Enzymatic encoding methods for efficient synthesis of large libraries

A split-and-mix synthesis method for synthesising a library of different bifunctional complexes comprising a small molecule and an identifier oligonucleotide identifying the reactants which have participated in the synthesis of the small molecule, the method comprising

• providing a plurality of nascent bifunctional complexes obtained after a first synthesis round and dividing the plurality into multiple fractions,
• reacting sequentially or simultaneously, in each fraction, the plurality of nascent bifunctional complexes with a different reactant and an oligonucleotide tag which identifies the different reactant, and
• forming a double stranded hybridization complex, in each fraction, comprising the small molecule or a portion thereof and a plurality of oligonucleotide tags and a plurality of complementary oligonucleotide tags;
• wherein oligonucleotide tags are covalently linked into an identifier oligonucleotide by chemical ligation, and at least two complementary oligonucleotide tags of the hybridization complex are covalently linked by the action of an enzyme comprising a ligase activity;
• wherein the plurality of oligonucleotide tags and complementary oligonucleotide tags in the hybridization complex are at least partly hybridised to each other, thereby forming a double stranded substrate for the enzyme comprising a ligase activity when said enzyme is covalently linking said at least two complementary oligonucleotide tags;
• wherein the oligonucleotide tags are chemically ligated prior to enzymatic ligation of the at least two complementary tags.