Detection of markers in nascent proteins

• US 20070172849A1
• Filed: 10/30/2006
• Published: 07/26/2007
• Est. Priority Date: 05/11/1994
• Status: Abandoned Application

• Current Assignee: Trustees of Boston University
• Original Assignee: Trustees of Boston University
• Inventors: Olejnik, Jerzy, Rothschild, Kenneth, Sonar, Sanjay
• Application Number:
  US11/589,425
• Publication Number:
  US 20070172849A1
• Time in Patent Office: Days
• US Class Current: 435/6
• 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 1/68 involving nucleic acids
• CPC Class Codes: G01N 33/532 Production of labelled immunochemicals
• CPC Class Codes: G01N 33/582 with fluorescent label

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 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 conjugate, comprising a substrate covalently coupled to a detectable moiety through a cleavable linker, said substrate selected from the group consisting of nucleosides and nucleotides, said detectable moiety having the property of fluorescence.

2. The conjugate of claim 1, wherein said substrate is a nucleotide selected from the group consisting of NTP, dNTP and ddNTP.

3. The conjugate of claim 2, wherein said nucleotide is part of an oligonucleotide.

4. The conjugate of claim 1, wherein said detectable moiety comprises coumarin.

5. The conjugate of claim 1, wherein said detectable moiety is selected from the group consisting of rhodamines and fluoresceins.

6. A method, comprising:

a) providing i) nucleic acid, ii) the conjugate of claim 1, and iii) a polymerase; and

b) mixing said nucleic acid and said polymerase in the presence of said conjugate under conditions such that said conjugate is incorporated into said nucleic acid to produce labeled nucleic acid.

7. The method of claim 6, wherein said nucleic acid is RNA.

8. The method of claim 6, wherein said nucleic acid is DNA.