Diagnosing genetic disorders

• US 6,949,341 B2
• Filed: 10/03/2002
• Issued: 09/27/2005
• 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 S
• Application Number:
  US10/264,126
• Publication Number:
  US 20030162198A1
• Time in Patent Office: 1,090 Days
• Field of Search: 435/6, 435/91.1, 435/91.2
• 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 1/6823 Release of bound markers
• CPC Class Codes: C12Q 1/6876 Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
• CPC Class Codes: C12Q 2523/319 Photocleavage, photolysis, photoactivation
• CPC Class Codes: G01N 33/5306 Improving reaction conditions, e.g. reduction of non-specific binding, promotion of specific binding
• CPC Class Codes: G01N 33/532 Production of labelled immunochemicals
• CPC Class Codes: G01N 33/58 involving labelled substances G01N33/53 takes precedence

Abstract

This invention relates to agents and conjugates that can be used to detect and isolate target components from complex mixtures such as nucleic acids from biological samples, cells from bodily fluids, and nascent proteins from translation reactions. Agents comprise a detectable moiety bound to a photoreactive moiety. Conjugates comprise agents coupled to substrates by covalent bounds which can be selectively cleaved with the administration of electromagnetic radiation. Targets substances labeled with detectable molecules can be easily identified and separated from a heterologous mixture of substances. Exposure of the conjugate to radiation releases the target in a functional form and completely unaltered. Using photocleavable molecular precursors as the conjugates, label can be incorporated into macromolecules, the nascent macromolecules isolated and the label completely removed. The invention also relates to targets isolated with these conjugates which may be useful as pharmaceutical agents or compositions that can be administered to humans and other mammals. Useful compositions include biological agents such as nucleic acids, proteins, lipids and cytokines. Conjugates can also be used to monitor the pathway and half-life of pharmaceutical composition in vivo and for diagnostic, therapeutic and prophylactic purposes. The invention also relates to kits comprised of agents and conjugates that can be used for the detection of diseases, disorders and nearly any individual substance in a complex background of substances.

Claims

1. A method, comprising:

a) providing a tRNA molecule, nucleic acid derived from a biological sample, and a marker;

b) aminoacylating said tRNA molecule with said marker to create a misaminoacylated tRNA;

c) introducing said misaminoacylated tRNA and said nucleic acid derived from a biological sample into a translation system under conditions such that said marker is incorporated into a nascent protein; and

d) screening for disease, said screening comprising detecting said marker.

2. The method of claim 1, wherein the nascent protein is selected from recombinant gene products, gene fusion products, enzymes, cytokines, carbohydrate and lipid binding proteins, nucleic acid binding proteins, hormones, immunogenic proteins, human proteins, viral proteins, bacterial proteins, parasitic proteins and fragments and combinations thereof.

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, primary cells, cells in vivo, isolated immortalized cells, 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, frog oocyte lysates, dog pancreatic lysates, human cell lysates, mixtures of purified or semi-purified translation factors and combinations thereof.

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

7. The method of claim 1, wherein said disease is selected from the group consisting of infections, neoplasias and genetic disorders.

8. The method of claim 1, wherein said biological sample is selected from the group consisting of blood, serum, tissue, urine, stool, nasal cells and spinal fluid.

9. The method of claim 1, wherein prior to step (c) said nucleic acid is amplied in a polymerase chain reaction.

10. The method of claim 1, wherein said marker comprises a detectable label.

11. The method of claim 1, wherein said marker comprises a coupling agent.

12. The method of claim 11, wherein said coupling agent is selected from the group consisting of biotin and derivatives thereof.

13. The method of claim 11, wherein said coupling agent comprises an antigenic site.

14. The method of claim 11, wherein said coupling agent binds to surfaces, said surfaces selected from the group consisting of resin surfaces, bead surfaces, ceramic surfaces, glass surfaces, particle surfaces, and polymer surfaces.

15. The method of claim 1, wherein said marker is a photocleavable marker.

16. The method of claim 15, wherein said photocleavable marker is photocleavable biotin.

17. The method of claim 16, wherein said photocleavable marker comprises an amino acid coupled via a photocleavable linkage to a detectable label.

18. The method of claim 15, wherein said photocleavable marker comprises an amino acid coupled via a photocleavable linkage to a coupling agent.

19. The method of claim 1, wherein two or more different misaminoacylated tRNAs are introduced to the translation system at step (c).

20. The method of claim 1, wherein said detecting of step (d) comprises using an electromagnetic field.

21. A method of screening for a genetic disorder, comprising:

a) providing a tRNA molecule, nucleic acid derived from a biological sample, and a detectable label;

b) aminoacylating said tRNA molecule with said detectable label to create a misaminoacylated tRNA;

c) introducing said misaminoacylated tRNA and said nucleic acid derived from a biological sample into a translation system under conditions such that said detectable label is incorporated into a nascent protein; and

d) screening for a genetic disorder, said screening comprising detecting said detectable label.

22. The method of claim 21, wherein the nascent protein is selected from recombinant gene products, gene fusion products, enzymes, cytokines, carbohydrate and lipid binding proteins, nucleic acid binding proteins, hormones, immunogenic proteins, human proteins, viral proteins, bacterial proteins, parasitic proteins and fragments and combinations thereof.

23. The method of claim 21, wherein the translation system comprises a cellular or cell-free translation system.

24. The method of claim 23, wherein the cellular translation system is selected from the group consisting of tissue culture cells, primary cells, cells in vivo, isolated immortalized cells, human cells and combinations thereof.

25. The method of claim 23, 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, frog oocyte lysates, dog pancreatic lysates, human cell lysates, mixtures of purified or semi-purified translation factors and combinations thereof.

26. The method of claim 21, wherein the tRNA molecule is aminoacylated by chemical or enzymatic misaminoacylation.

27. The method of claim 21, wherein said genetic disorder is familial adenomatous polyposis.

28. The method of claim 21, wherein said biological sample is selected from the group consisting of blood, serum, tissue, urine, stool, nasal cells and spinal fluid.

29. The method of claim 21, wherein prior to step (c) said nucleic acid is amplied in a polymerase chain reaction.

30. The method of claim 21, wherein said detectable label comprises a fluorescent label.

31. The method of claim 21, wherein said detectable label comprises a coupling agent.

32. The method of claim 31, wherein said coupling agent is selected from the group consisting of biotin and derivatives thereof.

33. The method of claim 31, wherein said coupling agent comprises an antigenic site.

34. The method of claim 31, wherein said coupling agent binds to surfaces, said surfaces selected from the group consisting of resin surfaces, bead surfaces, ceramic surfaces, glass surfaces, particle surfaces, and polymer surfaces.

35. The method of claim 31, wherein two or more different misaminoacylated tRNAs are introduced to the translation system at step (c).

36. The method of claim 31, wherein said detecting of step (d) comprises using an electromagnetic field.