Detecting targets in heterologous mixtures

• US 20040053217A1
• Filed: 03/25/2003
• Published: 03/18/2004
• Est. Priority Date: 05/11/1994
• Status: Active Grant

• Current Assignee: Trustees of Boston University
• Original Assignee: Trustees of Boston University
• Inventors: Olejnik, Jerzy, Rothschild, Kenneth J., Sonar, Sanjay M.
• Granted Patent: US 7,195,874 B2
• Time in Patent Office: Days
• US Class Current: 435/5
• 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 conjugate comprising a detectable moiety linked to a photoreactive moiety, wherein said detectable moiety comprises a biotinyl moiety, wherein said photoreactive moiety comprises at least one group covalently bound to a substrate, wherein said substrate is selected from proteins, peptides, amino acids, lipids, cells, virus particles, fatty acids, nucleic acids, nucleotides, nucleosides, polysaccharides and inorganic molecules; and

b) treating said conjugate with electromagnetic radiation to release said detectable moiety.

2. The method of claim 1, further comprising c) detecting said released detectable moiety.

3. The method of claim 1, wherein said biotinyl moiety is linked to said photoreactive moiety with a spacer arm to form a photocleavable biotinyl moiety.

4. The method of claim 1, wherein said photoreactive moiety comprises a substituted aromatic ring containing at least one polyatomic group.

5. The method of claim 4, wherein said aromatic ring is a six-membered ring.

6. The method of claim 4, wherein said aromatic ring is a five-membered ring.

7. A method, comprising:

a) providing

i) a heterologous mixture comprising a target molecule, and ii) a conjugate comprising a detectable moiety linked to a photoreactive moiety, wherein said detectable moiety comprises a biotinyl moiety, wherein said photoreactive moiety comprises at least one group covalently bound to a substrate, wherein said substrate is selected from proteins, peptides, amino acids, lipids, cells, virus particles, fatty acids, nucleic acids, nucleotides, nucleosides, polysaccharides and inorganic molecules;

b) contacting said conjugate with said heterologous mixture under conditions such that said substrate binds to said target molecule to form a coupled conjugate; and

c) treating said coupled conjugate with electromagnetic radiation to release said detectable moiety.

8. The method of claim 7, further comprising c) detecting said released detectable moiety.

9. The method of claim 7, wherein said biotinyl moiety is linked to said photoreactive moiety with a spacer arm to form a photocleavable biotinyl moiety.

10. The method of claim 7, wherein said photoreactive moiety comprises a substituted aromatic ring containing at least one polyatomic group.

11. The method of claim 10, wherein said aromatic ring is a six-membered ring.

12. The method of claim 10, wherein said aromatic ring is a five-membered ring.

13. A method, comprising:

a) providing

i) a sample comprising a plurality of target molecules, and ii) a conjugate preparation, said conjugate comprising a detectable moiety linked to a photoreactive moiety, wherein said detectable moiety comprises a biotinyl moiety, wherein said photoreactive moiety comprises at least one group covalently bound to a substrate, wherein said substrate is selected from proteins, peptides, amino acids, nucleic acids, nucleotides, and nucleosides;

b) contacting said sample with a first portion of said conjugate preparation under conditions such that said substrate binds to at least one of said plurality of target molecules to form a first coupled conjugate;

c) treating said first coupled conjugate with electromagnetic radiation to release said detectable moiety;

d) contacting said sample with a second portion of said conjugate preparation under conditions such that said substrate binds to at least one of said plurality of target molecules to form a second coupled conjugate; and

e) treating said second coupled conjugate with electromagnetic radiation to release said detectable moiety.

14. The method of claim 13, further comprising f) detecting said released detectable moiety.

15. The method of claim 13, wherein said biotinyl moiety is linked to said photoreactive moiety with a spacer arm to form a photocleavable biotinyl moiety.

16. The method of claim 13, wherein said photoreactive moiety comprises a substituted aromatic ring containing at least one polyatomic group.

17. The method of claim 16, wherein said aromatic ring is a six-membered ring.

18. The method of claim 16, wherein said aromatic ring is a five-membered ring.

22. The method of claim 13, wherein said photoreactive moiety comprises a substituted aromatic ring containing at least one polyatomic group.

23. The method of claim 22, wherein said aromatic ring is a six-membered ring.

24. The method of claim 22, wherein said aromatic ring is a five-membered ring.

19. A method, comprising:

a) providing

i) a sample comprising a plurality of target molecules, and ii) a conjugate preparation, said conjugate comprising a detectable moiety linked to a photoreactive moiety, wherein said detectable moiety comprises a fluorescent moiety, wherein said photoreactive moiety comprises at least one group covalently bound to a substrate, wherein said substrate is selected from proteins, peptides, amino acids, nucleic acids, nucleotides, and nucleosides;

b) contacting said sample with a first portion of said conjugate preparation under conditions such that said substrate binds to at least one of said plurality of target molecules to form a first coupled conjugate;

c) treating said first coupled conjugate with electromagnetic radiation to release said detectable moiety;

d) contacting said sample with a second portion of said conjugate preparation under conditions such that said substrate binds to at least one of said plurality of target molecules to form a second coupled conjugate; and

e) treating said second coupled conjugate with electromagnetic radiation to release said detectable moiety.

20. The method of claim 19, further comprising f) detecting said released detectable moiety.

21. The method of claim 19, wherein said fluorescent moiety is linked to said photoreactive moiety with a spacer arm to form a photocleavable fluorescent moiety.