Template directed split and mix synthesis of small molecule libraries

Abstract

The invention combines the advantages of split and mix synthesis with the advantages of template directed synthesis. The method comprises the steps of: a) adding a linker molecule L to one or more reaction wells; b) adding a molecule fragment to each of said reaction wells; c) adding an oligonucleotide identifier to each of said reaction wells; d) subjecting said wells to conditions sufficient to allow said molecule fragments and said oligonucleotide identifiers to become attached to said linker molecule, or conditions sufficient for said molecule fragments to bind to other molecule fragments and sufficient for said oligonucleotide identifiers to bind to other oligonucleotide identifiers; e) combining the contents of said one or more reaction wells; and f) contacting the resulting bifunctional molecule(s) of step e) with one or more (oligonucleotide) templates each capable of hybridizing to at least one of the oligonucleotide identifiers added in step c).

1 . A method for synthesizing an encoded molecule comprising the steps of:
 a) Adding a linker molecule L to one or more reaction wells;   b) Adding a molecule fragment to each of said reaction wells;   c) Adding an oligonucleotide identifier to each of said reaction wells;   d) Subjecting said wells to:
 conditions sufficient to allow said molecule fragments and said oligonucleotide identifiers to become attached to said linker molecule, or 
 conditions sufficient for said molecule fragments to bind to other molecule fragments and sufficient for said oligonucleotide identifiers to bind to other oligonucleotide identifiers; 
   e) Combining the contents of said one or more reaction wells;   wherein at least one reactive group of the linker molecule L reacts with a reactive group in the molecule fragment, or with a reactive group in the oligonucleotide;   wherein at least one reactive group of the molecule fragments reacts with a reactive group in the linker molecule L, or with a reactive group in another molecule fragment,   wherein at least one reactive group of the oligonucleotide identifiers reacts with a reactive group in the linker L, or with a reactive group in another oligonucleotide identifier; and   wherein the oligonucleotide identifier added to each well in step c) identifies the molecule fragment added to the same well in step b).   
     
     
         2 . A method for synthesizing an encoded molecule comprising the steps of:
 a) Adding a linker molecule L to one or more reaction wells;   b) Adding a molecule fragment to each of said reaction wells;   c) Adding an oligonucleotide identifier to each of said reaction wells;   d) Subjecting said wells to conditions sufficient to allow said molecule fragments and said oligonucleotide identifiers to become attached to said linker molecule, or conditions sufficient for said molecule fragments to bind to other molecule fragments and sufficient for said oligonucleotide identifiers to bind to other oligonucleotide identifiers, so as to form bi-functional molecules consisting of an encoded molecule and an oligonucleotide;   e) Combining the contents of said one or more reaction wells, to produce an admixture of said bi-functional molecules;   f) Optionally, distributing the combined product to one or more new reaction wells;   g) Optionally, repeating steps b) to f) one or more times; and   h) Optionally, contacting the resulting bifunctional molecule(s) of step e) or g) with one or more templates each capable of hybridizing to at least one of the oligonucleotide identifiers added in step c);   
       wherein
 the linker molecule L contains at least one reactive group capable of reacting with a reactive group in the molecule fragment and at least one reactive group capable of reacting with a reactive group in the oligonucleotide; 
 the molecule fragments each contain at least one reactive group capable of reacting with a reactive group in the linker molecule L or a reactive group in another molecule fragment, and the reactive groups of each molecule fragment may be the same or different; 
 the oligonucleotide identifiers each contain at least one reactive group capable of reacting with a reactive group in the linker L or a reactive group in another oligonucleotide identifier, and the reactive groups of each oligonucleotide identifier may be the same or different; 
 the region of the oligonucleotide identifier added to each well in step c) which hybridizes to said template identifies the molecule fragment added to the same well in step b); 
 the steps a) to d) may be performed in any order; 
 the steps b) to d) in step g) may also be performed in any order; 
 the number of wells in steps a) and f) may be the same or different; 
 the oligonucleotide template optionally is associated with a reactive group. 
 
     
     
         3 . The method according to  claim 2 , wherein reactive groups of a molecule fragment of each of two or more bi-functional molecules hybridized to the same template are reacted, or wherein the reactive group of a molecule fragment of one bi-functional molecule is reacted with a reactive group associated with the template to which it is hybridized. 
     
     
         4 . The method according to  claim 2 , wherein the number of wells in step a) is m and the number of wells in step f) is n, and wherein for each repetition of steps b) to f) in step g) n may be the same or different; and
 the structure of the encoded molecule is
   O p,q —( . . . —(O 2,q —(O 1,q —Z)))—Y—(((X—R 1,q )—R 2,q )— . . . )—R p,q ;
 
   wherein
 X, Y and Z are components of the linker molecule, L, X being adapted for reaction with a molecule fragment, Z being adapted for reaction with an oligonucleotide and Y being a flexible linker connecting X and Z; 
 O 2,q  is the oligonucleotide identifier added in repetition number 1 of steps b) to f) in well number q; 
 O p,q  is the oligonucleotide identifier added in repetition number (p−1) of steps b) to f) in well number q; 
 R 2,q  is the molecule fragment added in repetition number 1 of steps b) to f) in well number q; 
 R p,q  is the molecule fragment added in repetition number (p−1) of steps b) to f) in well number q; 
 p is an integer of at least 2; 
 m and n are integers of at least 2, and 
 for O 1,q , O 2,q , and R 1,q , R 2,q , q is in the range 1 to m, for O p,q  and R p,q  where p is greater than 1, q is in the range 1 to n. 
   
     
     
         5 . The method according to  claim 2 , wherein the number of wells in step a) is m and the number of wells in step f) is n, and wherein for each repetition of steps b) to f) in step g) n may be the same or different; and
 the structure of the encoded molecule is
   O p,q — . . . —O 2,q —O 1,q -(((L-R 1,q )—R 2,q )— . . . )—R p,q ;
 
   wherein
 O p,q  is the oligonucleotide identifier added in repetition number (p−1) of steps b) to f) in well number q; 
   O 2,q  is the oligonucleotide identifier added in repetition number 1 of steps b) to f) in well number q;
 R 2,q  is the molecule fragment added in repetition number 1 of steps b) to f) in well number q; 
   R p,q  is the molecule fragment added in repetition number (p−1) of steps b) to f) in well number q;
 p is an integer of at least 1; 
 m and n are integers of at least 2, and 
 for O 1,q , O 2,q , R 1,q , and R 2,q , q is in the range 1 to m, for O p,q  and R p,q  where p is greater than 1, q is in the range 1 to n. 
   
     
     
         6 . A method for synthesizing an encoded molecule comprising the steps of:
 a) Dispensing aliquots of a nascent linker molecule L into each of m reaction wells;   b) Dispensing into each of said m reaction wells a corresponding aliquot of an m th  molecule fragment, R 1,m  and a corresponding aliquot of an m th  oligonucleotide, O 1,m  identifier;   c) Combining all of the nascent bi-functional molecules from all m reaction wells to produce an admixture of nascent bi-functional molecules;   d) Optionally, Dispensing said admixture of nascent bi-functional molecules into n reaction wells;   e) Optionally, dispensing into each of the n reaction wells of step d) a corresponding aliquot of an m th  molecule fragment, R p,q , and a corresponding aliquot of an m th  oligonucleotide identifier, O p,q ;   f) Optionally, combining all of the nascent bi-functional molecules from all n reaction wells in step e) to produce an admixture of nascent bi-functional molecules;   g) Optionally repeating steps d) to f) one or more times;   h) Optionally, contacting a resulting bi-functional molecule of step f) or g) with one or more templates, said one or more templates optionally being associated with a reactive group, under conditions to allow for hybridization of each of the templates to one or more of said nascent bi-functional molecule generated in step f) or g); and   i) Optionally, reacting reactive groups of a molecule fragment of two or more nascent bi-functional molecules hybridized to the same template, or reacting the reactive group of a molecule fragment of one nascent bi-functional molecule with the reactive group associated with the template to which it is hybridized;   
       Wherein:
 the linker molecule L contains at least one reactive group capable of reacting with a reactive group in the molecule fragment and at least one reactive group capable of reacting with a reactive group in the oligonucleotide; 
 the molecule fragments each contain at least one reactive group capable of reacting with a reactive group in the linker molecule L or a reactive group in another molecule fragment, and the reactive groups of each molecule fragment may be the same or different; 
 the oligonucleotide identifiers each contain at least one reactive group capable of reacting with a reactive group in the linker L or a reactive group in another oligonucleotide identifier, and the reactive groups of each oligonucleotide identifier may be the same or different; 
 the oligonucleotide identifier added to each well in step b) and e) identifies the molecule fragment added to the same well in the respective step; 
 the steps a) and b) as well as the steps d) and e) may be performed in any order; and 
 the steps d) and e) in step g) may also be performed in any order, 
 wherein O p,q  is the oligonucleotide identifier added in repetition number (p−1) of steps b) to f) in well number q, and R p,q  is the molecule fragment added in repetition number (p−1) of steps b) to f) in well number q. 
 
     
     
         7 . The method according to  claim 6 , wherein the number of wells in step a) is m and the number of wells in step e) is n, and wherein for each repetition of steps d) to f) in step g) n may be the same or different; and
 the structure of the encoded molecule is
   O p,q —( . . . —(O 2,q —(O 1,q —Z)))—Y—(((X—R 1,q )—R 2,q )— . . . )—R p,q ;
 
   wherein
 X, Y and Z are components of the linker molecule, L, X being adapted for reaction with a molecule fragment, Z being adapted for reaction with an oligonucleotide and Y being a flexible linker connecting X and Z; 
 O p,q  is the oligonucleotide identifier added in repetition number (p−1) of steps b) to f) in well number q; 
 O 2,q  is the oligonucleotide identifier added in repetition number 1 of steps b) to f) in well number q; 
 R 2,q  is the molecule fragment added in repetition number 1 of steps b) to f) in well number q; 
 R p,q  is the molecule fragment added in repetition number (p−1) of steps b) to f) in well number q; 
 p is an integer of at least 2; 
 m and n are integers of at least 2; and 
 for O 1,q , O 2,q , R 1,q , and R 2,q , q is in the range 1 to m, for O p,q  and R p,q  where p is greater than 1, q is in the range 1 to n. 
   
     
     
         8 . The method according to  claim 6 , wherein for each repetition of steps d) to f) in step g) n may be the same or different; and
 the structure of the encoded molecule is
   O p,q — . . . —O 2,q —O 1,q -(((L-R 1,q )—R 2,q )— . . . )—R p,q ;
 
   wherein
 O p,q  is the oligonucleotide identifier added in repetition number (p−1) of steps b) to f) in well number q; 
 O 2,q  is the oligonucleotide identifier added in repetition number 1 of steps b) to f) in well number q; 
 R 2,q  is the molecule fragment added in repetition number 1 of steps b) to f) in well number q; 
 R p,q  is the molecule fragment added in repetition number (p−1) of steps b) to f) in well number q; 
 p is an integer of at least 1; 
 m and n are integers of at least 2; and 
 for O 1,q , O 2,q , R 1,q , and R 2,q , q is in the range 1 to m, for O p,q  and R p,q  where p is greater than 1, q is in the range 1 to n. 
   
     
     
         9 . The method according to  claim 6 , wherein the structure of the nascent bi-functional molecules resulting from step b is
   O 1,q -L-R 1,q ;   and the structure of the nascent bi-functional molecule obtained after repeating the process steps defined in step g) p−1 times is
   O p,q —( . . . )—O 1,q -L-R 1,q —( . . . )—R p,q ;
 
   and wherein p is greater than or equal to 1.   
     
     
         10 . The method of  claim 6  in which steps h) and i) have been omitted. 
     
     
         11 . A method for identifying a molecule with desired characteristics, said method comprising synthesizing a library of encoded molecules by a method as described in  claim 2 , and subjecting the library to a partitioning or enrichment procedure, to identify and optionally increase the relative amount of an encoded molecule having one or more desired characteristics. 
     
     
         12 . A method for synthesising one or more encoded molecules, comprising the following steps:
 step 1: dispensing aliquots of a nascent linker molecule L comprising the components X, Y, and Z, where X is adapted for reaction with a molecule fragment, Z is adapted for reaction with an oligonucleotide and Y is a flexible linker connecting X and Z, into each of m reaction wells; then   step 2: dispensing into each of the m reaction wells of said step 1 a corresponding aliquot of the mth molecule fragment R1, m and a corresponding aliquot of the mth oligonucleotide identifier 01, m to allow reaction between the molecule fragment and X of the linker, and reaction between the oligonucleotide and Z of the linker, to produce a product bi-functional molecule R1, m-L-O1, m where the produced bi-functional molecule comprises a reactive group; then   step 3: combining all of the nascent bi-functional molecules from all m reaction wells produced in said step 2 for producing an admixture of nascent bi-functional molecules; then   step 4: dispensing equal aliquots of the admixture of nascent bi-functional molecules from the prior step into each of m reaction wells, then   step 5: dispensing into each of the m reaction wells of said step 4 a corresponding aliquot of the mth molecule fragment represented by Rn, m and a corresponding aliquot of the mth identifier molecule represented by On, m for producing a nascent bi-functional molecule represented by:
   Rn,m-( . . . )-R1,m-L-O1,m-( . . . )-On,m 
   wherein n is greater than or equal to 2; then   step 6: combining all of the elongated nascent bi-functional molecules from all m reaction wells of step 5 for producing an admixture of elongated nascent bi-functional molecules; then   step 7: repeating steps 4-6 until the desired bi-functional carrier molecules, each formed from the reaction of n molecule fragments and n oligonucleotide identifiers, are produced, and where each bi-functional carrier molecule generated comprises one or more reactive units; then   step 8: optionally providing one or more templates, which one or more templates optionally have a reactive unit associated therewith; then   step 9: optionally contacting one or more carrier molecules of step 7 with said one or more templates under conditions to allow for specific hybridisation of the oligonucleotide identifiers of the one or more carrier molecules to the one or more templates; then   step 10: optionally reacting the reactive units of the molecule fragments of at least two carrier molecules hybridised to the same template, or reacting the reactive unit of a carrier molecule with the reactive unit associated with the template to which it is hybridised,   to generate one or more encoded molecules.   
     
     
         13 . The method of  claim 12  wherein the oligonucleotide identifiers of two or more carrier molecules are covalently linked together prior to, during or after step 9 or 10. 
     
     
         14 . The method of  claim 12  where m does not have the same value in different repetitions of step 5. 
     
     
         15 . The method of  claim 12  where in step 2 or 5 the oligonucleotide identifiers are linked to the nascent bi-functional molecule by a ligase enzyme. 
     
     
         16 . The method of  claim 12  where in step 2 or 5 the oligonucleotide identifiers are linked to the nascent bi-functional molecule without the use of a ligase enzyme. 
     
     
         17 . The method of  claim 12  wherein a library of more than one bi-functional molecule is generated, the method further comprising enriching for library members comprising an encoded molecule displaying a desired property. 
     
     
         18 . The method of  claim 12  wherein steps 4-7 have been eliminated. 
     
     
         19 . The method of  claim 12  wherein steps 8-10 have been eliminated. 
     
     
         20 . The library of 10,000,000 or more bi-functional molecules generated by the method of  claim 12 .