Enzymatic encoding methods for efficient synthesis of large libraries

A method for the synthesis of a plurality of different bifunctional complexes, said method comprising the steps of

• i) providing a plurality of display oligonucleotides each attached to
• a) one or more chemical reaction site(s) comprising one or more reactive groups and
• b) one or more priming site(s) for enzymatic addition of a tag,
• ii) providing a plurality of first reactants each comprising one or more chemical entities and one or more reactive groups, each first reactant being capable of reacting with
• c) the one or more chemical reaction site(s) of the display oligonucleotide, and/or
• d) one or more reactive groups of a first further reactant comprising one or more chemical entities, wherein said first further reactant is provided simultaneously or sequentially in any order with the first reactant,
• iii) providing a plurality of first oligonucleotide tags each capable of hybridising to part of a first oligonucleotide anti-tag, wherein each first oligonucleotide tag identifies a first reactant and, optionally, a further first reactant,
• iv) providing a plurality of first oligonucleotide anti-tags each capable of hybridizing to at least part of a first oligonucleotide tag provided in step iii) and to at least part of a display oligonucleotide provided in step i),
• v) reacting each of the first reactants provided in step ii) with c) the one or more chemical reaction site(s) of the display oligonucleotides and/or with d) the one or more reactive groups of a first further reactant comprising one or more chemical entities,
• wherein the reaction of complementary reactive groups result in the formation of a covalent bond, and
• wherein one or more reactive group reactions of step v) result in the formation of one or more covalent bond(s) between the one or more chemical reaction site(s) of the display oligonucleotides and at least one chemical entity of at least one reactant selected from the group consisting of a first reactant and a further first reactant,
• vi) hybridising the first anti-tags to display oligonucleotides and to first oligonucleotide tags, wherein method steps v) and vi) are simultaneous or sequential in any order,
• vii) enzymatically ligating display oligonucleotides and first oligonucleotide tags to form a plurality of nascent bifunctional complexes,
• viii) dividing the plurality of nascent bifunctional complexes obtained in step vii) into a plurality of different compartments,
• ix) providing in each different compartment a plurality of different second reactants each comprising one or more chemical entities and one or more reactive groups capable of reacting with
• c) the one or more chemical reaction site(s) of each of the display oligonucleotides, and/or
• d) one or more reactive groups of one or more reactant(s) having reacted in a previous synthesis round, and/or
• e) one or more reactive groups of a second further reactant comprising one or more chemical entities, wherein said second further reactants are provided simultaneously or sequentially in any order with the second reactants,
• x) providing in each different compartment a plurality of second oligonucleotide tags each capable of hybridising to part of a second oligonucleotide anti-tag, wherein different second oligonucleotide tags are provided in each different compartment, and wherein each different second oligonucleotide identifies a different second reactant and, optionally, a further second reactant which may be the same or a different further second reactant in each different compartment,
• xi) providing in each different compartment a plurality of second oligonucleotide anti-tags capable of hybridising to part of a first oligonucleotide tag provided in step iii) and to part of a second oligonucleotide tag provided in step x),
• xii) reacting in each different compartment each of the different second reactants provided in step ix) with c) the one or more chemical reaction site(s) of a display oligonucleotide and/or d) one or more reactive groups of one or more reactant(s) having reacted in a previous synthesis round and/or e) one or more reactive groups of a further second reactant comprising one or more chemical entities,
• wherein said one or more reactions result in the formation of different bifunctional complexes in each different compartment,
• wherein the reaction of complementary reactive groups result in the formation of a covalent bond, and wherein one or more reactive group reactions of step xii) result in
• f) the formation of one or more covalent bond(s) between the one or more chemical reaction site(s) and at least one chemical entity of at least one reactant selected from the group consisting of second reactants and further second reactants, and/or
• g) the formation of one or more covalent bond(s) between a reactant having reacted in a previous synthesis round and at least one chemical entity of at least one reactant selected from the group consisting of second reactants and further second reactants,
• xiii) hybridising anti-tags to first oligonucleotide tags and second oligonucleotide tags in each different compartment,
• wherein method steps xii) and xiii) are simultaneous or sequential in any order,
• xiv) enzymatically ligating in each different compartment first and second oligonucleotide tags in the absence of ligation first and second anti-tag oligonucleotides to provide a bifunctional complex comprising an oligonucleotide identifier having ligated first and second oligonucleotide tags hybridized with unligated first and second anti-tag oligonucleotides
• xv) displacing in each compartment the unligated anti-tags from the bifunctional complex(es) of step (xiv) to provide bifunctional complexes comprising a molecule and a single stranded oligonucleotide identifier comprising tags identifying the reactants which participated in the synthesis of the molecule,
• xv) hybridising a primer to the 3′-end of the single stranded oligonucleotide identifier comprising tags identifying the reactants which participated in the synthesis of the molecule, and
• xvi) extending the primer by a polymerase extension reaction to generate a double stranded identifier oligonucleotide.