Method for the synthesis of a bifunctional complex
Disclosed is a method for obtaining a bifunctional complex comprising a display molecule part and a coding part, wherein a nascent bifunctional complex comprising a chemical reaction site and a priming site for enzymatic addition of a tag is reacted at the chemical reaction site with one or more reactants, and provided with respective tag(s) identifying the reactant(s) at the priming site is using one or more enzymes.
1. A split-and-mix method for generating a library of bifunctional complexes comprising a display molecule part and a coding part, which method comprises the steps of:
providing, separate compartments, nascent bifunctional complexes, each comprising a chemical reaction site and a priming site for enzymatic addition of an oligonucleotide tag;
performing a round of reaction in each compartment comprising, in any order, a reaction between the chemical reaction site and one or more reactants to provide a new chemical reaction site, and an addition of one or more respective oligonucleotide tags identifying the one or more reactants at the priming site using one or more enzymes to provide a new priming site;
pooling together the content of two or more compartments and subsequently splitting the pooled contents into an array of compartments for a new round of reaction;
performing the new round of reaction in each compartment comprising, in any order, a reaction between the new chemical reaction site and one or more reactants to provide a reactant specific reaction product, and an addition of one or more respective oligonucleotide tags identifying the one or more reactants at the new priming site using one or more enzymes,
obtaining the library of bifunctional complexes in which each member of the library comprises a display molecule part comprising a reactant specific reaction product and a coding part comprising respective oligonucleotide tags which code for the identity of each of the reactants that have participated in the formation of the reactant specific reaction product.
2. The method of claim 1 , wherein oligonucleotide tags are double stranded during a reaction of the chemical reaction site with one or more reactants.
3. The method of claim 1 , wherein two or more oligonucleotide tags consist of a DNA backbone structure.
4. The method of claim 3 , wherein oligonucleotide tags consisting of a DNA backbone structure are added to the priming site by a DNA ligase.
5. The method of claim 4 , wherein ligation of oligonucleotide tags is performed in a double stranded state.
6. The method of claim 5 , wherein the enzyme is a T4 DNA ligase or a Taq DNA ligase.
7. The method of claim 1 , wherein the coding part of the bifunctional complexes consist of double stranded DNA.
8. The method of claim 1 , wherein the coding part comprising all the oligonucleotide tags is transformed to a double stranded form by an extension method in which a primer is annealed to the 3′ end of an oligonucleotide and extended using a polymerase.
9. The method of claim 8 , wherein the extension method is performed by a combination of a ligase and a polymerase.
10. The method of claim 1 , wherein the library contains from 10 3 to 10 6 bifunctional complexes.
11. The method of claim 1 , wherein the library contains from 10 3 to 10 10 bifunctional complexes.
12. The method of claim 11 comprising the further step of partitioning the library of different bifunctional complexes, said method comprising the step of targeting a target entity and selecting from the library of bifunctional complexes those complexes which have an affinity for said target.
13. The method of claim 12 , wherein the selection is repeated one or more additional times.
14. The method of claim 12 , wherein the target is immobilized.
15. The method of claim 12 , wherein the partitioning step involves size exclusion chromatography.
16. The method of claim 12 comprising the further step of amplifying the oligonucleotide tags of the coding part of the selected bifunctional complexes.
17. The method of claim 12 comprising the further step of sequencing the oligonucleotide tags of the coding part.
18. The method of claim 11 comprising the further step of partitioning the library of different bifunctional complexes, said method comprising the step of targeting a target entity and selecting from the library of bifunctional complexes those complexes which have an affinity for said target.