Method for identifying high-affinity complexes made of two ligands and one receptor, device for implementing the method and self-assembling chemical library for use in the method

Abstract

The present invention relates to a method for the sensitive identification of high-affinity complexes made of two ligands (2, 3, 4, 5, 6, 7) and one receptor (1). A large number of different ligands (2, 3, 4, 5, 6, 7) of a chemical library are hereby contacted with at least one receptor (1) in a solution. The ligands of the library have a single-strand DNA (8, 9) or RNA with a base length of 2 to 10 bases or alternatively more than 10 bases. In addition, the solution is incubated for a specific period of time and complexes made of two ligands (2, 3, 4, 5, 6, 7) and one receptor (1) are identified.

1 - 29 . (canceled) 
     
     
         30 . A method for sensitive identification of high-affinity complexes made of two ligands and one receptor, comprising the steps of:
 i) interaction of a large number of different complexes made of ligands of a chemical library with at least one receptor in a solution, the ligands of the library having a single-strand DNA or RNA with a base length of more than 2 bases, which is bonded chemically covalently to the ligands, and only 2 to 10 bases of the single-strand DNA or RNA of a first part of the ligands being complementary to bases of the single-strand DNA or RNA of a second part of the ligands, and the ligands being complexed to form ligand complexes via hybridisation of the DNA or RNA;   ii) incubation of the solution for a specific period of time, complexes made of ligand complexes and the receptor being produced;   iii) identification of the resulting complexes made of ligand complexes and the receptor, wherein the solution is incubated at a temperature at which, between the first part of the ligands and the second part of the ligands, an equilibrium of hybridisation to form ligand complexes and dissociation to form free ligands arises.   
     
     
         31 . The method according to  claim 30 , wherein only 3 to 9 bases of the single-strand DNA or RNA of a first part of the ligands are complementary to the single-strand DNA or RNA of a second part of the ligands. 
     
     
         32 . The method according to  claim 30 , wherein the length of the DNA or RNA of the first part of the ligands and/or the length of the DNA or RNA of the second part of the ligands is at least 20 bases. 
     
     
         33 . The method according to  claim 30 , wherein the first part of the ligands has L different ligands and the second part of the ligands has M different ligands so that L M difference ligand complexes are formed. 
     
     
         34 . The method according to  claim 30 , wherein the solution is incubated in step ii) at a temperature of 1° C. to 50° C. 
     
     
         35 . The method according to  claim 30 , wherein the solution is incubated in step ii) for a period of time of 0.1 to 48 hours. 
     
     
         36 . The method according to  claim 30 , wherein the receptor is immobilized on a substrate selected from the group consisting of glass, ceramic, biopolymer, sepharaose, synthetic polymer and hydrogel. 
     
     
         37 . The method according to  claim 30 , wherein the receptor comprises or consists of a protein, a DNA, a RNA, a cell and/or an organic molecule with a molecular mass ≤200 kDa. 
     
     
         38 . The method according to  claim 30 , wherein the ligand comprise or consist of a molecule selected from the group consisting of protein, peptide, lipid, carbohydrate, dsDNA, ssDNA, dsRNA and ssRNA, aptamer, organic molecule with a molecular mass ≤200 kDa. 
     
     
         39 . The method according to  claim 30 , wherein the complexes made of ligand complexes and the receptor are identified via an analytical method selected from the group consisting of mass spectrometry, HPLC, gas chromatography, IR spectroscopy and DNA sequencing of a DNA or RNA barcode. 
     
     
         40 . The method according to  claim 30 , wherein the single-strand DNA or RNA in the first part of the ligands and/or the single strand DNA or RNA in the second part of the ligands comprises a base sequence which codes for the chemically covalently-bonded ligands. 
     
     
         41 . The method according to  claim 30 , wherein the single-strand DNA or RNA of the ligands of the library is hybridized in regions to form a complementary base sequence of a further single-stranded DNA or RNA, 2 to bases on the further single-strand DNA or RNA of a first part of the ligands being complementary to bases of the single-strand DNA or RNA of a second part of the ligands and binding to the complementary bases, during the method, a Y-form being configured, and a ligase being added during the method, which ligates chemically covalently the further single-strand DNA or RNA of the first part of the ligands to the further single-strand DNA or RNA of the second part of the ligands. 
     
     
         42 . The method according to  claim 41 , wherein, in the case of a single-strand DNA or RNA of a first part of the ligands,
 i) which has the ligand at the 5′ end thereof, has the bases which are complementary to the bases of the single-strand DNA or RNA of a second part of the ligands, at the 5′ end thereof, and the 2 to 10 bases of the further single-strand DNA or RNA, which are complementary to bases of the single-stranded DNA or RNA of a second part of the ligands, are disposed at the 3′ end of the further single-strand DNA or RNA; or   ii) which has the ligand at the 3′ end thereof, has the bases, which are complementary to the bases of the single-strand DNA or RNA of a second part of the ligands, at the 3′ end thereof, and the 2 to 10 bases of the further single-strand DNA or RNA, which are complementary to bases of the single-strand DNA or RNA of a second part of the ligands, are disposed at the 5′ end of the further single-strand DNA or RNA.   
     
     
         43 . A chemical library, comprising ligands which have bonded chemically covalently a single-strand DNA or RNA with a base length of more than 2 bases, wherein only 2 to 10 bases of the single-strand DNA or RNA of a first part of the ligands are complementary to bases of the single-strand DNA or RNA of a second part of the ligands. 
     
     
         44 . The library according to  claim 43 , wherein only 3 to 9 bases of the single-stranded DNA or RNA of a first part of the ligands are complementary to the single-strand DNA or RNA of a second part of the ligands. 
     
     
         45 . The library according to  claim 43 , wherein the length of the DNA or RNA of the first part of the ligands and/or the length of the DNA or RNA of the second part of the ligands is at least 20 bases. 
     
     
         46 . The library according to  claim 43 , wherein the first part of the ligands has L different ligands and the second part of the ligands has M different ligands so that the library has LM different ligand complexes. 
     
     
         47 . The library according to  claim 43 , wherein the ligands comprise or consist of a molecule selected from the group consisting of protein, peptide, lipid, carbohydrate, dsDNA, ssDNA, dsRNA and ssRNA, aptamer, organic molecule with a molecular mass ≤200 kDa. 
     
     
         48 . The library according to  claim 43 , wherein the single-strand DNA or RNA of the ligands of the library is hybridised respectively in regions to form a complementary base sequence of a further single-strand DNA or RNA, 2 to 10 bases of a first further single-strand DNA or RNA of a first part of the ligands being complementary to bases of the second single-strand DNA or RNA of a second part of the ligands. 
     
     
         49 . The library according to  claim 48 , wherein the single-strand DNA or RNA of a first part of the ligands,
 i) which has the ligand at the 5′ end thereof, has the bases, which are complementary to the bases of the single-strand DNA or RNA of a second part of the ligands, at the 5′ end thereof, and the 2 to 10 bases of the further single-strand DNA or RNA, which are complementary to bases of the second single-strand DNA or RNA of a second part of the ligands, are disposed at the 3′ end of the further single-strand DNA or RNA; or   ii) which has the ligand at the 3′ end thereof, has the bases, which are complementary to the bases of the single-strand DNA or RNA of a second part of the ligands, at the 3′ end thereof, and the 2 to 10 bases of the further single-strand DNA or RNA, which are complementary to bases of the single-strand DNA or RNA of a second part of the ligands, are disposed at the 5′ end of the further single-strand DNA or RNA.   
     
     
         50 . A method in which the library according to  claim 43  is used for selective and sensitive identification of high-affinity ternary ligand-receptor complexes.