Nucleic acid-protein fusion molecules and libraries

• US 6,281,344 B1
• Filed: 02/05/1999
• Issued: 08/28/2001
• Est. Priority Date: 01/21/1997
• Status: Expired due to Term

• Current Assignee: Adnexus Therapeutics, Inc. (Bristol-Myers Squibb Company)
• Original Assignee: The General Hospital Corporation (Mass General Brigham, Inc.)
• Inventors: Szostak, Jack W., Liu, Rihe, Roberts, Richard W.
• Primary Examiner(s): Zitomer, Stephanie W.
• Assistant Examiner(s): FORMAN, BETTY J
• Application Number:
  US09/244,796
• Time in Patent Office: 935 Days
• Field of Search: 435/6, 435/68.1, 435/69.1, 536/23.1, 536/24.2, 536/24.1, 530/300
• US Class Current: 536/23.1
• CPC Class Codes: C07B 2200/11 Compounds covalently bound to a solid support
• CPC Class Codes: C07K 14/82 Translation products from oncogenes
• CPC Class Codes: C07K 2319/00 Fusion polypeptide
• CPC Class Codes: C12N 15/1048 SELEX
• CPC Class Codes: C12N 15/1062 mRNA-Display, e.g. polypeptide and encoding template are connected covalently
• CPC Class Codes: C40B 30/04 by measuring the ability to specifically bind a target molecule, e.g. antibody-antigen binding, receptor-ligand binding
• CPC Class Codes: G01N 33/6845 Methods of identifying protein-protein interactions in protein mixtures

Abstract

Described herein are methods and reagents for the selection of protein molecules that make use of RNA-protein fusions.

Claims

1. A molecule comprising a deoxyribonucleic acid (DNA) covalently bonded to a protein through a peptide acceptor, said peptide acceptor being a molecule that can be added to the C-terminus of a growing protein chain by the catalytic activity of a ribosomal peptidyl transferase, wherein said molecule further comprises a nucleic acid sequence regulating the initiation and start of translation, said nucleic acid sequence being operably linked to a protein coding sequence.

2. The molecule of claim 1, wherein said peptide acceptor is puromycin.

3. The molecule of claim 1, wherein said protein coding sequence is a ribonucleic acid (RNA) sequence.

4. The molecule of claim 3, wherein said DNA is positioned between said RNA and said protein.

5. The molecule of claim 1, wherein said molecule is immobilized on a solid support.

6. The molecule of claim 5, wherein said solid support is a chip.

7. A molecule comprising a double-stranded nucleic acid covalently bonded to the problem which it encodes through a puromycin, wherein said double-stranded nucleic acid comprises a nucleic acid sequence regulating the initiation and start of translation, said nucleic acid sequence being operably linked to a protein coding sequence.

8. The molecule of claim 7, wherein said double-stranded nucleic acid comprises an RNA-DNA hybrid.

9. The molecule of claim 8, wherein said RNA-DNA hybrid is produced by reverse transcription.

10. The molecule of claim 7, wherein said molecule is immobilized on a solid support.

11. The molecule of claim 10, wherein said solid support is a chip.

12. A protein-encoding RNA covalently bonded to its encoded DNA molecule.

13. A library of molecules, each comprising a deoxyribonucleic acid (DNA) covalently bonded to a protein through a peptide acceptor, said peptide acceptor being a molecule that can be added to the C-terminus of a growing protein chain by the catalytic activity of a ribosomal peptidyl transferase, wherein each of said molecules further comprises a nucleic acid sequence regulating the initiation and start of translation, said nucleic acid sequence being operably linked to a protein coding sequence.

14. The library of claim 13, wherein said peptide acceptor is puromycin.

21. The library of any one of claims 13 or 15, wherein said library comprises at least 10⁹ molecules.

22. The library of any one of claims 13, 15, or 18, wherein said library comprises at least 10¹³ molecules.

23. The library of any one of claims 13, 15, or 18, wherein said library is immobilized on a solid support.

24. The library of claim 23, wherein said solid support is a chip.

15. A library of molecules, each comprising a double-stranded nucleic acid covalently bonded to a protein through a puromycin, wherein each double-stranded nucleic acid comprises a nucleic acid sequence regulating the initiation and start of translation, said nucleic acid sequence being operably linked to a protein coding sequence.

16. The library of claim 15, wherein said double-stranded nucleic acid comprises an RNA-DNA hybrid.

17. The library of claim 16, wherein said RNA-DNA hybrid is produced by reverse transcription.

20. The library of claim 15, wherein said nucleic acid comprises the coding sequence of said protein.

18. A library of at least 10⁹ molecules, each molecule comprising a protein-encoding nucleic acid linked to said encoded protein through a DNA covalent bond.

19. The library of claim 18, wherein said nucleic acid is RNA.

25. A molecule comprising a double-stranded nucleic acid covalently bonded to a protein through a puromycin.

26. The molecule of claim 25, wherein said double-stranded nucleic acid encodes said protein.

27. The molecule of claim 25, wherein said double-stranded nucleic acid comprises an RNA-DNA hybrid.

28. The molecule of claim 27, wherein said RNA-DNA hybrid is produced by reverse transcription.

29. The molecule of claim 25, wherein said molecule is immobilized on a solid support.

30. The molecule of claim 29, wherein said solid support is a chip.

31. A library of molecules, each comprising a double-stranded nucleic acid covalently bonded to a protein through a puromycin.

32. The library of claim 31, wherein said double-stranded nucleic acid encodes said protein.

33. The library of claim 31, wherein said double-stranded nucleic acid comprises an RNA-DNA hybrid.

34. The library of claim 33, wherein said RNA-DNA hybrid is produced by reverse transcription.

35. The library of claim 31, wherein said molecules are immobilized on a solid support.

36. The library of claim 35, wherein said solid support is a chip.