Methods and Compositions Comprising Non-Natural Amino Acids

Abstract

Disclosed herein are methods of detecting non-natural amino acids and polypeptides that include at least one non-natural amino acid. The non-natural amino acids, by themselves or as a part of a polypeptide, can include a wide range of functionalities, including but not limited to oxime, carbonyl, and/or hydroxylamine groups. Also disclosed herein are non-natural amino acid polypeptides that are further modified post-translationally, and methods for detecting such polypeptides.

1 . A method of detecting a polypeptide comprising detecting a non-naturally encoded amino acid side chain in the polypeptide. 
     
     
         2 . The method of  claim 1  wherein the polypeptide is detected using a technique selected from the group consisting of immunoassays, microarrays, microscopy, fluoroscent microscopy, electron microscopy, electrophoresis, spectroscopy, chromogenic reactions, radio-detection, radio-transmission, subatomic particle detection, metal binding, chelating, structure or conformation changes, enzymatic activity, specific binding, photography, magnetic field measurement, sensors, electromagnetic energy detection, gene expression, visible or invisible light detection, temperature detection, and chemical detection. 
     
     
         3 . The method of  claim 2  wherein the immunoassays are selected from the group consisting of Radioimmunoassay, Enzyme-linked immunosorbent assay, Enzyme Multiplied Immunoassay, Microparticle Enzyme Immunoassay, luminescent immunoassay, and fluorescent immunoassay. 
     
     
         4 . The method of  claim 2  wherein the spectroscopy is selected from the group consisting of SELDI, MALDI, fluorescence spectroscopy, NMR, UV-Vis, and X-ray Crystallography. 
     
     
         5 . The method of  claim 2  wherein the electrophoresis is selected from the group consisting of Gel Electrophoresis, Zone Electrophoresis, Isoelectric Focusing Electrophoresis, Capillary Electrophoresis, Capillary Zone Electrophoresis, Capillary Gel Electrophoresis, Capillary Isotachophoresis, Capillary Isoelectric Focusing Electrophoresis, and Capillary Electrochromatography. 
     
     
         6 . The method of  claim 1 , wherein the polypeptide is ribosomally synthesized. 
     
     
         7 . The method of  claim 1 , wherein the non-natural amino acid polypeptide is post-translationally modified. 
     
     
         8 . A method of detecting a polypeptide comprising detecting a non-naturally encoded amino acid side chain in the polypeptide that has been post-translationally modified. 
     
     
         9 . The method of  claim 8  wherein the polypeptide is detected using a technique selected from the group consisting of immunoassays, microarrays, microscopy, fluorescent microscopy, electron microscopy, electrophoresis, spectroscopy, chromogenic reactions, radio-detection, radio-transmission, sub-atomic particle detection, metal binding, chelating, structure or conformation changes, enzymatic activity, specific binding, photography, magnetic field measurement, sensors, electromagnetic energy detection, gene expression, visible or invisible light detection, temperature detection, and chemical detection. 
     
     
         10 . The method of  claim 9  wherein the immunoassays are selected from the group consisting of Radioimmunoassay, Enzyme-linked immunosorbent assay, Enzyme Multiplied Immunoassay, Microparticle Enzyme Immunoassay, luminescent immunoassay, and fluorescent immunoassay. 
     
     
         11 . The method of  claim 9  wherein the spectroscopy is selected from the group consisting of SELDI, MALDI, fluorescence spectroscopy, NMR, UV-Vis, and X-ray Crystallography. 
     
     
         12 . The method of  claim 9  wherein the electrophoresis is selected from the group consisting of Gel Electrophoresis, Zone Electrophoresis, Isoelectric Focusing Electrophoresis, Capillary Electrophoresis, Capillary Zone Electrophoresis, Capillary Gel Electrophoresis, Capillary Isotachophoresis, Capillary Isoelectric Focusing Electrophoresis, and Capillary Electrochromatography. 
     
     
         13 . The method of  claim 8 , wherein the polypeptide is ribosomally synthesized. 
     
     
         14 . A method of detecting a non-naturally encoded amino acid side chain in said polypeptide, comprising contacting the non-naturally encoded amino acid side chain with a molecule comprising a functional group that specifically interacts with the non-naturally encoded amino acid side chain. 
     
     
         15 . A method for screening a library of molecules, comprising:
 a) combining a polypeptide comprising a non-naturally encoded amino acid with the library molecules under conditions to allow interaction of the library molecules with the polypeptide comprising a non-naturally encoded amino acid,   b) identifying the library, molecules which interact with the polypeptide comprising a non-naturally encoded amino acid.   
     
     
         16 . The method of  claim 15 , wherein the non-natural amino acid polypeptide is ribosomally synthesized. 
     
     
         17 . The method of  claim 15 , wherein the side chain of the non-naturally encoded amino acid is post-translationally modified. 
     
     
         18 . The method of  claim 17 , wherein the non-natural amino acid polypeptide is ribosomally synthesized. 
     
     
         19 . The method of  claim 15  wherein the library is chemical or biological. 
     
     
         20 . The method of  claim 19  wherein the chemical library is organic or inorganic. 
     
     
         21 . The method of  claim 19  wherein the biological library molecules are selected from the group consisting of protein, peptide polypeptide, DNA, RNA, virus, ribosomes, translation complexes, bacteriophage, bacteria, and yeast. 
     
     
         22 . The method of  claim 15  wherein the interaction of the library molecules with the polypeptide is specific binding of the library molecules to the polypeptide. 
     
     
         23 . The method of  claim 15  wherein the interaction of the library molecules with the polypeptide is covalent bond formation or complex formation of the library molecules to the polypeptide. 
     
     
         24 . The method of  claim 15  further comprising the steps:
 c) recovering a library molecule that interacts with the non-natural amino acid polypeptide; and   d) separating the non-natural amino acid polypeptide from the library molecule, thereby obtaining isolated library molecules.   
     
     
         25 . A library of ribosomally made polypeptides comprising a plurality of polypeptides having different amino acid sequences, wherein each polypeptide comprises a non-natural amino acid. 
     
     
         26 . The library of  claim 25 , wherein each polypeptide comprises the same non-natural amino acid. 
     
     
         27 . The library of  claim 25 , wherein each polypeptide comprises a different non-natural amino acid. 
     
     
         28 . The library of  claim 25 , wherein at least one polypeptide is post-translationally modified. 
     
     
         29 . The library of  claim 27 , wherein the polypeptides are identical except for the non-naturally encoded amino acid. 
     
     
         30 . The library of  claim 25 , wherein each polypeptide is homologous to a polypeptide comprised of natural amino acids. 
     
     
         31 . The library of  claim 26 , wherein each polypeptide is identical except for the location of the non-natural amino acid. 
     
     
         32 . A method of purifying a polypeptide having a non-naturally encoded amino acid in the polypeptide chain, comprising contacting the polypeptide with a substance that interacts with the non-naturally encoded amino acid side chain in the polypeptide. 
     
     
         33 . The method of  claim 32  wherein the substance is a chromatography matrix selected from the group consisting of liquid chromatography, gas chromatography and supercritical fluid chromatography. 
     
     
         34 . The method of  claim 33  wherein the liquid chromatography is selected from the group consisting of partition chromatography, adsorption chromatography, ion exchange chromatography, size exclusion chromatography, thin layer chromatography and affinity chromatography. 
     
     
         35 . The method of  claim 33  wherein the liquid chromatography is selected from the group consisting of HPLC, column chromatography and Batch treatment. 
     
     
         36 . The method of  claim 32  wherein the contacting of the polypeptide with the substance occurs in a microfluidic device. 
     
     
         37 . The method of  claim 32  wherein the contacting of the polypeptide with the substance occurs in a nanofluidic device. 
     
     
         38 . The method of  claim 34  wherein the partition chromatography is selected from the group consisting of normal phase chromatography, reversed phase chromatography and ion-pair chromatography. 
     
     
         39 . The method of  claim 34  wherein the thin layer chromatography is selected from the group consisting of paper chromatography, thin-layer chromatography and electrochromatography. 
     
     
         40 . The method of  claim 34  wherein the affinity chromatography is selected from the group consisting of ligand chromatography, dye chromatography, metal chelate chromatography, immunoaffinity chromatography and hydrophobic interaction chromatography. 
     
     
         41 . A method of purifying a polypeptide having a non-naturally encoded amino acid in the polypeptide chain, comprising precipitation of the polypeptide, wherein the non-naturally encoded amino acid alters the solubility of the polypeptide when compared to the solubility of the polypeptide without a non-naturally encoded amino acid in the polypeptide chain. 
     
     
         42 . The method of  claim 41  wherein the precipitation is performed by a compound selected from the group consisting of salts, acid, base, and polymers. 
     
     
         43 . The method of  claim 41  wherein the purifying is by immunoprecipitation. 
     
     
         44 . The method of  claim 32  wherein the substance is a magnetic substance. 
     
     
         45 . A method of purifying a ribosomally made polypeptide having a non-naturally encoded amino acid in the polypeptide side chain, comprising electrophoresis of the polypeptide, wherein the non-naturally encoded amino acid alters the electrophoretic mobility of the polypeptide when compared to the electrophoretic mobility of the polypeptide without a non-naturally encoded amino acid in the polypeptide chain. 
     
     
         46 . The method of  claim 45  where the electrophoresis is selected from the group consisting of Gel Electrophoresis and Capillary Electrophoresis. 
     
     
         47 . The method of  claim 46  wherein the Gel Electrophoresis, is selected from the group consisting of Zone Electrophoresis and Isoelectric Focusing Electrophoresis. 
     
     
         48 . The method of  claim 46  wherein the Capillary Electrophoresis is selected from the group consisting of Capillary Zone Electrophoresis, Capillary Gel Electrophoresis, Capillary Isotachophoresis, Capillary Isoelectric Focusing Electrophoresis, Capillary Electrochrormatography, Micellar electrokinetic capillary chromatography, Isotachophoresis, and Transient Isotachophoresis. 
     
     
         49 . A method of purifying a ribosomally made polypeptide having a non-naturally encoded amino acid in the polypeptide side chain, comprising dialysis of the polypeptide, wherein the non-naturally encoded amino acid alters the diffusion rate of the polypeptide when compared to the diffusion rate of the polypeptide without a non-naturally encoded amino acid in the polypeptide chain. 
     
     
         50 . The method of  claim 49  wherein the dialysis is electrodialysis. 
     
     
         51 . A method of purifying a non-natural amino acid polypeptide comprising purifying the polypeptide by ultrafiltration. 
     
     
         52 . The method of  claim 32 , wherein the polypeptide is expressed in a microorganism. 
     
     
         53 . The method of  claim 32 , wherein the polypeptide is made in a microorganism selected from the group consisting of  Escherichia coli, Pseudomonas fluorescens, Bacillus subtilis , yeast, mammalian cells and insect cells. 
     
     
         54 . The method of  claim 53  wherein the non-natural amino acid polypeptide is a hybrid peptide that contain and affinity tag. 
     
     
         55 . The method of  claim 32 , wherein the non-natural amino acid polypeptide is post-translationally modified. 
     
     
         56 . The method of  claim 55 , wherein the post-translational modification is by an oxime exchange reaction. 
     
     
         57 . The method of  claim 32 , wherein the non-natural amino acid is posttranslationally modified to comprise an oxime group. 
     
     
         58 . A method, comprising:
 a) substituting a non-naturally encoded amino acid for a naturally encoded amino acid at a single pre-selected site in a pre-selected polypeptide having at least one known biological activity; and   b) measuring a biological activity of the pre-selected polypeptide comprising the non-naturally encoded amino acid; and   c) comparing the biological activity of the pre-selected polypeptide of step b) with the pre-selected polypeptide having a non-naturally encoded amino acid substituted for a naturally encoded amino acid at a different position in the pre-selected polypeptide chain or with the pre-selected polypeptide without a substituted non-naturally encoded amino acid in the polypeptide chain.   
     
     
         59 . The method of  claim 58  wherein the pre-selected positions in the polypeptide chain for substitution with a non-naturally encoded amino acid are sequential. 
     
     
         60 . The method of  claim 58  wherein the substituting of the non-naturally encoded amino acid for a naturally encoded amino acid at a single site in the pre-selected polypeptide chain is repeated for every individual position in the polypeptide chain. 
     
     
         61 . The method of  claim 58  wherein the same non-naturally encoded amino acid is substituted. 
     
     
         62 . The method of  claim 58  wherein a different non-naturally encoded amino acid is substituted. 
     
     
         63 . The method of  claim 58  wherein more than one position of the pre-selected polypeptide chain is substituted with a non-naturally encoded amino acid. 
     
     
         64 . A method for selecting a position for post-translational modification of a pre-selected polypeptide comprising:
 a) substituting a non-naturally encoded amino acid for a naturally encoded amino acid at a single pre-selected site in a pre-selected polypeptide having at least one known biological activity; and   b) measuring a biological activity of the pre-selected polypeptide comprising the non-naturally encoded amino acid; and   c) comparing the biological activity of the pre-selected polypeptide of step b) with the pre-selected polypeptide having a non-naturally encoded amino acid substituted for a naturally encoded amino acid at a different position in the pre-selected polypeptide chain or with the pre-selected polypeptide without a substituted non-naturally encoded amino acid in the polypeptide chain.   
     
     
         65 . The method of  claim 64  wherein the post-translational modification is coupling to a water soluble polymer. 
     
     
         66 . A composition comprising a polypeptide covalently linked to a nucleic acid molecule at one or more specific amino acid positions of said polypeptide, wherein said polypeptide and nucleic acid molecule are covalently linked to an amino acid side chain of one or more non-naturally encoded amino acids of said polypeptide.