Encoded self-assembling chemical libraries (esachel)

Abstract

The invention concerns a chemical compound comprising a chemical moiety (p) capable of performing a binding interaction with a target molecule (e.g. a biological target) and further comprising an oligonucleotide (b) or functional analogue thereof. In a first embodiment according to the invention, the chemical compound is characterized in that the oligonucleotide (b) or functional analogue comprises at least one self-assembly sequence (b1) capable of performing a combination reaction with at least one self-assembly sequence (b1′) of a complementary oligonucleotide or functional analogue bound to another chemical compound comprising a chemical moiety (q). In a second embodiment according to the invention, the chemical compound which comprises a coding sequence (b1) coding for the identification of the chemical moiety (p) is characterized in that the chemical compound further comprises at least one self-assembly moiety (m) capable of performing a combination reaction with at least one self-assembly moiety (m′) of a similar chemical compound comprising a chemical moiety (q). The invention comprises corresponding libraries of chemical compounds as well as methods of biopanning of target molecules and of identifying such targets.

1 . A combination reaction product of at least two chemical compounds, each one of these chemical compounds comprising: 
 a) a chemical moiety (p,q,r,s) potentially capable of performing a binding interaction with a single target molecule;    b) an oligonucleotide (b,b′,b″,b′″) or functional analogue thereof with at least one self-assembly moiety (m,m′,m″,m′″);    the chemical compounds being bound to each other by their self-assembly moieties (m,m′,m″,m′″), characterized in that the combination reaction product is stable in the absence of said target molecule, wherein the oligonucleotides (b,b′,b″,b′″) or functional analogues of at least one of the chemical compounds comprise a variable, unique coding sequence (b2,b2′,b2″,b2′″) individually coding for the identification of the particular chemical moiety) (p,q,r,s).    
     
     
         2 . The combination reaction product of  claim 1 , characterized in that the self-assembly moieties (m,m′,m″,m′″) are a self-assembly sequences (b1,b1′,b1″,b1′″) of the oligonucleotides (b,b′,b″,b′″), functional analogues thereof, ligands (1) capable to perform a complex reaction with a specific ion (i), or peptides capable of association with other molecules.  
     
     
         3 . The combination reaction product of  claim 1 , characterized in that the at least two chemical compounds each comprise a chemical group by which they are covalently linked together after the stable combination reaction product had been formed.  
     
     
         4 . The combination reaction product of  claim 1 , characterized in that the oligonucleotides (b,b′,b″,b′″) or functional analogues thereof are covalently and directly linked to the chemical moieties (p,q,r,s).  
     
     
         5 . The combination reaction product of  claim 1 , characterized in that the oligonucleotides (b,b′,b″,b′″) or functional analogues thereof further, comprise a linking portion (b3,b3′,b3″,b3′″) which is situated between the self-assembly sequence (b1,b1′,b1″,b1′″) and the chemical moiety (p,q,r,s).  
     
     
         6 . The combination reaction product of  claim 1 , characterized in that the coding sequence (b2,b2′,b2″,b2′″) of oligonucleotide or the functional analogue thereof is situated between the chemical moiety (p,q,r,s) and the self-assembly sequence (b1,b1′,b1″,b1′″).  
     
     
         7 . The combination reaction product of  claim 1 , characterized in that it is a dimer, trimer or tetramer exhibiting chemical moieties (p,q,r,s).  
     
     
         8 . A chemical library comprising combination reaction products of at least two chemical compounds, each one of these chemical compounds comprising; 
 a) a chemical moiety (p,q,r,s) potentially capable of performing a binding interaction with a single target molecule;    b) an oligonucleotide (b,b′,b″,b′″) or functional analogue thereof with at least one self-assembly moiety (m,m′,m″,m′″);    the chemical compounds being bound to each other by their self-assembly moieties (m,m′,m″,m′″), characterized in that the combination reaction product is stable in the absence of said target molecule, wherein the oligonucleotides (b,b′,b″,b′″) or functional analogues of at least one of the chemical compounds comprise a variable, unique coding sequence (b2,b2′,b2″,b2′″) individually coding for the identification of the particular chemical moiety (p,q,r,s).    
     
     
         9 . The chemical library of  claim 8 , characterized in that the self-assembly moieties (m,m′,m″,m′″) are a self-assembly sequences (b1,b1′,b1″,b1′″) of the oligonucleotides (b,b′,b″,b′″), functional analogues thereof, ligands (l) capable to perform a complex reaction with a specific ion (i), or peptides capable of association with other molecules.  
     
     
         10 . The chemical library of  claim 8 , characterized in that the at least two chemical compounds each comprise a chemical group by which they are covalently linked together after the stable combination reaction product had been formed.  
     
     
         11 . The chemical library according to one of  claim 8 , characterized in that it comprises combination reaction products according to any one of  claims 4  to  7 .  
     
     
         12 . The chemical library according to  claim 8 , characterized in that its individual combinations of moieties (p,q,r,s) is derived by forming heteroduplexes, heterotriplexes or heteroquadruplexes of the self-assembly sequences (b1,b1′,b1″,b1′″) of the oligonucleotides (b,b′,b″,b′″).  
     
     
         13 . The chemical library according to  claim 8  characterized in that its individual combinations of moieties (p,q,r,s) is derived by chelation of the self-assembly moieties (m,m′,m″m′″) with specific ions (i).  
     
     
         14 . The chemical library according to  claim 12 , characterized in that it comprises individually encoded sub-libraries (A) and (B), whereas sub-library (A) comprises n compounds coupled to the 3 extremity of a different DNA oligonucleotides (b) and sub-library (B) comprises m compounds coupled to the 5′ extremity of m different DNA oligonucleotides (b′).  
     
     
         15 . The chemical library according to  claim 14 , characterized in that in sub-library (A) or in sub-library (B) respectively, iodoacetamido- or maleimido-derivatives of n or m chemical entities have been coupled to individual DNA oligonucleotides which carry a thiol group at the 3′ or 5′ end.  
     
     
         16 . The chemical library according to  claim 14 , characterized in that in sub-library (A) or in sub-library (B) respectively, amide derivatives—forming chemical structures such as —P(O) 2 —O—(CH 2 ) n —NH—CO—R, where R may correspond to a number of different chemical entities, and n may range between 1 and 10—have been coupled to the oligonucleotides carrying a phosphodiester bond at one extremity.  
     
     
         17 . The chemical library according to  claim 14 , characterized in that in sub-library (A) the self-assembly sequence (b1) is interrupted by a d-spacer in opposite position to a code (B), the d-spacer preventing any undesired pairing to the bases of code (B) which encodes sub-library (B), whereas the oligonucleotide (b) of sub-library (A) has its distinctive code (A) towards the 5′ extremity.  
     
     
         18 . A method of biopanning ligands specific for target molecules, wherein a combination reaction product is incubated with a target molecule, the combination reaction product comprising at least two chemical compounds, each one of these chemical compounds comprising: 
 c) a chemical moiety (p,q,r,s) potentially capable of performing a binding interaction with a single target molecule;    d) an oligonucleotide (b,b′,b″,b′″) or functional analogue thereof with at least one self-assembly moiety (m,m′,m″,m′″);    wherein the chemical compounds are bound to each other b) their self-assembly moieties (m,m′,m″,m′″), characterized in that the combination reaction product is stable in the absence of said target molecule, wherein the oligonucleotides (b,b′,b″,b′″) or functional analogues of at least one of the chemical compounds comprise a variable, unique coding sequence (b2,b2′,b2″,b2′″) individually coding for the identification of the particular chemical moiety (p,q,r,s).    
     
     
         19 . The method of  claim 18 , characterized in that combination reaction products according to at least one of  claims 1  to  7  are utilized for biopanning.  
     
     
         20 . The method of  claim 18 , characterized in that a chemical library of combination reaction products according to at least one of  claims 8  to  17  is used for biopanning.  
     
     
         21 . A method to identify a target molecule with a combination reaction product comprising a chemical moiety (p,q,r,s) capable of performing a binding interaction with this target molecule and further comprising an oligonucleotide (b,b′,b″,b′″) or functional analogue thereof, characterized in that the combination reaction product is bound to a target by biopanning according to at least one of  claims 18  to  20 .  
     
     
         22 . The method of  claim 21 , characterized in that PCR-fragments are generated by polymerase chain reaction (PCR), each of which carries the code of pairs of sub-library members (A) and (B), whereas sub-library (A) comprises a compounds coupled to the 3′ extremity of n different DNA oligonucleotides (b) and sub-library (B) comprises m compounds coupled to the 5′ extremity of m different DNA oligonucleotides (b′).  
     
     
         23 . The method of  claim 22 , characterized in that in sub-library (A) or in sub-library (B) respectively, iodoacetamido- or maleido-derivatives of a or m chemical entities are coupled to individual DNA oligonucleotides, which carry a thiol group at the 3′ or 5′ end.  
     
     
         24 . The method of  claim 23 , characterized in that in sub-library (A) the self-assembly sequence (b1) is interrupted by a d-spacer in opposite position to a code (B), the d-spacer preventing any undesired pairing, to the bases of code (B) which encodes sub-library (B) whereas the oligonucleotide (b) of sub-library (A) has its distinctive code (A) towards the 5′ extremity.  
     
     
         25 . The method of  claim 22  characterized in that the length of the PCR-fragments are checked and their sequence identity is established by digesting the PCR-fragments with a restriction site for a specific endopeptidase (e.g. EcoRI), followed by cloning into a suitable plasmid and sequencing.  
     
     
         26 . The method of  claim 22  where several specific binding members are isolated at the end of a biopanning experiment, characterized in that concatenamers are created, starting from the various PCR fragments present in the reaction mixture, the concatenated sequences are “read” by sequencing, revealing both the identity and the frequency of pairs of code (A) and code (B).  
     
     
         27 . The method of  claim 22  where several specific binding members are isolated at the end of a biopanning experiment and sub-libraries (A) and/or (B) carry chemical moieties at the extremities of partially-annealing oligonucleotides characterized in that unpaired DNA strands are hybridized with target oligonucleotides (e.g. DNA oligonucleotides) being immobilized on one or more chips.  
     
     
         28 . The method of  claim 27 , characterized in that by using chip (A) or chip (B) respectively, the reading of the identity and/or frequency of members of sub-library (A) or sub-library (B) respectively, rescued after a biopanning experiment, is carried out and by decoding on chip (A) and (B) candidate components of sub-libraries (A) and (B), to be re-annealed and screened in a successive round of biopanning are suggested.  
     
     
         29 . The method of  claim 28 , characterized in that increasingly stringent binding to the target is mirrored by a reduction in the number of (A) and/or (B) members as identified on the respective chip and the possible combinations of the candidate (A) and (B) members are assembled individually or in smaller pools and assayed for binding to the target.  
     
     
         30 . The method of  claim 27 , characterized in that libraries are allowed to self-assemble in order to form trimeric or tetrameric complexes by using three or four chips, respectively, which carry distinctive target oligonucleotides for decoding.  
     
     
         31 . The method of  claim 27 , characterized in that the DNA of selected binding moieties is PCR amplified prior to chip hybridization.