Method for synthesising templated molecules

• US 20050221316A1
• Filed: 03/14/2003
• Published: 10/06/2005
• Est. Priority Date: 03/15/2002
• Status: Active Grant

• Current Assignee: Nuevolution A/S (Amgen Inc.)
• Original Assignee: Nuevolution A/S (Amgen Inc.)
• Inventors: Haahr, Alex, Franch, Thomas, Pedersen, Henrik, Holtmann, Anette, Felding, Jakob
• Granted Patent: US 7,413,854 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C07B 2200/11 Compounds covalently bound to a solid support
• CPC Class Codes: C07D 405/04 directly linked by a ring-member-to-ring-member bond
• CPC Class Codes: C07H 21/00 Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• CPC Class Codes: C07H 21/04 with deoxyribosyl as saccharide radical
• 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 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C40B 40/06 Libraries containing nucleotides or polynucleotides, or derivatives thereof
• CPC Class Codes: C40B 40/14 Libraries containing macromolecular compounds and not covered by groups C40B40/06 - C40B40/12

Abstract

The invention relates to a method for synthesizing templated molecules attached to the templated which directed the synthesis thereof. The method involves a template, a scaffold functional entity and a functional entity attached to a building block, which, in turn, is attached the template. The scaffold functional entity and the functional entity of the building block are both provided with complementary dimerization domains allowing the functional entities to come into close proximity when the complementary domains interact with to each other. The method may be used for generating libraries of templated molecules which may be selected for biological activity.

Claims

1. A method for synthesizing a templated molecule, comprising the steps of:

a) providing at least one template comprising one or more codons,

b) providing a first functional entity attached to a zipping domain, said zipping domain comprising a first part of a molecule pair, said first part being capable of reversible interaction with a second part of the molecule pair,

c) providing one or more building blocks, each comprising an anti-codon, a further functional entity and a linker connecting the anti-codon and the functional entity, wherein the anti-codon complements a codon of a template, and the functional entity is connected to a zipping domain comprising the second part of said molecule pair and is capable of being chemically connected to the first functional entity,

d) contacting the components of step a), b), and c) with each other under conditions allowing specific hybridization of the anti-codon(s) to the codon(s) of the template(s) and dimerization of the two parts of the molecule pair,

e) allowing the functional entity of the building block to form a chemical connection to the first functional entity,

f) optionally, cleaving one or more linkers, provided that at least one linker remains to connect the functional entities with the template,

g) obtaining a templated molecule attached to the template which directed the synthesis thereof.

2. The method according to claim 1, wherein steps d) through f) are repeated one or more times.

3. The method according to claim 2, wherein the repetition is conducted using the templated molecule attached to the template which directed the synthesis thereof according to step g) as the first functional entity attached to a zipping domain in the contacting step according to step d).

4. The method according to claim 1, wherein the first functional entity is covalently connected to the template.

5. The method according to claim 1, wherein the first functional entity is connected by hybridization to the template.

6. The method according to claim 1, wherein the first functional entity is part of a building block.

7. The method according to claim 6, wherein the zipper domain polarity of the building block harboring the first functional entity is reverse compared to the zipper domain polarity of the building block harboring the further functional entity.

8. The method according to claim 1, wherein the zipping domain of the first functional entity is present in the template.

9. The method according to claim 1, wherein the molecule pair comprises two complementary sequences of nucleic acids or nucleic acid analogs.

10. The method according to claim 1, wherein the first functional entity is connected to a sequence of nucleic acids complementing a sequence of nucleic acids harbored by the template.

11. The method according to claim 1, wherein the zipping domain is a part of the linker of the building block.

12. The method according to claim 8, wherein the zipping domain is proximal to the functional entity.

13. The method according to claim 11, wherein the zipping domain is separated from the functional entity by no more than 2 nucleic acid monomers.

14. The method according to claim 13, wherein the zipping domain and the first functional entity are separated by no more than 2 nucleic acid monomers.

15. The method according to claim 1, wherein the zipping domain of the further functional entity of the building block and the first functional entity are distanced from the respective functional entities by the same number of nucleic acid monomers.

16. The method according to claim 1, wherein the zipping domain sequence is 3 to 20 nucleic acid monomers.

17. The method according to claim 16, wherein the zipping domain sequence is 4 to 16 nucleic acid monomers.

18. The method according to claim 17, wherein the zipping domain sequence is 5 to 10 nucleic acid monomers.

19. The method according to claim 1, wherein the linker between the anti-codon and the zipping domain is a single bond.

20. The method according to claim 1, wherein the annealing temperature of the codon:

anti-codon is higher than the annealing temperature of the zipping domain hybrid.

21. The method according to claim 20, wherein the difference between the annealing temperatures is 10°

C. or above.

22. The method according to claim 20, wherein the difference between the annealing temperatures is 25°

C. or above.

23. The method according to claim 1, wherein the conditions for allowing specific hybridization of the anti-codon(s) to the codon(s) of the template(s) are distinct from the conditions allowing for optimal dimerisation of the two pairs of the molecule pair.

24. The method according to claim 23, wherein the conditions during specific hybridization of the anti-codon(s) to the codon(s) include a concentration of codons and/or anti-codons, which is higher than the concentration of codons and/or anti-codons used during dimerisation of the two pairs of the molecule pair.

25. The method according to claim 24, wherein the concentration during hybridization of codon(s) and anti-codons is at least 10 times higher compared to the concentration used for dimerisation of the two pairs of the zipping domain.

26. The method according to claim 1, wherein the contacting according to step d) is performed by alternating the temperature below and above the annealing temperature of the zipping domain.

27. The method according to claim 26, wherein the alternating is performed a plurality of times.

28. The method according to claim 26, wherein the highest temperature is below the annealing temperature of the codon:

anti-codon hybrid.

29. A templated molecule obtainable according to claim 1.