Method for Making an Enriched Library

Abstract

A method for making an enriched library comprising specific nucleic acid sequence information allowing to identifying at least one binding entity that binds to at least one target wherein the specific binding entity has been present in an in vitro display library.

1 . A method for making an enriched library comprising specific nucleic acid sequence information allowing to identifying at least one binding entity that binds to at least one target wherein the specific binding entity has been present in an in vitro display library and wherein the method comprises the steps of:
 (i): making an in vitro display library of at least 100 different binding entities, wherein each binding entity is attached to a nucleic acid molecule and the nucleic acid molecule comprises specific nucleic acid sequence information allowing to identify the binding entity once one knows the specific nucleic acid sequence information of the nucleic acid molecule one directly knows the structure of the specific binding entity attached to the nucleic acid molecule—the structure of the binding entity, attached to the nucleic acid molecule, is herein termed B-structure;   (ii): making nucleic acid molecules with at least one target T n  attached to a nucleic acid molecule and the nucleic acid molecule comprises specific nucleic acid sequence information allowing to identify the specific target, wherein the target is capable of binding to at least one of the binding entities present in the library of step (i)—the structure of the target attached to the nucleic acid molecule is herein termed T-structure;   and wherein the method is characterized by that:   (iii): mixing a solution comprising X, wherein X is a number greater than 10 4 , numbers of B-structures of the library of step (i) with a solution comprising Y, wherein Y is a number greater than 10 2 , numbers of T-structures of step (ii) under binding conditions, i.e. conditions where a B-structure containing a binding entity capable of binding to a target molecule, binds more efficiently to the corresponding T-structure, than a B-structure containing a binding entity not capable of binding to the same target do and wherein one gets binding of at least one of the binding entities to at least one target thereby creating a complex comprising a B-structure bound to a T-structure, herein termed B BoundTo T-structure;   (iv): applying an in vitro compartmentalization system—under binding conditions, i.e. conditions where a B-structure containing a binding entity capable of binding to a target molecule, binds more efficiently to the corresponding T-structure, than a B-structure containing a binding entity not capable of binding to the same target do—wherein the compartmentalization system comprises at least 2 times more individual compartments than the Y number of T-structures present in step (iii) under conditions wherein the B-structures, T-structures and B BoundTo T-structures enter randomly into the individual compartments; and   (v): fusing the nucleic acid molecules of a B-structure and a T-structure which are both present within the same individual compartment—i.e. fusing the nucleic acid molecule of the B-structure to the nucleic acid molecule of the T-structure—this structure is herein termed BT Fused -structure and the BT Fused -structure comprises the specific nucleic acid sequence information allowing to identify the binding entity of step (i) and the specific nucleic acid sequence information allowing to identify the specific target of step (ii); and   (vi): combining the content of the individual compartments of step (v) under conditions wherein there is no fusing of the nucleic acid molecules of a B-structure and a T-structure—i.e. there is not created any new BT Fused -structure not already created in step (v)—in order to get a library of BT Fused -structures, wherein the library is an enriched library of species of BT Fused -structures originating from binding pairs of target and binder entity when compared to BT Fused -structures originating from nonbinding pairs of target and binder entity; and   wherein the method is further characterized by at least one of (a) or (b) below:   (a) B BoundTo T-structures remain suspended in solution in the individual compartments of step (iv) of the first aspect;   (b) the method does not rely on target immobilization on a solid support.   
     
     
         2 . The method of  claim 1 , wherein the binding entity of step (i) is attached to the nucleic acid molecule by a covalent binding and wherein the target of step (ii) is attached to the nucleic acid molecule by a covalent binding and wherein the nucleic acid molecule of the B-structure is DNA and the nucleic acid molecule of the T-structure is DNA. 
     
     
         3 . The method of  claim 2 , wherein the DNA nucleic acid molecule in the B-structure is a double stranded nucleic acid molecule and wherein the DNA nucleic acid molecule in the T-structure is a double stranded nucleic acid molecule. 
     
     
         4 . The method of  claim 1 , wherein the nucleic acid molecule attached to the binding entity in the B-structure contains a PCR priming site and wherein the nucleic acid molecule attached to the target in the T-structure contains a PCR priming site. 
     
     
         5 . The method of  claim 1 , wherein the in vitro library of step (i) comprises at least 10 5  different binding entities and wherein the binding entities of step (i) are chemical compounds with an average molecular weight MW below 5000 dalton. 
     
     
         6 . The method of  claim 1 , wherein there is at least two different targets in step (ii). 
     
     
         7 . The method of  claim 1 , wherein at least one target is a protein. 
     
     
         8 . The method of  claim 1 , wherein there in step (iii) is at least 10 5  copies of a T-structure of interest, “Y” is at least 10 5  and wherein the concentration of T-structures in the “mixing step (iii)” is at least 10 −9  M. 
     
     
         9 . The method of  claim 1 , wherein step (iii) is performed under binding conditions, wherein a B-structure containing a binding entity capable of binding to a target molecule, binds 100 fold more efficiently to the corresponding T-structure, than a B-structure containing a binding entity not capable of binding to the same target do. 
     
     
         10 . The method of  claim 1 , wherein said method comprises an additional step (iii-b) that is performed before the step (iv), comprising:
 (iii-b): diluting the solution of step (iii) at least 100 fold under binding conditions, i.e. conditions where a B-structure containing a binding entity capable of binding to a target molecule, binds more efficiently to the corresponding T-structure, than a B-structure containing a binding entity not capable of binding to the same target do.   
     
     
         11 . The method of  claim 1 , wherein there in step (iv) is at least 100 times more individual compartments than the Y number of T-structures present in step (iii) and wherein there in step (iv) there is at least the square root of 10, 3.16, times more individual compartments than the X number of B-structures in step (iii). 
     
     
         12 . The method of  claim 1 , wherein the in vitro compartmentalization system of step (iv) is a water-in-oil emulsion system and wherein the average compartments volume is less than 10 −12  liter. 
     
     
         13 . The method of  claim 1 , wherein the fusing of the nucleic acid molecules of a B-structure and a T-structure which are both present within the same individual compartment of step (v) is:
 (a): done by a DNA ligase where a phosphodiester bond between a 3′-OH and a 5′-phosphate groups is formed; or   (b): by using a DNA polymerase using emulsion PCR.   
     
     
         14 . The method of  claim 1 , wherein the in vitro compartmentalization system of step (iv) is a water-in-oil emulsion and wherein the content of the individual compartments of step (v) is combined in step (vi) by a method, wherein the oil compartments are disrupted. 
     
     
         15 : The method of  claim 1 , wherein there is an extra step (vii), wherein the BT Fused -structures present in the enriched library of step (vi) is amplified by PCR and thereafter subjected to DNA sequencing to identify at least one individual binding entity that binds to at least one target of interest. 
     
     
         16 : The method of  claim 1 , wherein there is an extra step (vii) comprising use the enriched library of step (vi) to identify at least one individual binding entity that binds to at least one target of interest. 
     
     
         17 - 18 . (canceled)