Methods for synthesis of encoded libraries

Abstract

The present invention provides a method of synthesizing libraries of molecules which include an encoding oligonucleotide tag.

1 . A method of synthesizing a library of compounds, wherein the compounds comprise a functional moiety comprising two or more building blocks which is operatively linked to an initial oligonucleotide which identifies the structure of the functional moiety, said method comprising the steps of
 (a) providing a solution comprising m initiator compounds, wherein m is an integer of 1 or greater, where the initiator compounds consist of a functional moiety comprising n building blocks, where n is an integer of 1 or greater, which is operatively linked to an initial oligonucleotide which identifies the n building blocks;   (b) dividing the solution of step (a) into r reaction vessels, wherein r is an integer of 2 or greater, thereby producing r aliquots of the solution;   (c) reacting the initiator compounds in each reaction vessel with one of r building blocks, thereby producing r aliquots comprising compounds consisting of a functional moiety comprising n+1 building blocks operatively linked to the initial oligonucleotide; and   (d) reacting the initial oligonucleotide in each aliquot with one of a set of r distinct incoming oligonucleotides in the presence of an enzyme which catalyzes the ligation of the incoming oligonucleotide and the initial oligonucleotide, under conditions suitable for enzymatic ligation of the incoming oligonucleotide and the initial oligonucleotide;   thereby producing r aliquots comprising molecules consisting of a functional moiety comprising n+1 building blocks operatively linked to an elongated oligonucleotide which encodes the n+1 building blocks.   
     
     
         2 . The method of  claim 1 , further comprising the step of
 (e) combining two or more of the r aliquots, thereby producing a solution comprising molecules consisting of a functional moiety comprising n+1 building blocks, which is operatively linked to an elongated oligonucleotide which encodes the n+1 building blocks.   
     
     
         3 . The method of  claim 2  wherein r aliquots are combined. 
     
     
         4 . The method of  claim 2  wherein the steps (a) to (e) are conducted one or more times to yield cycles 1 to i, where i is an integer of 2 or greater, wherein in cycle s+1, where s is an integer of i−1 or less, the solution comprising m initiator compounds of step (a) is the solution of step (e) of cycle s. 
     
     
         5 . The method of either  claim 1  or  claim 2  wherein in at least one of cycles 1 to step (d) precedes step (c). 
     
     
         6 . The method of  claim 2  wherein at least one of building blocks is an amino acid. 
     
     
         7 . The method of  claim 1 , wherein the enzyme is DNA ligase, RNA ligase, DNA polymerase, RNA polymerase or topoisomerase. 
     
     
         8 . The method of  claim 2  wherein the initial oligonucleotide is a double-stranded oligonucleotide. 
     
     
         9 . The method of  claim 8  wherein the incoming oligonucleotide is a double-stranded oligonucleotide. 
     
     
         10 . The method of  claim 2  wherein the initiator compounds comprise a linker moiety comprising a first functional group adapted to bond with a building block, a second functional group adapted to bond to the 5′ end of an oligonucleotide, and a third functional group adapted to bond to the 3′-end of an oligonucleotide. 
     
     
         11 . The method of  claim 10  wherein the linker moiety is of the structure 
       
         
           
           
               
               
           
         
         wherein 
         A is a functional group adapted to bond to a building block; 
         B is a functional group adapted to bond to the 5′-end of an oligonucleotide; 
         C is a functional group adapted to bond to the 3′-end of an oligonucleotide; 
         S is an atom or a scaffold; 
         D is a chemical structure that connects A to S; 
         E is a chemical structure that connects B to S; and 
         F is a chemical structure that connects C to S. 
       
     
     
         12 . The method of  claim 11  wherein:
 A is an amino group; 
 B is a phosphate group; and 
 C is a phosphate group. 
 
     
     
         13 . The method of  claim 11  wherein D, E and F are each, independently, an alkylene group or an oligo(ethylene glycol) group. 
     
     
         14 . The method of  claim 11  wherein S is a carbon atom, a nitrogen atom, a phosphorus atom, a boron atom, a phosphate group, a cyclic group or a polycyclic group. 
     
     
         15 . The method of  claim 14  wherein the linker moiety is of the structure 
       
         
           
           
               
               
           
         
         wherein each of n, m and p is, independently, an integer from 1 to about 20. 
       
     
     
         16 . The method of  claim 15  wherein each of n, m and p is independently an integer from 2 to eight. 
     
     
         17 . The method of  claim 16  wherein each of n, m and p is independently an integer from 3 to 6. 
     
     
         18 . The method of  claim 15  wherein the linker moiety has the structure 
       
         
           
           
               
               
           
         
       
     
     
         19 . The method of  claim 1 , wherein each of said initiator compounds comprises a reactive group and wherein each of said r building blocks comprises a complementary reactive group which is complementary to said reactive group. 
     
     
         20 . The method of  claim 19  wherein the reactive group and the complementary reactive group are selected from the group consisting of an amino group; a carboxyl group; a sulfonyl group; a phosphonyl group; an epoxide group; an aziridine group; and an isocyanate group. 
     
     
         21 . The method of  claim 19  wherein reactive group and the complementary reactive group are selected from the group consisting of a hydroxyl group; a carboxyl group; a sulfonyl group; a phosphonyl group; an epoxide group; an aziridine group; and an isocyanate group. 
     
     
         22 . The method of  claim 19  wherein the reactive group and the complementary reactive group are selected from the group consisting of an amino group and an aldehyde or ketone group. 
     
     
         23 . The method of  claim 19  wherein the reaction between the reactive group and the complementary reactive group is conducted under reducing conditions. 
     
     
         24 . The method of  claim 19  wherein the reactive group and the complementary reactive group are selected from the group consisting of a phosphorous ylide group and an aldehyde or ketone group. 
     
     
         25 . The method of  claim 19  wherein the reactive group and the complementary reactive group react via cycloaddition to form a cyclic structure. 
     
     
         26 . The method of  claim 25  wherein the reactive group and the complementary reactive group are selected from the group consisting of an alkyne and an azide. 
     
     
         27 . The method of  claim 19  wherein the reactive group and the complementary functional group are selected from the group consisting of a halogenated heteroaromatic group and a nucleophile. 
     
     
         28 . The method of  claim 27  wherein the halogenated heteroaromatic group is selected from the group consisting of chlorinated pyrimidines, chlorinated triazines and chlorinated purines. 
     
     
         29 . The method of  claim 27  wherein the nucleophile is an amino group. 
     
     
         30 . The method of  claim 2 , further comprising following cycle i, the step of:
 (f) cyclizing one or more of the functional moieties.   
     
     
         31 . The method of  claim 30  wherein a functional moiety of step (f) comprises an azido group and an alkynyl group. 
     
     
         32 . The method of  claim 31  wherein the functional moiety is maintained under conditions suitable for cycloaddition of the azido group and the alkynyl group to form a triazole group, thereby forming a cyclic functional moiety 
     
     
         33 . The method of  claim 32  wherein the cycloaddition reaction is conducted in the presence of a copper catalyst. 
     
     
         34 . The method of  claim 33  wherein at least one of the one or more functional moieties of step (f) comprises at least two sulfhydryl groups, and said functional moiety is maintained under conditions suitable for reaction of the two sulfhydryl groups to form a disulfide group, thereby cyclicizing the functional moiety. 
     
     
         35 . The method of  claim 1  wherein the initial oligonucleotide comprises a PCR primer sequence. 
     
     
         36 . The method of  claim 2 , wherein the incoming oligonucleotide of cycle i comprises a PCR closing primer. 
     
     
         37 . The method of  claim 2 , further comprising following cycle i, the step of
 (d) ligating an oligonucleotide comprising a closing PCR primer sequence to the encoding oligonucleotide.   
     
     
         38 . The method of  claim 37  wherein the oligonucleotide comprising a closing PCR primer sequence is ligated to the encoding oligonucleotide in the presence of an enzyme which catalyzes said ligation.