Dual markers

• US 6,596,481 B1
• Filed: 06/17/1999
• Issued: 07/22/2003
• Est. Priority Date: 05/11/1994
• Status: Expired due to Fees

• Current Assignee: Ambergen Inc.
• Original Assignee: Ambergen Inc.
• Inventors: Sonar, Sanjay M., Olejnik, Jerzy, Rothschild, Kenneth J.
• Primary Examiner(s): KETTER, JAMES S
• Application Number:
  US09/335,018
• Time in Patent Office: 1,496 Days
• Field of Search: 435/69.1, 435/6, 435/68.1
• 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 1/6876 Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
• CPC Class Codes: G01N 33/532 Production of labelled immunochemicals
• CPC Class Codes: Y02A 50/30 Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

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 misarminoacylated tRNAs which can be utilized in pharmaceutical compositions for the treatment of diseases and disorders in humans and other mammals, and kits which may be used for the detection of diseases and disorders.

Claims

1. A method for incorporating a marker into a protein, comprising:

a) providing a misaminoacylated initiator tRNA molecule which only recognizes the first AUG codon that serves to initiate protein synthesis, said misaminoacylated initiator tRNA molecule comprising a fluorescent marker; and

b) introducing said misaminoacylated initiator tRNA to a translation system under conditions such that said fluorescent marker is incorporated into a nascent protein.

wherein said nascent protein, after step b) is complete, comprises a fluorescent marker and an affinity marker.

2. The method of claim 1, further comprising, after step b), isolating said nascent protein.

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

4. The method of claim 3, wherein said cell-free translation system is selected from wheat germ extracs, E. Coli cell lysates, and rabbit reticulocyte lysates.

5. The method of claim 1, wherein, after step b), said fluorescent marker is detected.

6. A method for incorporating a marker into a protein, comprising:

a) providing;

i) a sample suspected of containing nucleic acid having one or more chain truncating mutations, and ii) a misaminoacylated tRNA molecule, said misaminoacylated tRNA molecule comprises a first marker; and

b) introducing said nucleic acid sample and said misaminoacylated tRNA into a translation system under conditions such that said first marker is incorporated into a nascent protein.

7. The method of claim 6, wherein said sample contains a chain truncating mutation and said nascent protein is truncated.

8. The method of claim 6, wherein said chain truncating mutation comprises a frameshift mutation.

9. The method of claim 6, wherein said first marker comprises a detectable label.

10. The method of claim 9, wherein said label has a detectable electromagnetic spectral property.

11. The method of claim 9, wherein said label comprises a fluorescent moiety.

12. The method of claim 6, wherein said nascent protein, after step b) is complete, comprises first and second markers.

13. The method of claim 12, wherein said second marker comprises a detectable marker.

14. The method of claim 12, wherein said second marker comprises an affinity marker.

15. The method of claim 6, wherein said first marker comprises an affinity marker.

16. The method of claim 6, further comprising, after step b), isolating said nascent protein.

17. The method of claim 6, wherein said translation system comprises a cell-free translation system.

18. The method of claim 17, wherein said cell-free translation system is selected from wheat germ extracs, E. coli cell lysates, and rabbit reticulocyte lysates.

19. The method of claim 6, wherein, after step b), said first marker is detected.

20. The method of claim 6, wherein said misaminoacylated tRNA is an initiator tRNA.

21. The method of claim 6, wherein said misaminoacylated tRNA is a suppressor tRNA.

22. A method for incorporating a marker into a protein, comprising:

a) providing;

i) a sample suspected of containing nucleic acid having one or more frameshift mutations, and ii) a misaminoacylated tRNA molecule, said misaminoacylated tRNA molecule comprises a first marker; and

b) introducing said nucleic acid sample and said misaminoacylated tRNA into a translation system under conditions such that said first marker is incorporated into a nascent protein.

23. The method of claim 22, wherein said transcript comprises a frameshift mutation which generates a stop codon resulting in chain termination.

24. The method of claim 22, wherein said transcript comprises a frameshift mutation and said nascent protein comprises an amino acid sequence that is altered.

25. The method of claim 22, wherein said first marker comprises a detectable label.

26. The method of claim 25, wherein said label has a detectable electromagnetic spectral property.

27. The method of claim 25, wherein said label comprises a fluorescent moiety.

28. The method of claim 22, wherein said nascent protein, after step b) is complete, comprises first and second markers.

29. The method of claim 28, wherein said second marker comprises a detectable marker.

30. The method of claim 28, wherein said second marker comprises an affinity marker.

31. The method of claim 22, wherein said first marker comprises an affinity marker.

32. The method of claim 22, further comprising, after step b), isolating said nascent protein.

33. The method of claim 22, wherein said translation system comprises a cell-free translation system.

34. The method of claim 33, wherein said cell-free translation system is selected from wheat germ extracs, E. coli cell lysates, and rabbit reticulocyte lysates.

35. The method of claim 22, wherein, after step b), said first marker is detected.

36. The method of claim 22, wherein said misaminoacylated tRNA is an initiator tRNA.

37. The method of claim 22, wherein said misaminoacylated tRNA is a suppressor tRNA.

38. A method for incorporating a marker into a protein, comprising:

a) providing a misaminoacylated tRNA molecule which only recognizes the first codon designed to serve to initiate protein synthesis, said misaminoacylated tRNA molecule comprising a fluorescent marker; and

b) introducing said misaminoacylated tRNA to a translation system under conditions such that said fluorescent marker is incorporated into a nascent protein,

wherein said nascent protein, after step) b) is complete comprises a fluorescent marker and an affinity marker.

39. The method of claim 38, further comprising, after step b), isolating said nascent protein.

40. The method of claim 38, wherein said translation system comprises a cell-free translation system.

41. The method of claim 40, wherein said cell-free translation system is selected from wheat germ extracs, E. coli cell lysates, and rabbit reticulocyte lysates.

42. The method of claim 38, wherein, after step b), said fluorescent marker is detected.

43. The method of claim 38, wherein said misaminoacylated tRNA is an initiator tRNA.

44. The method of claim 38, wherein said misaminoacylated tRNA is a suppressor tRNA.