DNA-protein fusions and uses thereof

• US 6,416,950 B1
• Filed: 12/02/1999
• Issued: 07/09/2002
• Est. Priority Date: 12/02/1998
• Status: Expired due to Term

• Current Assignee: Bristol-Myers Squibb Company
• Original Assignee: Phylos, Inc. (Bristol-Myers Squibb Company)
• Inventors: Kurz, Markus, Lohse, Peter
• Primary Examiner(s): Jones, W. Gary
• Assistant Examiner(s): Chakrabarti, Arun K.
• Application Number:
  US09/453,190
• Time in Patent Office: 950 Days
• Field of Search: 435/6, 435/91.1, 435/91.53, 435/320.1, 436/6, 514/44, 536/23.1, 536/23.5
• US Class Current: 435/6.12
• CPC Class Codes: C07B 2200/11 Compounds covalently bound to a solid support
• CPC Class Codes: C07H 19/20 with the saccharide radical esterified by phosphoric or polyphosphoric acids
• CPC Class Codes: C07H 21/00 Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• CPC Class Codes: C07K 14/00 Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
• CPC Class Codes: C12N 15/1062 mRNA-Display, e.g. polypeptide and encoding template are connected covalently
• CPC Class Codes: C12N 15/1075 by coupling phenotype to genotype, not provided for in other groups of this subclass
• CPC Class Codes: C12N 15/1096 cDNA Synthesis; Subtracted cDNA library construction, e.g. RT, RT-PCR
• 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: C40B 40/00 Libraries per se, e.g. arrays, mixtures
• CPC Class Codes: C40B 40/08 Libraries containing RNA or DNA which encodes proteins, e.g. gene libraries
• CPC Class Codes: C40B 40/10 Libraries containing peptides or polypeptides, or derivatives thereof
• CPC Class Codes: C40B 50/14 Solid phase synthesis, i.e. wherein one or more library building blocks are bound to a solid support during library creation; Particular methods of cleavage from the solid support

Abstract

Disclosed herein are molecules that include a deoxyribonucleic acid (DNA) covalently bonded to a protein and uses thereof.

Claims

1. A method for generating a DNA-protein fusion, said method comprising:

(a) covalently bonding a nucleic acid reverse-transcription primer to an RNA, said reverse-transcription primer being bound to a peptide acceptor;

(b) translating said RNA to produce a protein product, said protein product being covalently bound to said reverse-transcription primer; and

(c) reverse transcribing said RNA to produce a DNA-protein fusion.

3. The method of claim 1 or 2, said method further comprising treating the product of step (c) to remove said RNA.

4. The method of claim 3, wherein said treating comprises contacting the product of step (c) with RNase H under conditions sufficient to digest said RNA.

5. The method of claim 1 or 2, wherein said nucleic acid reverse-transcription primer is a DNA primer.

6. The method of claim 1 or 2, wherein said translating step is carried out in vitro.

7. The method of claim 1 or 2, wherein said peptide acceptor is puromycin.

8. The method of claim 1, wherein said nucleic acid reverse-transcription primer has a hairpin structure.

9. The method of claim 1 or 2, wherein said nucleic acid reverse-transcription primer further comprises a photocrosslinking agent.

10. The method of claim 9, wherein said photocrosslinking agent is psoralen.

11. The method of claim 9, wherein said crosslinked to an oligonucleotide which is bound to said peptide acceptor.

12. The method of claim 9, wherein said nucleic acid reverse-transcription primer is hybridized to said RNA molecule and said linking step is carried out by photocrosslinking.

2. A method for generating a DNA-protein fusion, said method comprising:

(a) generating an RNA-protein fusion;

(b) hybridizing a nucleic acid reverse-transcription primer to said fusion;

(c) covalently bonding said primer to said fusion; and

(d) reverse transcribing the RNA of said RNA-protein fusion to produce a DNA-protein fusion.

13. A method for generating a DNA-protein fusion, said method comprising:

(a) providing an RNA molecule covalently bonded to a peptide acceptor;

(b) covalently bonding a nucleic acid reverse-transcription primer to the molecule of step (a);

(c) translating said RNA molecule to produce a protein, and

(d) reverse transcribing said RNA molecule to produce a DNA-protein fusion.

14. The method of claim 2 or 13, wherein the protein of said DNA-protein fusion is covalently bonded to the RNA through a non-RNA linker and said nucleic acid reverse-transcription primer is covalently bonded to said linker.