Method for producing second-generation library

• US 9,096,951 B2
• Filed: 07/08/2011
• Issued: 08/04/2015
• Est. Priority Date: 02/21/2003
• Status: Expired due to Term

• Current Assignee: Nuevolution A/S (Amgen Inc.)
• Original Assignee: Nuevolution A/S (Amgen Inc.)
• Inventors: Freskgard, Per-Ola, Gouliaev, Alex Haahr, Thisted, Thomas, Olsen, Eva Kampmann
• Primary Examiner(s): Vivlemore, Tracy
• Assistant Examiner(s): Weiler, Karen S
• Application Number:
  US13/179,283
• Publication Number:
  US 20120028812A1
• Time in Patent Office: 1,488 Days
• Field of Search: None
• US Class Current: 1/1
• CPC Class Codes: B01J 19/0046 Sequential or parallel reactions, e.g. for the synthesis of polypeptides or polynucleotides; Apparatus and devices for combinatorial chemistry or for making molecular arrays synthesis methods per se C40B50/00
• CPC Class Codes: B01J 2219/00592 Split-and-pool, mix-and-divide processes
• CPC Class Codes: C12N 15/1068 Template (nucleic acid) mediated chemical library synthesis, e.g. chemical and enzymatical DNA-templated organic molecule synthesis, libraries prepared by non ribosomal polypeptide synthesis [NRPS], DNA/RNA-polymerase mediated polypeptide synthesis
• CPC Class Codes: C12Q 1/6811 Selection methods for production or design of target specific oligonucleotides or binding molecules
• CPC Class Codes: C12Q 2531/113 PCR
• CPC Class Codes: C12Q 2565/501 being an array of oligonucleotides
• CPC Class Codes: C40B 40/08 Libraries containing RNA or DNA which encodes proteins, e.g. gene libraries
• CPC Class Codes: C40B 50/04 using dynamic combinatorial chemistry techniques
• CPC Class Codes: C40B 50/06 Biochemical methods, e.g. using enzymes or whole viable microorganisms
• CPC Class Codes: C40B 50/10 involving encoding steps

Abstract

The present invention relates to a method for generating a second-generation library. In a first step, a library of encoded molecules associated with an identifier nucleic acid comprising codons identifying chemical entities that have participated in the formation of the encoded molecule is provided. In a second step, the library is partitioned and encoded molecules having a certain property are selected. Codons of identifiers of selected encoded molecules are subsequently identified, and a second-generation library is prepared using at least some of the chemical entities coded for by the identified codons. The new focussed library may be used for another partition step to select encoded molecules with a certain property.

Claims

1. A method for producing a focused library comprising a plurality of different molecules, wherein each molecule is linked to an identifier oligonucleotide comprising tags identifying a plurality of reactants that participated in formation of the molecule, the method comprising the steps of:

i) producing an initial library comprising a plurality of different initial molecules, wherein each initial molecule is linked to an identifier oligonucleotide comprising tags identifying a plurality of reactants that participated in formation of the initial molecule, the initial library having a higher diversity than the focused library, and wherein said initial library is produced by a split-and-mix method comprising the steps of;

a. providing, in separate compartments, a plurality of nascent bifunctional complexes each comprising a chemical reaction site and a priming site, and reacting the nascent bifunctional complexes at their chemical reaction sites with one or more reactants;

b. reacting the priming sites of the plurality of nascent bifunctional complexes with one or more tags, wherein the reaction between the priming site and the one or more tags is catalyzed by an enzyme;

wherein steps (a) and (b) provide a plurality of intermediate bifunctional complexes having a modified chemical reaction site containing a structural entity formed from the chemical reaction site and the one or more reactants and a modified priming site containing the one or more tags;

c. pooling the intermediate bifunctional complexes into a single compartment to provide a mixture, and splitting the mixture into separate compartments

d. reacting each compartment containing a mixture of intermediate bifunctional complexes with one or more additional reactants at the modified reactant sites and one or more additional tags identifying the one or more additional reactants at the modified priming sites using the methods of steps (a) and (b); and

e. optionally repeating steps (c) and (d) as many times as desired

ii) subjecting said initial library to a partitioning step, wherein said partitioning step provides a partitioned library synthesized from some, but not all, of the plurality of reactants used in the synthesis of the initial library, and

iii) identifying the reactants used in the synthesis of at least some of the molecules of the partitioned library,

iv) producing a focused library having a lower diversity than the initial library, wherein said focused library is produced by a split-and-mix method comprising the steps of;

a. providing at least some of the reactants used in the synthesis of the molecules of the partitioned library,

b. providing either i) at least some, but not all, of the reactants used in the synthesis of the initial library but not used in the synthesis of the partitioned library, and/or ii) further reactants not used in the synthesis of the initial library,

c. reacting the reactants provided in steps (iv)(a) and (iv)(b) in a split-and-mix method comprising the steps cited in (i)(a)-(i)(e); and

d. producing the focused library comprising a plurality of different molecules.

2. The method of claim 1 comprising the further step of partitioning the focused library.

3. The method of claim 1, wherein the molecules are covalently associated with the identifier oligonucleotides.

4. The method of claim 1, wherein identifier oligonucleotide tags identifying individual chemical entities each have 4 or more nucleotides.

5. The method of claim 1, wherein identifier oligonucleotide tags are separated by a framing sequence.

6. The method of claim 1, wherein the identifier oligonucleotide has at least three tags.

7. The method of claim 1, wherein the focused library is prepared using reactants used to synthesize the molecules appearing in the initial library and reactants that are different than those used in the synthesis of the initial library.

8. The method of claim 1, wherein a reactant is allowed in every position of a reaction sequence used to generate the focused library.

9. The method of claim 1, wherein a reactant is allowed in one position of a reaction sequence used to generate the focused library.

10. The method of claim 1, wherein the identifier comprises 3 or more codons.

11. The method of claim 1, wherein identifier oligonucleotide tags are separated by a framing sequence and wherein the identifier oligonucleotide tags and the framing sequence has a total length of 15 or more nucleotides.

12. The method of claim 1, wherein the initial library contains from 10⁴ to 10²⁰ different molecules.