Method for screening of an in vitro display library within a cell

A method for screening of an in vitro display library for binding within a cell of a small-molecule chemical compound binding entity of the library to a protein or RNA target of interest in order to identify at least one individual chemical compound binding entity of the library that is capable of binding within the cell to the protein or RNA target of interest and wherein the method comprises the steps of:

• (i):
• expressing at least one target Tn, wherein n=1 or more, in a cell and wherein the at least one target is a protein or RNA—the structure of the target is herein termed T-structure;
• (ii):
• introducing an in vitro display library into the cell of (i), wherein:
• (a):
• the library is a library of at least 1000 different binding entities B., wherein n=1000 or more, 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—wherein 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 and wherein the binding entities of the library are chemical compounds with an average molecular weight MW below 10000 dalton—the structure of the binding entity attached to the nucleic acid molecule is herein termed B-structure;
• and
• (b):
• the library is introduced into the cell under binding conditions, which are 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 within the cell gets binding of at least one of the binding entities to at least one target thereby creating within the cell a complex comprising a B-structure bound to a T-structure, which is termed BBoundToT-structure;
• (iii):
• making lysis of the cell of (ii) to breaking down the membrane of cells in order to:
• (A):
• get the BBoundToT-structures of (ii) out of the cell and thereby obtain a solution comprising BBoundToT-structures, wherein the BBoundToT-structures are not within the cells;
• or
• (B):
• get B-structures out of the cells that had bound to target in step (ii) and have been marked before step (iii) in a way that makes it possible to distinguish the marked B-structures from a B-structure that has not bound to target in step (ii) and thereby obtain a solution comprising binder marked B-structures, wherein the binder marked B-structures are not within the cells;
• and
• (iv):
• identifying, via use of the solution of (iii) and the nucleic acid molecules that comprises the specific nucleic acid sequence information allowing to identify the binding entity of the B-structures, at least one individual binding entity that binds within the cell of (ii) to at least one target of interest.