CA2278786A1 - Selection of proteins using rna-protein fusions - Google Patents

Selection of proteins using rna-protein fusions Download PDF

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Publication number
CA2278786A1
CA2278786A1 CA002278786A CA2278786A CA2278786A1 CA 2278786 A1 CA2278786 A1 CA 2278786A1 CA 002278786 A CA002278786 A CA 002278786A CA 2278786 A CA2278786 A CA 2278786A CA 2278786 A1 CA2278786 A1 CA 2278786A1
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Prior art keywords
protein
rna
candidate
population
sequence
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Granted
Application number
CA002278786A
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French (fr)
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CA2278786C (en
Inventor
Jack W. Szostak
Richard W. Roberts
Rihe Liu
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General Hospital Corp
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The General Hospital Corporation
Jack W. Szostak
Richard W. Roberts
Rihe Liu
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Application filed by The General Hospital Corporation, Jack W. Szostak, Richard W. Roberts, Rihe Liu filed Critical The General Hospital Corporation
Publication of CA2278786A1 publication Critical patent/CA2278786A1/en
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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C12N15/1062—Isolating an individual clone by screening libraries mRNA-Display, e.g. polypeptide and encoding template are connected covalently
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/82—Translation products from oncogenes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C12N15/1048—SELEX
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B30/00—Methods of screening libraries
    • C40B30/04—Methods of screening libraries by measuring the ability to specifically bind a target molecule, e.g. antibody-antigen binding, receptor-ligand binding
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • G01N33/6845—Methods of identifying protein-protein interactions in protein mixtures
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
    • C07B2200/00—Indexing scheme relating to specific properties of organic compounds
    • C07B2200/11—Compounds covalently bound to a solid support
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Abstract

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

Claims (43)

1. A method for the selection of 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 operably linked to a peptide acceptor at the 3 end of said candidate protein coding sequence;
b) in vitro or in situ translating said candidate protein coding sequences to produce a population of candidate RNA-protein fusions; and c) selecting a desired RNA-protein fusion, thereby selecting said desired protein.
2. 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 operably linked to a peptide acceptor at the 3 end of said candidate protein coding sequence;
b) in vitro or in situ translating said candidate protein coding sequences to produce a population of candidate RNA-protein fusions;
c) selecting a desired RNA-protein fusion; and d) generating from said RNA portion of said fusion a DNA molecule which encodes said desired protein.
3. A method for the selection of 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 DNA templates, said candidate DNA templates each having a candidate protein coding sequence which differs from said 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 operably linked to a peptide acceptor at the 3 end;

b) in vitro or in situ translating said candidate protein coding sequences to produce a population of candidate RNA-protein fusions; and c) selecting an RNA-protein fusion having an altered function, thereby selecting said protein having said altered function.
4. 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 DNA templates each having a candidate protein coding sequence which differs from said 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 operably linked to a peptide acceptor at the 3 end;
b) in vitro or in translating said candidate protein coding sequences to produce a population of RNA-protein fusions;
c) selecting an RNA-protein fusion having an altered function; and d) generating from said RNA portion of said fusion a DNA molecule which encodes said protein having said altered function.
5. 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 operably linked to a peptide acceptor at the 3 end of said candidate protein coding sequence;
b) in vitro or in situ translating said candidate protein coding sequences to produce a population of candidate RNA-protein fusions; and c) selecting a desired RNA-protein fusion, thereby selecting said desired RNA.
6. The method of any of claims 1-5, wherein said peptide acceptor is puromycim.
7. The method of any of claims 1-5, wherein each of said candidate RNA
molecules further comprises a pause sequence or further comprises a DNA or DNA
analog sequence covalently bonded to the 3 end of said RNA molecule.
8. The method of any of claims 1-5, wherein said population of candidate RNA molecules comprises at least 10 13 different RNA molecules.
9. The method of any of claims 1-5, wherein said in vitro translation reaction is carried out in a lysate prepared from a eukaryotic cell or portion thereof.
10. The method of claim 9, wherein said in vitro translation reaction is carried out in a reticulocyte lysate.
11. The method of claim 9, wherein said in vitro translation reaction is carried out in a wheat germ lysate.
12. The method of any of claims 1-5; wherein said in vitro translation reaction is carried out in a lysate prepared from a bacterial cell or portion thereof.
13. The method of any of claims 1-5, wherein said selection step comprises binding of said desired protein to an immobilized binding partner.
14. The method of any of claims 1-5, wherein said selection step comprises assaying for a functional activity of said desired protein.
15. The method of claims 2 or 4, wherein said DNA molecule is amplified.
16. -The method of claims 1, 3, or 5, wherein said method further comprises repeating step (a) through (c).
17. The method of claims 2 or 4, wherein said method further comprises transcribing an RNA molecule from said DNA molecule and repeating steps (a) through (d).
18. The method of any of claims 1-5, wherein said RNA is covalently bonded though an amide bond to said protein in said RNA-protein fusion.
19. The method of any of claims 1-5, wherein said RNA is covalently bonded to said protein in said RNA-protein fusion, said covalent bond being resistant to cleavage by a ribosome.
20. The method of any of claims 1-5, wherein, following the in vitro translating step, an incubation is carried out in the presence of 50-100 mM
Mg2+.
21. The method of any of claims 1-5, wherein said RNA-protein fusion further comprises a nucleic acid or nucleic acid analog sequence positioned proximal to said peptide acceptor which increases flexibility.
22. An RNA-protein fusion selected by any of the methods of claims 1-5.
23. A molecule comprising a ribonucleic acid covalently bonded though an amide bond to a protein.
24. The molecule of claim 23, wherein said protein is encoded by said ribonucleic acid.
25. The molecule of claim 23, wherein said ribonucleic acid is a messenger RNA.
26. A molecule comprising a ribonucleic acid covalently bonded to a protein, said protein being entirely encoded by said ribonucleic acid.
27. The molecule of claim 26, wherein said ribonucleic acid is messenger RNA.
28. A molecule comprising a ribonucleic acid covalently bonded to a protein, said covalent bond being resistant to cleavage by a ribosome.
29. The molecule of claim 28, wherein said ribonucleic acid is messenger RNA.
30. A ribonucleic acid comprising a translation initiation sequence and a start codon operably linked to a candidate protein coding sequence, said ribonucleic acid being covalently bonded to a peptide acceptor at the 3' end of said candidate protein coding sequence.
31. A method for the selection of a desired protein or desired RNA, comprising:
(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 operably linked to a peptide acceptor at the 3' end of the candidate protein coding sequence;
(b) in vitro or in situ translating the candidate protein coding sequences to produce a population of candidate RNA-protein fusions;

(c) contacting said population of RNA-protein fusions with a binding partner specific for either the RNA portion or the protein portion of said RNA-protein fusion under conditions which substantially separate said binding partner-RNA-protein fusion complex from unbound members of said population;
(d) releasing said bound RNA-protein fusions from said complex; and (e) contacting said population of RNA-protein fusions from step (d) with a binding partner specific for the protein portion of said desired RNA-protein fusion under conditions which substantially separate said binding partner-RNA-protein fusion complex from unbound members of said population, thereby selecting the desired protein and the desired RNA.
32. The method of claim 31, wherein said method further comprises repeating steps (a) through (e).
33. The method of claim 31, wherein said peptide acceptor is puromycin.
34. The method of claim 31, wherein each of said candidate RNA
molecules further includes a pause sequence or further comprises a DNA or DNA
analog sequence covalently bonded to the 3' end of said RNA molecule.
35. The method of claim 31, wherein said population of candidate RNA
molecules includes at least 10 13 different RNA molecules.
36. The method of claim 31, wherein said in vitro translation reaction is carried out in a lysate prepared from a eukaryotic cell or portion thereof.
37. The method of claim 36, wherein said in vitro translation reaction is carried out in a reticulocyte lysate or wheat germ lysate.
38. The method of claim 31, wherein said in vitro translation reaction is carried out in an extract prepared from a prokaryotic cell or portion thereof.
39. The method of claim 31, wherein at least one of said binding partners is immobilized-on a solid support.
40. The method of claim 31, wherein, following the in vitro translating step, an incubation is carried out in the presence of 50-100 mM Mg2+.
41. The method of claim 31, wherein said RNA-protein fusion further comprises a nucleic acid or nucleic acid analog sequence positioned proximal to said peptide acceptor which increases flexibility.
42. A microchip comprising an array of immobilized single-stranded nucleic acids, said nucleic acids being hybridized to RNA-protein fusions.
43. The microchip of claim 42, wherein said protein is encoded by said RNA.
CA2278786A 1997-01-21 1998-01-14 Selection of proteins using rna-protein fusions Expired - Lifetime CA2278786C (en)

Applications Claiming Priority (5)

Application Number Priority Date Filing Date Title
US3596397P 1997-01-21 1997-01-21
US60/035,963 1997-01-21
US6449197P 1997-11-06 1997-11-06
US60/064,491 1997-11-06
PCT/US1998/000807 WO1998031700A1 (en) 1997-01-21 1998-01-14 Selection of proteins using rna-protein fusions

Publications (2)

Publication Number Publication Date
CA2278786A1 true CA2278786A1 (en) 1998-07-23
CA2278786C CA2278786C (en) 2010-07-20

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US (5) US6258558B1 (en)
EP (2) EP1712623B1 (en)
JP (1) JP3692542B2 (en)
KR (1) KR100566859B1 (en)
CN (1) CN1238366C (en)
AT (2) ATE332368T1 (en)
AU (1) AU738328B2 (en)
CA (1) CA2278786C (en)
DE (1) DE69835143T2 (en)
DK (2) DK0971946T3 (en)
ES (2) ES2373110T3 (en)
HK (1) HK1027577A1 (en)
IL (1) IL131016A (en)
PT (2) PT971946E (en)
RU (1) RU2233878C2 (en)
TW (1) TW589323B (en)
WO (1) WO1998031700A1 (en)

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