Detection of disease related genes

• US 7,169,558 B2
• Filed: 03/27/2003
• Issued: 01/30/2007
• Est. Priority Date: 05/11/1994
• Status: Expired due to Fees

• Current Assignee: Trustees of Boston University
• Original Assignee: Trustees of Boston University
• Inventors: Olejnik, Jerzy, Rothschild, Kenneth J., Sonar, Sanjay M.
• Primary Examiner(s): Ketter; James
• Application Number:
  US10/401,251
• Publication Number:
  US 20040033514A1
• Time in Patent Office: 1,405 Days
• Field of Search: None
• US Class Current: 435/6.13
• CPC Class Codes: A61K 38/00 Medicinal preparations containing peptides peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00
• CPC Class Codes: A61K 41/0042 Photocleavage of drugs in vivo, e.g. cleavage of photolabile linkers in vivo by UV radiation for releasing the pharmacologically-active agent from the administered agent; photothrombosis or photoocclusion
• CPC Class Codes: C07C 205/45 the carbon skeleton being further substituted by at least one doubly—bound oxygen atom, not being part of a —CHO group
• CPC Class Codes: C07C 205/57 having nitro groups and carboxyl groups bound to carbon atoms of six-membered aromatic rings of the carbon skeleton
• CPC Class Codes: C07D 207/404 with only hydrogen atoms or radicals containing only hydrogen and carbon atoms directly attached to other ring carbon atoms, e.g. succinimide
• CPC Class Codes: C07D 311/16 substituted in position 7
• CPC Class Codes: C07D 405/12 linked by a chain containing hetero atoms as chain links
• CPC Class Codes: C07D 495/04 Ortho-condensed systems
• CPC Class Codes: C07H 19/04 Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
• 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 1/1077 by covalent attachment of residues other than amino acids or peptide residues, e.g. sugars, polyols, fatty acids
• CPC Class Codes: C07K 1/13 Labelling of peptides
• CPC Class Codes: C12P 21/00 Preparation of peptides or proteins single cell protein C12N1/00
• CPC Class Codes: C12Q 1/6883 for diseases caused by alterations of genetic material
• CPC Class Codes: G01N 33/58 involving labelled substances G01N33/53 takes precedence

Abstract

The invention is directed to methods for the non-radioactive labeling, detection, quantitation and isolation of nascent proteins translated in a cellular or cell-free translation system. tRNA molecules are misaminoacylated with non-radioactive markers which may be non-native amino acids, amino acid analogs or derivatives, or substances recognized by the protein synthesizing machinery. Markers may comprise cleavable moieties, detectable labels, reporter properties wherein markers incorporated into protein can be distinguished from unincorporated markers, or coupling agents which facilitate the detection and isolation of nascent protein from other components of the translation system. The invention also comprises proteins prepared using misaminoacylated tRNAs which can be utflized in pharmaceutical compositions for the treatment of diseases and disorders in humans and other maninials, and kits which may be used for the detection of diseases and disorders.

Claims

1. A method for detecting nascent proteins synthesized in a translation system comprising the steps of:

a) misaminoacylating a tRNA molecule with a non-radioactive marker;

b) introducing the misaminoacylated tRNA to a translation system, wherein said translation system comprises nucleic acid corresponding to a disease-related gene;

c) incubating the system to incorporate marker into a nascent protein corresponding to at least a portion of said nucleic acid; and

d) detecting said nascent protein containing said marker.

2. The method of claim 1, wherein said disease-related gene is the APC gene and said disease is familial adenomatous polyposis.

3. The method of claim 1, wherein the translation system comprises a cellular or cell-free translation system.

4. The method of claim 3, wherein the cellular translation system is selected from the group consisting of tissue culture cells, isolated primary cells, isolated immortalized cells, isolated human cells and combinations thereof.

5. The method of claim 3, wherein the cell-free translation system is selected from the group consisting of Escherichia coli lysates, wheat germ extracts, insect cell lysates, rabbit reticulocyte lysates, rabbit oocyte lysates, dog pancreatic lysates, human cell lysates, mixtures of purified or semi-purified translation factors and combinations thereof.

6. The method of claim 3, wherein the cell-free translation system is a continuous flow system.

7. The method of claim 1, wherein said marker has a detectable electromagnetic spectrum.

8. The method of claim 1, wherein said marker has a defined mass.

9. The method of claim 1, wherein said marker is a photocleavable marker which is cleaved by electromagnetic radiation.

10. The method of claim 9, wherein the electromagnetic radiation is selected from the group consisting of x-rays, ultraviolet rays, visible light, infrared rays, microwaves, radio waves, electric fields and combinations thereof.

11. The method of claim 1, wherein the tRNA molecule is aminoacylated by chemical misaminoacylation.

12. The method of claim 1, wherein two or more different misaminoacylated tRNAs are introduced to the translation system.

13. The method of claim 1, wherein said nascent protein is detected by spectrometry.

14. The method of claim 1, wherein the tRNA molecule is an initiator tRNA molecule.

15. The method of claim 1, wherein the tRNA molecule is a suppressor tRNA molecule.

16. The method of claim 1, further comprising the step of isolating said nascent protein containing said marker.

17. The method of claim 16, wherein said nascent protein is isolated by a process selected from the group consisting of electrophoresis, electroelution, precipitation, chemical extraction, chromatography, magnetic separation and combinations thereof.

18. A method for detecting nascent proteins synthesized in a translation system comprising the steps of:

a) misaminoacylating a tRNA molecule with a non-radioactive marker;

b) introducing the misaminoacylated tRNA to a translation system, wherein said translation system comprises nucleic acid corresponding to a disease-related gene having a reading frame, wherein said nucleic acid contains a stop codon within said reading frame;

c) incubating the system to incorporate marker into a truncated nascent protein corresponding to the portion of said nucleic acid prior to said stop codon; and

d) detecting said truncated nascent protein containing said marker.

19. The method of claim 18, wherein said disease-related gene is the APC gene and said disease is familial adenomatous polyposis.

20. The method of claim 18, wherein the translation system comprises a cellular or cell-free translation system.

21. The method of claim 18, wherein said marker is a photocleavable marker which is cleaved by electromagnetic radiation.

22. The method of claim 18, further comprising the step of isolating said truncated nascent protein containing said marker.

23. The method of claim 22, wherein said nascent protein is isolated by a process selected from the group consisting of electrophoresis, electroelution, precipitation, chemical extraction, chromatography, magnetic separation and combinations thereof.