) n is optionally present.
- the CU(X) n XXXAA sequence comprises
- the leucyl O-tRNA can include a stem region comprising matched base pairs and a conserved discriminator base (position 73). See, e.g., Figure 4, Panel B. This position is indicated in e.g., Figure 4, Panel A.
- the leucyl O-tRNA also optionally includes a C:G base pair at position 3:70.
- the CU(X) n XXXAA sequence comprises a CUUCCUAA sequence, typically when the selector codon is a four-base codon. See, e.g., Figure 5.
- the leucyl O-tRNA can also include a first pair selected from T28:A42, G28:C42 and/or C28:G42, and a second pair selected from G:49:C65 or C49:G65, where the numbering corresponds to that indicated in Figure 4, Panel A.
- C28:G42 is the first pair and C49:G65 is the second pair.
- the selector codon is an opal codon
- the CU(X) n XXXAA sequence can comprises a CUUCAAA sequence.
- a leucyl O-tRNA of the invention comprises at least about a 25% suppression activity in presence of a cognate synthetase in response to a selector codon, as compared to a control lacking the cognate synthetase.
- Suppression activity can be determined by any of a number of assays known in the art. For example, a ?-galactosidase reporter assay can be used.
- a derivative of a plasmid that expresses lacZ gene under the control of promoter is used, e.g., where the Leu-25 of the peptide VVLQRRDWEN of lacZ is replaced by a selector codon, e.g., TAG, TGA, AGGA, etc.
- lacZ plasmid is introduced into cells from an appropriate organism (e.g., an organism where the orthogonal components can be used) along with plasmid comprising a O-tRNA of the invention.
- a cognate synthetase can also be introduced (either as a polypeptide or a polynucleotide that encodes the cognate synthetase when expressed).
- the cells are grown in media to a desired density, e.g., to an OD 6 oo of about 0.5., and ⁇ -galactosidase assays are performed, e.g., using the BetaFluorTM ⁇ -Galactosidase Assay Kit (Novagen). Percent suppression is calculated as the percentage of activity for a sample relative to a comparable control, e.g., the value observed from the derivatived lacZ construct, where the construct has a corresponding sense codon at desired position rather than a selector codon.
- Examples of leucyl O-tRNAs of the invention are transcribed from any one of SEQ RO NO.: 1-7 and/or 12.
- RNA and DNA versions of a tRNA are often referred to simply by reference to the DNA sequence that corersponds to the RNA form of the tRNA. Any time a DNA form of a tRNA is given, one of skill will easily be able to derive the RNA (or vice versa) by strandard transcription (or reverse transcription). In addition, additional modifications to the bases can be present.
- the invention also includes conservative variations of leucyl O-tRNA.
- conservative variations of leucyl O-tRNA include those molecules that function like the leucyl O-tRNA of any one of SEQ RO NO.: 1-7 and 12 and maintain the tRNA L-shaped structure, but do not have the same sequence (and are other than wild type leucyl tRNA molecules). See also, the section herein entitled “Nucleic acids and Polypeptides Sequence and Variants.”
- composition comprising a leucyl O-tRNA can further include an orthogonal leucyl aminoacyl-tRNA synthetase (leucyl O-RS), where the leucyl O-RS preferentially aminoacylates the leucyl O-tRNA with a selected amino acid (e.g., an unnatural amino acid).
- a composition that includes a leucyl O- tRNA can further include a translation system (e.g., in vitro or in vivo).
- a nucleic acid that comprises a polynucleotide that encodes a polypeptide of interest, where the polynucleotide comprises a selector codon that is recognized by the leucyl O-tRNA, or a combination of one or more of these can also be present in the cell. See also, the section herein entitled “Orthogonal aminoacyl-tRNA synthetases.”
- O-tRNA orthogonal tRNA
- An O-tRNA, e.g., a leucyl O-tRNA, produced by the method is also a feature of the invention.
- a method includes mutating an anticodon loop of members of a pool of tRNAs (e.g., a pool of leucyl tRNAs) to allow recognition of a selector codon (e.g., an amber codon, an opal codon, a four base codon, etc.), thereby providing a plurality of potential O-tRNAs; and analyzing secondary structure of a member of the plurality potential O-tRNA to identify non-canonical base pairs in the secondary structure, and optionally mutating the non-canonical base pairs (e.g., the non- canonical base pairs are mutated to canonical base pairs).
- the non-canonical base pairs can be located in stem region of the secondary structure.
- a leucyl O-tRNA possesses an improvement of orthogonality for a desired organism compared to the starting material, e.g., the plurality of tRNA sequences, while preserving its affinity towards a desired RS.
- the methods optionally include analyzing the homology of sequences of tRNAs and/or aminoacyl-tRNA synthetases to determine potential candidates for an O- tRNA, O-RS and/or pairs thereof, that appear to be orthogonal for a specific organism.
- Computer programs known in the art and described herein can be used for the analysis.
- a prokaryotic organism a synthetase and/or a tRNA is chosen that does not display unusual homology to prokaryotic organisms.
- the pool of tRNAs can also be produced by a consensus strategy.
- the pool of tRNAs is produced by aligning a plurality of tRNA sequences (see e.g., Figure 1, Panel C); determining a consensus sequence (see e.g., Figure 1, Panel C); and generating a library of tRNAs using at least a portion, most of, or the entire consensus sequence.
- a consensus sequence can be compiled with a computer program, e.g., the GCG program pileup.
- degenerate positions determined by the program are changed to the most frequent base at those positions.
- a library is synthesized by techniques known in the art using the consensus sequence.
- overlap extension of oligonucleotides in which each site of the tRNA gene can be synthesized as a doped mixture of 90% the consensus sequence and 10% a mixture of the other 3 bases can be used to provide the library based on the consensus sequence.
- Other mixtures can also be used, e.g., 75% the consensus sequence and 25% a mixture of the other 3 bases, 80% the consensus sequence and 20% a mixture of the other 3 bases, 95% the consensus sequence and 5% a mixture of the other 3 bases, etc.
- the library of mutant tRNAs can be generated using various mutagenesis techniques known in the art.
- the mutant tRNAs can be generated by site- specific mutations, random point mutations, homologous recombination, DNA shuffling or other recursive mutagenesis methods, chimeric construction or any combination thereof.
- Additional mutations can be introduced at a specific position(s), e.g., at a nonconservative position(s), or at a conservative position, at a randomized position(s), or a combination of both in a desired loop or region of a tRNA, e.g., an anticodon loop, the acceptor stem, D arm or loop, variable loop, T ⁇ C arm or loop, other regions of the tRNA molecule, or a combination thereof.
- mutations in a leucyl tRNA include introducing a CU(X) n XXXAA sequence into the anticodon loop, where X refers to any nucleotide, and (X) n is optionally present.
- the n refers to number of bases the anticodon loop needs to be extended based on the selector codon, e.g., an extended codon, such as a four-, five-, six- base pair, etc.
- mutations include matched base pairs in the stem region.
- mutations include a first pair selected from T28:A42, G28:C42; C28:G42, etc. and a second pair selected from G49:C65 or C49:G65.
- an O-tRNA is obtained by subjecting to negative selection a population of cells of a first species, where the cells comprise a member of the plurality of potential O-tRNAs.
- the negative selection eliminates cells that comprise a member of the plurality of potential O-tRNAs that is aminoacylated by an aminoacyl-tRNA synthetase (RS) that is endogenous to the cells. This provides a pool of tRNAs that are orthogonal to the cell of the first species.
- RS aminoacyl-tRNA synthetase
- a selector codon(s) is introduced into polynucleotide that encodes a negative selection marker, e.g., an enzyme that confers antibiotic resistance, e.g., ⁇ -lactamase, an enzyme that confers a detectable product, e.g., ⁇ -galactosidase, chloramphenicol acetyltransferase (CAT), e.g., a toxic product, such as barnase, at a nonessential position, etc.
- Screening/selection can be done by growing the population of cells in the presence of a selective agent (e.g., an antibiotic, such as ampicillin). In one embodiment, the concentration of the selection agent is varied.
- a selection system is used that is based on the in vivo suppression of selector codon, e.g., nonsense or frameshift mutations introduced into a polynucleotide that encodes a negative selection marker, e.g., a gene for ⁇ -lactamase (.bid).
- selector codon e.g., nonsense or frameshift mutations introduced into a polynucleotide that encodes a negative selection marker, e.g., a gene for ⁇ -lactamase (.bid).
- polynucleotide variants e.g., bla variants, with, e.g., TAG, AGGA, and TGA, at a certain position (e.g., A184), are constructed.
- Cells e.g., bacteria, are transformed with these polynucleotides.
- antibiotic resistance e.g., ampicillin resistance
- a bacteria transformed with no plasmid should be about or less than that for a bacteria transformed with no plasmid. If the leucyl tRNA is not orthogonal, or if a heterologous synthetase capable of charging the tRNA is co-expressed in the system, a higher level of antibiotic, e.g., ampicillin, resistance is be observed.
- Cells e.g., bacteria, are chosen that are unable to grow on LB agar plates with antibiotic concentrations about equal to cells transformed with no plasmids.
- a toxic product e.g., ribonuclease or barnase
- a member of the plurality of potential leucyl tRNAs is aminoacylated by endogenous host, e.g., Escherichia coli synthetases (i.e., it is not orthogonal to the host, e.g., Escherichia coli synthetases)
- the selector codon is suppressed and the toxic polynucleotide product produced leads to cell death.
- Cells harboring orthogonal leucyl tRNAs or non-functional tRNAs survive.
- the pool of tRNAs that are orthogonal to a desired organism are then subjected to a positive selection in which a selector codon is placed in a positive selection marker, e.g., encoded by a drug resistance gene, such a ⁇ -lactamase gene.
- a positive selection marker e.g., encoded by a drug resistance gene, such a ⁇ -lactamase gene.
- the positive selection is performed on cell comprising a polynucleotide encoding or comprising a member of the pool of tRNAs, a polynucleotide encoding a positive selection marker, and a polynucleotide encoding a cognate RS.
- a selection agent e.g., ampicillin.
- Leucyl tRNAs are then selected for their ability to be aminoacylated by the coexpressed cognate synthetase and to insert an amino acid in response to this selector codon.
- these cells show an enhancement in suppression efficiency compared to cells harboring nonfunctional tRNA(s), or tRNAs that cannot efficiently be recognized by the synthetase of interest.
- the cell harboring the non-functional tRNAs or tRNAs that are not efficiently recognized by the synthetase of interest, are sensitive to the antibiotic.
- leucyl tRNAs that: (i) are not substrates for endogenous host, e.g., Escherichia coli, synthetases; (ii) can be aminoacylated by the synthetase of interest; and (iii) are functional in translation, survive both selections.
- the stringency of the selection e.g., the positive selection, the negative selection or both the positive and negative selection, in the above described-methods, optionally include varying the selection stringency.
- the stringency of the negative selection can be controlled by introducing different numbers of selector codons into the barnase gene and/or by using an inducible promoter.
- the concentration of the selection or screening agent is varied (e.g., ampicillin concentration).
- the stringency is varied because the desired activity can be low during early rounds. Thus, less stringent selection criteria are applied in early rounds and more stringent criteria are applied in later rounds of selection.
- the negative selection, the positive selection or both the negative and positive selection can be repeated multiple times. Multiple different negative selection markers, positive selection markers or both negative and positive selection markers, can be used. In certain embodiments, the positive and negative selection marker can be the same.
- Other types of selections/screening can be used in the invention for producing orthogonal translational components, e.g., a leucyl O-tRNA, a leucyl O-RS, and a leucyl O-tRNA/O-RS pair.
- the negative selection marker, the positive selection marker or both the positive and negative selection markers can include a marker that fluoresces or catalyzes a luminescent reaction in the presence of a suitable reactant.
- a product of the marker is detected by fluorescence-activated cell sorting (FACS) or by luminescence.
- FACS fluorescence-activated cell sorting
- the marker includes an affinity based screening marker. See, Francisco, J. A., et al., (1993) Production and fluorescence-activated cell sorting of Escherichia coli expressing a functional antibody fragment on the external surface. Proc Natl Acad Sci U S A. 90: 10444-8.
- a leucyl O-RS of the invention preferentially aminoacylates a leucyl O- tRNA with a selected amino acid in vitro or in vivo.
- a leucyl O-RS of the invention can be provided to the translation system, e.g., a cell, by a polypeptide that includes a leucyl O-RS and/or by a polynucleotide that encodes a leucyl O-RS or a portion thereof.
- a leucyl O-RS is encoded by a polynucleotide sequence as set forth in any one of SEQ RO NO.: 13-14, or a complementary polynucleotide sequence thereof.
- a leucyl O-RS comprises an amino acid sequence as set forth in any one of SEQ RO NO.: 15-16, or a conservative variation thereof. See, e.g., Table 3 and Example 2 herein for sequences of exemplary leucyl O-RS molecules.
- O-RS orthogonal aminoacyl-tRNA synthetase
- a method includes subjecting to positive selection a population of cells of a first species, where the cells individually comprise: 1) a member of a plurality of aminoacyl-tRNA synthetases (RSs), where the plurality of RSs comprise mutant RSs, RSs derived from a species other than the first species or both mutant RSs and RSs derived from a species other than the first species; 2) the orthogonal tRNA (O-tRNA) from a second species; and 3) a polynucleotide that encodes a positive selection marker and comprises at least one selector codon.
- RSs aminoacyl-tRNA synthetases
- Cells are selected or screened for those that show an enhancement in suppression efficiency compared to cells lacking or with a reduced amount of the member of the plurality of RSs.
- Cells having an enhancement in suppression efficiency comprise an active RS that aminoacylates the O-tRNA.
- a level of aminoacylation (in vitro or in vivo) by the active RS of a first set of tRNAs from the first species is compared to the level of aminoacylation (in vitro or in vivo) by the active RS of a second set of tRNAs from the second species.
- the level of aminoacylation can be determined by a detectable substance (e.g., a labeled amino acid or unnatural amino acid).
- the active RS that more efficiently aminoacylates the second set of tRNAs compared to the first set of tRNAs is selected, thereby providing an efficient (optimized) orthogonal aminoacyl-tRNA synthetase for use with the O-tRNA.
- An O-RS e.g., a leucyl O-RS, identified by the method, is also a feature of the invention.
- any of a number of assays can be used to determine aminoacylation. These assays can be performed in vitro or in vivo. For example, in vitro aminoacylation assays are described in, e.g., Hoben, P., and Soil, D. (1985) Methods Enzymol. 113:55-59. Aminoacylation can also be determined by using a reporter along with orthogonal translation components and detecting the reporter in a cell expressing a polynucleotide comprising at least one selector codon that encodes a protein. See also, WO 2002/085923, entitled “IN VIVO INCORPORATION OF UNNATURAL AMINO ACIDS;" and, USSN 60/479,931 entitled “EXPANDING THE EUKARYOTIC GENETIC CODE.”
- Identified leucyl O-RS can be further manipulated to alter the substrate specificity of the synthetase, so that only a desired unnatural amino acid, but not any of the common 20 amino acids are charged to the leucyl O-tRNA.
- Methods to generate an orthogonal leucyl aminoacyl tRNA synthetase with a substrate specificity for an unnatural amino acid include mutating the synthetase, e.g., at the active site in the synthetase, at the editing mechanism site in the synthetase, at different sites by combining different domains of synthetases, or the like, and applying a selection process.
- a strategy is used, which is based on the combination of a positive selection followed by a negative selection.
- the positive selection suppression of the selector codon introduced at a nonessential position(s) of a positive marker allows cells to survive under positive selection pressure.
- survivors thus encode active synthetases charging the orthogonal suppressor tRNA with either a natural or unnatural amino acid.
- the negative selection suppression of a selector codon introduced at a nonessential position(s) of a negative marker removes synthetases with natural amino acid specificities.
- Survivors of the negative and positive selection encode synthetases that aminoacylate (charge) the orthogonal suppressor tRNA with unnatural amino acids only. These synthetases can then be subjected to further mutagenesis, e.g., DNA shuffling or other recursive mutagenesis methods.
- the library of mutant leucyl O-RSs can be generated using various mutagenesis techniques known in the art.
- the mutant RSs can be generated by site-specific mutations, random point mutations, homologous recombination, DNA shuffling or other recursive mutagenesis methods, chimeric construction or any combination thereof.
- a library of mutant leucyl RSs can be produced from two or more other, e.g., smaller, less diverse "sub-libraries.” Chimeric libraries of RSs are also included in the invention.
- libraries of tRNA synthetases from various organism such as microorganisms such as eubacteria or archaebacteria
- libraries that comprise natural diversity see, e.g., U.S. Patent No. 6,238,884 to Short et al; U.S. Patent No. 5,756,316 to Schallenberger et al; U.S. Patent No. 5,783,431 to Petersen et al; U.S. Patent No. 5,824,485 to Thompson et al; U.S. Patent No. 5,958,672 to Short et al
- synthetases are subject to the positive and negative selection/screening strategy, these synthetases can then be subjected to further mutagenesis.
- a nucleic acid that encodes the leucyl O-RS can be isolated; a set of polynucleotides that encode mutated leucyl O-RSs (e.g., by random mutagenesis, site- specific mutagenesis, recombination or any combination thereof) can be generated from the nucleic acid; and, these individual steps or a combination of these steps can be repeated until a mutated leucyl O-RS is obtained that preferentially aminoacylates the leucyl O- tRNA with the unnatural amino acid.
- the steps are performed multiple times, e.g., at least two times.
- Additional levels of selection/screening stringency can also be used in the methods of the invention, for producing leucyl O-tRNA, leucyl O-RS, or pairs thereof.
- the selection or screening stringency can be varied on one or both steps of the method to produce an O-RS. This could include, e.g., varying the amount of selection/screening agent that is used, etc. Additional rounds of positive and/or negative selections can also be performed.
- Selecting or screening can also comprise one or more positive or negative selection or screening that includes, e.g., a change in amino acid permeability, a change in translation efficiency, a change in translational fidelity, etc.
- the one or more change is based upon a mutation in one or more gene in an organism in which an orthogonal tRNA-tRNA synthetase pair is used to produce protein.
- the translational components of the invention can be derived from non- eukaryotic organisms.
- the orthogonal O-tRNA can be derived from a non- eukaryotic organism, e.g., an archaebacterium, such as Methanococcus jannaschii,
- Methanobacterium thennoautotrophicum Halobacterium such as Haloferax volcanii and Halobacterium species NRC-1 , Archaeoglobus fulgidus, Pyrococcus furiosus, Pyrococcus horikoshii, Aeuropyrum pernix, or the like, or a eubacterium, such as Escherichia coli, Thermus thennophilus, Bacillus stearothennp ilus, or the like, while the orthogonal O-RS can be derived from a non-eukaryotic organism, e.g., Methanobacterium thennoautotrophicum, Halobacterium such as Haloferax volcanii and Halobacterium species NRC-1, Archaeoglobus fulgidus, Pyrococcus furiosus, Pyrococcus horikoshii, Aeuropyrum pemix, or the like, or a eubacterium, such as Escherichia coli, Thermus thermo
- eukaryotic sources can also be used, e.g., plants, algae, protists, fungi, yeasts, animals (e.g., mammals, insects, arthropods, etc.), or the like.
- the individual components of a leucyl O-tRNA O-RS pair can be derived from the same organism or different organisms.
- the leucyl O-tRNA/O- RS pair is from the same organism.
- the leucyl O-tRNA and the leucyl O-RS of the leucyl O-tRNA/O-RS pair are from different organisms.
- the leucyl O- tRNA can be derived from, e.g., a Halobacterium sp NRC-1, and the leucyl O-RS can be derived from, e.g., a Methanobacterium thermoautrophicum.
- the leucyl O-tRNA, leucyl O-RS or leucyl O-tRNA O-RS pair can be selected or screened in vivo or in vitro and/or used in a cell, e.g., a non-eukaryotic cells (such as E. coli cell), or a eukaryotic cell, to produce a polypeptide with a selected amino acid (e.g., an unnatural amino acid).
- a non-eukaryotic cells such as E. coli cell
- a eukaryotic cell e.g., an unnatural amino acid
- a non-eukaryotic cell can be from a variety of sources, e.g., Methanobacterium thennoautotrophicum, Halobacterium such as Haloferax volcanii and Halobacterium species NRC-1 , Archaeoglobus fulgidus, Pyrococcus furiosus, Pyrococcus horikoshii, Aeuropyrum pen ⁇ x, or the like, or a eubacterium, such as Escherichia coli, Thermus thennophilus, Bacillus stearothennphilus, or the like.
- sources e.g., Methanobacterium thennoautotrophicum, Halobacterium such as Haloferax volcanii and Halobacterium species NRC-1 , Archaeoglobus fulgidus, Pyrococcus furiosus, Pyrococcus horikoshii, Aeuropyrum pen ⁇ x, or the like, or a eubacterium, such as Escherichia coli,
- a eukaryotic cell can be from any of a variety of sources, e.g., a plant (e.g., complex plant such as monocots, or dicots), an algae, a protist, a fungus, a yeast (e.g., Saccharomyces cerevisiae), an animal (e.g., a mammal, an insect, an arthropod, etc.), or the like.
- a plant e.g., complex plant such as monocots, or dicots
- an algae e.g., a protist, a fungus, a yeast (e.g., Saccharomyces cerevisiae)
- an animal e.g., a mammal, an insect, an arthropod, etc.
- Compositions of cells with translational components of the invention are also a feature of the invention. [0101] See also, International Application Number PCT/US2004/011786, filed
- Selector codons of the invention expand the genetic codon framework of protein biosynthetic machinery.
- a selector codon includes, e.g., a unique three base codon, a nonsense codon, such as a stop codon, e.g., an amber codon (UAG), or an opal codon (UGA), an unnatural codon, at least a four base codon, a rare codon, or the like.
- a number of selector codons can be introduced into a desired gene, e.g., one or more, two or more, more than three, etc.
- the methods involve the use of a selector codon that is a stop codon for the incorporation of a selected amino acid, e.g., an unnatural amino acids, in vivo in a cell.
- a selector codon that is a stop codon for the incorporation of a selected amino acid, e.g., an unnatural amino acids, in vivo in a cell.
- a leucyl O-tRNA is produced that recognizes the stop codon and is aminoacylated by a leucyl O-RS with a selected amino acid.
- This leucyl O-tRNA is not recognized by the naturally occurring host's aminoacyl-tRNA synthetases.
- Conventional site-directed mutagenesis can be used to introduce the stop codon at the site of interest in a polypeptide of interest. See, e.g., Sayers, J.R., et al.
- a stop codon used as a selector codon is an amber codon, UAG, and/or an opal codon, UGA.
- SEQ RO NO: 3 for an example of a leucyl O-tRNA that recognizes an amber codon
- SEQ RO NO: 7 for an example of a leucyl O-tRNA that recognizes an opal codon.
- a genetic code in which UAG and UGA are both used as a selector codon can encode 22 amino acids while preserving the ochre nonsense codon, UAA, which is the most abundant termination signal.
- the incorporation of selected amino acids, e.g., unnatural amino acids, in vivo can be done without significant perturbation of the host cell.
- selected amino acids e.g., unnatural amino acids
- the suppression efficiency for the UAG codon depends upon the competition between the O-tRNA, e.g., the amber suppressor tRNA, and the release factor 1 (RF1) (which binds to the UAG codon and initiates release of the growing peptide from the ribosome)
- the suppression efficiency can be modulated by, e.g., either increasing the expression level of O-tRNA, e.g., the suppressor tRNA, or using an RF1 deficient strain.
- the suppression efficiency for the UAG codon depends upon the competition between the O-tRNA, e.g., the amber suppressor tRNA, and a eukaryotic release factor (e.g., eRF) (which binds to a stop codon and initiates release of the growing peptide from the ribosome), the suppression efficiency can be modulated by, e.g., increasing the expression level of O-tRNA, e.g., the suppressor tRNA.
- O-tRNA e.g., the amber suppressor tRNA
- a eukaryotic release factor e.g., eRF
- Unnatural amino acids can also be encoded with rare codons.
- the rare arginine codon, AGG has proven to be efficient for insertion of Ala by a synthetic tRNA acylated with alanine.
- the synthetic tRNA competes with the naturally occurring tRNAArg, which exists as a minor species in Escherichia coli. Some organisms do not use all triplet codons.
- An unassigned codon AGA in Micrococcus luteus has been utilized for insertion of amino acids in an in vitro transcription/translation extract. See, e.g., Kowal and Oliver, Nucl. Acid. Res.. 25:4685 (1997).
- Components of the present invention can be generated to use these rare codons in vivo.
- Selector codons can also comprise extended codons, e.g., four or more base codons, such as, four, five, six or more base codons.
- four base codons include, e.g., AGGA, CUAG, UAGA, CCCU, and the like.
- five base codons include, e.g., AGGAC, CCCCU, CCCUC, CUAGA, CUACU, UAGGC, and the like.
- Methods of the invention include using extended codons based on frameshift suppression.
- Four or more base codons can insert, e.g., one or multiple selected amino acids, e.g., unnatural amino acids, into the same protein.
- the four or more base codon is read as single amino acid.
- the anticodon loops can decode, e.g., at least a four-base codon, at least a five-base codon, or at least a six-base codon or more.
- N can be U, A, G, or C
- the quadruplet UAGA can be decoded by a tRNALeu with a UCUA anticodon with an efficiency of 13 to 26% with little decoding in the 0 or -1 frame.
- extended codons based on rare codons or nonsense codons can be used in invention, which can reduce missense readthrough and frameshift suppression at other unwanted sites.
- a selector codon can also include one of the natural three base codons, where the endogenous system does not use (or rarely uses) the natural base codon. For example, this includes a system that is lacking a tRNA that recognizes the natural three base codon, and/or a system where the three base codon is a rare codon.
- Selector codons optionally include unnatural base pairs. These unnatural base pairs further expand the existing genetic alphabet. One extra base pair increases the number of triplet codons from 64 to 125.
- Third base pairs include stable and selective base pairing, efficient enzymatic incorporation into DNA with high fidelity by a polymerase, and the efficient continued primer extension after synthesis of the nascent unnatural base pair.
- Descriptions of unnatural base pairs which can be adapted for methods and compositions include, e.g., Hirao, et al., (2002) An unnatural base pair for incorporating amino acid analogues into protein, Nature Biotechnology, 20: 177-182. See, also, Wu, Y., et al, (2002) J. Am. Chem. Soc. 124:14626-14630. Other relevant publications are listed herein.
- the unnatural nucleoside is membrane permeable and is phosphorylated to form the corresponding triphosphate.
- the increased genetic information is stable and not destroyed by cellular enzymes.
- Previous efforts by Benner and others took advantage of hydrogen bonding patterns that are different from those in canonical Watson-Crick pairs, the most noteworthy example of which is the iso-C:iso-G pair. See, e.g., Switzer et al., (1989) J. Am. Chem. Soc, 111:8322; and Piccirilli et al., (1990) Nature. 343:33; Kool, (2000) Curr'. Opin. Chem. Biol., 4:602.
- a PICS:PICS self-pair is found to be more stable than natural base pairs, and can be efficiently incorporated into DNA by Klenow fragment of Escherichia coli DNA polymerase I (KF). See, e.g., McMinn et al., (1999) J. Am. Chem. Soc. 121:11586; and Ogawa et al, (2000) J. Am. Chem. Soc, 122:3274.
- a 3MN:3MN self -pair can be synthesized by KF with efficiency and selectivity sufficient for biological function. See, e.g., Ogawa et al, (2000) J. Am. Chem. Soc. 122:8803. However, both bases act as a chain terminator for further replication.
- a mutant DNA polymerase has been recently evolved that can be used to replicate the PICS self pair.
- a 7AI self pair can be replicated.
- a novel metallobase pair, DipicPy has also been developed, which forms a stable pair upon binding Cu(II). See, Meggers et al, (2000) J. Am. Chem. Soc. 122:10714. Because extended codons and unnatural codons are intrinsically orthogonal to natural codons, the methods of the invention can take advantage of this property to generate orthogonal tRNAs for them.
- a translational bypassing system can also be used to incorporate a selected amino acid, e.g., an unnatural amino acid, in a desired polypeptide.
- a translational bypassing system a large sequence is inserted into a gene but is not translated into protein. The sequence contains a structure that serves as a cue to induce the ribosome to hop over the sequence and resume translation downstream of the insertion.
- a selected amino acid refers to any desired naturally occurring amino acid or unnatural amino acid.
- a naturally occurring amino acid includes any one of the twenty genetically encoded alpha-amino acids: alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine.
- the selected amino acid is incorporated into a growing polypeptide chain with high fidelity, e.g., at greater than 75% efficiency for a given selector codon, at greater than about 80% efficiency for a given selector codon, at greater than about 90% efficiency for a given selector codon, at greater than about 95% efficiency for a given selector codon, or at greater than about 99% or more efficiency for a given selector codon.
- an unnatural amino acid refers to any amino acid, modified amino acid, or amino acid analogue other than selenocysteine and or pyrrolysine and the following twenty genetically encoded alpha-amino acids: alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine.
- the generic structure of an alpha-amino acid is illustrated by Formula I: I
- An unnatural amino acid is typically any structure having Formula I wherein the R group is any substituent other than one used in the twenty natural amino acids. See, e.g., Biochemistry by L. Stryer, 3 rd ed. 1988, Freeman and Company, New York, for structures of the twenty natural amino acids. Note that, the unnatural amino acids of the invention can be naturally occurring compounds other than the twenty alpha-amino acids above.
- the unnatural amino acids of the invention typically differ from the natural amino acids in side chain only, the unnatural amino acids form amide bonds with other amino acids, e.g., natural or unnatural, in the same manner in which they are formed in naturally occurring proteins. However, the unnatural amino acids have side chain groups that distinguish them from the natural amino acids.
- the unnatural amino acids of the invention typically differ from the natural amino acids in side chain, the unnatural amino acids form amide bonds with other amino acids, e.g., natural or unnatural, in the same manner in which they are formed in naturally occurring proteins. However, the unnatural amino acids have side chain groups that distinguish them from the natural amino acids.
- R in Formula I optionally comprises an alkyl-, aryl-, acyl-, keto-, azido-, hydroxyl-, hydrazine, cyano-, halo-, hydrazide, alkenyl, alkynl, ether, thiol, seleno-, sulfonyl-, borate, boronate, phospho, phosphono, phosphine, heterocyclic, enone, inline, aldehyde, ester, thioacid, hydroxylamine, amine, and the like, or any combination thereof.
- the unnatural amino acids have a photoactivatable cross-linker that is used, e.g., to link a protein to a solid support.
- the unnatural amino acids have a saccharide moiety attached to the amino acid side chain.
- unnatural amino acids In addition to unnatural amino acids that contain novel side chains, unnatural amino acids also optionally comprise modified backbone structures, e.g., as illustrated by the structures of Formula II and 1TJ:
- Z typically comprises OH, NH 2 , SH, NH-R', or S-R';
- X and Y which can be the same or different, typically comprise S or O, and R and R', which are optionally the same or different, are typically selected from the same list of constituents for the R group described above for the unnatural amino acids having Formula I as well as hydrogen.
- unnatural amino acids of the invention optionally comprise substitutions in the amino or carboxyl group as illustrated by Formulas II and IH.
- Unnatural amino acids of this type include, but are not limited to, ⁇ -hydroxy acids, -thioacids ⁇ -aminothiocarboxylates, e.g., with side chains corresponding to the common twenty natural amino acids or unnatural side chains.
- substitutions at the -carbon optionally include L, D, or ⁇ - ⁇ - disubstituted amino acids such as D-glutamate, D-alanine, D-methyl-O-tyrosine, aminobutyric acid, and the like.
- Other structural alternatives include cyclic amino acids, such as proline analogues as well as 3,4,6,7,8, and 9 membered ring proline analogues, ⁇ and ⁇ amino acids such as substituted ⁇ -alanine and ⁇ -amino butyric acid.
- many unnatural amino acids are based on natural amino acids, such as tyrosine, glutamine, phenylalanine, and the like.
- Tyrosine analogs include para- substituted tyrosines, ortho-substituted tyrosines, and meta substituted tyrosines, wherein the substituted tyrosine comprises an acetyl group, a benzoyl group, an amino group, a hydrazine, an hydroxyamine, a thiol group, a carboxy group, an isopropyl group, a methyl group, a C 6 - C 20 straight chain or branched hydrocarbon, a saturated or unsaturated hydrocarbon, an O-methyl group, a polyether group, a nitro group, or the like.
- multiply substituted aryl rings are also contemplated.
- Glutamine analogs of the invention include, but are not limited to, -hydroxy derivatives, ⁇ -substituted derivatives, cyclic derivatives, and amide substituted glutamine derivatives.
- Example phenylalanine analogs include, but are not limited to, para-substituted phenylalanines, ortho-substituted phenyalanines, and meta-substituted phenylalanines, wherein the substituent comprises a hydroxy group, a methoxy group, a methyl group, an allyl group, an aldehyde or keto group, or the like.
- unnatural amino acids include, but are not limited to, ap- acetyl-L- phenylalanine, a p-propargyl-phenylalanine, O-methyl -L-tyrosine, an L-3-(2- naphthyl)alanine, a 3-methyl-phenylalanine, an O-4-allyl-L-tyrosine, a 4-propyl-L-tyrosine, a tri-O-acetyl-GlcNAc ⁇ -serine, an L-Dopa, a fluorinated phenylalanine, an isopropyl-L- phenylalanine, a p-azido-L-phenylalanine, ap-acyl-L-phenylalanine, a /?-benzoyl-L- phenylalanine, an L-phosphoserine, a phosphonoserine, a phosphonotyrosine, ap
- Unnatural Amino Acids Many of the unnatural amino acids provided above are commercially available, e.g., from Sigma (USA) or Aldrich (Milwaukee, WI, USA). Those that are not commercially available are optionally synthesized as provided in various publications or using standard methods known to those of skill in the art. For organic synthesis techniques, see, e.g., Organic Chemistry by Fessendon and Fessendon, (1982, Second Edition, Willard Grant Press, Boston Mass.); Advanced Organic Chemistry by March (Third Edition, 1985,
- Unnatural amino acid uptake by a cell is one issue that is typically considered when designing and selecting unnatural amino acids, e.g., for incorporation into a protein. For example, the high charge density of ⁇ -amino acids suggests that these compounds are unlikely to be cell permeable.
- Natural amino acids are taken up into the cell via a collection of protein-based transport systems often displaying varying degrees of amino acid specificity. A rapid screen can be done which assesses which unnatural arnino acids, if any, are taken up by cells. See, e.g., the toxicity assays in, e.g., International Application Number PCT/US03/41346, entitled "Protein Arrays," filed on December 22,
- biosynthetic pathways already exist in cells for the production of amino acids and other compounds. While a biosynthetic method for a particular unnatural amino acid may not exist in nature, e.g., in a cell, the invention provides such methods.
- biosynthetic pathways for unnatural amino acids are optionally generated in host cell by adding new enzymes or modifying existing host cell pathways. Additional new enzymes are optionally naturally occurring enzymes or artificially evolved enzymes.
- the biosynthesis of p-aminophenylalanine relies on the addition of a combination of known enzymes from other organisms.
- the genes for these enzymes can be introduced into a cell by transforming the cell with a plasmid comprising the genes.
- the genes when expressed in the cell, provide an enzymatic pathway to synthesize the desired compound.
- Examples of the types of enzymes that are optionally added are provided in the examples below. Additional enzymes sequences are found, e.g., in Genbank. Artificially evolved enzymes are also optionally added into a cell in the same manner. In this manner, the cellular machinery and resources of a cell are manipulated to produce unnatural amino acids. [0122] Indeed, any of a variety of methods can be used for producing novel enzymes for use in biosynthetic pathways, or for evolution of existing pathways, for the production of unnatural amino acids, in vitro or in vivo.
- DNA shuffling is optionally used to develop novel enzymes and/or pathways of such enzymes for the production of unnatural amino acids (or production of new synthetases), in vitro or in vivo. See, e.g., Stemmer (1994), Rapid evolution of a protein in vitro by DNA shuffling, Nature 370(4):389-391; and, Stemmer, (1994), DNA shuffling by random fragmentation and reassembly: In vitro recombination for molecular evolution, Proc. Natl.
- Another approach uses exponential ensemble mutagenesis to produce libraries of enzyme or other pathway variants that are, e.g., selected for an ability to catalyze a biosynthetic reaction relevant to producing an unnatural amino acid (or a new synthetase).
- small groups of residues in a sequence of interest are randomized in parallel to identify, at each altered position, amino acids which lead to functional proteins. Examples of such procedures, which can be adapted to the present invention to produce new enzymes for the production of unnatural amino acids (or new synthetases) are found in Delegrave & Youvan (1993) Biotechnology Research 11 : 1548-1552.
- random or semi-random mutagenesis using doped or degenerate oligonucleotides for enzyme and/or pathway component engineering can be used, e.g., by using the general mutagenesis methods of e.g., Arkin and Youvan (1992) "Optimizing nucleotide mixtures to encode specific subsets of amino acids for semi-random mutagenesis" Biotechnology 10:297-300; or Reidhaar-Olson et al. (1991) "Random mutagenesis of protein sequences using oligonucleotide cassettes" Methods
- Enzymol. 208:564-86 Yet another approach, often termed a "non-stochastic" mutagenesis, which uses polynucleotide reassembly and site-saturation mutagenesis can be used to produce enzymes and/or pathway components, which can then be screened for an ability to perform one or more synthetase or biosynthetic pathway function (e.g., for the production of unnatural amino acids in vivo). See, e.g., Short "Non-Stochastic Generation of Genetic Vaccines and Enzymes" WO 00/46344.
- An alternative to such mutational methods involves recombining entire genomes of organisms and selecting resulting progeny for particular pathway functions (often referred to as “whole genome shuffling”).
- This approach can be applied to the present invention, e.g., by genomic recombination and selection of an organism (e.g., an E. coli or other cell) for an ability to produce an unnatural amino acid (or intermediate thereof).
- an organism e.g., an E. coli or other cell
- methods taught in the following publications can be applied to pathway design for the evolution of existing and/or new pathways in cells to produce unnatural amino acids in vivo: Patnaik et al. (2002) “Genome shuffling of lactobacillus for improved acid tolerance” Nature Biotechnology. 20(7): 707-712; and Zhang et al. (2002) “Genome shuffling leads to rapid phenotypic improvement in bacteria” Nature, February 7, 415(6872): 644-646.
- the unnatural amino acid produced with an engineered biosynthetic pathway of the invention is produced in a concentration sufficient for efficient protein biosynthesis, e.g., a natural cellular amount, but not to such a degree as to significantly affect the concentration of other cellular amino acids or to exhaust cellular resources.
- concentrations produced in vivo in this manner are about 10 mM to about 0.05 mM.
- nucleic acid polynucleotide sequences and polypeptide amino acid sequences e.g., leucyl O-tRNAs and leucyl O-RSs
- compositions, systems and methods comprising said sequences.
- said sequences e.g., leucyl O-tRNAs and leucyl O-RSs are disclosed herein (see, Table 3, e.g., SEQ RO NO. 1-7, 12-16).
- Table 3 e.g., SEQ RO NO. 1-7, 12-16.
- the invention is not limited to those sequences disclosed herein, e.g., as in the Examples.
- the invention provides many related and unrelated sequences with the functions described herein, e.g., encoding a leucyl O-tRNA or a leucyl O-RS.
- the invention provides polypeptides (leucyl O-RSs) and polynucleotides, e.g., leucyl O-tRNA, polynucleotides that encode leucyl O-RSs or portions thereof, oligonucleotides used to isolate aminoacyl-tRNA synthetase clones, etc.
- Polynucleotides of the invention include those that encode proteins or polypeptides of interest of the invention with one or more selector codon.
- polynucleotides of the invention include, e.g., a polynucleotide comprising a nucleotide sequence as set forth in any one of SEQ ID NO.: 1-2, 4-7 and 12; a polynucleotide that is complementary to or that encodes a polynucleotide sequence thereof.
- a polynucleotide of the invention also includes a polynucleotide that encodes a polypeptide of the invention.
- a nucleic acid that hybridizes to a polynucleotide indicated above under highly stringent conditions over substantially the entire length of the nucleic acid is a polynucleotide of the invention.
- a composition in one embodiment, includes a polypeptide of the invention and an excipient (e.g., buffer, water, pharmaceutically acceptable excipient, etc.).
- excipient e.g., buffer, water, pharmaceutically acceptable excipient, etc.
- the invention also provides an antibody or antisera specifically immunoreactive with a polypeptide of the invention.
- a polynucleotide of the invention also includes a polynucleotide that is, e.g., at least 75%, at least 80%, at least 90%, at least 95%, at least 98% or more identical to that of a naturally occurring leucyl tRNA and comprises an anticodon loop comprising a CU(X) n XXXAA sequence, an stem region lacking noncanonical base pairs and a conserved discriminator base at position 73.
- a polynucleotide also includes a polynucleotide that is , e.g., at least 75%, at least 80%, at least 90%, at least 95%, at least 98% or more identical to that of a naturally occurring leucyl tRNA and comprises an anticodon loop comprising a CUUCCUAA sequence, a first pair selected from T28:A42, G28:C42 and/or C28:G42, and a second pair selected from G:49:C65 or C49:G65, wherein the numbering corresponds to that indicated in Figure 4, Panel A.
- a vector (e.g., a plasmid, a cosmid, a phage, a virus, etc.) comprises a polynucleotide of the invention.
- the vector is an expression vector.
- the expression vector includes a promoter operably linked to one or more of the polynucleotides of the invention.
- a cell comprises a vector that includes a polynucleotide of the invention.
- variants of the disclosed sequences are included in the invention. For example, conservative variations of the disclosed sequences that yield a functionally identical sequence are included in the invention. Variants of the nucleic acid polynucleotide sequences, wherein the variants hybridize to at least one disclosed sequence, are considered to be included in the invention. Unique subsequences of the sequences disclosed herein, as determined by, e.g., standard sequence comparison techniques, are also included in the invention. Conservative variations
- substitutions i.e., substitutions in a nucleic acid sequence which do not result in an alteration in an encoded polypeptide
- conservative amino acid substitutions in one or a few amino acids in an amino acid sequence are substituted with different amino acids with highly similar properties, are also readily identified as being highly similar to a disclosed construct. Such conservative variations of each disclosed sequence are a feature of the present invention.
- Constant variations of a particular nucleic acid sequence refers to those nucleic acids which encode identical or essentially identical amino acid sequences, or, where the nucleic acid does not encode an amino acid sequence, to essentially identical sequences.
- nucleic acid sequences or, where the nucleic acid does not encode an amino acid sequence, to essentially identical sequences.
- substitutions, deletions or additions which alter, add or delete a single amino acid or a small percentage of amino acids (typically less than 5%, more typically less than 4%, 2% or 1%) in an encoded sequence are "conservatively modified variations" where the alterations result in the deletion of an amino acid, addition of an amino acid, or substitution of an amino acid with a chemically similar amino acid.
- “conservative variations” of a listed polypeptide sequence of the present invention include substitutions of a small percentage, typically less than 5%, more typically less than 2% or 1%, of the amino acids of the polypeptide sequence, with a conservatively selected amino acid of the same conservative substitution group.
- substitutions of a small percentage, typically less than 5%, more typically less than 2% or 1%, of the amino acids of the polypeptide sequence with a conservatively selected amino acid of the same conservative substitution group.
- sequences which do not essentially alter the encoded activity of a nucleic acid molecule is a conservative variation of the basic nucleic acid.
- Conservative substitution tables providing functionally similar amino acids are well known in the art.
- Conservative Substitution Groups Nonpolar and/or Polar, Positively Negatively Aromatic Aliphatic Side Uncharged Charged Side Charged Side Side Chains Chains Side Chains Chains Chains Glycine Serine Alanine Threonine Phenylalanine Lysine Valine Cysteine Aspartate Tyrosine Arginine Leucine Methionine Glutamate Tryptophan Histidine Isoleucine Asparagine Proline Glutamine
- Comparative hybridization can be used to identify nucleic acids of the invention, such as SEQ JD NO.: 1-2, 4-7 and 12, including conservative variations of nucleic acids of the invention, and this comparative hybridization method is a preferred method of distinguishing nucleic acids of the invention.
- target nucleic acids which hybridize to the nucleic acids represented by SEQ O NO: 1-2, 4-7 and 12 under high, ultra-high and ultra-ultra high stringency conditions are a feature of the invention.
- nucleic acids examples include those with one or a few silent or conservative nucleic acid substitutions as compared to a given nucleic acid sequence.
- a test nucleic acid is said to specifically hybridize to a probe nucleic acid when it hybridizes at least ⁇ as well to the probe as to the perfectly matched complementary target, i.e., with a signal to noise ratio at least V2 as high as hybridization of the probe to the target under conditions in which the perfectly matched probe binds to the perfectly matched complementary target with a signal to noise ratio that is at least about 5x-10x as high as that observed for hybridization to any of the unmatched target nucleic acids.
- Nucleic acids hybridize due to a variety of well characterized physico-chemical forces, such as hydrogen bonding, solvent exclusion, base stacking and the like.
- An extensive guide to the hybridization of nucleic acids is found in Tijssen (1993) Laboratory Techniques in Biochemistry and Molecular Biology-Hybridization with Nucleic Acid Probes part I chapter 2, “Overview of principles of hybridization and the strategy of nucleic acid probe assays,” (Elsevier, New York), as well as in Ausubel, supra.
- An example of stringent hybridization conditions for hybridization of complementary nucleic acids which have more than 100 complementary residues on a filter in a Southern or northern blot is 50% formalin with 1 mg of heparin at 42°C, with the hybridization being carried out overnight.
- An example of stringent wash conditions is a 0.2x SSC wash at 65°C for 15 minutes (see, Sambrook, supra for a description of SSC buffer). Often the high stringency wash is preceded by a low stringency wash to remove background probe signal.
- An example low stringency wash is 2x SSC at 40°C for 15 minutes. In general, a signal to noise ratio of 5x (or higher) than that observed for an unrelated probe in the particular hybridization assay indicates detection of a specific hybridization.
- Stringent hybridization wash conditions in the context of nucleic acid hybridization experiments such as Southern and northern hybridizations are sequence dependent, and are different under different environmental parameters. An extensive guide to the hybridization of nucleic acids is found in Tijssen (1993), supra, and in Hames and Higgins, 1 and 2. Stringent hybridization and wash conditions can easily be determined empirically for any test nucleic acid. For example, in determining highly stringent hybridization and wash conditions, the hybridization and wash conditions are gradually increased (e.g., by increasing temperature, decreasing salt concentration, increasing detergent concentration and/or increasing the concentration of organic solvents such as formalin in the hybridization or wash), until a selected set of criteria are met. For example, the hybridization and wash conditions are gradually increased until a probe binds to a perfectly matched complementary target with a signal to noise ratio that is at least 5x as high as that observed for hybridization of the probe to an unmatched target.
- the hybridization and wash conditions are gradually increased until a probe binds to a perfectly matched complementary target with a signal to
- “Very stringent” conditions are selected to be equal to the thermal melting point (T m ) for a particular probe.
- T m is the temperature (under defined ionic strength and pH) at which 50% of the test sequence hybridizes to a perfectly matched probe.
- “highly stringent” hybridization and wash conditions are selected to be about 5° C lower than the T m for the specific sequence at a defined ionic strength and pH.
- Ultra high-stringency hybridization and wash conditions are those in which the stringency of hybridization and wash conditions are increased until the signal to noise ratio for binding of the probe to the perfectly matched complementary target nucleic acid is at least lOx as high as that observed for hybridization to any of the unmatched target nucleic acids.
- a target nucleic acid which hybridizes to a probe under such conditions, with a signal to noise ratio of at least V ⁇ that of the perfectly matched complementary target nucleic acid is said to bind to the probe under ultra-high stringency conditions.
- even higher levels of stringency can be determined by gradually increasing the hybridization and/or wash conditions of the relevant hybridization assay. For example, those in which the stringency of hybridization and wash conditions are increased until the signal to noise ratio for binding of the probe to the perfectly matched complementary target nucleic acid is at least lOx, 20X, 50X, 100X, or 500X or more as high as that observed for hybridization to any of the unmatched target nucleic acids.
- a target nucleic acid which hybridizes to a probe under such conditions, with a signal to noise ratio of at least l that of the perfectly matched complementary target nucleic acid is said to bind to the probe under ultra-ultra-high stringency conditions.
- nucleic acids which do not hybridize to each other under stringent conditions are still substantially identical if the polypeptides which they encode are substantially identical. This occurs, e.g., when a copy of a nucleic acid is created using the maximum codon degeneracy permitted by the genetic code.
- Unique subsequences [0143] In one aspect, the invention provides a nucleic acid that comprises a unique subsequence in a nucleic acid selected from the sequences of leucyl O-tRNAs and leucyl O-
- the unique subsequence is unique as compared to a nucleic acid corresponding to any known leucyl O-tRNA or leucyl O-RS nucleic acid sequence.
- Alignment can be performed using, e.g., BLAST set to default parameters. Any unique subsequence is useful, e.g., as a probe to identify the nucleic acids of the invention.
- the invention includes a polypeptide which comprises a unique subsequence in a polypeptide selected from the sequences of leucyl O-RSs disclosed herein.
- the unique subsequence is unique as compared to a polypeptide corresponding to any of known polypeptide sequence.
- the invention also provides for target nucleic acids which hybridizes under stringent conditions to a unique coding oligonucleotide which encodes a unique subsequence in a polypeptide selected from the sequences of leucyl O-RSs wherein the unique subsequence is unique as compared to a polypeptide corresponding to any of the control polypeptides (e.g., parental sequences from which synthetases of the invention were derived, e.g., by mutation). Unique sequences are determined as noted above.
- nucleic acid or polypeptide sequences refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same, when compared and aligned for maximum correspondence, as measured using one of the sequence comparison algorithms described below (or other algorithms available to persons of skill) or by visual inspection.
- nucleic acids or polypeptides refers to two or more sequences or subsequences that have at least about 60%, about 80%, about 90-95%, about 98%, about 99% or more nucleotide or amino acid residue identity, when compared and aligned for maximum correspondence, as measured using a sequence comparison algorithm or by visual inspection.
- substantially identical sequences are typically considered to be “homologous,” without reference to actual ancestry.
- the "substantial identity” exists over a region of the sequences that is at least about 50 residues in length, more preferably over a region of at least about 100 residues, and most preferably, the sequences are substantially identical over at least about 150 residues, or over the full length of the two sequences to be compared.
- sequence comparison and homology determination typically one sequence acts as a reference sequence to which test sequences are compared.
- test and reference sequences are input into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated.
- sequence comparison algorithm then calculates the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters.
- Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson & Lipman, Proc. Nat'L Acad. Sci. USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or by visual inspection (see generally, Ausubel et al, infra).
- HSPs high scoring sequence pairs
- initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them.
- the word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always > 0) and N (penalty score for mismatching residues; always ⁇ 0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached.
- the BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment.
- the BLASTP program uses as defaults a wordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff (1989) Proc Natl. Acad. Sci. USA 89:10915).
- the BLAST algorithm In addition to calculating percent sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul, Proc. Nafl. Acad. Sci. USA 90:5873-5787 (1993)).
- One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance.
- a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0J, more preferably less than about 0.01, and most preferably less than about 0.001.
- Polynucleotide and polypeptides of the invention and used in the invention can be manipulated using molecular biological techniques.
- molecular biological techniques include Berger and Kimmel, Guide to Molecular Cloning
- mutagenesis e.g., to mutate tRNA molecules, to produce libraries of leucyl tRNAs, to produce libraries of leucyl synthetases, to insert selector codons that encode a selected amino acid in a protein or polypeptide of interest.
- mutagenesis include but are not limited to site-directed, random point mutagenesis, homologous recombination, DNA shuffling or other recursive mutagenesis methods, chimeric construction, mutagenesis using uracil containing templates, oligonucleotide- directed mutagenesis, phosphorothioate-modified DNA mutagenesis, mutagenesis using gapped duplex DNA or the like, or any combination thereof.
- Additional suitable methods include point mismatch repair, mutagenesis using repair-deficient host strains, restriction- selection and restriction-purification, deletion mutagenesis, mutagenesis by total gene synthesis, double-strand break repair, and the like.
- Mutagenesis e.g., involving chimeric constructs, is also included in the present invention.
- mutagenesis can be guided by known information of the naturally occurring molecule or altered or mutated naturally occurring molecule, e.g., sequence, sequence comparisons, physical properties, crystal structure or the like.
- Host cells are genetically engineered (e.g., transformed, transduced or transfected) with the polynucleotides of the invention or constructs which include a polynucleotide of the invention, e.g., a vector of the invention, which can be, for example, a cloning vector or an expression vector.
- a vector of the invention which can be, for example, a cloning vector or an expression vector.
- the coding regions for the orthogonal tRNA, the orthogonal tRNA synthetase, and the protein to be derivatized are operably linked to gene expression control elements that are functional in the desired host cell.
- Typical vectors contain transcription and translation terminators, transcription and translation initiation sequences, and promoters useful for regulation of the expression of the particular target nucleic acid.
- the vectors optionally comprise generic expression cassettes containing at least one independent terminator sequence, sequences permitting replication of the cassette in eukaryotes, or prokaryotes, or both (e.g., shuttle vectors) and selection markers for both prokaryotic and eukaryotic systems.
- Vectors are suitable for replication and/or integration in prokaryotes, eukaryotes, or preferably both. See, Giliman & Smith, Gene 8:81 (1979); Roberts, et al, Nature, 328:731 (1987); Schneider, B., et al, Protein Expr. Purifi 6435:10 (1995); Ausubel, Sambrook, Berger (all supra).
- the vector can be, for example, in the form of a plasmid, a bacterium, a virus, a naked polynucleotide, or a conjugated polynucleotide.
- the vectors are introduced into cells and/or microorganisms by standard methods including electroporation (From et al., Proc. Natl. Acad. Sci. USA 82, 5824 (1985), infection by viral vectors, high velocity ballistic penetration by small particles with the nucleic acid either within the matrix of small beads or particles, or on the surface (Klein et al., Nature 327. 70-73 (1987)), and/or the like.
- a catalogue of Bacteria and Bacteriophages useful for cloning is provided, e.g., by the ATCC, e.g., The ATCC Catalogue of Bacteria and Bacteriophage (1992) Gherna et al. (eds) published by the ATCC. Additional basic procedures for sequencing, cloning and other aspects of molecular biology and underlying theoretical considerations are also found in Watson et al. (1992) Recombinant DNA Second Edition Scientific American Books, NY.
- nucleic acid and virtually any labeled nucleic acid, whether standard or non-standard
- the engineered host cells can be cultured in conventional nutrient media modified as appropriate for such activities as, for example, screening steps, activating promoters or selecting transformants. These cells can optionally be cultured into transgenic organisms.
- Other useful references, e.g. for cell isolation and culture include Freshney (1994) Culture of Animal Cells, a Manual of Basic Technique, third edition, Wiley- Liss, New York and the references cited therein; Payne et al (1992) Plant Cell and Tissue Culture in Liquid Systems John Wiley & Sons, Inc.
- a method includes growing, in an appropriate medium, the cell, where the cell comprises a nucleic acid that comprises at least one selector codon and encodes a protein; and, providing the selected amino acid; where the cell further comprises: an orthogonal leucyl-tRNA (leucyl-O-tRNA) that functions in the cell and recognizes the selector codon; and, an orthogonal aminoacyl- tRNA synthetase (O-RS) that preferentially aminoacylates the leucyl-O-tRNA with the selected amino acid.
- an orthogonal leucyl-tRNA leucyl-O-tRNA
- O-RS orthogonal aminoacyl- tRNA synthetase
- the leucyl-O-tRNA comprises at least about a 25%, 50%, 75%, 80%, 85%, 90%, 95% or 98% suppression activity in the presence of a cognate synthetase in response to a selector codon as compared to a control lacking the selector codon (and, typically, the cognate synthetase).
- a protein produced by this method is also a feature of the invention.
- the invention also teaches variant orthogonal leucyl-tRNAs and variant orthogonal aminoacyl-tRNA synthetase species that display suppression activity, where the suppression activity is measured relative to the suppression activity of a leucyl-O-tRNA nucleotide sequence or an O-RS amino acid sequence provided by the present invention.
- the invention teaches variant leucyl-O-tRNA species that display suppression activity that is at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% as effective as a leucyl-O-tRNA sequence provided by the examples herein (e.g., SEQ RO NOs: 1-7 and 12).
- the invention teaches variant O-RS species that display suppression activity that is at least 50%, 60%, 70%, 75%, 80%, 85%, 90%), 95%, 98% or 99% as effective as an O-RS sequence provided by the examples herein (e.g., SEQ RO NO: 15 and 16).
- the invention teaches variant orthogonal leucyl-tRNAs and variant orthogonal aminoacyl-tRNA synthetase species that display suppression activity that is equal to or greater than the suppression activity of a leucyl-O-tRNA nucleotide sequence or an O-RS amino acid sequence provided by the present specification.
- the invention teaches variant leucyl-O-tRNA species that display suppression activity that is at least 100% as effective as a leucyl-O-tRNA sequence provided by the examples herein (e.g., SEQ ID NO: 1-7 and 12).
- compositions of the invention and compositions made by the methods of the invention optionally are in a cell.
- the leucyl O-tRNA/O-RS pairs or individual components of the invention can then be used in a host system's translation machinery, which results in a selected amino acid, e.g., unnatural amino acid, being incorporated into a protein.
- the International Application Number PCT/US2004/011786, filed April 16, 2004, and WO 2002/085923, entitled “IN VIVO INCORPORATION OF UNNATURAL AMINO AC OS” describe this process, and is incorporated herein by reference.
- compositions of the present invention can be in an in vitro translation system, or in an in vivo system(s).
- any protein (or portion thereof) that includes a selected amino acid, e.g., an unnatural amino acid, (and any corresponding coding nucleic acid, e.g., which includes one or more selector codons) can be produced using the compositions and methods herein. No attempt is made to identify the hundreds of thousands of known proteins, any of which can be modified to include one or more unnatural amino acid, e.g., by tailoring any available mutation methods to include one or more appropriate selector codon in a relevant translation system. Common sequence repositories for known proteins include GenBank EMBL, DDB J and the NCBI. Other repositories can easily be identified by searching the internet.
- the proteins are, e.g., at least 60%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, or at least 99% or more identical to any available protein (e.g., a therapeutic protein, a diagnostic protein, an industrial enzyme, or portion thereof, and the like), and they comprise one or more selected amino acid.
- a therapeutic protein e.g., a diagnostic protein, an industrial enzyme, or portion thereof, and the like
- examples of therapeutic, diagnostic, and other proteins that can be modified to comprise one or more selected amino acid, e.g., an unnatural amino acid can be found, but not limited to, those in International Application Number PCT/US2004/011786, filed April 16, 2004, and WO 2002/085923, entitled "IN VIVO INCORPORATION OF UNNATURAL AMI-NO ACROS.”
- the protein or polypeptide of interest (or portion thereof) in the methods and/or compositions of the invention is encoded by a nucleic acid.
- the nucleic acid comprises at least one selector codon, at least two selector codons, at least three selector codons, at least four selector codons, at least five selector codons, at least six selector codons, at least seven selector codons, at least eight selector codons, at least nine selector codons, ten or more selector codons.
- Genes coding for proteins or polypeptides of interest can be mutagenized using methods well-known to one of skill in the art and described herein under "Mutagenesis and Other Molecular Biology Techniques" to include, e.g., one or more selector codon for the incorporation of a selected amino acid, e.g., an unnatural amino acid.
- a nucleic acid for a protein of interest is mutagenized to include one or more selector codon, providing for the insertion of the one or more selected amino acids, e.g., unnatural amino acids.
- the invention includes any such variant, e.g., mutant, versions of any protein, e.g., including at least one selected amino acid.
- the invention also includes corresponding nucleic acids, i.e., any nucleic acid with one or more selector codon that encodes one or more selected amino acid.
- Host cells are genetically engineered (e.g., transformed, transduced or transfected) with one or more vectors that express the orthogonal leucyl tRNA, the orthogonal leucyl tRNA synthetase, and a vector that encodes the protein to be derivatized.
- Each of these components can be on the same vector, or each can be on a separate vector, two components can be on one vector and the third component on a second vector.
- the vector can be, for example, in the form of a plasmid, a bacterium, a virus, a naked polynucleotide, or a conjugated polynucleotide.
- polypeptides of the invention provide a variety of new polypeptide sequences (e.g., comprising selected amino acids (e.g., unnatural amino acids) in the case of proteins synthesized in the translation systems herein, or, e.g., in the case of the novel synthetases, novel sequences of standard amino acids), the polypeptides also provide new structural features which can be recognized, e.g., in immunological assays.
- selected amino acids e.g., unnatural amino acids
- antisera which specifically bind the polypeptides of the invention, as well as the polypeptides which are bound by such antisera, are a feature of the invention.
- antibody includes, but is not limited to a polypeptide substantially encoded by an immunoglobulin gene or immunoglobulin genes, or fragments thereof which specifically bind and recognize an analyte (antigen). Examples include polyclonal, monoclonal, chimeric, and single chain antibodies, and the like. Fragments of immunoglobulins, including Fab fragments and fragments produced by an expression library, including phage display, are also included in the term “antibody” as used herein. See, e.g., Paul, Fundamental Immunology, 4th Ed., 1999, Raven Press, New York, for antibody structure and terminology.
- one or more of the immunogenic polypeptides is produced and purified as described herein.
- recombinant protein can be produced in a recombinant cell.
- An inbred strain of mice (used in this assay because results are more reproducible due to the virtual genetic identity of the mice) is immunized with the immunogenic protein(s) in combination with a standard adjuvant, such as Freund's adjuvant, and a standard mouse immunization protocol (see, e.g., Harlow and Lane (1988) Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, New York, for a standard description of antibody generation, immunoassay formats and conditions that can be used to determine specific immunoreactivity.
- a standard adjuvant such as Freund's adjuvant
- a standard mouse immunization protocol see, e.g., Harlow and Lane (1988) Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, New York, for a standard description of antibody generation, immunoassay formats and conditions that can be used
- Kits are also a feature of the invention.
- a kit for producing a protein that comprises at least one selected amino acid, e.g., an unnatural amino acid, in a cell is provided, where the kit includes a container containing a polynucleotide sequence encoding an leucyl O-tRNA, and/or an leucyl O-tRNA, and/or a polynucleotide sequence encoding an leucyl O-RS, and/or an leucyl O-RS.
- the kit further includes at least selected amino acid.
- the kit further comprises instructional materials for producing the protein.
- EXAMPLE 1 ADAPTATION OF AN ORTHOGONAL ARCHAEAL LEUCYL-TRNA AND SYNTHETASE PAIR FOR FOUR-BASE. AMBER. AND OPAL SUPPRESSION
- an amber suppressor tRNA-aminoacyl tRNA synthetase pair derived from the tyrosyl-tRNA synthetase of Methanococcus jannaschii can be used to genetically encode unnatural amino acids in response to the amber nonsense codon, TAG.
- This pair is unable to decode either the opal nonsense codon, TGA, or the four-base codon, AGGA.
- NRC-1 was adapted as an orthogonal tRNA-synthetase pair in E. coli to decode amber (TAG), opal (TGA) and four-base (AGGA) codons.
- TAG decode amber
- TGA opal
- AGGA four-base codons.
- the synthetase should not cross-react with any of the endogenous tRNAs (40 in E. coli), and the orthogonal tRNA should not be aminoacylated by any of the endogenous synthetases (21 in E. coli).
- the tRNA should decode only a specific new codon that is not decoded by any endogenous tRNA, and the synthetase should charge its tRNA with only a specific unnatural amino acid.
- This system has been used to incorporate a series of unnatural amino acids including keto amino acids (Wang et al., (2003) Proc Natl. Acad. Sci. U. S. A. 100, 56-61), photocrosslinking amino acids (Chin et al., (2002) Proc Natl. Acad. Sci. U. S. A. 99, 11020-11024; Chin et al, (2002) J. Am. Chem. So 124, 9026-9027), and heavy atom containing amino acids selectively into proteins in response to the TAG codon. [0175] Several other orthogonal pairs have been reported. Glutaminyl (Liu and Schultz (1999) Proc Natl. Acad. Sci. U. S. A.
- a desired property of any orthogonal pair are a codon that is unique within the genetic code and that will not cross-react with noncognate tRNAs.
- TAG amber stop codon
- TGA opal nonsense codon
- UAA ochre nonsense codon
- Unnatural amino acids have been incorporated in response to novel codons containing the unnatural base (iso-dC)AG (Piccirilli et al., (1990) Nature 343, 33- 37) or pyridin-2-one (Hirao et al., (2002) Nat. Biotechnol. 20, 177-182) using an in vitro translation system.
- Adaptation of unnatural base pairs for the incorporation of unnatural amino acids into proteins in vivo need the faithful replication and transcription of unnatural base pairs in DNA and RNA (Wu et al., (2002) J. Am. Chem. Soc 124, 14626-14630).
- Another codon that can used to encode additional amino acids are four- and five-base codons.
- tRNA anticodon loop is a major identity element for recognition by most synthetases, one must identify a synthetase that does not recognize these identity elements in order to generate suppressor tRNAs for these unusual codons.
- the leucyl-, seryl-, and alanyl-tRNA synthetases of E. coli are well known to tolerate extensive substitutions in the anticodon loop (Shimizu et al., (1992) J. Mol. ⁇ vol. 35, 436-443; Kleina (1990) J. Mol. Biol. 213, 705-717; Sampson and Saks (1993) Nucleic Acids Res. 21, 4467-4475).
- Some homologous archaeal or eukaryotic synthetases may have similar properties.
- NRC-1 that act as orthogonal tRNA- synthetase pairs for the amber codon in E. coli.
- information gained in these studies, together with multiple sequence alignments of native archaeal tRNA sequences allowed us to design efficient orthogonal suppressor tRNAs of opal codons and a four-base codon, AGGA.
- Genomic DNA was either purchased from ATCC or was prepared from a cell pellet purchased from ATCC. Genomic DNA was extracted using the DNeasy kit (Qiagen). Synthetase genes were amplified from genomic DNA by PCR then subcloned into the Ncol and either EcoRI, Kpnl, or PvuH sites of plasmid pKQ. More details on the cloning of these genes can be found Table 2 and is also available on the Internet at http://pubs.acs.org.
- Plasmid pKQ contains the ribosome binding site, multiple cloning site, and rniB terminator from plasmid pB AD-Myc/HisA (Invitrogen) under control of a constitutive glutamine promoter.
- the plasmid also contains a Col ⁇ l origin of replication, and a kanamycin resistance gene for plasmid maintenance.
- Beta-lactamase reporter plasmids were constructed from plasmid pACKO-Bla. This plasmid was constructed with a pl5a origin, a chloramphenicol resistance gene, and unique sites for insertion of a gene for ⁇ -lactamase and a tR ⁇ A under control of the strong, constitutive Ipp promoter.
- Site A184 of the ⁇ - lactamase gene was changed to TAG, AGGA, or TGA by an overlap PCR strategy, and the genes were subcloned into the Aatll and Xmal sites of pACKO-Bla to give plasmids pACKO-A184TAG, pACKO-A184AGGA, and pACKO-A184TGA.
- Constructions of tRNA plasmids Genes for individual tRNAs and for tRNA libraries were constructed by extension reactions and subcloned into the EcoRI and Pstl sites of pACKO-Bla derivatives. All libraries represented at least 10-fold more members than the theoretical size of the library to ensure high coverage. [0182] Measurement of suppression efficiency.
- a series of LB agar plates were prepared with 25 ⁇ g/mL of kanamycin, 25 ⁇ g/mL of chloramphenicol, and concentrations of ampicillin between 5 and 1000 ⁇ g/mL. Synthetase and tRNA plasmids were cotransformed and plated at densities below 100 cells per plate. Suppression efficiency was reported as the highest concentration at which cells survived to form colonies among a series of plates for which the next highest and lowest concentrations would be within 20% of the reported value.
- Beta-galactosidase reporter assays The full-length lacZ gene of plasmid pBAD-Myc/His/lacZ (Invitrogen) was amplified by PCR and subcloned into plasmid pLASC to obtain plasmid pLASC-lacZ.
- This pSC 101 -derived plasmid expresses lacZ gene under the control of an Ipp promoter and has an ampicillin resistance gene for plasmid maintenance.
- Percent suppression was calculated as the percentage of activity for a sample relative to the value observed from the pLASC-lacZ construct with the corresponding sense codon at position 25.
- Cells containing pLASC-lacZ plasmids with sense codons at position 25 were also assayed by 2-nitrophenyl- ⁇ -D-galactopyranoside assays (Miller (1972) Experiments in molecular genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.), and activity was calculated in Miller units.
- Purification of synthetase proteins Synthetase genes were cloned in frame with the C-terminal myc/his tag of pB AD-Myc/HisA (Invitrogen).
- Aeropyrum pernix K1 BspHI/EcoRI (subcloned into Ncol/EcoRI sites)
- Pyrococcus horikoshii Ncol/EcoRV (subcloned into Ncol/Pvull sites)
- Methanobacterium thennoautotrophicum BsmBI (subcloned into Ncol/EcoRI sites)
- Plasmids derived from pACKO-Bla can also express tRNA genes under the control of a strong Ipp promoter.
- pACKO-A184TAG which encodes the A184TAG variant of bid
- host bacteria survive at an ampicillin concentration of 1000 ⁇ g/mL.
- ampicillin resistance should be less than 5 ug/mL.
- the tRNA is not orthogonal, or if a heterologous synthetase capable of charging the tRNA is co-expressed in the system, a higher level of ampicillin resistance should be observed.
- NRC-1 HhLRS
- Methanococcus jannaschii MjLRS
- Methanobacterium thennoautotrophicum MtLRS
- Pyrococcus horikoshi PhLRS
- the genes for these synthetases were cloned under the control of a constitutive glutamine promoter on the high copy plasmid, pKQ, which was constructed from pBR322 and contains a kanamycin resistance gene.
- the leuS gene from E. coli ( ⁇ cLRS) was also cloned as a negative control.
- An in vivo suppression screen can distinguish active and inactive aminoacyl-tRNA synthetases, but it cannot distinguish an orthogonal synthetase from one that cross-reacts with E. coli tRNA.
- the synthetases were overexpressed, purified, and then subjected to in vitro aminoacylation assays to measure their ability to charge E. coli tRNA.
- AfLRS, MjLRS, and MtLRS were purified from an arabinose promoter over-expression system by Ni-NTA affinity chromatography in yields of 14, 8, and 3 mg/L respectively.
- In vitro aminoacylation assays were performed with tritium- labeled leucine and either E. coli or Halobacterium NRC-1 total tRNA ( Figure 3, Panels A and B). Based on the charging of 10 ⁇ M crude total tRNA, MtLRS and AfLRS charge halobacterial tRNA 54- and 21- fold more efficiently than E. coli tRNA, respectively.
- the MjLRS enzyme shows only a 6-fold preference for halobacterial tRNA.
- the E. coli enzyme was 100-fold more efficient at charging E.
- MtLRS and AfLRS are good candidates for orthogonal aminoacyl-tRNA synthetases with respect to E. coli tRNA, but MjLRS is not. Since
- MtLRS showed a higher level of suppression with HL(TAG)1 in vivo than did AfLRS, the MtLRS/HL(TAG)l pair was carried forward as a potential new orthogonal pair for use in E. coli.
- HhL4 has a G at position 37, therefore substitution of G37 to A might be expected to improve suppression efficiency.
- a library was constructed in which the 7 positions of the anticodon loop (positions 32-38, see Figure 4, Panel A) in HhL4 were replaced with degenerate bases and subcloned into pACKO-A184TAG.
- the library of tRNAs was cotransformed with pKQ-MtLRS and subjected to ampicillin selection initially at 35 ⁇ g/mL ampicillin for two rounds of selection, then plated on a series of plates with increasing ampicillin concentration in the third round of selection.
- ⁇ -Galactosidase activity was determined for tRNA reporter plasmids derived from pACKO-Bla cotransformed with the appropriate pLASC-lacZ mutant and either a synthetase-expressing plasmid or a plasmid with no synthetase. Activity is reported as the percentage of activity observed relative to the value observed from the pLASC-lacZ construct with a leucyl (wild-type), seryl, or tyrosyl sense codon at position 25. In each case, the codon at position 25 of lacZ is designated in parentheses. J17, the M.
- jannaschii tyrosyl amber suppressor tRNA with improved orthogonality was expressed in plasmid pACKO-A184TAG in the presence of pLASC- lacZ(TAG) and either pKQ or pBK-JYRS.
- a library in which the 3 terminal base pairs of the acceptor stem and the discriminator base were randomized was constructed from the HL(TAG)2 mutant tRNA and subcloned into pACKO- A184TAG. [0195] To identify members of this tRNA library that retained activity but were even poorer substrates for endogenous synthetases, a selection strategy was adopted from previous work on the M. jannaschii system (Wang and Schultz (2001) Chem. Biol. 8, 883- 890).
- the tRNA library in which the acceptor stem was randomized was cotransformed with pKQ-MtLRS and subjected to two rounds of positive selection at 500 ⁇ g/mL ampicillin. Six clones surviving the positive selection were sequenced, and all were unique and conserved the discriminator base, A73 ( Figure 4, Panels A and B). In all cases the stem positions had standard Watson-Crick base pairs.
- tRNA-expressing plasmids were transferred into cells containing a barnase reporter plasmid, pSCB2.
- This plasmid contains the gene for the RNase, barnase, with two TAG codons at permissive positions 2 and 44, under control of the arabinose promoter, as well as the gene for ⁇ -lactamase. Any tRNA that is aminoacylated by an endogenous E. coli synthetase will result in suppression of the nonsense codons and cell death.
- the cells were plated on LB plates containing 25 ⁇ g/mL of chloramphenicol,
- mutant HL(TAG)3 gave the highest level of suppression in the presence of MtLRS (600 ⁇ g/mL ampicillin) and only survived to 7.5 ⁇ g/mL ampicillin without the synthetase. These values correspond to 33.2% suppression in the presence of MtLRS and 1.5% in the absence of the synthetase as determined by ⁇ -galactosidase amber suppression assays (see Table 1).
- the mutant M. jannaschii suppressor tRNA, J17 gives values of 18.5% and 0.2% with and without the M. jannaschii tyrosine synthetase, respectively.
- Identification of AGGA suppressors To expand the list of codons that can be used for unnatural amino acid mutagenesis, a tRNA that could efficiently suppress a four-base codon was sought. Previous studies indicated that the four-base codon AGGA can be efficiently suppressed in E. coli, and tRNAs with 8 nucleotide anticodon loops were the most efficient suppressors of AGGA codons (Magliery et al., (2001) J. Mol. Biol. 307, 755-769).
- a ⁇ -lactamase reporter plasmid analogous to the TAG reporter was constructed but with A 184 replaced by AGGA instead of TAG. Normal translation in the absence of a +1 frameshift suppressor tRNA should result in missense errors downstream of position 184 and premature truncation of the protein.
- a library of tRNAs derived from the HhL4 tRNA was constructed in which the 7 nucleotide anticodon loop was replaced with 8 random nucleotides. The library was subcloned into pACKO-A184AGGA, cotransformed with pKQ-MtLRS, and then subjected to ampicillin selection.
- HL(AGGA)1 At the highest concentration of ampicillin at which growth was observed, 75 ⁇ g/mL, only one clone, HL(AGGA)1, was found. This clone had the anticodon loop sequence CUUCCUAA. As was the case with the bla A184TAG reporter plasmid, cells transformed with pACKO-A 184 AGGA can survive to only 5 ⁇ g mL ampicillin in the absence of a suppressor tRNA. Therefore, the clone identified, HL(AGGA)1, is a weak suppressor of AGGA codons. [0197] During these experiments, serendipitous mutants capable of surviving up to
- the anticodon loop was changed to CUUCCUAA since this sequence was already shown to be the optimal sequence for an AGGA suppressor derived from HhL4.
- the final sequence used as the consensus sequence is shown in Figure 5.
- a library was synthesized by overlap extension of oligonucleotides in which each site of the tRNA gene was synthesized as a doped mixture of 90% the consensus sequence and 10% a mixture of the other 3 bases.
- the library was subcloned into pACKO-A 184 AGGA. Sequencing of 24 naive clones revealed that the average number of mutations per clone was 5.9, and these mutations were randomly distributed throughout the tRNA sequence.
- the most efficient suppressor designated HL(AGGA)3, can survive to 300 ⁇ g/mL ampicillin in the presence of pKQ-MtLRS but to only 30 ⁇ g/mL in the absence of the synthetase, which correspond to 35.5% and 7.4% suppression, respectively, as determined by ⁇ -galactosidase assays (Table 1).
- HhL4 a library was prepared in which the 8 nucleotide anticodon loop was randomized with 7 nucleotides in HL(AGGA)3, the most robust AGGA suppressor identified from the consensus sequence.
- the 8 nucleotide anticodon loop was randomized with 7 nucleotides in HL(AGGA)3, the most robust AGGA suppressor identified from the consensus sequence.
- HL(TGA)1 At the highest concentrations of ampicillin at which growth was observed (300 ⁇ g/mL) only one clone, designated HL(TGA)1, with the sequence CUUCAAA was found.
- the clone can survive to 350 ⁇ g/mL ampicillin when coexpressed with pKQ-MtLRS, but can survive to only 30 ⁇ g/mL without the synthetase plasmid, which corresponds to 60.8% suppression as determined by ⁇ -galactosidase assays (Table 1).
- the beneficial effects of using the consensus sequence are not limited
- orthogonal tRNA-synthetase pairs are designed to adapt eukaryotic or archaeal synthetases and tRNAs for use in E. coli.
- yeast synthetases notably glutamine, aspartic acid, arginine, and tyrosine, have been shown not to recognize E. coli tRNA, and might therefore be useful for the construction of orthogonal tRNA-synthetase pairs..
- many eukaryotic synthetases express poorly or have low specific activity in E. coli.
- Eukaryotic synthetases particularly the mammalian enzymes, are often organized into large complexes (Mirande et al., (1982) EMBO J. 1, 733-736), and the low activity often observed may be related to the inability to form these complexes in E. coli.
- archaeal tRNA synthetases are more similar to their eukaryotic than prokaryotic counterparts in terms of homology and tRNA recognition elements. Unlike their eukaryotic counterparts, however, there is currently no evidence for their higher order assembly into structured multimers (Tumbula et al., (1999) Genetics 152. 1269-1276; Woese et al, (2000) Microbiol. Mol. Biol. Rev. 54, 202-236). Moreover, since most archaea are thermophiles, active synthetases from archaea can be expressed in good yields in E. coli and can be readily purified in active form.
- Another design issue in the construction of orthogonal tRNA-synthetase pairs is the ability of the aminoacyl-tRNA synthetase to recognize mutants of the cognate tRNA with altered anticodon loops (i.e., nonsense or missense suppressors).
- Aminoacyl- tRNA synthetases frequently use the anticodon loop as a major positive identity element, and mutations in this region of the tRNA frequently result in impaired synthetase recognition.
- the leucyl-tRNA synthetases frequently lack strong anticodon recognition elements, and a leucyl orthogonal tRNA-synthetase pair can therefore be able to decode a variety of codons, including amber, opal and four-base codons.
- a leucyl orthogonal tRNA-synthetase pair can therefore be able to decode a variety of codons, including amber, opal and four-base codons.
- the enzyme from Haloferax volcanii has been thoroughly investigated (Soma et al, (1999) J. Mol. Biol. 293, 1029-1038).
- the synthetase does not recognize bases in the anticodon loop; instead, a highly conserved pattern of mismatches within the variable loop is the primary recognition element for the synthetase.
- the MjL2 and PfL5 suppressors fail to give an enhancement in suppression when coexpressed with a cognate or noncognate archaeal synthetase.
- these tRNAs may be expressed as functional suppressor tRNAs in E. coli but are unable to be charged due to incompatibility with both cognate and noncognate synthetases.
- the suppressor is derived from the natural substrate for MjLRS, so it seems unlikely that the tRNA would not be charged, when other tRNAs are efficiently charged by MjLRS. Another explanation might be that the tRNAs are efficiently charged but are incompatible with the E.
- tyrosine system gives levels of amber suppression comparable to the levels observed for strong native amber suppressors such as supD or supF.
- the reporter plasmid for this study was a medium-copy plasmid
- cells containing the original glutamine, aspartate, and tyrosine orthogonal amber suppressor tRNAs can survive to 140, 60, and 1220 ug/mL ampicillin, respectively (Pastrnak et al., (2000) Helv. Chim. Acta 83, 2277- 2286; Wang et al., (2000) J. Am.
- a high level of suppression may be critical to the successful modification of the amino acid specificity of synthetases using a double-sieve selection strategy (Liu and Schultz (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 4780-4785).
- a high level of suppression is required for the production of protein containing unnatural amino acids. Therefore, a great deal of attention has been paid to those features of orthogonal tRNAs that give rise to robust suppression.
- the anticodon loop sequence CUCUAAA afforded the highest suppression efficiency corresponding to six-fold and five-fold enhancements in the concentration of ampicillin at which growth is observed for the glutamine and aspartate systems, respectively.
- tRNAs with the anticodon loop sequence CUCUAAA have emerged as the most efficient amber suppressors.
- the anticodon loop sequence CUUCCUAA was found to be the most efficient sequence for a leucyl AGGA suppressor. Selection experiments on tRNAs with randomized anticodon loops in E. coli tRNA, er similarly converged on the anticodon loop sequence CUUCCUAA for AGGA suppression (Magliery et al, (2001) J. Mol. Biol.
- the most efficient suppressor tRNAs had bases at positions 32, 33, 37, and 38 which differed from the consensus sequence.
- the most efficient suppressors of the codon CUAG had an anticodon loop with the sequence CGCTAGGA, deviating at both U33 and A37.
- some synthetases employ position 37 as a strong positive determinant for recognition, in which case a CU(X)XXXAA anticodon loop sequence can prove to be non-optimal.
- This library of tRNAs would therefore contain all combinations of mismatched and Watson-Crick base pairs. In fact, 98.4% of the theoretical members of this library should have at least one mismatched base pair. Nevertheless, in the 9 active acceptor stem mutants outlined in Figure 4, Panel B, all positions are occupied by Watson-Crick base pairs. Similarly, the D, T ⁇ C, anticodon, and acceptor stems of the yeast glutamine amber suppressor tRNA have been individually randomized and subjected to positive selection (J.C.A. and P.G.S., unpublished results). In all surviving clones, every position in these stem regions was occupied by a Watson-Crick pair. In the parent tRNA, the 6:67 base pair is U:G.
- tRNAs with mispaired bases are not readily folded into the correct cloverleaf structure and therefore are not readily processed and modified (Furdon et al., (1983) Nucleic Acids Res. 11, 1491-1505).
- a quantitative analysis of the ratio of charged to uncharged species and of the ratio of fully processed to unprocessed tRNA present in the cell could enhance our understanding of the mechanisms by which these poorly-suppressing tRNAs are impaired.
- An analysis of multiple sequence alignments of many families of tRNAs reveal multiple examples of conserved non-Watson-Crick pairings. For example, a G3:U70 base pair is a conserved positive determinant for recognition by E.
- these and other variations from the consensus sequence of the family of tRNAs present in individual isoacceptors may be present as a result of subtle, species-specific adaptations in positive or negative synthetase recognition, optimal processing and modification, or interactions with elongation factors. Alternatively, these variations may simply be the result of neutral evolutionary drift.
- Af, Ap, Hh, Mj, Mt , Pf, Ph, and Ec Archaeoglobus fulgidus, Aeuropyrum pemix, Halobacterium sp. NRC-1, Methanococcus jannaschii, Methanobacterium thennoautotrophicum, Pyrococcus furiosus, Pyrococcus horikoshi, and Escherichia coli, respectively; LRS, leucyl-tRNA synthetase; bla, gene for ⁇ -lactamase; lacZ, gene for ⁇ -galactosidase.
- EXAMPLE 2 EXEMPLARY LEUCYL O-RSs AND LEUCYL O-tRNAs.
- Exemplary O-fRNAs comprise, e.g., SEQ ID NO.: 1-7 and 12 (See, Table 3).
- Exemplary O-RSs include, e.g., SEQ ID NOs.: 15 and 16 (See, Table 3).
- Exemplary polynucleotides that encode O-RSs or portions thereof include, e.g., SEQ JD NOs.: 13 and 14.
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Also Published As
Publication number | Publication date |
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EP1704242A4 (en) | 2008-06-18 |
EP1704242A2 (en) | 2006-09-27 |
WO2005007870A3 (en) | 2007-11-15 |
US20060160175A1 (en) | 2006-07-20 |
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