DNA-protein fusions and uses thereof

• US 7,195,880 B2
• Filed: 06/26/2002
• Issued: 03/27/2007
• Est. Priority Date: 12/02/1998
• Status: Expired due to Term

• Current Assignee: Bristol-Myers Squibb Company
• Original Assignee: Adnexus Therapeutics, Inc. (Bristol-Myers Squibb Company)
• Inventors: Lohse, Peter, Kurz, Markus
• Primary Examiner(s): WESSENDORF, TERESA D
• Application Number:
  US10/180,819
• Publication Number:
  US 20020177158A1
• Time in Patent Office: 1,735 Days
• Field of Search: 435/7.1, 435/6, 435/5, 435/4, 435/DIG.16, 435/DIG.17, 435/91.1, 435/DIG.15, 435/DIG.14, 536/23.4, 536/23.1, 536/24.33, 530/350
• US Class Current: 435/7.1
• 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 the selection of a desired protein or its encoding DNA, said method comprising the steps of:

a) producing a population of candidate DNA-protein fusions, wherein each of the candidate DNA-protein fusions is generated by a method selected from;

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

(b) translating in vitro 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;

ii) (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; and

iii) (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; and

b) selecting a desired DNA-protein fusion that binds to a binding partner, thereby selecting a desired protein or DNA.

2. The method of claim 1, wherein said DNA-protein fusion further comprises a ribonucleic acid covalently bonded to the DNA.

3. The method of claim 2, wherein the protein of said DNA-protein fusion is encoded by said ribonucleic acid.

4. The method of claim 1, wherein the protein of said DNA-protein fusion comprises at least 10 amino acids.

5. The method of claim 4, wherein the protein of said DNA-protein fusion comprises at least 30 amino acids.

6. The method of claim 5, wherein the protein of said DNA-protein fusion comprises at least 100 amino acids.

7. The method of claim 1, wherein the DNA of said DNA-protein fusion is covalently bonded to the protein through a peptide acceptor.

8. The method of claim 7, wherein said peptide acceptor is puromycin.

9. The method of claim 1, wherein said population of candidate DNA-protein fusions comprises at least 10⁵ different DNA-protein fusions.

10. The method of claim 9, wherein said population of candidate DNA-protein fusions comprises at least 10¹⁴ different DNA-protein fusions.

11. The method of claim 1, wherein said selecting step (b) comprises assaying said candidate DNA-protein fusions for their ability to bind to an immobilized binding partner.

12. The method of claim 1, wherein said method further comprises repeating steps (a) and (b).

13. The method of claim 1, wherein said DNA is double stranded.