Combinatorial selection of oligonucleotide aptamers

• US 20030027184A1
• Filed: 04/11/2002
• Published: 02/06/2003
• Est. Priority Date: 10/26/1998
• Status: Active Grant

• Current Assignee: Allen R. Brasier, Daniel A. Ventura, David G. Gorenstein, David J. King
• Original Assignee: Allen R. Brasier, Daniel A. Ventura, David G. Gorenstein, David J. King
• Inventors: Brasier, Allen R., King, David J., Gorenstein, David G., Ventura, Daniel A.
• Granted Patent: US 7,179,894 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: A61K 38/00 Medicinal preparations containing peptides peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00
• 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: C12N 15/1048 SELEX
• CPC Class Codes: C12N 15/115 Aptamers, i.e. nucleic acids binding a target molecule specifically and with high affinity without hybridising therewith ; Nucleic acids binding to non-nucleic acids, e.g. aptamers
• CPC Class Codes: C12N 2310/313 Phosphorodithioates
• CPC Class Codes: C12N 2310/315 Phosphorothioates
• CPC Class Codes: C40B 40/00 Libraries per se, e.g. arrays, mixtures

Abstract

A random combinatorial selection method is disclosed for the construction of oligonucleotide aptamers in which nuclease resistance is conferred by the inclusion of modified nucleotides. The modified nucleotides are incorporated during PCR amplification to form achiral modified oligonucleotides. Thio-substituted aptamers are provided that bind tightly to the nuclear factor for human IL6 (NF-IL6).

Claims

1. A method of identifying an aptamer comprising the steps of:

(a) synthesizing a random phosphodiester oligonucleotide combinatorial library wherein constituent oligonucleotides comprise at least a set of 5′ and

/or 3′

PCR primer nucleotide sequences flanking a randomized nucleotide sequence;

(b) amplifying the library enzymatically using a mix of four nucleotides, wherein at least a percentage of one of the nucleotides is modified, to form a modified oligonucleotide combinatorial library;

(c) contacting the modified oligonucleotide combinatorial library with a target molecule and isolating a subset of oligonucleotides binding to the target molecule;

(e) amplifying the subset of binding oligonucleotides enzymatically using a mix of four nucleotide substrates, wherein at least one nucleotide substrate is modified, to form a modified oligonucleotide sub-library; and

(f) repeating steps (c)-(e) iteratively until at least one aptamer comprising a modified oligonucleotide population of defined sequence is obtained.

2. The method of claim 1, wherein the modified nucleotide comprises a phosphorothioate.

3. The method of claim 1, wherein the modified nucleotide comprises a phosphorodithioate.

4. The method of claim 1, wherein the modified nucleotide is selected from the group consisting of DATP(α

S), dTTP(α

S), dCTP(α

S) and dGTP(α

S).

5. The method of claim 1, wherein no more than three adjacent phosphate sites are replaced with phosphorothioate groups.

6. The method of claim 1, wherein at least a portion of non-adjacent phosphate sites are replaced with phosphorothioate groups.

7. The method of claim 1, wherein no more than three adjacent phosphate sites are replaced with phosphorodithioate groups.

8. The method of claim 1, wherein at least a portion of non-adjacent phosphate sites are replaced with phosphorodithioate groups.

9. The method of claim 1, wherein the target is a nucleic acid binding protein.

10. The method of claim 1, wherein the target is NF-IL6.

23. An aptamer prepared by the method of claim 1.

25. A pharmaceutical preparation comprising an aptamer prepared in accordance with the method claim 1.

11. A method of thiophosphate aptamer selection comprising:

(a) generating a library of aptamers having a sequence of sufficient length to bind a desired target molecule, wherein at least a portion of the oligonucleotide phosphate groups are replaced with thio-modified nucleotides;

(b) contacting the aptamer library with the target under conditions sufficient to allow complexation between the aptamer library and the target;

(c) removing aptamer library members that bind weakly to the target molecule to yield an aptamer sub-library characterized by enhanced binding to the target;

(d) amplifying the aptamer sub-library enzymatically, wherein the amplifying includes incorporation thio-modified nucleotides into members of the amplified aptamer sub-library; and

(e) repeating steps (b) through (d), for a sufficient number of cycles to yield at least one aptamer of defined sequence and binding specificity for the target.

12. The method of claim 11, wherein said portion of the oligonucleotide phosphate groups are replaced with achiral thio-modified nucleotides.

13. The method of claim 11, wherein said portion of the oligonucleotide phosphate groups are replaced with chiral thio-modified nucleotides.

14. The method of claim 11, wherein the thio-modified nucleotide comprises a phosphoromonothioate.

15. The method of claim 11, wherein the thio-modified nucleotide comprises a phosphorodithioate.

16. The method of claim 11, wherein the thio-modified nucleotide is selected from the group consisting of dATP(α

S), dTTP(α

S), dCTP(α

S) and dGTP(α

S).

17. The method of claim 11, wherein no more than three adjacent phosphate sites are replaced with phosphorothioate groups.

18. The method of claim 11, wherein at least a portion of non-adjacent phosphate sites are replaced with phosphorothioate groups.

19. The method of claim 11, wherein no more than three adjacent phosphate sites are replaced with phosphorodithioate groups.

20. The method of claim 11, wherein at least a portion of non-adjacent phosphate sites are replaced with phosphorodithioate groups.

21. The method of claim 11, wherein the target is a nuclear binding protein.

22. The method of claim 11, wherein the target is NF-IL6.

24. An aptamer prepared by the method of claim 11.

26. A pharmaceutical preparation comprising an aptamer prepared in accordance with the method claim 11.

27. An aptamer specific for NF-IL6 essentially homologous to the sequences of oligonucleotides selected from the group consisting of SEQ ID NOS.:

5 through 10.

28. The aptamer of claim 27, wherein one or more nucleotides have at least one thiophosphate or dithiophosphate group.

29. An NF-IL6 binding aptamer comprising a sequence of nucleotides essentially homologous to a nucleotide sequence of the formula:

5′

d GGGCCC GCTGN₁ ACN₂N₃G C ACACG

wherein substantially all dA nucleotides are phosphothioated and N₁ is a C or T;

N₂ is an A or G; and

N₃ is a T or C.

30. An NF-IL6 binding aptamer comprising a sequence of nucleotides essentially homologous to a nucleotide sequence of the formula:

31. An NF-IL6 binding aptamer comprising the sequence of nucleotides:

G C T G T A C A T G (SEQ ID NO.;

     12)

wherein one or more nucleotides have at least one thio-modified phosphate group.

32. The aptamer of claim 31, wherein substantially all dA phosphate groups are thiophosphate groups.

33. The aptamer of claim 31, wherein substantially all dA phosphate groups are dithiophosphate groups.

34. A NF-IL6 binding oligonucleotide comprising at least the sequence of nucleotides:

G T T G T C C C A C (SEQ ID NO.;

     13)

wherein one or more nucleotides have at least one thio-modified phosphate group.

35. A NF-IL6 binding oligonucleotide comprising at least the sequence of nucleotides:

G T T G T T G T C C (SEQ ID NO.;

     14)

wherein one or more nucleotides have at least one thio-modified phosphate group.