Methods of incorporating amino acid analogs into proteins

Abstract

The invention provides a method of incorporating nonstandard amino acids into a protein by utilizing a modified aminoacyl-tRNA synthetase to charge the nonstandard amino acid to a modified tRNA, which forms strict Watson-Crick base-pairing with a codon that normally forms wobble base-pairing with natural tRNAs.

1 . A polynucleotide encoding a modified tRNA, wherein said modified tRNA comprises a modified anticodon sequence that forms Watson-Crick base-pairing with a wobble degenerate codon for a natural amino acid.  
     
     
         2 . The polynucleotide of  claim 1 , wherein the interaction between the modified tRNA and the wobble degenerate codon at 37° C. is at least about 1.0 kcal/mole more favorable than the interaction between the wild-type tRNA and the wobble degenerate codon.  
     
     
         3 . The polynucleotide of  claim 1 , wherein said modified tRNA is derived from tRNA Phe , said wobble degenerate codon is UUU, and said unnatural amino acid is L-3-(2-naphthyl)alanine (NaI).  
     
     
         4 . The polynucleotide of  claim 1 , wherein said modified tRNA further comprises a mutation at the fourth, extended anticodon site for increasing translation efficiency.  
     
     
         5 . A method for incorporating an unnatural amino acid into a target protein at one or more specified position(s), the method comprising: 
 (1) providing to a translation system a first polynucleotide of  claim 1 , or the modified tRNA encoded thereby;    (2) providing to the translation system a second polynucleotide encoding a modified AminoAcyl tRNA Synthetase (AARS) with relaxed substrate specificity, or the modified AARS, wherein the modified AARS is capable of charging the modified tRNA with said unnatural amino acid;    (3) providing to the translation system the unnatural amino acid;    (4) providing to the translation system a template polynucleotide encoding the target protein, wherein the codon(s) on the template polynucleotide for said specified position(s) forms Watson-Crick base-pairing with the modified tRNA; and,    (5) allowing translation of the template polynucleotide, thereby incorporating the unnatural amino acid into the target protein at the specified position(s), wherein steps (1)-(4) are effectuated in any order.    
     
     
         6 . The method of  claim 5 , wherein the translation system is a cell.  
     
     
         7 . The method of  claim 5 , wherein step (3) is effectuated by contacting the translation system with a solution containing the unnatural amino acid.  
     
     
         8 . The method of  claim 7 , wherein the unnatural amino acid: 
 is an analog of said natural amino acid;    or is an analog of at least one amino acid different from said natural amino acid;    or is not an analog of any natural amino acids;    or comprises a side-chain R group selected from: alkyl-, aryl-, acyl-, keto-, azido-, hydroxyl-, hydrazine, cyano-, halo-, hydrazide, alkenyl, alkynl, ether, thiol, seleno-, sulfonyl-, borate, boronate, phospho, phosphono, phosphine, heterocyclic, enone, imine, aldehyde, ester, thioacid, hydroxylamine, amino group, or the like or any combination thereof;    or comprises a photoactivatable cross-linker, or is a spin-labeled amino acid, a fluorescent amino acid, a metal-binding amino acid, a metal-containing amino acid, a radioactive amino acid, an amino acid with novel functional group(s), an amino acid that covalently or noncovalently interacts with other molecules, a photocaged and/or photoisomerizable amino acid, an amino acids comprising biotin or a biotin analog, a glycosylated amino acid comprising a sugar-substituted serine, a carbohydrate-modified amino acid, a keto-containing amino acid, an amino acid comprising polyethylene glycol or polyether, a heavy atom-substituted amino acid, a chemically cleavable and/or photocleavable amino acid, an amino acids with an elongated side-chain as compared to natural amino acids, a carbon-linked sugar-containing amino acid, a redox-active amino acid, an amino thioacid-containing amino acid, or an amino acid comprising one or more toxic moiety;    or is represented by Formula II or III:                          wherein    Z comprises —OH, —NH 2 , —SH, —NH—R′, or S—R′;    X and Y, which may be the same or different, comprise S or O, and    R and R′, which may be the same or different, are selected from: alkyl-, aryl-, acyl-, keto-, azido-, hydroxyl-, hydrazine, cyano-, halo-, hydrazide, alkenyl, alkynl, ether, thiol, seleno-, sulfonyl-, borate, boronate, phospho, phosphono, phosphine, heterocyclic, enone, imine, aldehyde, ester, thioacid, hydrogen, hydroxylamine, amino group, or the like or any combination thereof;    or is selected from: α-hydroxy acids, α-thioacids α-aminothiocarboxylates;    or is L, D, or α-α-disubstituted amino acid selected from D-glutamate, D-alanine, D-methyl-O-tyrosine, or aminobutyric acid;    or comprises a functional group selected from: bromo-, iodo-, ethynyl-, cyano-, azido-, acetyl, aryl ketone, photolabile, fluorescent, or heavy metal group;    or is a cyclic amino acid selected from: a 3-, 4-, 6-, 7-, 8-, and 9-membered ring proline analog; a P or y amino acid selected from substituted β-alanine or γ-amino butyric acid;    or is a Tyrosine analog selected from: a para-substituted tyrosine, an ortho-substituted tyrosine, a meta-substituted tyrosine, 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 C6-C20 straight chain or branched hydrocarbon, a saturated or unsaturated hydrocarbon, an O-methyl group, a polyether group, a nitro group, or multiply substituted aryl rings; a Glutamine analog selected from: α-hydroxy derivatives, β-substituted derivatives, cyclic derivatives, or amide-substituted glutamine derivatives; a Phenylalanine analog selected from: meta-substituted phenylalanines, wherein the substituent comprises a hydroxy group, a methoxy group, a methyl group, an allyl group, an acetyl group, or the like;    or is an 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-GlcNAcβ-serine, an L-Dopa, a fluorinated phenylalanine, an isopropyl-L-phenylalanine, a p-azido-L-phenylalanine, a p-acyl-L-phenylalanine, a p-benzoyl-L-phenylalanine, an L-phosphoserine, a phosphonoserine, a phosphonotyrosine, a p-iodo-phenylalanine, a p-bromophenylalanine, a p-amino-L-phenylalanine, or an isopropyl-L-phenylalanine;    or modifies one or more biological properties of a protein into which it is incorporated, said biological properties comprising: toxicity, biodistribution, solubility, thermal stability, hydrolytic stability, oxidative stability, resistance to enzymatic degradation, facility of purification and processing, structural properties, spectroscopic properties, chemical and/or photochemical properties, catalytic activity, redox potential, half-life, ability to react with other molecules either covalently or noncovalently.    
     
     
         9 . The method of  claim 5 , wherein said modified AARS with relaxed substrate specificity charges said modified tRNA with said unnatural amino acid.  
     
     
         10 . The method of  claim 9 , wherein the specificity constant (k cat /K M ) for activation of said unnatural amino acid by said modified AARS is at least 5-fold larger than that for said natural amino acid.  
     
     
         11 . The method of  claim 5 , wherein said modified tRNA is charged by an endogenous AARS at a rate no more than 1% of that of its cognate tRNA.  
     
     
         12 . The method of  claim 5 , wherein the unnatural amino acid is provided by introducing additional nucleic acid construct(s) into the translation system, wherein the additional nucleic acid construct(s) encode one or more proteins required for biosynthesis of the unnatural amino acid.  
     
     
         13 . The method of  claim 5 , wherein the first polynucleotide and/or the second polynucleotide further comprises either a constitutively active or an inducible promoter sequence that controls the expression of the modified tRNA or AARS, respectively.  
     
     
         14 . The method of  claim 5 , wherein the translation system is a cell, and the cell is auxotrophic for the natural amino acid encoded at the specified position.  
     
     
         15 . The method of  claim 5 , wherein the translation system: 
 (1) lacks endogenous tRNA that forms Watson-Crick base-pairing with the codon(s) at said specified position(s);    (2) is a cell, and the method further comprises disabling one or more genes encoding any endogenous tRNA that forms Watson-Crick base-pairing with the codon(s) at said specified position(s); or    (3) is a cell, and the method further comprises inhibiting one or more endogenous AARS that charges tRNAs that form Watson-Crick base-pairing with the codon(s) at said specified position(s).    
     
     
         16 . The method of  claim 5 , wherein the cell is a bacterial cell, an  E. coli  cell, an insect cell, a mammalian cell, a fungal cell, or a yeast cell.  
     
     
         17 . The method of  claim 5 , wherein the translation system is a cell, and the modified tRNA and/or the modified AARS are derived from an organism different from that of the cell.  
     
     
         18 . The method of  claim 5 , further comprising verifying the incorporation of the unnatural amino acid.  
     
     
         19 . The method of  claim 5 , wherein the analog is incorporated into the position at an efficiency of at least about 50%.  
     
     
         20 . A translation system comprising the polynucleotide of  claim 1 .  
     
     
         21 . The translation system of  claim 20 , further comprising a second polynucleotide encoding a modified AARS with relaxed substrate specificity, or the modified AARS, wherein the modified AARS is capable of charging the modified tRNA with an unnatural amino acid.  
     
     
         22 . The translation system of  claim 20 , comprising more than two different polynucleotides of  claim 1 , each said polynucleotides capable of carrying a different unnatural amino acid.  
     
     
         23 . The translation system of  claim 20 , which is a cell.  
     
     
         24 . The translation system of  claim 23 , wherein the modified tRNA is from an organism different from that of the cell.  
     
     
         25 . The translation system of  claim 24 , wherein the modified tRNA is from a yeast, and the cell is an  E. coli  bacterium.  
     
     
         26 . The translation system of  claim 23 , wherein the modified AARS and the tRNA are from the same organism, said organism is different from that of the cell.  
     
     
         27 . The translation system of  claim 24 , wherein the modified AARS and the tRNA are from a yeast, and the cell is an  E. coli  bacterium.