Method for producing second-generation library

Abstract

The present invention relates to a method for generating a second-generation library. In a first step, a library of encoded molecules associated with an identifier nucleic acid comprising codons identifying chemical entities that have participated in the formation of the encoded molecule is provided. In a second step, the library is partitioned and encoded molecules having a certain property are selected. Codons of identifiers of selected encoded molecules are subsequently identified, and a second-generation library is prepared using at least some of the chemical entities coded for by the identified codons. The new focused library may be used of another partition step to select encoded molecules with a certain property.

1 . A method for producing a composition of molecules with an improved desired property, comprising the steps of: 
 i) providing an initial library comprising a plurality of different encoded molecules associated with a corresponding identifier nucleic acid sequence, wherein each encoded molecule comprises a reaction product of multiple chemical entities and the identifier nucleic acid sequence comprises codons identifying said chemical entities,    ii) subjecting the initial library to a condition partitioning members having encoded molecules displaying a predetermined property from the remainder of the initial library,    iii) identifying codons of the identifier nucleic acid sequences of the partitioned members of the initial library, and    iv) preparing a second-generation library of encoded molecules using the chemical entities coded for by the codons of the partitioned members of the initial library or a part thereof.    
     
     
         2 . The method according to  claim 1 , wherein the second-generation library comprises a plurality of different encoded molecules associated with a corresponding identifier nucleic acid sequence, wherein each encoded molecule comprises a reaction product of multiple chemical entities and the identifier nucleic acid sequence comprises codons identifying said chemical entities.  
     
     
         3 . The method of  claim 1 , further comprising subjecting the second generation library to a condition partitioning members having encoded molecules displaying a predetermined property from the remainder of the second generation library.  
     
     
         4 - 6 . (canceled)  
     
     
         7 . The method according to  claim 1 , wherein the encoded molecule is covalently associated with the corresponding identifier nucleic acid sequence.  
     
     
         8 . (canceled)  
     
     
         9 . The method according to  claim 1 , wherein the chemical entities are reacted without enzymatic interaction to produce the encoded molecule.  
     
     
         10 . The method according to  claim 1 , wherein some or all chemical entities are not naturally occurring α-amino acids or precursors thereof.  
     
     
         11 . The method according to  claim 1 , wherein the encoded molecule is not an a-polypeptide.  
     
     
         12 . The method according to  claim 1 , wherein each codon comprises 4 or more nucleotides.  
     
     
         13 . The method according to  claim 1 , wherein the codons are separated by a framing sequence.  
     
     
         14 . The method according to  claim 13 , wherein the framing sequence positions the reaction of a chemical entity in the synthesis history of the encoded molecule.  
     
     
         15 . (canceled)  
     
     
         16 . The method according to  claim 1 , wherein the identifier nucleic acid sequence comprises three or more codons.  
     
     
         17 . The method according to  claim 1 , wherein the identifier nucleic acid sequence is amplifiable and comprises codons identifying chemical entities, which have participated in the formation of the encoded molecule.  
     
     
         18 . (canceled)  
     
     
         19 . The method according to  claim 1 , wherein the encoded molecule has a molecular weight less than 2000 Dalton, preferably less than 1000 Dalton, and more preferred less than 500 Dalton.  
     
     
         20 - 21 . (canceled)  
     
     
         22 . The method according to  claim 1 , wherein identifier nucleic acid sequence prior to step iii) is amplified.  
     
     
         23 . (canceled)  
     
     
         24 . The method according to  claim 1 , wherein the codons of the identifier nucleic acid sequences of the partitioned members of the initial library are identified by contacting said identifier nucleic acid sequences with a pool of nucleic acid fragments under conditions allowing for hybridisation.  
     
     
         25 - 33 . (canceled)  
     
     
         34 . The method according to  claim 24 , wherein nucleic acid fragments are primer oligonucleotides, and the identification involves subjecting the hybridisation complex between the primer oligonucleotides and the identifier nucleic acid sequences to a condition allowing for an extension reaction to occur when the primer is sufficient complementary to a part of the identifier nucleic acid sequence, and evaluating based on measurement of the extension reaction, the presence, absence, or relative abundance of one or more codons.  
     
     
         35 - 47 . (canceled)  
     
     
         48 . The method according to  claim 24 , wherein the nucleic acid fragment is associated with a chemical entity precursor capable of being transferred to a recipient reactive group.  
     
     
         49 - 59 . (canceled)  
     
     
         60 . The method according to  claim 1 , wherein the second generation library is formed by 
 a) mixing under hybridisation conditions, nascent bifunctional complexes comprising a chemical entity or a reaction product of chemical entities, and an identifier nucleic acid sequence comprising codon(s) identifying said chemical entities, with the recovered nucleic acid fragments, said fragments comprising an oligonucleotide sufficient complementary to at least a part of the identifier nucleic acid sequence to allow for hybridisation, a transferable chemical entity and an anticodon identifying the chemical entity, to form hybridisation products,    b) transferring the chemical entities of the nucleic acid fragments to the nascent bifunctional complexes through a reaction involving a reactive group of the nascent bifunctional complex, in conjunction with a transfer of the genetic information of the anticodon.    
     
     
         61 - 64 . (canceled)  
     
     
         65 . The method according to, wherein the second generation library are subjected to a partitioning according to step ii) of  claim 1 .  
     
     
         66 . The method according to  claim 1 , wherein, prior to the partitioning, the second generation library of complexes are contacted with sequences complementary to the identifier nucleic acid sequences, and the complexes which have hybridised with the complementary sequences are recovered.  
     
     
         67 - 68 . (canceled)  
     
     
         69 . The method according to  claim 1 , wherein the second-generation library is prepared using chemical entities appearing in the initial library and chemical entities foreign to the initial library.  
     
     
         70 - 75 . (canceled)