Selection of proteins using RNA-protein fusions

• US 6,207,446 B1
• Filed: 10/29/1999
• Issued: 03/27/2001
• 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: Liu, Rihe, Szostak, Jack W., Roberts, Richard W.
• Primary Examiner(s): Zitomer, Stephanie
• Assistant Examiner(s): FORMAN, BETTY J
• Application Number:
  US09/430,049
• Time in Patent Office: 515 Days
• Field of Search: 435/6, 435/68.1, 435/278.2, 536/23.1, 536/24.1, 530/402
• US Class Current: 435/287.2
• 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 microchip comprising an array of immobilized single-stranded nucleic acids, said nucleic acids being hybridized to RNA-protein fusions, wherein each of said RNA-protein fusions comprises an RNA covalently bonded at the RNA 3′

end to a protein through a peptide acceptor selected from the group consisting of puromycin, tRNA-puromycin conjugate, phenylalanyl-adenosine, tyrosyl adenosine, alanyl adenosine, phenylalanyl 3′

deoxy 3′

amino adenosine, alanyl 3′

deoxy 3′

amino adenosine, and tyrosyl 3′

deoxy 3′

amino adenosine, and wherein said protein is encoded by said covalently bonded RNA.

6. The microchip of claim 1, wherein said peptide acceptor is covalently bonded to said RNA through a dCdC moiety.

7. The microchip of claim 1, wherein each of said RNA-protein fusions further comprises a covalently bonded pause sequence, a covalently bonded DNA sequence, or a covalently bonded DNA analog sequence, positioned between the RNA 3′

end and the peptide acceptor.

8. The microchip of claim 7, wherein said pause sequence, said DNA sequence, or said DNA analog sequence is of a length sufficient to span the distance between the decoding site and the peptidyl transfer center of a ribosome.

9. The microchip of claim 7, wherein said pause sequence, said DNA sequence or said DNA analog sequence is approximately 60-70 Å

in length.

10. The microchip of claim 7, wherein said pause sequence, said DNA sequence, or said DNA analog sequence is less than approximately 80 nucleotides in length.

11. The microchip of claim 10, wherein said pause sequence, said DNA sequence, or said DNA analog sequence is less than approximately 45 nucleotides in length.

12. The microchip of claim 11, wherein said pause sequence, said DNA sequence, or said DNA analog sequence is between approximately 21-30 nucleotides in length.

13. The microchip of claim 7, wherein said pause sequences said DNA sequence, or said DNA analog sequence is joined to said RNA molecule using a DNA splint.

14. The microchip of claim 7, wherein said pause sequence, said DNA sequence, or said DNA analog sequence comprises a non-nucleotide moiety.

15. The microchip of claim 14, wherein said non-nucleotide moiety is one or more HO(CH₂CH₂O)₃PO₂ (polyethylene glycol phosphate) moieties.

2. A microchip comprising an array of immobilized single-stranded nucleic acids, said nucleic acids being hybridized to RNA-protein fusions, wherein each of said RNA-protein fusions comprises an RNA covalently bonded at the RNA 3′

end to a protein through a DNA pause sequence or a DNA analog pause sequence and a peptide acceptor, and wherein said proteins encoded by said covalently bonded RNA.

3. The microchip of claim 2, wherein said RNA is covalently bonded though an amide bond to said protein.

4. The microchip of claim 2, wherein said RNA is a messenger RNA.

5. The microchip of claim 2, wherein said peptide acceptor is puromycin.

16. The microchip of claim 2, wherein said DNA pause sequence or said DNA analog pause sequence comprises a non-nucleotide moiety.

17. The microchip of claim 16, wherein said non-nucleotide moiety is one or more HO(CH₂CH₂O)₃ PO₂ (polyethylene glycol phosphate) moieties.

18. The microchip of claim 2, wherein said peptide acceptor is covalently bonded to said DNA pause sequence or said DNA analog pause sequence through a dCdC moiety.

19. The microchip of claim 2, wherein said DNA pause sequence or said DNA analog pause sequence is of a length sufficient to span the distance between the decoding site and the peptidyl transfer center of a ribosome.

20. The microchip of claim 2, wherein said DNA pause sequence or said DNA analog pause sequence is approximately 60-70 Å

in length.

21. The microchip of claim 2, wherein said DNA pause sequence or said DNA analog pause sequence is less than approximately 80 nucleotides in length.

22. The microchip of claim 21, wherein said DNA pause sequence or said DNA analog pause sequence is less than approximately 45 nucleotides in length.

23. The microchip of claim 22, wherein said DNA pause sequence or said DNA analog pause sequence is between approximately 21-30 nucleotides in length.

24. The microchip of claim 2, wherein said DNA pause sequence or said DNA analog pause sequence is joined to said RNA molecule using a DNA splint.