Method for Obtaining Structural Information Concerning an Encoded Molecule and Method for Selecting Compounds

• US 20080305957A1
• Filed: 09/17/2004
• Published: 12/11/2008
• Est. Priority Date: 09/18/2003
• Status: Active Grant

• Current Assignee: Nuevolution A/S (Amgen Inc.)
• Original Assignee: Nuevolution A/S (Amgen Inc.)
• Inventors: Rasmussen, Torben Ravn, Thisted, Thomas, Freskgard, Per-Ola, Lundorf, Mikkel Dybro
• Granted Patent: US 11,118,215 B2
• Time in Patent Office: Days
• US Class Current: 506/4
• 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 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 2531/113 PCR
• CPC Class Codes: C12Q 2565/501 being an array of oligonucleotides

Abstract

In one aspect, the present invention relates to a method for obtaining structural information about an encoded molecule. The encoded molecule may be produced by a reaction of a plurality of chemical entities and may be capable of being connected to an identifier oligonucleotide containing codons informative of the identity of the chemical entities which have participated in the formation of the encoded molecule. In a certain embodiment, primers are designed complementary to the codons appearing on the identifier oligonucleotide, and the presence, absence or relative abundance of a codon is evaluated by mixing a primer with the identifier oligonucleotide in the presence of a polymerase and substrate (deoxy)ribonucleotide triphosphates and measuring the extension reaction. In another aspect, the invention provides a method for selecting compounds which binds to a target. More specifically, the invention relates to a method in which a target associated with an oligonucleotide initially is mixed with a library of complexes, each complex comprising a display molecule and an oligonucleotide identifying said display molecule. Next, due an increased proximity, the target oligonucleotide is coupled to the identifier oligonucleotide of complexes having a display molecule with affinity towards the target. In a final stage the coupled nucleotides are analysed to deduce at least the identity of the display molecule.

Claims

1. A method for identifying display molecule(s) having affinity towards molecular target(s), comprising the steps of

mixing one or more molecular target(s) associated with target oligonucleotide(s) and a library of bifunctional complexes, each bifunctional complex of the library comprising a display molecule attached to an identifier oligonucleotide, which codes for said display molecule,

coupling to the target oligonucleotide(s) the identifier oligonucleotide of complexes comprising display molecules binding to the target, and

deducing the identity of the binding display molecule(s) and/or the molecular target(s) from the coupled product between the identifier oligonucleotide(s) and the target oligonucleotide(s).

2. The method of claim 1, wherein the display molecule is a reaction product of two or more chemical entities and the identifier oligonucleotide comprises codons identifying the chemical entities.

3. The method of claim 1, wherein one or more members of the library are potentially binding compounds tagged with identifier oligonucleotides.

4. The method according to claim 1, wherein the chemical entities are precursors for a structural unit appearing in the display molecule.

5. The method according to claim 1, wherein some or all of the chemical entities are not naturally occurring α

-amino acids or precursors thereof.

6. The method according to claim 1, wherein each codon comprises 4 or more nucleotides.

7. The method according to claim 1, wherein the display molecules of the library complexes are non-α

-polypeptides.

8. The method according to claim 1, wherein the display molecules of the library complexes are non-nucleic acids.

9. The method according to claim 1, wherein the display molecule has a molecular weight less than 2000 Dalton, preferably less than 1000 Dalton, and more preferred less than 500 Dalton.

10. The method according to claim 1, wherein the identifier oligonucleotide uniquely identifies the display molecule.

11. The method according to claim 1, wherein one or more chemical entities are transferred to the nascent display molecule by a chemical building block further comprising an anti-codon.

12. The method according to claim 11, wherein the information of the anti-codon is transferred in conjunction with the chemical entity to the nascent complex.

13. The method according to claim 1, wherein the chemical entities are reacted without enzymatic interaction.

14. The method according to claim 1, wherein the codons are separated by a framing sequence.

15. The method according to claim 1, wherein the display molecule and the identifier oligonucleotide are joined by a selectively cleavable linker.

16. The method according to claim 15, wherein the linker is cleaved by irradiation.

17. The method according to claim 1, wherein the library comprises at least one complex and, if it comprises, two or more complexes, they are not identical.

18. The method according to claim 1, wherein the library comprises 1,000 or more different complexes.

19. The method according to claim 1, wherein the molecular target is of a biological origin.

20. The method according to claim 1, wherein the molecular target is immobilized on a solid support.