Nucleic acid ligand diagnostic biochip

• US 20030162216A1
• Filed: 02/27/2003
• Published: 08/28/2003
• Est. Priority Date: 12/15/1997
• Status: Active Grant

• Current Assignee: SomaLogic Operating Co., Inc. (Somalogic Incorporated)
• Original Assignee: Somalogic Incorporated
• Inventors: Gold, Larry, Zichi, Dominic A.
• Granted Patent: US 7,709,192 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C07B 2200/11 Compounds covalently bound to a solid support
• CPC Class Codes: C12Q 1/6834 Enzymatic or biochemical coupling of nucleic acids to a solid phase
• CPC Class Codes: C12Q 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 1/6839 Triple helix formation or other higher order conformations in hybridisation assays
• CPC Class Codes: C12Q 2525/205 Aptamer
• CPC Class Codes: C12Q 2541/101 Selex
• CPC Class Codes: C12Q 2565/101 Interaction between at least two labels
• CPC Class Codes: C12Q 2565/514 characterised by the use of the arrayed oligonucleotides as identifier tags, e.g. universal addressable array, anti-tag or tag complement array
• CPC Class Codes: C40B 40/00 Libraries per se, e.g. arrays, mixtures
• CPC Class Codes: Y10S 977/924 using nanostructure as support of dna analysis

Abstract

A nucleic acid ligand “biochip” is disclosed, consisting of a solid support to which one or more specific nucleic acid ligands is attached in a spatially defined manner. Each nucleic acid ligand binds specifically and avidly to a particular target molecule contained within a test mixture, such as a bodily fluid. The target molecules include, but are not limited to, proteins (cellular, viral, bacterial, etc.) hormones, sugars, metabolic byproducts, cofactor, and intermediates, drugs, and toxins. Contacting the test mixture with the biochip leads to the binding of a target molecule to its cognate nucleic acid ligand. The biochip may then be contacted with a reagent(s) that reacts covalently with proteins and not with nucleic acids. Each protein target in the test mixture may then detected by detecting the presence of the reagent at the appropriate address on the biochip.

Claims

1. A method for detecting the presence of a target molecule suspected of being contained in a test mixture, wherein said target molecule to be detected is a protein, the method comprising:

a) providing a biochip comprising a solid support, said solid support comprising a plurality of spatially defined addresses, each said address comprising at least one copy of a single species of nucleic acid ligand attached thereto, each said species of nucleic acid ligand having specific affinity for one of said target molecules suspected of being contained in said test mixture, and each said species of nucleic acid ligand binding specifically to said target molecule through non-Watson-Crick interactions;

b) contacting said biochip with said test mixture suspected of containing said target molecule;

c) removing non-specifically bound material from said biochip;

d) contacting said biochip with a reagent that reacts covalently with proteins and not with nucleic acids; and

e) detecting the presence of said target molecule by detecting the presence of said reagent at the appropriate address on said biochip.

2. The method of claim 1 wherein said reagent reacts with a primary amine.

3. The method of claim 2 wherein said primary amine is present on a lysine residue.

4. The method of claim 1 wherein said reagent comprises a fluorophore.

5. The method of claim 1 wherein said reagent comprises a radiolabel.

6. The method of claim 1 wherein said reagent is an N-hydroxysuccinimide ester.

7. The method of claim 1 wherein said reagent is an isothiocyanate.

8. The method of claim 1 wherein said reagent is an aldehyde.

9. The method of claim 1 wherein step c) is accomplished by washing said biochip.

10. A method for detecting the presence of a target molecule suspected of being contained in a test mixture, wherein said target molecule be to detected is a protein, the method comprising;

a) providing a biochip comprising a solid support, said solid support comprising a plurality of spatially defined addresses, each said address comprising at least one copy of a single species of nucleic acid ligand attached thereto, each said species of nucleic acid ligand having specific affinity for one of said target molecules suspected of being contained in said test mixture, each said species of nucleic acid ligand binding specifically to said target molecule through non-Watson-Crick interactions, and wherein said nucleic acid ligand having specific affinity for said target molecule to be detected is a photoreactive nucleic acid ligand;

b) contacting said biochip with said test mixture suspected of containing said target molecule, wherein a nucleic acid ligand-target molecule complex is formed if said target molecule is present;

c) irradiating said biochip, wherein said nucleic acid ligand-target molecule complex photocrosslinks;

d) removing non-specifically bound material from said biochip;

e) contacting said biochip with a reagent that reacts covalently with proteins and not with nucleic acids; and

f) detecting the presence of said target molecule by detecting the presence of said reagent at the appropriate address on said biochip.

11. The method of claim 10 wherein said step d) is accomplished by exposing said biochip to conditions that denature nucleic acids.

12. The method of claim 10 wherein step d) is accomplished by exposing said biochip to conditions that denature proteins.

13. The method of claim 10 wherein said reagent reacts with primary amines.

14. The method of claim 13 wherein primary amine is present on a lysine residue.

15. The method of claim 10 wherein said reagent comprises a fluorophore.

16. The method of claim 15 wherein the fluorophore is detected by its fluorescence emission.

17. The method of claim 10 wherein said reagent comprises a radiolabel.

18. The method of claim 10 wherein said reagent is an N-hydroxysuccinimide ester.

19. The method of claim 10 wherein said reagent is an isothiocyanate.

20. The method of claim 10 wherein said reagent is an aldehyde.

21. A method for detecting the presence of a target molecule suspected of being contained in a test mixture, wherein said target molecule is a protein, the method comprising;

a) providing a solid support, said solid support comprising a nucleic acid ligand having specific affinity for said target protein, said nucleic acid ligand binding specifically to said target protein through non-Watson-Crick interactions;

b) contacting said solid support with said test mixture suspected of containing said target molecule;

c) removing non-specifically bound material from said solid support;

d) contacting said solid support with a reagent that reacts with proteins and not with nucleic acids; and

e) detecting the presence of said target molecule by detecting the presence of said reagent on said solid support.

22. A method for detecting the presence of a target molecule suspected of being contained in a test mixture, wherein said target molecule is a protein, the method comprising;

a) providing a solid support, said solid support comprising a photoreactive nucleic acid ligand having specific affinity for said target protein, said photoreactive nucleic acid ligand binding specifically to said target molecule through non-Watson-Crick interactions;

b) contacting said solid support with said test mixture suspected of containing said target molecule, wherein a nucleic acid ligand-target molecule complex is formed if said target molecule is present;

c) irradiating said solid support, wherein said nucleic acid ligand-target molecule complex photocrosslinks;

d) removing non-specifically bound material from said solid support;

e) contacting said solid support with a reagent that reacts with proteins and not with nucleic acids; and

f) detecting the presence of said target molecule by detecting the presence of said reagent on said solid support.

23. A diagnostic biochip for the detection of a target molecule in a test mixture, the biochip comprising a solid support, said solid support comprising a plurality of spatially defined addresses, each said address comprising at least one copy of a single species of nucleic acid ligand attached thereto, each said species of nucleic acid ligand selected from the group consisting of photoreactive nucleic acid ligands and non-photoreactive nucleic acid ligands, and each said species of nucleic acid ligand having specific affinity for one of said target molecules suspected of being contained in said test mixture, and each said species of nucleic acid ligand binding specifically to said target molecule through non-Watson-Crick interactions.