Method for selection of proteins using RNA-protein fusions

• US 6,258,558 B1
• Filed: 01/14/1998
• Issued: 07/10/2001
• 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): Jones, W. Gary
• Assistant Examiner(s): FORMAN, BETTY J
• Application Number:
  US09/007,005
• Time in Patent Office: 1,273 Days
• Field of Search: 435/6, 435/69.1, 435/91.2
• US Class Current: 435/69.1
• 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 method for generating a DNA molecule which encodes a desired protein, comprising the steps of:

a) providing a population of candidate RNA molecules, each of which comprises a translation initiation sequence and a start codon operably linked to a candidate protein coding sequence and each of which is covalenty bonded to a peptide acceptor at the 3′

end of said candidate protein coding sequence said peptide acceptor being a molecule that can be added to the C-terminus of protein chain by the catalytic activity of a ribosomal peptidyl transferae;

b) in vitro translating said candidate protein coding sequences of said candidate RNA molecules to produce a population of candidate RNA-protein fusions;

c) selecting from said population a desired RNA-protein fusion based on fusion binding or activity; and

d) generating from said RNA portion of said selected fusion a DNA molecule, thereby generating a DNA molecule which encodes said desired protein.

9. The method of any one of claims 1, 2, 3, 6, and 7, wherein said population of candidate RNA-protein fusions are recovered from the in vitro translation reaction mixture prior to said selecting step.

10. The method of claim 9, wherein said population of candidate RNA-protein fusions are recovered by an RNA or protein chromatographic step, or a combination thereof.

11. The method of claim 9, wherein said population of candidate RNA-protein fusions are recovered by binding to dT-agarose or thiopropyl sepharose, or a combination thereof.

15. The method of any one of claims 1, 2, 3, 6, or 7, wherein said RNA is messenger RNA.

16. The method of any of claims 1, 2, 3, 6, and 7, wherein said peptide acceptor is puromycin.

17. The method of claim 16, wherein said puromycin is covalently bonded to said candidate protein coding sequence through a dCdC moiety.

18. The method of any one of claims 1, 2, 3, 6, and 7, wherein each of said candidate RNA molecules further comprises a covalently bonded pause sequence, a covalently bonded DNA sequence or, a covalently bonded DNA analog sequence, any one of which is positioned between said candidate protein coding sequence and said peptide acceptor.

19. The method of claim 18, 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.

20. The method of claim 18, wherein said pause sequence, said DNA sequence, or said DNA analog sequence is approximately 60-70 Å

in length.

21. The method of claim 18, wherein said pause sequence, said DNA sequence, or said DNA analog sequence is less than approximately 80 nucleotides in length.

22. The method of claim 18, wherein said pause sequence, said DNA sequence, or said DNA analog sequence is less than approximately 45 nucleotides in length.

23. The method of claim 18, wherein said pause sequence, said DNA sequence, or said DNA analog sequence is between approximately 21-30 nucleotides in length.

24. The method of claim 18, wherein said pause sequence, said DNA sequence, or said DNA analog sequence is covalently bonded to said RNA molecule using a DNA splint.

25. The method of claim 18, wherein said pause sequence, said DNA sequence, or said DNA analog sequence comprises a non-nucleotide moiety.

26. The method of claim 25, wherein said non-nucleotide moiety is one or more HO(CH₂CH₂O)₃PO₂ (polyethylene glycol phosphate) moieties.

27. The method of any one of claims 1, 2, 3, 6, and 7, wherein said population of candidate RNA molecules comprises at least 10¹³ different RNA molecules.

28. The method of any one of claims 1, 2, 3, 6, and 7, wherein said in vitro translation reaction is carried out in a lysate prepared from a eukaryotic cell or portion thereof.

29. The method of claim 28, wherein said in vitro translation reaction is carried out in a reticulocyte lysate.

30. The method of claim 28, wherein said in vitro translation reaction is carried out in a wheat germ lysate.

31. The method of any one of claims 1, 2, 3, 6, and 7, wherein said in vitro translation reaction is carried out in a lysate prepared from a bacterial cell or portion thereof.

32. The method of any one of claims 1, 2, 3, 6, and 7, wherein said selection step comprises binding of the protein of said desired RNA-protein fusion to an immobilized binding partner.

33. The method of any one of claims 1, 2, 4, 6, and 7, wherein said selection step comprises assaying for a functional activity of the protein of said desired RNA-protein fusion.

34. The method of any one of claims 1, 2, 6, or 7, wherein said DNA molecule is amplified.

35. The method of any one of claims 1, 2, 6, or 7, wherein said method further comprises transcribing an RNA molecule from said DNA molecule and repeating steps (a) through (d).

36. The method of any one of claims 1, 2, 3, 6 and 7, wherein said RNA is covalently bonded though an amide bond to said protein in said RNA-protein fusion.

37. The method of any one of claims 1, 2, 3, 6, and 7, wherein, following the in vitro translating step, an incubation is carried out in the presence of 50-100 mM Mg²⁺.

38. The method of claim 37, wherein said incubation is carried out at approximately −

20°

C.

39. The method of claim 37, wherein said incubation is carried out for approximately 12-48 hours.

40. The method of any one of claims 1, 2, 3, 6, and 7, wherein said RNA-protein fusion further comprises a covalently bonded nucleic acid sequence or covalently bonded nucleic acid analog sequence, either of which is positioned proximal to said peptide acceptor, said covalently bonded nucleic acid sequence or said covalently bonded nucleic acid analog sequence increasing the flexibility of said RNA-protein fusion relative to an RNA-protein fusion lacking said covalently bonded nucleic acid sequence or said covalently bonded nucleic acid analog sequence.

41. The method of any one of claims 1, 2, 3, 6, and 7, wherein step (b) results in the conversion of 40% or more of said candidate RNA molecules of step (a) into RNA-protein fusions.

2. A method for generating a DNA molecule which encodes a protein having an altered function relative to a reference protein, comprising the steps of:

a) producing a population of candidate RNA molecules from a population of candidate DNA templates, said candidate RNA molecules each having a candidate protein coding sequence which differs from the reference protein coding sequence, said RNA molecules each comprising a translation initiation sequence and a start codon operably linked to said candidate protein coding sequence and each being covalently bonded to a peptide acceptor at the 3′

end of said candidate protein coding sequence 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;

b) in vitro translating said candidate protein coding sequences of said candidate RNA molecules to produce a population of RNA-protein fusions;

c) selecting from said population an RNA-protein fusion having an altered function based on fusion binding or activity; and

d) generating from said RNA portion of said selected fusion a DNA molecule, thereby generating a DNA molecule which encodes said protein having said altered function.

3. A method for the selection of a desired RNA, comprising the steps of:

a) providing a population of candidate RNA molecules, each of which comprises a translation initiation sequence and a start codon operably linked to a candidate protein coding sequence and each of which is covalently bonded to a peptide acceptor at the 3′

end of said candidate protein coding sequence, 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;

b) in vitro translating said candidate protein coding sequences of said candidate RNA molecules to produce a population of candidate RNA-protein fusions; and

c) selecting from said population a desired RNA-protein fusion based on fusion binding or activity, thereby selecting said desired RNA.

4. The method of claim 3, wherein said RNA is reverse transcribed either before or after selecting step.

5. The method of claim 3, wherein said method further comprises repeating step (a) through (c).

6. A method for the selection of a DNA molecule which encodes a desired protein, comprising the steps of:

a) providing a population of candidate RNA molecules, each of which comprises a translation initiation sequence and a start codon operably linked to a candidate protein coding sequence and each of which is covalently bonded to a peptide acceptor at the 3′

end of said candidate protein coding sequence, 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;

b) in vitro translating said candidate protein coding sequences of said candidate RNA molecules to produce a population of candidate RNA-protein fusions;

c) generating a DNA copy of the RNA of said population of candidate RNA-protein fusions; and

d) selecting from said population a desired fusion based on fusion binding or activity, thereby selecting a DNA molecule which encodes said desired protein.

8. The method of claim 6 or 7, wherein said DNA copy is generated either before or after isolation of said population of RNA-protein fusions from the in vitro or in situ translation mixture.

12. The method of any one of claims 6 and 7, wherein said population of candidate RNA-protein fusions are recovered from the in vitro translation reaction mixture prior to said generating step.

13. The method of claim 12, wherein said population of candidate RNA-protein fusions are recovered by an RNA or protein chromatographic step, or a combination thereof.

14. The method of claim 12, wherein said population of candidate RNA-protein fusions are recovered by binding to dT-agarose or thiopropyl sepharose, or a combination thereof.

7. A method for the selection of a DNA molecule which encodes a protein having an altered function relative to a reference protein, comprising the steps of:

a) producing a population of candidate RNA molecules from a population of candidate DNA templates, said candidate RNA molecules each having a candidate protein coding sequence which differs from the reference protein coding sequence, said RNA molecules each comprising a translation initiation sequence and a start codon operably linked to said candidate protein coding sequence and each being covalently bonded to a peptide acceptor at the 3′

end of said candidate protein coding sequence, 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;

b) in vitro translating said candidate protein coding sequences of said candidate RNA molecules to produce a population of RNA-protein fusions;

c) generating a DNA copy of the RNA of said population of RNA-protein fusions; and

d) selecting from said population a fusion having an altered function based on fusion binding or activity, thereby selecting a DNA molecule which encodes said protein having said altered function.

42. A method for generating a DNA molecule which encodes a desired protein, comprising the steps of:

a) providing a population of candidate RNA molecules, each of which comprises a translation initiation sequence and a start codon operably linked to a candidate protein coding sequence and each of which is covalently bonded to a peptide acceptor at the 3′

end of said candidate protein coding sequence, said peptide acceptor being a molecule that can be added to the C-terminus of growing protein chain by the catalytic activity of a ribosomal peptidyl transferase;

b) in vitro or in situ translating said candidate protein coding sequences of said candidate RNA molecules to produce a population of candidate RNA-protein fusions;

c) selecting from said population a desired RNA-protein fusion based on fusion binding or activity; and

d) generating from said RNA portion of said selected fusion a DNA molecule, thereby generating a DNA molecule which encodes said desired protein.

43. A method for generating a DNA molecule which encodes a protein having an altered function relative to a reference protein, comprising the steps of:

a) producing a population of candidate RNA molecules from a population of candidate DNA templates, said candidate RNA molecules each having a candidate protein coding sequence which differs from the reference protein coding sequence, said RNA molecules each comprising a translation initiation sequence and a start codon operably linked to said candidate protein coding sequence and each being covalently bonded to a peptide acceptor at the 3′

end of said candidate protein coding sequence, 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;

b) in vito or in situ translating said candidate protein coding sequences of said candidate RNA molecules to produce a population of RNA-protein fusions;

c) selecting from said population an RNA-protein fusion having an altered function based on fusion binding or activity; and

d) generating from said RNA portion of said selected fusion a DNA molecule, thereby generating a DNA molecule which encodes said protein having said altered function.

44. A method for the selection of a desired RNA, comprising the steps of:

a) providing a population of candidate RNA molecules, each of which comprises a translation initiation sequence and a start codon operably linked to a candidate protein coding sequence and each of which is covalently bonded to a peptide acceptor at the 3′

end of said candidate protein coding sequence, 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;

b) in vitro or in situ translating said candidate protein coding sequences of said candidate RNA molecules to produce a population of candidate RNA-protein fusions; and

c) selecting from said population a desired RNA-protein fusion based on fusion binding or activity, thereby selecting said desired RNA.

45. A method for the selection of a DNA molecule which encodes a desired protein, comprising the steps of:

a) providing a population of candidate RNA molecules, each of which comprises a translation initiation sequence and a start codon operably linked to a candidate protein coding sequence and each of which is covalently bonded to a peptide acceptor at the 3′

end of said candidate protein coding sequence, 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;

b) in vitro or in situ translating said candidate protein coding sequences of said candidate RNA molecules to produce a population of candidate RNA-protein fusions;

c) generating a DNA copy of the RNA of said population of candidate RNA-protein fusions; and

d) selecting from said population a desired fusion based on fusion binding or activity, thereby selecting a DNA molecule which encodes said desired protein.

46. A method for the selection of a DNA molecule which encodes a protein having an altered function relative to a reference protein, comprising the steps of:

a) producing a population of candidate RNA molecules from a population of candidate DNA templates, said candidate RNA molecules each having a candidate protein coding sequence which differs from the reference protein coding sequence, said RNA molecules each comprising a translation initiation sequence and a start codon operably linked to said candidate protein coding sequence and each being covalently bonded to a peptide acceptor at the 3′

end of said candidate protein coding sequence, 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;

b) in vitro or in situ translating said candidate protein coding sequences of said candidate RNA molecules to produce a population of RNA-protein fusions;

c) generating a DNA copy of the RNA of said population of RNA-protein fusions; and

d) selecting from said population a fusion having an altered function based on fusion binding or activity, thereby selecting a DNA molecule which encodes said protein having said altered function.