Detecting molecular complexes

• US 20060204999A1
• Filed: 03/13/2006
• Published: 09/14/2006
• Est. Priority Date: 03/14/2005
• Status: Abandoned Application

• Current Assignee: Stephen Macevicz
• Original Assignee: Stephen Macevicz
• Inventors: MacEvicz, Stephen
• Application Number:
  US11/374,506
• Publication Number:
  US 20060204999A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/25 involving enzymes not classifiable in groups C12Q1/26 - C12Q1/66
• CPC Class Codes: C12Q 1/26 involving oxidoreductase
• CPC Class Codes: C12Q 1/485 involving kinase
• CPC Class Codes: G01N 2458/10 Oligonucleotides as tagging agents for labelling antibodies
• CPC Class Codes: G01N 33/53 Immunoassay; Biospecific binding assay; Materials therefor
• CPC Class Codes: G01N 33/532 Production of labelled immunochemicals
• CPC Class Codes: G01N 33/535 with enzyme label or co-enzymes, co-factors, enzyme inhibitors or enzyme substrates
• CPC Class Codes: G01N 33/542 with steric inhibition or signal modification, e.g. fluorescent quenching
• CPC Class Codes: G01N 33/566 using specific carrier or receptor proteins as ligand binding reagents where possible specific carrier or receptor proteins are classified with their target compounds
• CPC Class Codes: G01N 33/58 involving labelled substances G01N33/53 takes precedence
• CPC Class Codes: G01N 33/581 with enzyme label (including co-enzymes, co-factors, enzyme inhibitors or substrates)
• CPC Class Codes: G01N 33/68 involving proteins, peptides or amino acids involving lipoproteins G01N33/92
• CPC Class Codes: G01N 33/6845 Methods of identifying protein-protein interactions in protein mixtures

Abstract

The invention provides methods and compositions for detecting the presence of, or for measuring amounts of, molecular complexes, particularly complexes comprising two or more proteins, such as receptor complexes of cell surface membranes. In one aspect of the invention, reagent pairs are provided that comprise a cleaving probe that specifically binds to at least one component of a molecular complex and one or more signaling reagents that specifically bind to one or more components of the molecular complex, at least one of which is different from the component to which the cleaving probe is attached. Each signaling reagent comprises a binding compound specific for a component of the molecular complex and a signaling polynucleotide attached thereto by a cleavable linkage. When the reagent pairs are bound to the same molecular complex, the cleaving probe may be induced to generate a reactive species that is capable of cleaving cleavable linkages within an effective proximity, thereby releasing a signaling polynucleotide by which the molecular complex is detected.

Claims

1. A method of determining the presence or amount of a molecular complex in a sample, the molecular complex comprising at least a first component and a second component, the method comprising the steps of:

providing a binding compound specific for the first component, the binding compound having a polynucleotide attached thereto by a cleavable linkage;

providing a cleaving probe specific for the second component, the cleaving probe having a cleaving agent capable of generating a reactive species within an effective proximity, and the reactive species being capable of cleaving the cleavable linkage;

combining with the sample in an assay mixture the binding compound and the cleaving probe so that the binding compound and cleaving probe specifically bind to the first and second components, respectively, and so that whenever the first component and the second component are in a molecular complex, the cleavable linkage is within the effective proximity of the cleaving agent and are cleaved by the reactive species, and the polynucleotide is released; and

determining the presence or amount of the molecular complex by the released polynucleotide.

2. The method of claim 1 further comprising the steps of:

circularizing said released polynucleotide; and

digesting uncircularized polynucleotide by exonuclease treatment.

3. The method of claim 2 wherein said polynucleotide comprises a first end, an oligonucleotide tag, a second end, and a complementary functionality at the first end or the second end, and said cleavable linkage being attached to the polynucleotide by an end opposite to that of the complementary functionality, and upon cleavage generating on such end a reactive functionality.

4. The method of claim 3 wherein said step of circularizing includes reacting said functionality with said complementary functionality in a template-driven reaction wherein said first end of the polynucleotide and said second end of the polynucleotide hybridize to a template oligonucleotide complementary thereto.

5. The method of claim 4 wherein said cleavable linkage is a thioether and said reactive species is singlet oxygen.

6. The method of claim 5 wherein said reactive functionality is a thiol group.

7. The method of claim 6 wherein said complementary functionality is a thiol group and wherein said template-driven reaction converts said reactive functionality and said complementary functionality into a disulfide linkage, thereby circularizing said released polynucleotide.

8. The method of claim 1 wherein said step of determining includes capturing said binding compounds by a capture agent attached to a solid support so that said released polynucleotide can be eluted from the captured binding compounds.

9. A composition comprising a plurality of binding compounds each having a polynucleotide attached by a cleavable linkage and each having a specificity for an antigen, such that each different binding compound has a different polynucleotide attached, and such that a binding compound having specificity for a different antigen has a different polynucleotide attached.

10. The composition of claim 9 wherein said cleavable linkage is selected from the group consisting of olefins and thioethers.

11. The composition of claim 10 wherein each said different antigen is a different protein in a molecular complex.

12. A composition comprising:

(a) one or more binding compounds each having a polynucleotide attached by a cleavable linkage and each having a specificity for a molecular complex, such that each different binding compound has a different polynucleotide attached, and such that a binding compound having specificity for a different antigenic determinant of the molecular complex has a different polynucleotide attached; and

(b) a cleaving probe specific for the molecular complex, the cleaving probe having a cleaving agent for generating an reactive species for cleaving the cleavable linkage.

13. The composition of claim 12 wherein said one or more binding compounds is a plurality of binding compounds, wherein said reactive species is singlet oxygen, and wherein said cleavable linkage is cleavable by singlet oxygen.

14. The composition of claim 13 wherein said polynucleotide has a complementary functionality on an end distal from said binding compound, and wherein upon cleavage said cleavable linkage generates a reactive functionality on an end of said polynucleotide proximal to said binding compound, such that the reactive functionality and the complementary functionality are capable of reacting in a template-driven reaction to form a stable linkage, thereby circularizing said polynucleotide.