JPH11505527A - Identification of enantiomeric ligands - Google Patents

Abstract

(57) [Summary] Macromolecules (peptides, oligonucleotides, sugars, and Identification methods of high molecular complexes such as RNA-protein complexes and protein-lipid complexes).

Description

DETAILED DESCRIPTION OF THE INVENTION                          Identification of enantiomeric ligands Related application   This application is related to US Ser. No. 08 / 627,4, filed Mar. 28, 1996. Ninety-seven continuation-in-part applications, filed on May 3, 1995 Filed June 7, 1995, which is a continuation-in-part of application no. 08 / 433,572. US Patent Application Serial No. 08 / 482,309, issued in 1995, which is a continuation-in-part application. Partial continuation of US Provisional Application No. 60 / 001,067 filed July 11 And these teachings are incorporated herein by reference.Fund   The work described herein was sponsored by Howard Hughes Medical Institute / Life Sciences Research Funded by the fund.Background art   Genetically encoded libraries of peptides and oligonucleotides Suitable for identifying ligands for many macromolecules. However, biologically A major drawback of the encoded library is that the resulting ligand is a naturally occurring enzyme. It is to be applied to the elementary gradation. In addition, cellular proteolytic enzymes Due to their sensitivity to amino acids, peptides composed of naturally occurring L-amino acids The peptide is a helper T cell (T_HMajor histocompatibility complex class II restriction Through the presentation process effectively. As a result, L-peptides increase the activity of such drugs. Induces a vigorous vigorous humoral immune response   Natural enantiomers of left-right macromolecules are better drugs than natural left-right macromolecules Is prepared. In contrast to naturally occurring L-peptide and D-nucleic acid sequences, Enantiomers of these naturally occurring macromolecules (e.g., D-peptide and L- Nucleic acid) is a good substrate for naturally occurring proteases and nucleases. Absent. In addition, enantiomers of the natural molecule do not elicit an effective immune response.   It is desirable to have D-peptides and L-nucleic acids for use as drugs. No.Summary of the Invention   The present invention relates to non-natural left-right images (such as naturally occurring or wild-type molecules). Enantiomers that are ligands to other chiral macromolecules) Macromolecules (proteins, peptides, oligonucleotides, nucleic acids, sugars, and RNA- Protein complex, polymer complex such as protein-lipid complex) Or a desired polymer. The target or desired macromolecule is an oligonucleotide (D NA, RNA) and proteins such as hormones, enzymes, antibodies, antigens (eg, poly Peptide and peptide). In one aspect, the present invention provides a method for targeting or A method for identifying a D-amino acid peptide ligand that binds an L-amino acid peptide is there. In a second aspect, the present invention provides an oligonucleotide (RNA or DNA) ) Is a method for identifying a peptide consisting of D-amino acid residues that is a ligand for . In a further aspect, the invention relates to a chirality as opposed to a naturally occurring one. This is a method for identifying RNA or DNA oligonucleotides. DNA naturally Occurs as the D isoform.   In one embodiment, the invention relates to a natural left-right target polymer (eg, a peptide , Oligonucleotides) to produce non-natural left-right macromolecules And is performed as follows: a characteristic domain of or of the target macromolecule Of the natural left and right macromolecules and their enantiomers in the library Contact with a natural library of right and left macromolecules under conditions suitable for binding to the body Consequently, the enantiomers are a fraction of the natural left and right image present in the library. Combine children. Mirror image of natural left-right polymer bound to target polymer enantiomer The isomers are produced; the natural enantiomeric enantiomer of the right and left image is the natural left and right image marker. Is a non-natural left-right image polymer that binds to the target polymer. In other words, in the library Enantiomers of macromolecules that exist and bind to the enantiomer of the target molecule are produced; The result is a non-natural left-right macromolecule that binds the target molecule (natural right-left image). You.   In an embodiment where the target macromolecule is a protein (eg, a peptide), the target L pepti A D-amino acid peptide that binds to a peptide is produced. The method is as follows: D amino acid peptide and L-amino acid of target L macromolecule or its characteristic domain Prepare a library of noacid peptides. L amino acid peptide in library Under conditions suitable for binding to the D-amino acid peptide of The D-amino acid peptide of the offspring; Binds L-amino acid peptides present in the library. D amino acid peptide L amino acid peptides present in the linked library were identified and sequenced. Is done. Of the L amino acid peptide identified in the library or a characteristic thereof A D-amino acid peptide of the domain is produced; Binds to the natural left and right target L macromolecules. That is, if they exist in the library and The enantiomer of the L amino acid peptide that binds to the D amino acid peptide of a typical L macromolecule is The result is a D amino acid peptide that binds the L target macromolecule; In this embodiment, it is an L-amino acid peptide.   In embodiments where the target macromolecule is a protein (eg, a peptide), the target L protein An L oligonucleotide that binds to is produced. The method is performed as follows : D amino acid peptide and D of the target L macromolecule or of its characteristic domain Prepare a library of oligonucleotides. D-oligonucs in library Under conditions suitable for binding of the reotide to the D amino acid peptide, the library was Contacting the amino acid peptide so that the peptide is present in the library Conjugated D oligonucleotide. D-oligonus linked to D-amino acid peptide Nucleotides are identified and sequenced. D-origin identified in library The L-oligonucleotide of the gonucleotide or its characteristic domain is produced The L oligonucleotide binds to the target L macromolecule in the unnatural left-right image. One That is, it exists in the library and binds to the D amino acid peptide of the target L macromolecule The enantiomer of the D oligonucleotide is produced; An L oligonucleotide that binds, in this embodiment an L amino acid peptide is there.   In another aspect, the invention relates to a non-naturally occurring non-naturally occurring binding target macromolecule. The method for producing a left-right image polymer is performed as follows: target polymer Prepare the enantiomers of the target domain or of the characteristic domain of the target molecule in the library. Under conditions suitable for the binding of the enantiomeric polymer to the natural left-right image, the natural left-right image height Contacting with a library of molecules; as a result, enantiomers are Binds the natural left and right macromolecules present in Natural left and right bound to enantiomer The macromolecules in the image are identified and sequenced. Of natural left and right macromolecules or their characteristics Unnatural left-right enantiomer of a macromolecule bound to a symbolic domain enantiomer Is produced; the resulting enantiomer of the natural left-right macromolecule is It is a non-natural left-right image polymer that binds to the target polymer.   In a further aspect of the invention, an L-amino acid that binds a D-amino acid peptide of interest The amino acid peptide is identified as follows: L-amino acid displayed on the phage surface Prepare phage display library containing acid peptide and display on phage surface Conditions suitable for binding of the selected L amino acid peptide and the desired D amino acid peptide And contact with the desired D amino acid peptide. L amino acid pep displayed on the surface A phage having a D amino acid peptide of interest on its surface bound to a tide (ie, Having a D-amino acid peptide-presenting L-amino acid peptide complex on the surface) Is done. The presented L-amino acid peptide in the complex is the desired D-amino acid peptide. L-amino acid peptide that binds   If necessary, the amino acid sequence of the L amino acid peptide displayed on the surface of the phage Can be determined and the D amino acid corresponding to the amino acid sequence of the L amino acid peptide Acid peptides can be synthesized and L-amino acid peptides displayed on the phage surface. This results in the production of a D amino acid peptide corresponding to the tide. In one aspect As described herein, the L amino acid peptide displayed on the phage surface comprises: One class of proteins, specifically the SRC homology 3 domain (SH3 domain ) D-amino acid peptide.   A further aspect is any protein (polypeptide, A D amino acid corresponding to the target L amino acid protein This is a method for producing a protein. In this embodiment, the L-amido displayed on the phage surface Under conditions suitable for the binding of the amino acid peptide to the D-amino acid protein, A phage display library containing a mixture of the indicated proteins was cloned into the target L-amino acid. D corresponding to the acid protein or to the characteristic domain of the target L amino acid protein -Contact with an amino acid peptide. The mixture can be a target L amino acid protein or a protein thereof. Contains a characteristic L amino acid peptide domain. L amino acids displayed on the surface Phage with D amino acid peptide on the surface bound to the peptide are identified . The amino acid sequence of the L amino acid peptide displayed on the surface of the identified phage is The D amino acid protein corresponding to the amino acid sequence of the L amino acid peptide Resulting in the production of a D amino acid peptide corresponding to the target L amino acid protein become.   The present invention also relates to an L oligonucleotide that binds to an L amino acid peptide of interest. The present invention relates to a method for obtaining a nucleic acid sequence. In this method, a collection of D nucleic acid sequences ( For example, a DNA library is prepared, and the desired D amino acid peptide of D nucleic acid is prepared. Under the conditions suitable for binding to the target D amino acid peptide. D-amino The D nucleic acid that binds to the acid peptide is isolated, and the nucleotide sequence of the D nucleic acid is determined. You. The D nucleic acid sequence that binds to the target D amino acid peptide uses L nucleotides. Resulting in the production of L nucleic acid sequences that bind L amino acid peptides . A further aspect of the invention provides a collection of D nucleic acid sequences, wherein the D nucleic acid sequences are A method of obtaining an L nucleic acid sequence that binds a D nucleic acid, comprising the step of contacting the L nucleic acid sequence Related. Producing an L nucleic acid sequence, thereby a D nucleic acid sequence-L nucleic acid sequence complex, A D nucleic acid sequence that binds to is identified. Nucleic acid sequence of D nucleic acid binding to L nucleic acid sequence The otide sequence is determined. The D nucleic acid sequence is synthesized using L nucleotides, The result is the production of an L nucleic acid sequence that binds the nucleic acids.   The subject of the present invention also relates to the D-identified and produced by the method described herein. Synthetic D amino acid peptide such as amino acid peptide, but binds to SH3 domain Synthetic D peptide corresponding to all or part of the SH3 domain Amino acid peptides, and more commonly, domains of intracellular signaling proteins. Including, but not limited to, synthetic D amino acid peptides. Sa Furthermore, oligonucleotides and the like identified and produced by the method described herein Non-natural left-right oligonucleotides (RNA, DNA) are the subject of the present invention. is there.   The present invention also relates to a method for binding non-natural left and right images that binds the target macromolecule in the natural left and right images. The present invention relates to a method for producing a derivative of a child. The method can be performed using the methods described herein to produce non-natural Identify macromolecules in left and right images and modify or induce unnatural left and right macromolecules And producing a derivative thereof. The method of the invention described herein The derivatives obtainable by the above are also within the scope of the present invention.   The D amino acid peptides and L nucleic acid sequences of the present invention are useful as drugs. An example For example, D-amino acid peptides are good groups for naturally occurring proteases. L-amino acid peptide, not quality (ie, proteolytic degradation resistance) Does not elicit an immune response comparable to that elicited byBrief description of figures   The figure illustrates the identification of D-peptide ligands through mirror image phage display It is.Detailed description of the invention   Synthetic enantiomers of structural biopolymers are enantiomeric conformations of natural molecules To the first partner for the two-molecule complex. The two enantiomers of the parent also form a complex with mirror symmetry with the original. Book The invention relates to a natural left-right image binding that binds enantiomers of a chiral biological target molecule. Of the target (L-peptide, single-stranded D-oligonucleotide, etc.) Based on the discovery that it provides a method for identifying non-natural left-right macromolecules that binds. Such enantiomeric macromolecules determine and evaluate the ability to interfere with the biological activity of the target. Be valued. Thus, the present invention provides for the development of novel long-term therapeutic or diagnostic molecules. Provide a means of launch.   The invention is not naturally occurring or wild-type bilateral (ie, chirality). ), Against other chiral molecules (peptides, oligonucleotides and macromolecular complexes) Ligands, such as peptides, polypeptides, proteins, oligonucleotides, Saccharide and polymer complex (oligonucleotide-protein complex, protein-lipid complex) And a method for identifying a macromolecule of the enantiomer of the union. As defined herein As can be seen, the enantiomer of the natural left-right polymer is the natural left-right polymer But an unnatural left-right image. In the method of the present invention, naturally occurring Prepare enantiomers of target macromolecules and generate collections of naturally occurring macromolecules Et al. Used to isolate naturally occurring ligands that interact with enantiomers. It is. The enantiomeric form of the isolated naturally occurring ligand is the naturally occurring standard. (Eg, bind) with the target macromolecule.   The target polymer of the natural left and right images can be any polymer with one or more chiral centers. There may be. Target macromolecules (chiral targets) can be intracellular or extracellular For example, nucleic acids (DNA, RNA), proteins or proteins having a chiral center are often used. Represents its characteristic domain (eg, peptide), peptide, polypeptide, or Gonucleotide, carbohydrate, sugar, oligonucleotide-protein complex (RNA- Protein complexes), protein-lipid complexes, and phospholipids. It is not limited. Domains, domain fragments of these macromolecules Target. The target macromolecule can be of mammalian (eg, human) or non-mammalian origin (eg, Bacteria, fungi, viruses, protozoa).   Examples of target macromolecules that are proteins (polypeptides and peptides) include intracellular information. Signaling proteins and their domains (eg, SH3 domain, SH2 domain, PH domain) (Cohen, GB, et al., Cell, 80:23). 7-248 (1995)), chemokines (e.g., α-chemokines, β-chemokines). Mokine) (Clore, MG, et al., FASEB J., 9: 5). 7-62 (1995)), cytokines (eg, IL-1, TNF, phosphorus Photoxin-α, IL-1β, IL-6, M-CSF, TGFα), enzymes (eg, For example, protein kinase C, phospholipase C, phospholipase D) (Dive cha, N .; and Irvine, R.A. F. , Cell, 80: 269-27. 8 (1995)), tyrosine kinase (Marshall, CJ, Ce). 11, 80: 179-185 (1995)), polypeptide growth factors and The domain, growth factor receptor and its domain or fragment (eg, PDGF Family, EGF family, FGF family, IGF family, HFG Family, VEGF family, neurotrophin family, Eph fa Millie, Class I cytokine family, GH family, IL-3 family -, IL-6 family, IL-2 family, Class II cytokine family -, Growth factors and growth factor receptors of the TNF family), (Heldin, C -H. , Cell, 80: 213-223 (1995)); Ase, protein phosphatase, cyclin and Cdc protein (Hun ter, T .; , Cell, 80: 225-236 (1995)), transcription factors. And its domains (eg, Ets domain, bZIP, rel homology domain) , STAT, NF-AT, TCF, Fos, JAK) (Hill, CS An) d Treisman, R .; , Cell, 80: 199-211 (1995). And hormones. Other target polymers used in the present invention Examples include, for example, human CD2, human CD58 (LFA-3), human endothelium. Phosphorus, heregulin-α, human interleukin-1β converting enzyme (ICE), human Macrophage inflammatory protein 1-β, platet factor 4, human Melanoma growth stimulating activity, GRO / melanoma growth stimulating activity, MHC molecules, bacteria Muramidase, kringle domains (eg, plasminogen, apolipopro Tein), ras, ras-GAP, selections ( E-selectin, L-selectin, P-selectin), pleckstrin homologous domain In, stromelysin, thrombin, tissue factor, calmodulin, CD4, cola -Genase, dihydrofolate reductase, fibronectin, fibronectin Type III module, G-protein subunit, vasopressin, IX GLA domain, interleukin-8, (Clore, GM, et al. , Biochemistry, 29; 1689-1696 (199). 0)), thrombomodulin EGF-like domain (Lentz, SR, et. al. , J. et al. of Biological Chem. , 288 (20): 15. 312-1317 (1993)), GPII_b−III_aCytoplasmic domain (Muir , T .; W. , Et al. , Biochemistry, 33: 7701-77. 08 (1994)), Factor VIIa GLA domain, Factor IX EGF -Like domain (Yang, Y., Protein Science, 3: 1267) -1275 (1994)), human immunodeficiency virus (HIV) protein (for example, HIV protease, integrase, matrix, protein tyrosine phos Phatase, reverse transcriptase, nef, tat, rev, envelope, and other HIV protein, its domain, fragment or scaffold mimic), NH_Two Terminal SH3 domain GRB2, (Witekind, M., et al., Biochemistry, 33: 13531-13539 (1994)), C OOH-terminal SH3 domain GRB2 (Kohda, D., et al., Str. result, 2: 1029-1040 (1994)), P120.^GAPSH3 do Main (Yang, YS, et al., The EMBO Journal) 1, 13 (6): 1270-1279 (1984)), and vascular permeability factors and And vascular endothelial growth factor.   Examples of target macromolecules that are oligonucleotides (RNA, DNA) include HI V RRE (rev response element), HIV Tar, and BCR-AB1 A fusion DNA sequence.   Examples of target macromolecules that are phospholipids include phosphoinositide; phosphoinositide Phosphoinositide 3-kinase; Phosphatidylinositol; Sphatidylinositol 3-phosphate; phosphatidylinositol ( 4,5) bisphosphate; phosphatidylinositol (3,4,5) tripho Phosphate; phosphatidylcholine; phosphatidylethanolamine; Inazitol (1,4) bisphosphate; inositol (1,4) , 5) triphosphate; diacylglycerol; sphingosine; sphingo Synphosphate; sphingosine phosphocholine and ceramide (Divecha) , N .; and Irvine, R.A. F. , Cell, 80: 269-278 (1 995).   In the method of the present invention, a naturally occurring polymer enantiomer (eg, a target polymer Enantiomers of natural or left-right macromolecules identified in libraries Is prepared using routine methods. Enantiomers of naturally occurring polymers Can be prepared through the use of opposite left and right components from naturally occurring (Eg, use of D amino acids for the synthesis of mirror image peptides or mirror image Use of L-nucleic acid for synthesis of gonucleotides). For example, the D used in the present invention -The peptide is chemically synthesized as described in Example 1 and the affinity chromatography It can be purified using chromatography. Other preparations of enantiomers of naturally occurring polymers Methods are known in the art.   The entire enantiomer type of the target polymer or a part of the three-dimensional molecular surface of the target polymer It can be used in the method of the invention. For example, by itself, this domain in the whole polymer Sub-dose of the target macromolecule with a conformation similar to that of the enantiomeric form The main enantiomeric form can be prepared and used in the method of the invention (S Chumacer, T, et al., Science, 271, 1854-1857 ( 1996)). On the other hand, continuous or discontinuous fragments of the enantiomer type of the target polymer Of the fragment resembling the portion of the molecular surface of the entire enantiomeric form of the target polymer It can be prepared on a surface peptide or non-peptide backbone (Tolman , R, L, et al., Int. J. Pept. prot. Res. , 41, 455 (19 93); Muir, T .; W. et al, Biochem, 33, 7701 (1 994); McConnell, S.A. J and Hoess, R .; H, JMB, 2 50, 460-470 (1995); Ku, J et al., Proc. Natl. Aca d. Sci. USA, 92, 6552 (1995); Martin, F .; etc, EMBO J.C. , 5303-5309 (1994); Venturini, S .; Et al., Proteins and Peptides Lett. , 1,70 (1 994); Mutter, M .; Ang. Chem. Int. Ed. Engl. Cochran, A. et al., 24, 639 (1985); G and Kim, P .; S. , Science, 271, 1113-1116 (1996); O'Shea, E. FIG. K. Et al., Cell, 68, 699 (1992); G. FIG. Etc., Na cure, 336, 42 (1988)).   Identification and production of non-natural left-right macromolecules bound to target macromolecules This can be done with several collections of macromolecules in the right image. Stated The method comprises providing a biologically encoded library to a chiral target (or chiral " Decoy ”) is applicable to all conditions used to isolate structures that interact with (Scott, JK and Smith, GP, Science, 2 49, 386 (1990); Devlin, J. M .; J. Et al., Ibid, 249, 4. 04 (1990); E. FIG. Et al., Proc. Natl. Acad . Sci. USA, 87, 6378 (1990); G. FIG. Etc., P rc. Natl. Acad. Sci. , USA, 89, 1865 (1992). Mattheak, L .; C. , Et al, Proc. Natl. Acad. Sci. , USA, 91,9022 (1994)). As stated in the examples Biologically encoded, such as phage display libraries (mirror image phage display) The library can be used in the method of the present invention. Ribonucleotides And deoxyribonucleotides are also chiral recognized by nucleases Due to the inclusion of centers, this approach is useful for RNA and DNA libraries. (Bock, LC, et al., Nature, 355, 5). 64 (1992)). Thus, the type of library for which the present invention is useful is RNA (Eg, SELEX), DNA (eg, a DNA library) and pepti Drive Illary (eg, libraries based on in vitro transcription / translation, And multivalent phage libraries and “peptide on plasmid” libraries Lee). The use of a DNA library in the method of the invention is described in Example 5. It is. The exploration of the large amounts of structural space presented in these libraries is And new ligands for medically important proteins . As described in the examples, the D-enantiomers of naturally occurring (wild-type) macromolecules and And phage that specifically interact with the L-enantiomer were isolated.   Selection of natural left and right macromolecules of a library bound to the enantiomer of the target macromolecule The optional step can be performed in an achiral solvent (eg, water) or a chiral solvent. You. Furthermore, the interaction between naturally occurring polymers and enantiomers is It is unlikely to require additional chiral cofactors. Available options Illustrative examples are set forth in the Examples. Modification of the selection step may be performed by a method known in the art. Method.   In a specific embodiment, the ligand is for a naturally occurring L amino acid peptide. D amino acid peptides can be identified by the claimed method. Such a D-ami No acid peptides can be produced in the same manner as L-amino acid peptides. (Except that the constituent amino acid is D and not the L enantiomer), or Substitution, deletion of one or more constituent amino acids to produce derivatives of D amino acid peptides Can be altered by loss or modification or by the addition of one or more D amino acids. Wear.   In another embodiment, an L nucleic acid sequence that binds to a D nucleic acid sequence or an L amino acid peptide The columns are identified in the manner claimed. The L nucleic acid sequence is produced consistent with the D nucleic acid. (Except that the constituent nucleotides are L nucleotides) or Substitution of one or more compositional L nucleotides to produce a derivative of the selected L nucleic acid sequence; It can be modified by deletion or modification or addition of one or more L nucleic acids. You.   Using the methods described herein, the non-natural left-right image Modification of macromolecule A method for producing a derivative can also be included in the present invention. As used here, The term "derivative" refers to, for example, a target identified by the methods described herein. Non-natural left by binding to macromolecules and by the addition, deletion, substitution or modification of one or more components Includes non-natural left- and right-view macromolecules modified in a manner different from the right-view macromolecules.   In one aspect, the derivative is a modified D-peptide. D of the present invention -A derivative of a peptide is, for example, modified or altered (eg, increased, decreased) Affinity, specificity, membrane permeability, hydrophobicity, lipophilicity, oral bioavailability and And / or D-peptides having a biological half-life. Of D-peptide derivatives Manufacturing strategies include, for example, modifying the peptide backbone by N-methylation. (Ostresh, JM, et al., Proc. Natl. Acad. Sc. i. USA, 91, 11138-11142 (1994); Drug Dis. coverage Technologies. , C.I. R. Clark. eds. , John Wiley & Sons, (1990)) and / or pseudopeptides (Cho, CY et al., Science, 261, 1303 (1) Moran E. 993); J. Et al., K Biopolymers, 37, 21 3-219 (1995)).   On the other hand, the side chain of D-peptide induces unnatural amino acids and amino acid analogs (Combs, AP, et al., J. AM. Chem. S. oc. , 118, 287-288 (1996); E. FIG. et al, Proc. Natl. Acad. Sci. , USA, 93, 2031 (1 995); Munroe, J .; E. FIG. Etc., Bioorganic & Medic inal Chem. Lett. , 5, 2897-2902 (1995)). Special In a particularly preferred embodiment, the D-peptide comprises a non-naturally occurring side chain. Modified side An example of a derivative of a D-amino acid peptide having a chain is represented by the following formula: NH_Two-CHR-C OOH Wherein R can be linear or branched here, and one or more An alkyl group of about 1 to about 50 carbon atoms, which can contain double or triple bonds; A lower alkyl group (eg, methyl, ethyl). The lower Aki (Akyl) is -NH_Two, -OH, aryl (eg, phenyl), hetero Aryl (e.g. imidazole, indole), -COOH (e.g. (Nin) group and the like. In addition, lower Akill (E.g., phenyl, naphthyl), substituted aryl (e.g., -OH, halogen) ), A heteroaryl or substituted heteroaryl group.   Furthermore, the D-peptide of the present invention can be modified by generating cyclic and polycyclic derivatives. Can be decorated, for example, where the disulfide bond is a D-peptide Substitutions to optimize and limit the formation (Katz, BA, et al., J. Am. Chem. Soc. , 117, 8541 (1995); Ladner.   R. C. , TIBTECH, 13, 426-430 (1995)). For example, D-amino acid peptides identified as described herein are linked via peptide bonds. To form a ring as in cyclosporine.   Furthermore, derivatives of the D-peptides of the invention can be used, for example, in liposomes and / or Are lipid derivatives (Eichholtz, T. et al., J. Biol. Chem., 26 8 (3), 1982-1986, 1982 (1993)) and / or pep Tide and non-peptidic polymers (Drug Discovery Tech) nologies, C.I. R. Clark, eds. , John Wiley & S ons, (1990); Et al., Proc. Natl. Aca d. Sci. , USA, 92, 2056 (1995)). Conjugation or incorporation into a support to improve bioavailability Can be.   Further, the D-amino acid peptide identified by the method described herein can be: Especially when the target is an intracellular target, increasing the hydrophobicity of the D-amino acid peptide Thus, it can be derived (e.g., alkylation, e.g., D-amino acid Methylation of the peptide backbone). Benzene rings are attached to side chains to increase hydrophobicity. Can be added. On the other hand, D-amino acid peptides increase the hydrophobicity of the peptides. It can be incorporated into drug delivery systems such as liposomes for addition.   Further, the C- or N-terminus of the D-peptide can be modified with a protecting group. (Eg, Green, TH and Wuts, PGM, Prot) active Groups in Organic Synthesis, Chapter 2nd Edition, John Wiley & Sons, New York (1991)). For example, a lipophilic polymer can be attached to the N-terminus or C-terminus (eg, , C-terminal polyethylene glycol ester). On the other hand, one or more side chains are For lipophilic polymers such as alkylene glycol (eg, polyethylene glycol) Bonds or ether or ester bonds (eg, Forms an ester with the side chain carboxyl group of aspartic acid or glutamic acid) . For example, D-peptide produces a polymer-D-peptide conjugate Wherein the polymer is, for example, monomethoxy Polyethylene glycol (PEG) and / or polyoxyethylated glycerol (POG) (Therapeutic Peptides and P proteins, Marshak, D.M. and Liu, D.C. eds. , Col d Spring Harbor Laboratory (1989)).   In another aspect, the derivative is an L-nucleic acid. D-peptides described herein Strategies to modify are to optimize their pharmacological properties, Non-natural left-right modified RNA and DNA identified as described herein (Eg, Green, LS, et al., Chem. Biol). . , 2,683 (1995); Latham, J. et al. A. Nucleic A cids Res. , 22, 2817 (1994); Gold, L .; Etc., Ann . Rev .. Biochem. 64, 763 (1995)).   Synthetic and biologically encoded libraries provide libraries for a variety of macromolecules. It has proven to be extremely useful for the identification of gand and nucleic acid sequences. Pep obtained Is insensitive to proteolytic cleavage and does not induce an effective immune response Therefore, a synthetic peptide library composed of (D) -amino acids is available from Scott , J. et al. K. And Smith, G .; P. , Science, 249, 386 (1 990); Devlin, J. et al. J. Et al., Ibid, 249, 404 (1990). Cwirl, S .; E. FIG. Et al., Proc. Natl. Acad. Sci. , US A, 87, 6378 (1990); @peptide on plasmid ', Cull, M .; G. FIG. Et al., Proc. Natl. Acad. Sci. , USA , 89, 1865 (1992) and Mattheak, L .; C Proc. Natl. Acad. Sci. , USA, 91, 9022 (1 994), a gene-based technique such as an in vitro translation-based system is preferred. New In addition, peptides composed of (D) -amino acids can be absorbed in the gastrointestinal tract (Pappenheimer, JR et al., Proc. Natl. Acad. S. ci. USA 91, 1942 (1994)). Mainly N-methylated And cyclospor, an 11-residue cyclic peptide composed of (D) -amino acids Phosphorus A is an immunosuppressive inducer and is generally administered orally (Ptachc ins, R.S. J. Et al., Clin. Pharmacokinetics, 11, 1 07 (1986)).   Screening of (D) -amino acid libraries has recently shown analgesic activity Leading to the identification of peptides (Dooley, CT, et al, Science e, 266, 2019 (1994)). The percentage of available sequence space is generally determined through the use of biologically encoded systems. Only the fraction of what can be obtained. Because of this low degeneracy, and Importantly, because of the lack of the intermediate amplification process, The use of synthetic libraries is not necessarily as successful as phage display libraries. Not necessarily.   Proteins composed of (D) -amino acids are naturally occurring (L) -amino acid proteins. Has chiral specificity for substrates and inhibitors that are exactly opposite to those of the protein (Del Milton, RC, et al., Science, 257, 1445) (1992); Petsko, G .; A. Ibid, 256, 1403 (1992) ); Zawadzke, L .; E. FIG. and Berg, J.M. M. , J. et al. Am. Ch em. Soc. , 114, 4002 (1992)). In certain instances, (D) The amino acid ligand is formed by forming the (D) -enantiomer of the natural ligand (Fisher, PJ, et al., Nature, 368, 651 (1994)). ) Or by making the "reverse" (D) -enantiomer (Jameson , B. A. Et al., Nature, 368, 744 (1994); Guptasar. ma, TIBTECH, 14, 42-43 (1996)). However, such a method has no general applicability (Brady, L. et al. and Dodson, G, Nature, 368, 692 (1994); Ch. ovev, M .; and Goodman, M .; , TIBTECH, 14, 43- 44 (1996); Guichard, G .; Etc., TIBTECH, 14, 44- 45 (1996)).   A more general method for obtaining (D) -amino acid ligands is to use the desired protein Using the (D) -enantiomer of a protein from a biologically encoded library Must be the choice of the peptide. (D)-and (L) -proteins are Due to having exactly the chiral specificity for the opposite substrate and inhibitor, the (D) -tan Types of (D) -enantiomers of peptides displayed on phage that interact with protein Will interact with the natural left-right protein.   The effectiveness of this approach is dependent on the SRC homology 3 domain (SH3 domain). Demonstrated by the isolation of phage that interact specifically with the D-enantiomer. Example As described in 1 above, from the phage library, (D) -amino acid ligand Use for the mirror image relationship between (L)-and (D) -proteins for identification To test that is possible, the SH3 domain of c-Src tyrosine kinase (D) -Amino acid version was synthesized. SH3 domain is an intracellular effector Is a 55-70 amino acid protein domain found in the diversity of chlessinger, J .; , Curr. Opin. Genet. Dev. , 4, 25 (1994)). Bone whose c-SRC activity mediated osteoclasts Disruption of SRC function is valuable in the treatment of osteoporosis because it is essential for resorption (Soriano, P et al, Cell, 64, 69) 3 (1992); Lowe, C.I. Proc. Natl. Acad. Sci. , ScL USA, 90, 4485 (1993); F. , Science ce and Medicine, 2, 48 (1995)). SH3 domain In their intracellular target forming a type II polyproline helix of 8-10 residues (Rickles, RJ, et al., EMB) OJ. , 13, 5598 (1994); Sparks, A .; B. , J. et al. Bio l. Chem. , 269, 23853 (1994): Beadle, C .; et   al, Ibid, 269, 24034 (1994); Yu, H .; , Et al . , Cell, 76, 933 (1994); Feng, S .; , Et al. , S science 266, 1241 (1994); A. et al. , Nature, 372, 375 (1994)). Intracellular cell signaling tan Mediating protein interactions in proteins and proteins containing SH3 domains Interference with quality signaling would be desirable. For the diversity of SH3 domains Ligand and substrate were isolated from a phage display library (Rickl es, R. J. , Et al. , EMBO J .; , 13, 5598 (1994). Sparks, A .; B. , J. et al. Biol. Chem. , 269, 23853 ( 1994); , Et al. , Ibid, 39, 2403 4 (1994)), from a synthetic (L) -amino acid peptide library. Identification of such sequences is only possible with previously known sequences of preferred ligands (Yu , H .; , Et al. , Cell, 76, 933 (1994)). in this way, Equivalent of (D) -amino acid ligand to SH3 domain from synthetic library Would not be successful without the sequence or structural information of the possible ligand U.   The L- and D-enantiomers of the chicken c-SRC domain, Prepared by physical expression and chemical synthesis. Biotinylated synthetic 60 amino acids The D-SH3 domain is refolded and the known pepti against the SH3 domain Affinity chromatography with doligand D-amino acid version (Yu, H., et al., 76, 933 (1994)). As expected, bacterially expressed L-SH3 is the L-enantiomer of this peptide. Remained on the affinity column with isomers but with D-enantiomer Did not remain. This indicates that the interaction between the SH3 domain and its substrate is stereospecific. Show that   The phage library contains 10-residue peptide sequences at random NH of pIII protein of ophage fd_Two-Constructed to be expressed at the end (Scott, JK and Smith, GP, Science, 249,386 (1990)). Immunosuppressant cyclosporine and cancer promote Many natural bioactive peptides, such as termicrocystin, are cyclic and The library contains many sequences with properties for disulfide bond formation. Designed (Smith, GP and Scott, JK, Meth ods Enzymol. , 217, 228 (1993)). L-SH3 Domain Screen a phage display library for interacting peptide sequences. Disulfide-free ports identified by others when used for Liproline-type sequences were isolated (Yu, H., et al., Cell, 76, 9). Rickles, R. 33 (1994); J. , Et al. , EMBO J .; , 13, 5598 (1994); Sparks, A .; B. et al. , J. et al. B iol. Chem. , 269, 23853 (1994); , Et al. , Ibid, p. 24034)).   When the same phage display was screened with the D-SH3 domain, L-SH A series of peptide sequences that did not show an obvious sequence similar to the three binding sequences were isolated. , Grouped into three classes (Table 1). All of these peptides, Since it was eluted with the (D) -YGGRELPPLPRF peptide (SEQ ID NO: 2), Interacted with the substrate binding site of the SH3 domain. Binds to D-SH3 domain The peptides displayed on these phages contain conserved leucine and glycine Characterized by a combination of residues and conserved arginine or lysine residues Attached. In contrast to the L-peptide ligand for the L-SH3 domain (Y u, H .; , Et al. , Cell, 76, 933 (1994); Rickle. s, R.S. J. , Et al. , EMBO J .; , 13, 5598 (1994); Sparks, A .; B. , Et al. , J. et al. Biol. Chem. , 269, 23,853 (1994); , Et al. , Ibid, p . 24034)), the positively charged residue in the ligand for the D-SH3 domain The group is located in the middle of the conserved residue range, which means that the form of ligand binding is Suggests a difference in morphology. In addition, all for the D-SH3 domain Ligands contain a pair of cysteine residues and interact with the L-SH3 domain Including properties not observed in L-peptides (Yu, H., et al., Cel. 1, 76, 933 (1994); J. , Et al. , E MBOJ. , 13, 5598 (1994); Sparks, A .; B. , Et al. , J. et al. Biol. Chem. , 269, 23853 (1994), Che. adle, C.I. , Et al. , Ibid. , P. 24034)). Disulfi Binding, the affinity of these peptides for the D-SH3 domain is May be increased by reducing the number of cases.   The D-amino acid enantiomer of the peptide expressed by these phage particles The conformation that interacts with all L-amino acid SH3 domains is standard And detection methods. As described in Example 2, D-SH3 Pep-, a mirror image of one of the peptides displayed on the phage that binds to the domain L-SH3 domain synthesized from D-peptide designated D1 and expressed in the bacterium The interaction with was investigated. (D) -SH3 domain is a group of (L) -SH3 domain Use an indirect binding assay to demonstrate binding to the Was.   Binding of the D-peptide in the SH3 domain as described in Example 3. To determine the site, in the absence and presence of Pep-D1^FifteenN-labeled Heteronuclear magnetic resonance (NMR) experiments were performed using the SH3 domain. Pep-D1 Residues in the SH3 domain that interact with Amide¹H or^FifteenIdentified through changes during the N chemical shift.   Ligands isolated through this method will, in all cases, The function that impairs the activity of the target object is significantly reduced or unaffected. For example Ligands isolated by the methods described herein are nucleotide-based ligands. RNase and DNase activities (Ashley, GW, J. Am. Chem. Soc. , 114, 9731 (1992); Urata, H .; , Et   al. , J. et al. Am. Chem. Soc. , 113, 8174 (1991)) Proteolytic degradation of peptide-based ligands and activation of immune responses (Gill, TJ et al., Nature, 197, 746 (1963). Mauer, P .; H. , J. et al. Exp. Med. , 121, 339 (1965). Borek, F .; , Et al. , Biochem. , J. et al. , 96,577 (1965); Janeway, C .; A. and Sela, M .; , Immun logic 13, 29 (1967); Ditzis, H .; M. , Et al. , Proteins, 16, 306 (1993)), or Not affected.   The approach described here for isolating the non-naturally occurring right-and-left ligand involves this All Approaches Used to Introduce Only Natural Isomorphic Ligand Isolation Only for the isolation of oligonucleotide-based ligands Things. As a result, such "conventional" ligands are naturally occurring enzymes Affected by decomposition. In contrast, synthetic peptides composed of D-amino acids Synthesis and screening of a library is technically possible. But However, many screens can be screened in biologically encoded library systems. Number of compounds (several orders of magnitude higher than can achieve a synthetic peptide library) The technique described here, due to both the beneficial effects of the intermediate amplification steps used for the When combined with, such systems produce excellent results.   In particular, strategies based on synthetic peptides have limitations in peptide solubility and Assays used (libraries of synthetic combinations and other solution-based protocols) (For peptide libraries), or based on solid phase by considering volume With an upper limit of degeneracy determined by the detection limit of any of the libraries. Second, the intermediate amplification process used for the biologically encoded library system In situations where background binding is high (if it is the first screeni If it constitutes an easily detectable part of the total pool of molecules recovered after Only a specific ligand is identified in a system that does not carry out the amplification process). Allows identification of gand. Third, phage display and other biologically encoded The library system that is used is an error-prone PCR or the like. Allows maturation of affinity by mutation of the encoding DNA. Most Later, phage libraries were compared to libraries based on synthetic peptides. , For inserts of significant length. This is a different class of Riga of different size The short ligand contained within these inserts allows for possible isolation of the The complexity of the sliding (sliding windows) Significantly increased).   Interacts with the specific targets described herein, as described in Example 4. Identification of D-amino acid peptide ligands was biased toward (D) -amino acid peptides and To provide guidelines for designing libraries based on peptides and peptides used. The library was then used to isolate new ligands.   The present invention is further illustrated by the following examples, which are not limited to any method. Does not meanExample 1   Identification of phages that specifically interact with D-amino acid peptides Preparation of L-SH3 domain   The residue numbered system is the full length of the chicken c-SRC protein. Chick Residues 81-140 of the avian c-SRC were replaced with the plasmid pMMHb (Staley , J. et al. P. And Kim, P .; S. , Protein Science, 3:18. 22 (1994)) at another Hind III-Bam HI site. I did it. In this plasmid, the methionine residue is replaced by leucine, The stain residue is replaced with alanine (Staley, JP and Kim, P. et al. S. , Protein Science, 3: 1822 (1994)), and Trp with 9 histidine residues added at the carboxyl terminus of the leader sequence The protein is expressed as a fusion protein with a modified LE leader sequence. 600n When the absorbance at m is 0.6, 0.4 mM isopropyl-β-D-thiogalact Topylanoside (IPTG) (Research Organics) was transformed into E. coli BL21- (DE 3) The plasmid pMMHb-S was added to pLys S cells (Stratagene). Expression of the fusion protein encoded by RC SH3 was induced. Next to 2 hours guidance process The cells were then centrifuged to isolate inclusion bodies. Recombinant protein, inclusion bodies, 6 Resuspended in M guanidine-HCl, 0.2 M Tris, pH 8.7 (buffer A) Ni²⁺Column (Ni²⁺-NTA-agarose; Cuiagen) The product was purified by attaching it to a column. After elution, dialyzed against water and lyophilized The fusion protein was dissolved in 70% formic acid and cleaved with CNBr (Stanley). , J. et al. P. And Kim P.S. S. , Protein Science, 3:18. 22 (1994)). The dialyzed and freeze-dried one is then dissolved in buffer A And Ni²⁺Purified by chromatography on a column (after cleavage) In this case, the isolated SH3 domain passes through this column, while The uncleaved fusion protein and the truncated TrpLE leader sequence are retained). Perform dialysis (against low ionic strength PBS buffer and finally against water) And then lyophilized and analyzed by high performance liquid chromatography (HPLC) at neutral pH. Analysis and laser desorption mass spectrometry (theory is 6686 daltons; measured The value was 6,683 daltons), confirming the purity and identity of the SH3 domain.Synthesis of all D-Src SH3 domains   All D amino acids SH3 domain, sequence S-COOH-terminal (SEQ ID NO: 1), residues 81- of the chicken c-SRC protein 140, the ABI 431A peptide synthesizer and the ABI fast mock cycle (The properties of Fmoc are activated by HBTU and capped with acetic anhydride) Was synthesized on HMP resin (ABI / Perkin Elmer) using protected D-amino acids are available from Bachem California, Bachem Bioscience, Adva Obtained from Sudochemtech and Nova Biochem. D-Ile and D-Thr As for the side chains, those having a mirror image are used, and the chirality of the side chains is also natural L- Thr and L-Ile are opposite. After the synthesis is completed, the amino terminal The ends were modified with NHS-LC-Biotin II (Pierce). After cutting, the pepti Is freeze-dried and dissolved in 6M guanidine hydrochloride at pH 6.0. Using a dialysis tube having a molecular weight of 3500 daltons (D) (Spectra / pore), 100 mM NaHPO at pH 6.0_FourDialysis against 100 mM NaCl Was. After dialysis, this is centrifuged for a short time to remove insoluble residues. Afterwards, the supernatant was dialyzed against 5% acetic acid and lyophilized. This peptide was A salt solution containing otine and buffered with Tris (50 mM Tris, pH 7.0). 5, 150 mM NaCl) to give a concentration of 3.3 mg / mL. all The biotin treated, 60 amino acid long (D) -SH3 domain product And all of the known substrates for the Src SH3 domain are in the (D) form (D) -YGGRELPPLPRF (SEQ ID NO: 2), which is biotinylated And immobilized on a streptavidin-agarose column (Pierce). Purification was carried out by affinity chromatography (Yu, H . Et al., Cell, 76: 933 (1994)). This peptide is all (L) All are derivatives of the (L) -peptide that show binding to the SH3 domain (Yu, H. et al., Cell, 76: 933-945 (1994)), amino powder. YGG was added to the end to facilitate concentration determination (H. Edelhoch) , Biochemistry, 6: 1948 (1967)). Similarly, amino powder The L-peptide with YGG added to the end is useful as an experimental control ligand. Was for As expected, bacterially expressed (L) -SH3 (chicken) (Residues 81-140 of c-SRC were expressed) were retained on the (L) -enantiomer. Although not possible on the (D) -enantiomer of this peptide, This indicates that the interaction of the SH3 domain with its substrate is stereospecific. Things.   Chromatographic fractions are collected on a Voyager mass spectrometer ( Laser desorption mass spectrometry by Perceptive Biosystems) Analyzed. Of expected mass (theoretical 7027 dalton; actual 7 027-7035 daltons) and collect against water for 72 Dialyzed for hours, lyophilized, and dissolved in water to a concentration of 107 μg / mL. did.Production of phage library   The phage library is a fusion of the amino terminus with the filamentous phage pill protein. It was designed to randomly express the peptide as a product. Typically, the file There are 3-5 copies per page particle and weak / medium affinity ligand It enabled isolation.   (Smith, GP and Scott, JK, Methods Enz. ymol, 217: 228 (1993)). With 10 residues inserted randomly between serine or cysteine (S / C-X)_Ten-S / C) (SEQ ID NO: 20) has 85 residues (Smith, G., "Cloat"). ning in Fuse vectors ", Division of Bi logical Sciences, University of Miss ouri (February 10, 1992 edition)).   Insert design is: NH_Two-ADGGAS / CX_Ten-S / CG- AGA-PIII (SEQ ID NO: 3). 85 residue oligonucleotide: Where S is C / G         N is A / T / C / G (Equimolar mixture) After purification of the PCR product and cleavage with Bgl I, it was coated with streptavidin. The end portion was removed using agarose beads (Pierce). Then P Precipitate the CR product with ethanol and swim on a polyacrylamide gel Analyzed by motion.   Ligation of random PCR products into Sfi I-cut Fuse5 vector This library was prepared by the following procedure. After ligation, the reaction mixture Is extracted with phenol and chloroform, precipitated with ethanol and 10 It was dissolved in mM Tris / 1 mM EDTA (pH 8.0). Then, by Using an Orad E. coli pulsar and a 0.1 cm cuvette, Transfer the ligation product to Bio-Rad electrocompetent cells Was. After one hour of non-restrictive cultivation, the cells are plated on a plate containing tetracycline. A portion of the transformed cells were placed and the efficiency of the transformation was examined.⁸Pieces An initial library of transformants was obtained. The transformation mixture was then diluted, Culture in 400 mL LB containing μg / mL tetracycline, The culture was continued for a while. Continuously use the above culture supernatant with polyethylene glycol (PEG) And precipitated twice to prepare a phage stock. Finally, this phage Stocks were washed with Tris buffered saline / NaN_ThreeResuspended in K91 -Titer was determined by infection of kan cells (A21), a total of 2.8 x 10¹¹Trait Conversion units were obtained. By examining the sequence of individual clones, you can eliminate insertion irregularities. confirmed. Then 4 × 10^TenOf K91-kan cells using the transformed unit And amplified the library. After 18 hours, precipitation with PEG is repeated three times To purify the phage, TBS / NaN_Three10 mL of amplified phage Library was obtained (1.2-10¹²Transformation units / mL). 1) It is a lectin Concanavalin A (ConA) (Oldenburg, KR et al., Proc. Natl. Acad. Sci. USA, 89: 5393 (1992); Scot. t, J. K. 5398; 2) a mouse MHC class I heavy chain Species mouse monoclonal antibodies; and 3) bacterial Src SH3 domain By selecting phage that express the insert that interacts with the expression, I confirmed the quality of the library. All screens show expected results Was.Screening at ConA   After the third time Screening of phage library   D-SH3 domain and a series of peptide sequences, these sequences are L-SH3-linked The phage display does not show clear similarity to the sex sequence And isolated (see Table 1).   96-well high binding E.E. I. A. / R. I. A. Plate (Costa -) Of 100 μL of 100 mM NaHCO_Three10μg in Of streptavidin (Pierce) at 4 ° C. overnight. Wash once with water After cleaning, the wells were treated with 100 μL (10.7 μg) of biotinylated (D) -SH3. Both were incubated at 20 ° C for 1 hour and dialyzed at 30mg / mL 100 mM NaHCO of bovine serum albumin (BSA)_ThreeBlow with solution for 2 hours And treated again with 100 μL (10.7 μg) of biotinylated (D ) Incubation for 1 hour with -SH3. 5 mM biotin, Add 8 μL of salt solution (TBS) buffered with squirrel and add ligand for 30 minutes Blocking of streptavidin not bound to was performed. Then, phosphoric acid The wells were buffered with a saline solution (PBS) and buffered with 0.1% Tween-20. Washed twice, 50 μL of TBS / NaN_ThreePhage stock and 50 μL of T BS, 0.1% Tween-20, 1 mg / mL BSA and 0.05% NaN_Three And overnight. Then, when the selection procedure comes later Under conditions of longer incubation times, 200 μL of TBS, 0 . The wells were washed by adding 1% Tween-20, 1 mg / mL BSA six times. Was. Then, 100 μL of the D-SH3 ligand peptide, the sequence of which was D-YGGRE LPPLPRF-amide (SEQ ID NO: 2), (715 μM) were added, and The phage was brought to a final peptide concentration of 700-1000 μM for 5 minutes. (D) -SH3 bound to the particles was eluted. Phage in this screen Due to the acid elution, selective binding to D-SH3 coated wells , Cannot be detected after the fourth selection. This eluate was then used for K91-kan cells. Used for infection. 100 μL of eluate was added to 100 μL (prepared above) Gently mix with K91 Terrific Broth cells and incubate at room temperature for 20 minutes. Was performed. The mixture was then added to 20 mL of LB / 0.2 μg / mL tet Transferred to Erlenmeyer flask containing lacycline. Shake at 37 ° C for 1 hour Perform incubation and add tetracycline to a final concentration of 20 μg / mL. The plate was added to the plate and diluted appropriately, and the plate containing tetracycline (20 μg / Check the eluate titer by spreading on top of Incubation was performed. From the supernatant by two PEG precipitations To isolate the phage stock and determine the yield from the titer. , And for subsequent rounds of selection. In the fourth selection, the phage were -Ink in wells coated with SH3 domain or in uncoated wells And the specificity of capture was determined. Washing conditions and yields at different times The quantities were as follows: From these data, 1) the yield was high despite the apparently severe washing conditions. Because of the increase, phages that bind more strongly are selected later. 2) eluted by incubation with substrate for this domain For this reason, and more importantly, the fourth round of phage particle recovery Is at least 20 times higher in the presence of the D-SH3 domain than in the absence ( Yield is higher from plates coated with D-SH3 domains) It is clear that this binding is specific for the D-SH3 domain. Was. Numerous phage particles are present in the presence rather than in the absence of the D-SH3 domain. Held in.   (D) The case where the -SH3 domain was used for screening this library. In this case, a series of peptides were isolated and divided into three classes. (D) -YGGR These vectors were eluted with the ELPPLPRF peptide (SEQ ID NO: 2). All peptides interact with the substrate binding site of the SH3 domain. Surprisingly, all All peptides interact with a pair of cysteine residues and the (L) -SH3 domain Have properties that are not observed with certain polyproline peptides. This disulfi Binding reduces these peptides by reducing the total number of possible conformers. It seems to increase the affinity of the C-type for the (D) -SH3 domain. Group I and III A group of peptides are those that can form a salt bridge with the aspartic acid 99 of the SH3 domain. Src-binding polyproline peptide having at least one residue of luginin (Feng, S., et al., Science 266: 1241 (1994)).   Conserved residues between the different members of Groups I and III are shown in bold and semi-conserved residues are Shown by lines. For all members of Groups I and III, the Note that the conserved residue positions are preserved. (D) -Src SH3 After the fourth to fifth selection for binding to the main, all three groups of inserts are presented Individual phage clones were analyzed. All clones compared to control wells At least 150 times, 0.5 μg of streptavidin and 1.3 μg of Sr c Binds to wells coated with SH3 domain. Low stringency and high In both stringency screens, after the fourth selection, individual colonies Was analyzed. Scripts of both high and low stringency Learning was based on the same initial selection. High stringency and low The difference between both screens of stringency was due to the That the concentration of (D) -Src SH3 used is two times lower, Short incubation time (16 hours to 1 hour) and plate Long incubation time for 6 washes (low stringency screening) Leaning: 2nd, 3rd and 4th times, 3 minutes, 5 minutes and 10 minutes each; high stringency -: 2nd to 4th times, all for 10 minutes incubation).   Phage display using the L-SH3 domain was also evaluated. (L) -SH3 domestic The phage library (A12) is screened for sequence interactions using When leaning, the polyproline sequences discovered by others are isolated ( The results were as follows, showing that the presentation worked: Decoy After the fourth GCN4 leucine zipper: short (33 residues) but low probability c-Src SH-3 domain: 60 residues, binding to peptide                               (Type II Poly Pro) GCN4 Leucine Zipper: Positively synthesized, expected CD after 5th round                               +/- No difference in zipper recovery                               Test 59 independent clones, +/- zipper                               No difference in Src SH3 domain: Src (L-) SH3 is a substrate in a stereospecific manner                               Join to                               Src (L-) SH3 was obtained from the library                               Select Pro sequence   After a fourth selection using the L-SH3 domain, sequence analysis of a small number of isolates Clarified the following two peptide sequences: CLARSRLPAIPS SEQ ID NO: 10) (9 isolates) and SRMSPLVPLRNS (SEQ ID NO: 2) 1) (1 isolate). The sequence of these peptides is the class I of the SH3 domain. And features consistent with those described for class II ligands ( Analysis of single phage clone   Obtained after 4 rounds of selection to analyze the specificity of this interaction more precisely Six clones were grown in LB / 20 μg / ml tetracycline. Fa Particles were isolated from 1.3 ml of supernatant by PEG precipitation and 500 μl TB Resuspend in S, estimated concentration 6.5 × 10^TenTransformation units / ml were obtained. 0.2μ 1 part (about 1.3 × 10⁷TU) is coated as described above, having a streptavidin of 0.5 instead of 1; 50 μl TB in wells with or without 1.3 μg D-SH3 S / 0.1% Tween incubated in 20/1 mg / ml BSA . After incubation with the phage, unbound phage was collected at 6 (3 minutes). 150 μl of TBS / 0.1% Tween 20/1 mg / ml BS Washed with A. The bound phage particles were then treated with 40 μl glycinecin HCl Elution was performed at 4 ° C. for 10 minutes using pH 2.2 / 1 mg / ml BSA. Eluate To neutral pH and then drop onto K91 kan cells as described above. Specified.   After 4 and 5 rounds of selection, individual clones were analyzed. At the next round, Increased incubation time (between 0, 3, 5, 1 and 5 times, respectively) 0 and 10 minutes time). After four rounds of selection, 29 clones were sequenced. , Of which only seven correspond to Group III in Table 1. chosen Phage is not bound to streptavidin or streptavidin- Composite table formed by streptavidin formed by D-SH3 complex Use the fifth selection as a matrix to ensure that it is not Using neutravidin (Pierce) I went. Sequence analysis of the clones after this fifth selection was performed using the fdSRC-2- Only the sequence of Ip is shown. Preliminary studies were performed on the parent of the corresponding peptide, Pep-D2. This suggests that the compatibility is similar to that of Pep-D1. Pep-D1 is fdS Similar to RC-1 inserted CLSGLRLGLVPC (SEQ ID NO: 16) (Table 1), Has the COOH-terminal alanine present in all flanking sequences ( See Example 2). Other phage isolates obtained after the four rounds of selection were: One of the sequences was expressed: CKRFVWRGQALC (SEQ ID NO: 13) (10 Isolate) and CWYLGYWPGQEC (SEQ ID NO: 15) (12 isolates) Stuff). The beginnings of these sequences are based on the background isolated using various biotinylated ligations. It is similar to the arrangement of the grounds (Smith, GP and Scott, JK. , Methods in Enzymol. , 217: 228 (1993)), Previously monochrome against myohemerythrin It is similar to the sequence isolated using a null antibody, but Not similar to recognition motifs (Smith, GP and Scott, JK . , Science, 249: 386 (1990)). Therefore, this array has S It is likely to bind some components in systems other than the H3 domain. In fact, The D-amino acid version of this sequence, as judged by ELISA and NMR experiments, Cannot bind to L-SH3 domain. Other sequences taken after the four selections begin. Shows limited similarity to the sequence and has not been investigated further.Example 2   The entire D amino acid Src SH3 domain is the L Src SH3 domain. Join   In one mirror image of a phage displayed peptide that binds to the D-Src SH3 domain (D) -amino acid peptide showing Pep-D1, (D) -RCLSGLR Synthesis of LGLVPCA (SEQ ID NO: 11, a representative sequence of group III sequence) And tested its interaction with the bacterially expressed (L) -SH3 domain. Pep-D1 is CLSGLRLGLVPC with fdSRC-1 insertion (SEQ ID NO: 1). 6) COOH terminus similar to (Table 1) and present in all flanking sequences It has alanine. Arginine immediately before the first cysteine residue was replaced with fdS Observed in the RC-3 sequence (Table 1). NH of secreted and transmembrane proteins_Two Presence of arginine and lysine residues close to the ends is negative for protein migration (Boyd, D. and Beckwith, J., Cell, 62: 1031 (1990)). In addition, the NH of the phage pill fusion_TwoEnd Selection for partial arginine residues was observed (Cunningham, B. C. et al. , EMBO J .; , 13: 2508 (1994)). Follow In this clone, the mutation from alanine to arginine was detected in the D-SH3 domain. Can increase the affinity of the inserted sequence for the peptide and improve the solubility of the peptide. Therefore, it was included in the synthetic peptide. For affinity measurements, NH_TwoEnd Terminal D-tyrosine was added to the peptide for densitometry (Edelhoch, H. Biochemistry, 6: 1948 (1967). NH_TwoTerminal Chiro 100 mM Tris, pH 8.5, 1 m with and without peptide Air oxidation at a concentration of g / ml for 48 hours. The oxidized peptide is subjected to reverse phase HPLC. Gives C₁₈Water-acetonitrile gradient in column and 0.1% trifluoroacetic acid And purified using The identity of the product was confirmed by laser desorption mass spectrometry.   Reduced form of Pep-D1 shows no detectable binding activity in this assay ( K_d>> 800 μM), which indicates that disulfide formation is required for effective binding. Is shown. Compete for affinity of Pep-D1 for L-SH3 domain Determined by a competitive enzyme-linked immunosorbent assay (ELISA). 96 Welp One well of the rate was coated with 5 μg of L-SH3 domain (Scott , J. et al. K. And Smith, G .; P. , Science, 249: 386 (1 990); Smith, G .; P. And Scott, J. et al. K. , Methods   in Enzymol. , 217: 228 (1993)). Well is BSA Blocked, L-SH3-linked insertion CLARSRLPAIPS (SEQ ID NO: 1) Phage expressing 0 mM NaOH in the presence of increasing amounts of competitor peptides. HPO_FourPH 7.2, 15 mM NaCl, 1 mg / ml BSA, 0.05 % NaN_Three, And 0.1% Tween 20. Phage binding was with rabbit M13 antibody (Stratagene) Phosphatase-labeled goat antibody against rabbits and rabbits (Pierce) Quantified using a fresh solution of p-nitrophenol phosphate as substrate . 410 nm absorbance was measured using a Dynatech microtiter plate reader. Were determined. L-peptide ligand YGGRELPPLPRF amide (SEQ ID NO: 2) and the D-peptide ligand Pep-D1 YRCLSGLRLGLVPC A (SEQ ID NO: 22) in the presence and absence of 25 mM dithiothreitol A titration curve (three-point method) was obtained in the presence. K_dThe relative values for were obtained as described above. (Minor, DL, Jr. and Kim, PS, Nature, 36. 7: 660 (1994)). L-peptide YGGRELPPLPRF-amide ( K in SEQ ID NO: 2)_dTryptophan fluorescence spectroscopy directly to 6.0 μM Determined by The peptide solution was diluted with 15 mM NaCl and 10 mM NaHP. O_FourWas titrated into a 1 μM SH3 solution in pH 7.2. Tryptophan fluorescence Induction by excitation at 295 nm (5 nm slit width), Hitachi F-450 Emission was measured at 339 nm (10 nm slit width) using a 0 fluorescence spectrometer. Solution The separation constant was determined by Scatchard analysis.   Lubredoxin (45 amino acids) and human immunodeficiency virus (HIV) pro The synthesis of both (D) -enantiomer forms of thease (99 amino acids) is by Del Mi Iton, R .; C. , Et al. , Science, 256: 1445 (19 92); Petsko, G .; A. , Ibid, 256: 1403 (1992); Zawadzke, L .; E. FIG. And Berg, J .; M. , J. et al. Am. Chem. Soc. , 114: 4002 (1992); Zawadzke, L .; E. FIG. and Berg, J.M. M. , Proteins, 16: 301 (1993)). Most proteins due to size limitations on the prospects of successful chemical synthesis. The synthesis of the full (D) -enantiomer form of white matter is not possible. However, Both intracellular and extracellular proteins are often autonomous domains of less than 100 amino acids. See). This size range is compatible with mobile solid phase peptide synthesis technology and chemistry. Of unprotected protein fragments within the scope of recent advances The ligation strategy promises to synthesize even larger protein domains ing. Therefore, resources for interesting proteins (eg, multi-domain proteins) Isolation of Gand was performed using its constituent domains as described herein for the SH3 domain. It may be achieved through synthesis and screening of one of the main.Example 3   Determination of D-peptide binding site in SH3 domain   Heterogeneous nuclear magnetic resonance (NMR) experiments were performed using this D-peptidic protein in the SH3 domain. To determine the binding site of Pep-D1 in the presence and absence of Pep-D1^FifteenN- Performed on labeled SH3 domains. SH3 interacting with Pep-D1 Residues in the domain are converted to amides upon addition of the D-peptide ligand.¹H Or^FifteenIdentified through changes in N chemical shifts.   Ligand-binding portion of SH3 domain for its natural L-amino acid ligand The position consists of three pockets, both of which form a relatively shallow groove on one side of the molecule ( ). Pocket A is aspartic acid⁹⁹And tryptophan¹¹⁸Formed by the side chains of To provide the converted arginine residue, and pocket B and pocket C Hydrophobic surface adapted to aliphatic and proline residues in SH3 ligand To form ( ).   Uniform (≧^Three95%)^FifteenThe N-labeled SH3 domain is^FifteenNH_Four)_TwoSO_Four E. coli harboring plasmid pMMHb-SRC SH3 in M9 medium supplemented with Obtained by growing (99.7%^FifteenN; Isotec, Miamisb urge, Ohio). Until the cells reach an absorbance of 0.6 at 600 nm, the cells are treated with 0.4 Induction was performed for 4 hours with mM IPTG. Protein as described for unlabeled material Was purified. Spectra on Bruker AMX 500 MHz NMR spectrometer collected. The resonance budget is determined using standard methods ( (Yu, H. et al., FEBS LETT., 324: 8). 7 (1993)). Peptide Pep-D1 was treated with 10 mM phosphate, pH 6.0. During 0^Fifteen1.5: 1 (peptide: protein) in solution containing N-labeled SH3 domain White matter) at 298 K; heteronucleic acid single quantum binding (HSQC) spec. Ktor (Bodenhausen, G. and Rubin, DJ, Chem. . Phys. Lett. , 69: 185 (1980). Compared.¹0.04 p. p. m. Greater than Is^Fifteen0.17 p. p. m. Greater chemical shift differences There was no resonance. However, many resonances decrease in intensity and Or the complex was completely absent in the HSQC spectrum. Ligand-f HSQ, which significantly reduced Pep-D1 binding compared to the Lie spectrum Residues with the intensity of the C resonance were identified in the following way: For individual peaks, Determine the percentage of peak intensity in the absence and presence of the peptide and place it on a logarithmic scale. Changed. The distribution obtained around the median is significantly skewed to the left. Media That contain more than 90% of the residues with a higher percentage than the median Applied to residues with chemical shift. A lower percentage of residues than the median and this window Only those not included in were considered to be significantly perturbed (these criteria Therefore, only residues with a proportion reduced below 0.65 of the median are significant. Perturbation). These residues are residues 94, 97, 112, 115, 117, 119, 120, 131, 132 and 135, tryptophan¹¹⁹of Indole resonance and asparagine¹¹³And asparagine¹³⁵The side chain amide of Including. 95, 96, 98, 99, 100, 118 and 134 resonances and birds Putphan¹¹⁸Was not present in the presence of the ligand. L -Control experiment using peptide YGGRELPLPLPRF amide (SEQ ID NO: 2) Is¹In the H dimension^Three0.1 p. p. m. i or more, or^FifteenN dime In the region^Three0.5p. p. m. 17 resonances shifted so far (Residues 87, 89, 90, 92, 96, 98, 99, 100, 109, 111 , 114, 116, 119, 121, 131, 132 and 135; Fan¹¹⁹Indole resonance and asparagine¹¹³And asparagine¹³⁵Side of Chain amide). The five resonances (95, 97, 117, 118 and 134) are complex Was not present in the HSQC spectrum. Residues that interact with Pep-D1 In order to confirm the approach chosen to determine -Obtained using the peptide YGGRELPPLPRF amide (SEQ ID NO: 2) Applied to the spectrum. Using this approach, interaction with this peptide No new residues to use were identified. Proline¹³³On the chemical shift of In this type of experiment, the effect of the matching peptide, which forms the pocket B part, Could not be observed.   Binding of Pep-D1 results in a pocket A formation similar to fragments of adjacent residues. A perturbation of the chemical shift of the group results (FIG. 2C). Also, most of these residues are L -These chemical shifts change with respect to the binding of the peptide (Fig. 2B). Poke A represents the arginine residue in the L-amino acid ligand in the D-peptide. Arginine residues or lysines converted in a manner that is an analog to group recognition May interact with residues. Both L- and D-amino acid ligands and Site interactions explain the observed competition for binding of these two ligands I do.   Pep-D1 is contacted by a polyproline-type ligand for the SH3 domain It is clear that it only occupies the portion of the binding site that is performed (FIG. 3). pocket B and the residues forming the pocket C portion (tyrosine⁹⁰And tyrosine⁹²),Also Is the residue adjacent to this pocket (valine⁸⁷And leucine⁸⁹) Of Pep-D1 Perturbs the bond (FIGS. 2 and 3). Mutation analysis was performed on L-amino acid ligands. Suggest that interactions at these sites are required for high affinity binding ( Feng, S.M. et al. , Science, 266: 1241 (1994). . Thus, a high affinity D-peptide inhibitor is further added along the groove to the SH3 domain. Of an analog of Pep-D1 or Pep-D2 (Table 1) that expands into pocket C of By design or choice, it could potentially be obtained.Example 4   D-amino acid peptides for designing highly enriched libraries Use of ligand   Random synthetic libraries provide high affinity ligands for a given target. It does not cover enough structural space to always allow isolation. More likely Some strategies tend to favor structural components that are known to interact with interesting targets. The use of a library. In particular, use the strategies described herein. D-amino acid peptide sequences that interact with a given target -Oriented (D) -Amino acid peptide and peptide-based library design Guidelines. These prone libraries are: Used for the isolation of new ligands. For example, for the SH3 domain The class (D) -amino acid peptide ligand (Example 2) is an SH3-binding peptide. (D) -Amino acid peptides-Basic Useful for designing libraries. Such a library is an SH3 domain Useful for identifying peptides that bind to the SH3 domain Tend to have a certain percentage of peptides that are known to Is particularly useful in (L) -all SH3 peptides that interact with the amino acid peptide The main was examined for binding to ligands having similar structural components. In addition, SH3 A synthetic library based on the structure of the (D) -amino acid ligand for the domain , Rich in ligands for other SH3 domains. (D) for SH3 domain Libraries that tend to be based on the structure of amino acid ligands, Useful for isolating ligands to the main.   For example, as shown below, binding to the (D) -SH3 domain Based on the amino acid sequences of the three classes of peptides described in Example 2, Configure the library to be used. About 80% of the D-amino acid peptides in the library About 20% of the library has D-amino acid peptides with converted amino acid residues Chemical peptide of D-amino acid so that the amino acid has no converted amino acid residues. Prepare the library.   Therefore, a general structure known to bind to the (D) -SH3 domain Libraries that are quite prone (eg, 80%) for peptides with peptides Lee has an inverted structure and binds to other SH3 domains (eg, humans) Can be used to isolate other D-amino acid peptides. Furthermore, conversion Amino acid residues are changed (eg, from 20% of the D-amino acid peptide) and D-amino, such as binding to an SH3 domain of The acid peptide can be isolated.Example 5   Mirror Image Selection of Enantiomeric DNA Vasopressin Inhibitors   Vasopressin was prepared using a conventional solid phase peptide synthesis method.   In the first step to produce an antagonist of the peptide hormone L-vasopressin Single-stranded DNA (ssDNAs) that binds to synthetic D-vasopressin (DVP) In vitro selection was used to isolate. 10¹⁶Different 96-mers (96 -Mers) was synthesized on a DNA synthesizer. The molecule in each pool is 60 A central region with a random sequence site of I was in contact. Molecules in this starting pool that bind to DVP are identified by affinity chromatography. Concentrated by torography. Biotinylated to streptavidin agarose An affinity resin was prepared by coupling DVP. ssDN The A pool was radiolabeled, denatured, reconstituted in physiological buffer and passed through the resin . After extensive washing of the column, one molar excess of DV was added to molecules that bind to DVP. The column was eluted with P. Negative strand "primer-terminator", ie Central segment of non-nucleotide material that blocks further extension of the positive strand The elution pool was amplified by PCR using the oligonucleotide having ). Such PCR produces a substantial size difference between the two product strands, Facilitates gel purification of the ssDNA pool. This affinity chromatography The combination of PCR and PCR constituted one cycle of selective amplification.   After 13 selection amplification cycles, the pool was cloned. 56 Analysis of the loan showed only two different sequences, 96.2 and 96.4. Correct Each sequence of ssDNA binds to DVP, and both form very similar secondary structures Have the potential to Affinity chromatography studies of a series of deletion mutations One that was found to bind to DVP rather than the sequence, 69.1, was identified. This aptamer is converted to DVP resin by L-vasopressin. Is not eluted. 96.2 96.4 69.1   D-Vasopressin aptamer. A sequence fragment that is split into two prototype aptamers Locks are shown in uppercase. Defined array cells that touch the random array region Segment is shown in bold. La separated by the two arrays in the final pool Segments from random sequence sites are outlined. 69.1 Aptamer Is a 96.4 deficient derivative.   The information needed to complete the selective reflection technique has been described above. Enantio- The synthesis of the 69.1 sequence using deoxyribose phosphoramidite gives a natural Aptamer that binds to vasopressin but is less susceptible to nuclease degradation You. However, prior to synthesizing such aptamers, a reduction based on the 69.1 sequence was required. It is possible to perform a second selection experiment starting with the duplicate sequence set. this This makes it possible to isolate sequence variants that bind to DVP with high affinity. You. The data from this second selection is a reliable secondary guide to further deletion experiments. A final DVP aptamer that also provides a structural model and is smaller in size and more active -Get. This nuclease-resistant aptamer is combined with a ligand in vitro. And tested as an antagonist of vasopressin in vivo .Equivalent   Those skilled in the art will recognize, by mere routine experimentation, the invention described herein. Many equivalents to a particular embodiment can be recognized or confirmed. Would. Such equivalents fall within the scope of the following claims.

────────────────────────────────────────────────── ─── Continuation of front page    (31) Priority claim number 60 / 001,067 (32) Priority Date July 11, 1995 (33) Priority country United States (US) (31) Priority claim number 08 / 627,497 (32) Priority date March 28, 1996 (33) Priority country United States (US) (81) Designated countries EP (AT, BE, CH, DE, DK, ES, FI, FR, GB, GR, IE, IT, L U, MC, NL, PT, SE), CA, JP, US (72) Inventor Kim, Peter S.             United States Massachusetts             02173 Lexington, Middle By Law             C4

Claims (1)

[Claims] 1. A method for producing a non-natural left-right macromolecule that binds to a natural left-right macromolecule target polymer, comprising the steps of: a) providing an enantiomer of the target macromolecule or one having domain properties thereof; b) Providing a natural left-right image polymer library; c) the step b) under conditions suitable for binding of the natural left-right polymer in the library to the enantiomer of step a). Contacting the library with the enantiomer of step a), whereby the enantiomer of step a) binds to the natural left-right macromolecule present in the library; and d) step a) A) producing an enantiomer of a natural left-right macromolecule that binds to the enantiomer, wherein the enantiomer of step d) is a non-natural non-natural macromolecule that binds to the natural left-right macromolecule target polymer; It is a polymer of left and right images. 2. A method for producing a non-natural left-right macromolecule that binds to a natural left-right macromolecule target polymer, comprising the steps of: a) providing an enantiomer of the target macromolecule or one having domain properties thereof; b) Providing a natural left-right image polymer library; c) the step b) under conditions suitable for binding of the natural left-right polymer in the library to the enantiomer of step a). Contacting the library with the enantiomer of step a); thereby binding the enantiomer of step a) to the natural left-right macromolecule present in the library; d) step a) E) determining the sequence of the natural left and right macromolecules identified in step d); and e) the macromolecules identified in step d) or a domain thereof. Non-enantiomeric enantiomer Process for producing a polymer of natural handedness, wherein enantiomers of step e) is a polymeric handedness unnatural that binds to a target macromolecular natural handedness. 3. 3. The method according to claim 2, wherein the target polymer is a protein. 4. Protein is vasopressin, interleukin-8, thrombomodulin EGF-like domain, GPII _b -III _a cytoplasmic domain, Factor VIIa GLA domain, Factor IX EGF-like domain, HIV protease, N H _2-terminal SH3 domain GRB2, COOH terminal SH3 domain GRB2, P1 20 ^GAP SH3 domain method of claim 3, wherein those selected from the group consisting of vascular permeability factor and vascular endothelial growth factor. 5. 3. The method according to claim 2, wherein the target polymer is an oligonucleotide. 6. The method according to claim 5, wherein the oligonucleotide is selected from the group consisting of HIV RRE, HIV Tar and BCR-AB1 fusion DNA sequence. 7. A method for producing a D amino acid peptide that binds to a target L macromolecule, comprising the following steps: a) providing a D amino acid peptide of a target L macromolecule or a substance having domain properties thereof; b) providing a library of L amino acid peptides C) contacting the library of step b) with the D-amino acid peptide of step a) under conditions suitable for binding the L-amino acid peptide in the library with the D-amino acid peptide of step a); Binding the peptide of step a) to an L-amino acid peptide present in the library by: d) identifying an L-amino acid peptide that binds to a D-amino acid peptide of step a); e) identifying in step d) Determining the sequence of the identified L amino acid peptide; and e) having the L amino acid peptide identified in step d) or its domain characteristics. Process for producing a D-amino acid peptide but that, here, D amino acid peptide step e), binds to a target L polymer. 8. The peptide, vasopressin, interleukin-8, thrombomodulin EGF-like domain, GPII _b -III _a cytoplasmic domain, Factor VIIa GLA domain, Factor IX EGF-like domain, HIV protease, NH ₂ terminal SH3 domain GRB2, COOH-terminal SH3 domain GRB2, P 120 ^GAP SH3 domain the method of claim 7, wherein those selected from the group consisting of vascular permeability factor and vascular endothelial growth factor. 9. A method for producing an L oligonucleotide that binds to a target L macromolecule, comprising the steps of: a) providing a D amino acid peptide of a target L macromolecule or one having domain properties thereof; b) providing a library of D oligonucleotides C) contacting the library of step b) with the D-amino acid peptide of step a) under conditions suitable for binding of the D-oligonucleotide in the library to the D-amino acid peptide of step a); Binding the peptide of step a) to a D oligonucleotide present in the library by: d) identifying a D oligonucleotide that binds to the D amino acid peptide of step a); e) identifying in step d) Determining the sequence of the D oligonucleotide identified; and e) the D oligonucleotide identified in step d) or Process for producing L oligonucleotides but having in properties, where, L oligonucleotides step e) binds to the target L polymer. 10. Polymer is vasopressin, interleukin-8, thrombomodulin EGF-like domain, GPII _b -III _a cytoplasmic domain, Factor VIIa GLA domain, Factor IX EGF-like domain, HIV protease, NH ₂ terminal SH3 domain GRB2, COOH The method according to claim 9, which is selected from the group consisting of a terminal SH3 domain GRB2, a P120 ^GAP SH3 domain, a vascular permeability factor and a vascular endothelial growth factor. 11. A method for identifying an L amino acid peptide that binds to a target D amino acid peptide, comprising the following steps: a) providing a phage display library containing an L amino acid peptide displayed on the surface of a phage; b) displaying on a phage surface Contacting the phage display library of step a) with the desired D amino acid peptide under conditions suitable for binding the L amino acid peptide to the desired D amino acid peptide; Identifying the phage on the surface that binds to the L amino acid peptide displayed on the surface, thereby producing a D amino acid peptide-presenting L amino acid peptide complex, wherein the displayed L amino acid peptide is: L amino acid peptide that binds to the desired D amino acid peptide. 12. 12. The method according to claim 11, further comprising the step of preparing a D amino acid peptide corresponding to the identified L amino acid, and further comprising the following step: d) changing the amino acid sequence of the L amino acid peptide displayed on the surface of the phage. Determining; and e) synthesizing a D amino acid peptide corresponding to the amino acid sequence of the L amino acid peptide determined in step d), thereby producing a D amino acid peptide corresponding to the L amino acid peptide displayed on the surface. Process. 13. The method according to claim 11, wherein the L amino acid peptide displayed on the surface of the phage binds to the D amino acid peptide of the src SH3 domain. 14． src A synthetic amino acid peptide that binds to the SH3 domain. 15. src A synthetic D amino acid peptide corresponding to all or part of the SH3 domain. 16. A synthetic D amino acid peptide that binds to a domain of an intracellular signal protein. 17． A method for obtaining an L nucleic acid sequence that binds to a target L amino acid peptide, comprising the following steps: a) providing a collection of D nucleic acid sequences; b) conditions suitable for binding the D nucleic acid sequence to the target D amino acid peptide Contacting the D nucleic acid sequence of step a) with the D amino acid peptide of interest; c) isolating the D nucleic acid sequence that binds to the D amino acid peptide; d) the nucleotide sequence of the D nucleic acid sequence of step c) And e) preparing a nucleic acid sequence having the nucleotide sequence of step d) using L nucleotides, wherein the nucleic acid sequence of step e) is an L nucleic acid sequence that binds to an L amino acid peptide. is there. 18. A D amino acid peptide identified by the method of claim 7. 19. An L oligonucleotide identified by the method of claim 9. 20. 2. The non-natural right-and-left macromolecule produced by the method of claim 1, wherein the macromolecule binds to a natural right-and-left macromolecule target polymer, wherein the target macromolecule is selected from the group consisting of proteins, oligonucleotides and phospholipids. . 21. Binds to a target macromolecule, wherein the target macromolecule is selected from the group consisting of an intracellular signal protein and its domain; a chemokine; a cytokine; an enzyme; a growth factor; a growth factor receptor and its domain; a transcription factor and its domain; 21. The non-natural right-and-left image polymer according to claim 20, which is an isolated protein. 22. The target polymer is SH3 domain; SH2 domain; PH domain; α-chemokine; β-chemokine; IL-1; TNF; lymphotoxin-α; IL-1β; IL-6; M-CSF; Phospholipase C; Phospholipase D; PDGF family, EGF family, FGF family, IGF family, HGF family, VEGF family, Neurotrophin family, Eph family, Class I cytokine family, GH family, IL-3 family, IL-6 Growth factors and growth factor receptors in the family, IL-2 family, class II cytokine family or TNF family; protein kinases; protein phosphatases; cyclins; Cdc proteins; s domain; bZIP; rel homology domain; STATs; NF-ATs; TCF; Fos; JAKs; human CD2; human CD58; human endothelin; heregulin-α; human interleukin-1β converting enzyme; Protein 1-β; platelet factor 4; human melanoma growth stimulating activity, GRO / melanoma growth stimulating activity; MHC molecule; bacterial muramidase; kringle domain; ras; ras-GAP; (Pleckstrin) homology domain; stromelysin; thrombin; tissue factor; calmodulin; CD4; collagenase; dihydrofolate reductase; fibronectin; fibronectin type III module; ; Vasopressin; Factor IX GLA domain; factor; interleukin-8; thrombomodulin EGF-like domain; GPII _b -III _a cytoplasmic domain; Factor VIIa GLA domain; Factor IX EGF-like domain; human immunodeficiency virus (HIV) protein; NH ₂ 22. The non-natural right-left image polymer according to claim 21, which is selected from the group consisting of terminal SH3 domain GRB2; COOH terminal SH3 domain GRB2; P120 ^GAP SH3 domain; vascular permeability factor and vascular endothelial growth factor. . 23. 21. The non-natural left-right macromolecule according to claim 20, wherein the macromolecule binds to a target polymer, and the target polymer is an oligonucleotide selected from the group consisting of HIV RRE; HIV Tar; and a BCR-AB1 fusion DNA sequence. 24. Binds to a target macromolecule and the target macromolecule is phosphoinositide; phosphoinosidase C; phosphoinositide 3-kinase; phosphatidylinositol; phosphatidylinositol 3-phosphate; phosphatidylinositol (4,5) bisphosphate; Phosphatidylethanolamine; phosphatidic acid; inositol (1,4) bisphosphate; inositol (1,4,5) triphosphate; diacylglycerol; sphingosine; sphingosine phosphate; sphingosine phosphocholine and ceramide. 21. The non-natural right-and-left image polymer of claim 20, which is a phospholipid selected from the group. 25. The non-natural left-right image produced by the method according to claim 7, wherein the target polymer binds to a target polymer in the natural left-right image, and the target polymer is selected from the group consisting of proteins, oligonucleotides and phospholipids. Polymer. 26. Binds to a target macromolecule, wherein the target macromolecule is selected from the group consisting of an intracellular signal protein and its domain; a chemokine; a cytokine; an enzyme; a growth factor; a growth factor receptor and its domain; a transcription factor and its domain; 26. The non-natural left-right imaged macromolecule of claim 25, which is a modified protein. 27. The target polymer is SH3 domain; SH2 domain; PH domain; α-chemokine; β-chemokine; IL-1; TNF; lymphotoxin-α; IL-1β; IL-6; M-CSF; Phospholipase C; Phospholipase D; PDGF family, EGF family, FGF family, IGF family, HGF family, VEGF family, Neurotrophin family, Eph family, Class I cytokine family, GH family, IL-3 family, IL-6 Growth factors and growth factor receptors in the family, IL-2 family, class II cytokine family or TNF family; protein kinases; protein phosphatases; cyclins; Cdc proteins; s domain; bZIP; rel homology domain; STATs; NF-ATs; TCF; Fos; JAKs; human CD2; human CD58; human endothelin; heregulin-α; human interleukin-1β converting enzyme; Protein 1-β; platet factor 4; human melanoma growth stimulating activity; GRO / melanoma growth stimulating activity; MHC molecules; bacterial muramidase; kringle domain; ras; ras-GAP; (Pleckstrin) homology domain; stromelysin; thrombin; tissue factor; calmodulin; CD4; collagenase; dihydrofolate reductase; fibronectin; fibronectin type III module; Vasopressin; Factor IX GLA domain; factor; interleukin-8; thrombomodulin EGF-like domain; GPII _b -III _a cytoplasmic domain; Factor VIIa GLA domain; Factor IX EGF-like domain; human immunodeficiency virus (HIV) protein; NH ₂ terminus 27. The non-natural right-left image polymer according to claim 26, which is selected from the group consisting of SH3 domain GRB2; COOH H-terminal SH3 domain GRB2; P120 ^GAP SH3 domain; vascular permeability factor and vascular endothelial growth factor. 28. 26. The non-natural left-right image height of claim 25, wherein said target polymer is an oligonucleotide selected from the group consisting of HIV RRE; HIV Tar; and a BCR-AB1 fusion DNA sequence. molecule. 29. Binds to a target macromolecule and the target macromolecule is phosphoinositide; phosphoinosidase C; phosphoinositide 3-kinase; phosphatidylinositol; phosphatidylinositol 3-phosphate; phosphatidylinositol (4,5) bisphosphate; Phosphatidylethanolamine; phosphatidic acid; inositol (1,4) bisphosphate; inositol (1,4,5) triphosphate; diacylglycerol; sphingosine; sphingosine phosphate; sphingosine phosphocholine and ceramide. 26. The non-natural left-right image polymer of claim 25, which is a phospholipid selected from the group. 30. The method according to claim 9, wherein the target polymer is vasopressin. 31. The method according to claim 30, wherein the L oligonucleotide that binds to the target polymer is selected from the group consisting of SEQ ID NO: 23, SEQ ID NO: 234 and SEQ ID NO: 25. 32. A method for producing a non-natural left-right image polymer derivative binding to a natural left-right image target polymer, comprising the following steps: a) a non-natural left-right image polymer binding to a natural left-right image target polymer B) providing enantiomers of the target macromolecule or those having domain properties thereof; c) providing a library of natural left-right imaged macromolecules; d) native in the library Contacting the library of step c) with the enantiomer of step b) under conditions suitable for binding of the left and right image macromolecules with the enantiomer of step b) Binding the enantiomer to the natural left and right macromolecule present in the library; e) producing an enantiomer of the natural left and right macromolecule that binds to the enantiomer of step b) Step, wherein the enantiomer of step e) is the natural left It is a polymer of handedness unnatural that bind to target macromolecules image; and f) a step of producing a polymer derivative of left and right images of an unnatural step e). 33. A derivative obtainable by the method according to claim 20.