Methods of Constructing and Screening Libraries of Peptide Structures

Abstract

The present invention provides the means for producing libraries of peptide structures for drug screening applications that are capable of folding or assuming their native conformations independently of artificial scaffolds or flanking sequences in the proteins from which they are derived. The libraries can be highly diverse such that they are representative of the repertoire of protein structures existing in nature. The libraries can also be non-redundant or normalized such that the bias towards specific structures existing in source data sets and/or in nature is/are removed. In a particularly preferred embodiment, the present invention provides 30,000 independent fold structures produced by this method. The present invention also provides computer-readable media and systems comprising structural data in relation to the peptide libraries, and methods for displaying and screening the libraries.

1 . A method for producing a peptide library, said method comprising:
 (i) obtaining a plurality of amino acid sequences capable of independently-forming secondary structures and/or assemblies of secondary structures and/or folds:   (ii) producing peptides having the amino acid sequences obtained at (i); and   (iii) displaying the peptides at (ii) such that said peptides form secondary structures and/or assemblies of secondary structures and/or folds.   
     
     
         2 - 7 . (canceled) 
     
     
         8 . The method of  claim 1  wherein the peptides mimic tertiary structures produced by interaction of non-contiguous portions of native proteins. 
     
     
         9 . The method of  claim 1  further comprising size-selecting sequences at (i) to thereby identify a sub-set of sequences having the average length of an independent protein fold. 
     
     
         10 . The method of  claim 1  further comprising identifying, redundant sequences and removing or deleting redundant sequences to thereby leave a non-redundant or normalized plurality of amino acid sequences. 
     
     
         11 - 12 . (canceled) 
     
     
         13 . The method of  claim 1  further comprising mutating peptides that are predicted to form a secondary structure or assembly of secondary structures or fold. 
     
     
         14 - 15 . (canceled) 
     
     
         16 . The method of  claim 1  further comprising performing combinations selected from:
 (i) (a) mutating, peptides and (b) identifying, redundant sequences and removing, or deleting—redundant sequences to thereby leave a non-redundant or normalized plurality of amino acid sequences; 
 (ii) (a) identifying related sequences to the obtained plurality of amino acid sequences and adding, those sequences to the plurality of amino acid sequences and (b) identifying redundant sequences and removing, or deleting redundant sequences to thereby leave a non-redundant or normalized plurality of amino acid sequences; and 
 (iii) (a) mutating peptides and (b) identifying redundant sequences and removing, or deleting—redundant sequences to thereby leave a non-redundant or normalized plurality of amino acid sequences and (c) identifying related sequences to the obtained plurality of amino acid sequences and adding, those sequences to the plurality of amino acid sequences. 
 
     
     
         17 . (canceled) 
     
     
         18 . The method of  claim 1  wherein the peptides are produced by synthetic means. 
     
     
         19 . The method of  claim 1  wherein the peptides are produced by recombinant means. 
     
     
         20 . The method of  claim 1  wherein the peptides are displayed as arrays on a solid surface or a plurality of solid surfaces. 
     
     
         21 . The method of  claim 1  wherein the peptides are expressed on the surface of a phage or a cell or by ribosome display or by in vitro display or expressed within a cell or a plurality of cells. 
     
     
         22 . A method for producing a peptide library having low structure redundancy, said method comprising:
 (i) obtaining a plurality of amino acid sequences capable of independently-forming secondary structures and/or assemblies of secondary structures and/or folds;   (ii) identifying redundant structures from the plurality at (i) and removing or deleting redundant sequences capable of forming, the redundant structures to thereby leave a non-redundant plurality of amino acid sequences;   (iii) producing peptides having the amino acid sequences of the non-redundant plurality at (ii); and   (iv) displaying the peptides at (iii) such that said peptides form secondary structures and/or assemblies of secondary structures and/or folds.   
     
     
         23 . The method according, to  claim 22  wherein the non-redundant plurality includes related structures that differ in their ability to fold autonomously. 
     
     
         24 . The method according to  claim 22  wherein the non-redundant plurality includes related structures that differ in their ligand-binding affinities and/or association/dissociation constants for a ligand. 
     
     
         25 . (canceled) 
     
     
         26 . A method for producing a peptide library having low structure redundancy, said method comprising:
 (i) obtaining a plurality of amino acid sequences capable of forming independent secondary structures and/or assemblies of secondary structures and/or folds;   (ii) producing peptides having the amino acid sequences obtained at (i);   (iii) identifying redundant sequences from the peptides produced at (ii) and removing or deleting, peptides having the redundant sequences to thereby leave a non-redundant plurality of amino acid sequences; and   (iv) displaying the peptides at (iii) such that said peptides form secondary structures and/or assemblies of secondary structures and/or folds.   
     
     
         27 . A method for producing a peptide library, said method comprising:
 (i) identifying a plurality of amino acid sequences capable of folding, independently from other parts of the proteins in which they are contained in their native contexts;   (ii) size-selecting those sequences at (i) to thereby identify a sub-set of sequences having the average length of an independent protein fold;   (iii) identifying redundant sequences from the sequences selected at (ii) and removing or deleting redundant sequences to thereby leave a non-redundant plurality of amino acid sequences;   (iv) producing peptides from the non-redundant plurality of amino acid sequences at (iii); and   (v) displaying the peptides at (iv) such that said peptides form secondary structures and/or assemblies of secondary structures and/or folds.   
     
     
         28 . A method for producing a peptide library, said method comprising:
 (i) identifying a plurality of amino acid sequences capable of folding independently from other parts of the proteins in which they are contained in their native contexts;   (ii) size-selecting those sequences at (i) to thereby identify a sub-set of sequences having the average length of an independent protein fold;   (iii) identifying redundant sequences from the sequences selected at (ii) and removing or deleting, redundant sequences to thereby leave a non-redundant plurality of amino acid sequences;   (iv) producing a diverse pool of sequence by a process comprising identifying related sequences to the non-redundant plurality of amino acid sequences at (iii) and adding those sequences to the non-redundant plurality of amino acid sequences at (iii);   (v) producing peptides from the diverse pool of sequences at (iv); and   (vi) displaying the peptides at (v) such that said peptides form secondary structures and/or assemblies of secondary structures and/or folds.   
     
     
         29 - 33 . (canceled) 
     
     
         34 . A peptide library comprising a plurality of non-redundant amino acid sequences capable of forming, independent folds or a selected subset of said plurality. 
     
     
         35 - 37 . (canceled) 
     
     
         38 . A high-throughput system for drug screening comprising a solid support consisting essentially of or having a plurality of peptides bound directly or indirectly thereto, wherein said plurality of peptides comprises non-redundant amino acid sequences capable of forming independent folds or a subset of said plurality. 
     
     
         39 . (canceled) 
     
     
         40 . A process comprising:
 (i) performing the method of  claim 1  to thereby produce a peptide library; and   (ii) screening the peptide library so produced to thereby identify a peptide.   
     
     
         41 . (canceled) 
     
     
         42 . The process of  claim 40  further comprising isolating the identified peptide. 
     
     
         43 - 46 . (canceled)