Methods for producing 5′-nucleic acid-protein conjugates

• US 6,623,926 B1
• Filed: 06/01/2000
• Issued: 09/23/2003
• Est. Priority Date: 06/01/1999
• Status: Expired due to Term

• Current Assignee: Bristol-Myers Squibb Company
• Original Assignee: Phylos, Inc. (Bristol-Myers Squibb Company)
• Inventors: Wright, Martin C., McPherson, Michael, Lohse, Peter
• Primary Examiner(s): Low, Christopher S. F.
• Assistant Examiner(s): KAM, CHIH MIN
• Application Number:
  US09/585,207
• Time in Patent Office: 1,209 Days
• Field of Search: 530/395, 530/391.9, 530/391.5, 435/6, 435/320.1, 435/91.1, 435/69.7, 536/23.1, 424/179.1, 514/2
• US Class Current: 435/6.14
• 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: C07K 14/003 Peptide-nucleic acids (PNAs)
• CPC Class Codes: C07K 17/00 Carrier-bound or immobilised peptides carrier-bound or immobilised enzymes C12N11/00; Preparation thereof

Abstract

Disclosed herein is a method for generating a 5′-nucleic acid-protein conjugate, the method involving: (a) providing a nucleic acid which carries a reactive group at its 5′ end; (b) providing a non-derivatized protein; and (c) contacting the nucleic acid and the protein under conditions which allow the reactive group to react with the N-terminus of the protein, thereby forming a 5′-nucleic acid-protein conjugate. Also disclosed herein are 5′-nucleic acid-protein conjugates and methods for their use.

Claims

1. A method for generating a 5′

-nucleic acid-protein conjugate, said method comprising contacting a non-derivatized protein and a nucleic acid which carries a reactive group at its 5′

end under conditions that allow said reactive group to react with an N-terminus of said non-derivatized protein, thereby forming a 5′

-nucleic acid-protein conjugate, wherein said non-derivatized protein comprises an N-terminal cysteine and the nucleic acid reactive group is an aminothiol reacting group, or wherein said non-derivatized protein comprises an α

-helical tetracysteine motif located proximal to the N-terminus and the nucleic acid reactive group is a bisarsenical group that is reactive with the tetracysteine group.

2. The method of claim 1, wherein said nucleic acid is about 20 nucleotides or greater in length.

3. The method of claim 2, wherein said nucleic acid is about 120 nucleotides or greater in length.

4. The method of claim 1, wherein said nucleic acid is greater than 120 nucleotides in length.

5. The method of claim 1, wherein said protein is about 20 amino acids or greater in length.

6. The method of claim 5, wherein said protein is about 40 amino acids or greater in length.

7. The method of claim 1, wherein said protein is between 2-300 amino acids in length.

8. The method of claim 1, wherein said contacting step is carried out in a physiological buffer.

9. The method of claim 1, wherein said contacting step is carried out using a nucleic acid and a protein, both of which are present at a concentration of less than 1 mM.

10. The method of claim 1, wherein said nucleic acid is DNA.

11. The method of claim 1, wherein said nucleic acid is RNA.

12. The method of claim 11, wherein said RNA is mRNA.

13. The method of claim 1, wherein said nucleic acid comprises the coding sequence for said protein.

14. The method of claim 1, wherein said N-terminal cysteine is exposed by protein cleavage.

15. The method of claim 1, wherein said α

-helical tetracysteine motif comprises [c]Cys-[c]Cys-X-X-[c]Cys-[c]Cys (SEQ ID NO;

6), wherein X is any amino acid.

16. A 5′

-nucleic acid-protein conjugate produced by the method of claim 1.

17. A 5′

-nucleic acid-protein conjugate comprising;

(a) a nucleic acid covalently bound through a reactive group at its 5′

-terminus to an N-terminus of a non-derivatized protein, wherein said protein comprises an N-terminal cysteine and said reactive group is an aminothiol reacting group;

or

(b) a nucleic acid covalently bound through a 5′

-terminal reactive group to an N-terminus of a non-derivatized protein, wherein said protein comprises an α

-helical tetracysteine motif located proximal to the N-terminus and said reactive group is a bisarsenical group that is reactive with the tetracysteine group.

18. The conjugate of claim 17, wherein said conjugate is immobilized on a solid support.

19. The conjugate of claim 18, wherein said solid support is a bead or chip.

20. The conjugate of claim 18, wherein said conjugate is one of an array immobilized on said solid support.

21. The conjugate of claim 17, wherein said nucleic acid is about 20 nucleotides or greater in length.

22. The conjugate of claim 17, wherein said protein is about 20 amino acids or greater in length.

23. The conjugate of claim 17, wherein said nucleic acid is DNA.

24. The conjugate of claim 17, wherein said nucleic acid is RNA.

25. The conjugate of claim 17, wherein said nucleic acid comprises the coding sequence for said protein.

26. The conjugate of claim 17, wherein said α

-helical tetracysteine motif comprises [c]Cys-[c]Cys-X-X-[c]Cys-[c]Cys (SEQ ID NO;

6), wherein X is any amino acid.