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Wo 2007/059312 A2 (12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (19) World Intellectual Property Organization International Bureau (43) International Publication Date (10) International Publication Number 24 May 2007 (24.05.2007) PCT WO 2007/059312 A2 (51) International Patent Classification: (74) Agent: WALLEN, John, W.; AMBRX, INC., 10975 GOlN 33/53 (2006.01) NORTH TORREY PINES ROAD, SUITE 100, La Jolla, CA 92037 (US). (21) International Application Number: (81) Designated States (unless otherwise indicated, for every PCT/US2006/044682 kind of national protection available): AE, AG, AL, AM, AT,AU, AZ, BA, BB, BG, BR, BW, BY, BZ, CA, CH, CN, (22) International Filing Date: CO, CR, CU, CZ, DE, DK, DM, DZ, EC, EE, EG, ES, FI, 16 November 2006 (16.1 1.2006) GB, GD, GE, GH, GM, GT, HN, HR, HU, ID, IL, IN, IS, JP, KE, KG, KM, KN, KP, KR, KZ, LA, LC, LK, LR, LS, (25) Filing Language: English LT, LU, LV,LY,MA, MD, MG, MK, MN, MW, MX, MY, MZ, NA, NG, NI, NO, NZ, OM, PG, PH, PL, PT, RO, RS, (26) Publication Language: English RU, SC, SD, SE, SG, SK, SL, SM, SV, SY, TJ, TM, TN, TR, TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, ZW (30) Priority Data: (84) Designated States (unless otherwise indicated, for every 60/737,855 16 November 2005 (16.1 1.2005) US kind of regional protection available): ARIPO (BW, GH, GM, KE, LS, MW, MZ, NA, SD, SL, SZ, TZ, UG, ZM, (71) Applicant (for all designated States except US): AMBRX, ZW), Eurasian (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), INC. [US/US]; 10975 NORTH TORREY PINES ROAD, European (AT,BE, BG, CH, CY, CZ, DE, DK, EE, ES, FI, SUITE 100, La Jolla, CA 92037 (US). FR, GB, GR, HU, IE, IS, IT, LT, LU, LV,MC, NL, PL, PT, RO, SE, SI, SK, TR), OAPI (BF, BJ, CF, CG, CI, CM, GA, (72) Inventors; and GN, GQ, GW, ML, MR, NE, SN, TD, TG). (75) Inventors/Applicants (for US only): MIAO, Zhenwei [CN/US]; 15887 SUNNYHELD PLACE, San Diego, CA Published: 92127 (US). TIAN, Feng [CN/US]; 4168 CAMINO TI- — without international search report and to be republished CINO, San Diego, CA 92122 (US). HAYS, Anna-Maria upon receipt of that report [US/US]; 3187 VIA ALICANTE 251, La Jolla, CA 92037 For two-letter codes and other abbreviations, refer to the "G uid (US). BUECHLER, Ying [US/US]; 1343 CASSINS ance Notes on Codes and Abbreviations" appearing at the beg in STREET, Carlsbad, CA 9201 1 (US). ning of each regular issue of the PCT Gazette. (54) Title: METHODS AND COMPOSITIONS COMPRISING NON-NATURAL AMINO ACIDS (57) Abstract: Disclosed herein are methods of detecting non-natural amino acids and polypeptides that include at least one non-nat- ural amino acid. The non-natural amino acids, by themselves or as a part of a polypeptide, can include a wide range of functionali- ties, including but not limited to oxime, carbonyl, and/or hydroxy lamine groups. Also disclosed herein are non-natural amino acid polypeptides that are further modified post-translationally, and methods for detecting such polypeptides. M the s and Compositions Comprising Non-Natural Amino Acids RELATED APPLICATIONS [01] This application claims benefit of U.S. Provisional Application No. 60/737,855, entitled "Methods of Detecting Non-Natural Amino Acid Polypeptides in vivo and in vitro" filed on November 16, 2005. BACKGROUND OF THE INVENTION [02] The ability to incorporate non-genetically encoded amino acids (i.e., "non-natural amino acids") into proteins permits the introduction of chemical functional groups that could provide valuable alternatives to the naturally-occurring functional groups, such as the epsilon — NH2 of lysine, the sulfhydryl -SH of cysteine, the imino group of histidine, etc. Certain chemical functional groups are known to be inert to the functional groups found in the 20 common, genetically-encoded amino acids but react cleanly and efficiently to form stable linkages with functional groups that can be incorporated onto non-natural amino acids. [03] Methods are now available to selectively introduce chemical functional groups that are not found in proteins, that are chemically inert to all of the functional groups found in the 20 common, genetically-encoded amino acids and that may be used to react efficiently and selectively with reagents comprising certain functional groups to form stable covalent linkages. SUMMARY OF THE INVENTION [04] Described herein and incorporated by reference are methods, compositions, techniques and strategies for making, purifying, detecting, characterizing, and using non-natural amino acids, non-natural amino acid polypeptides and modified non-natural amino acid polypeptides. [05] This invention provides a method of detecting a polypeptide that comprises detecting a non-naturally encoded amino acid side chain in the polypeptide. In some embodiments, the polypeptide is ribosomally synthesized. The invention also provides methods of detecting a polypeptide that comprise detecting a non-naturally encoded amino acid side chain in the polypeptide that has been post-translationally modified. Also provided are methods of detecting a non-naturally encoded amino acid side chain in said polypeptide that comprise 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. Also provided are methods of purifying a polypeptide having a non-naturally encoded amino acid in the polypeptide chain. In some embodiments the method comprises contacting the polypeptide with a substance that interacts with the non-naturally encoded amino acid side chain in the polypeptide. In other embodiments, the method of purifying a polypeptide having a non- naturally encoded amino acid in the polypeptide chain comprises 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. Methods of purifying a ribosomally made polypeptide having a non-naturally encoded amino acid in the polypeptide side chain comprises 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 are also provided. In other embodiments, the method of purifying a ribosomally made polypeptide having a non-naturally encoded amino acid in the polypeptide side chain, comprises 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. [06] The invention also provides 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, and b) identifying the library molecules which interact with the polypeptide comprising a non-naturally encoded amino acid. In some embodiments, 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 is screened. [07] The invention also provides methoda, 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. In some embodiments, a method for selecting a position for post-translational modification of a pre-selected polypeptide comprises 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. [08] It is to be understood that the methods and compositions described herein and incorporated by reference are not limited to the particular methodology, protocols, cell lines, constructs, and reagents described herein and as such may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the methods and compositions described herein, which will be limited only by the appended claims. DEFINITIONS [09] As used herein and in the appended claims, the singular forms "a," "an," and "the" include plural reference unless the context clearly indicates otherwise. [10] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which the inventions described herein belong. Although any methods, devices, and materials similar or equivalent to those described herein can be used in the practice or testing of the inventions described herein, the preferred methods, devices and materials are now described.
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