Metallic solid supports modified with nucleic acids

• US 7,033,760 B2
• Filed: 08/03/2001
• Issued: 04/25/2006
• Est. Priority Date: 06/07/1995
• Status: Expired due to Fees

• Current Assignee: California Institute of Technology
• Original Assignee: California Institute of Technology
• Inventors: Meade, Thomas J., Kayyem, Jon F.
• Primary Examiner(s): Benzion, Gary
• Assistant Examiner(s): STRZELECKA, TERESA E
• Application Number:
  US09/921,645
• Publication Number:
  US 20020034759A1
• Time in Patent Office: 1,726 Days
• Field of Search: 435/6, 435/287.2, 536/24.3
• US Class Current: 435/6.11
• CPC Class Codes: B82Y 30/00 Nanotechnology for materials or surface science, e.g. nanocomposites
• 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: C07H 23/00 Compounds containing boron, silicon, or a metal, e.g. chelates, vitamin B12 esters with inorganic acids C07H11/00; metal salts, see parent compounds
• CPC Class Codes: C12Q 1/6818 involving interaction of two or more labels, e.g. resonant energy transfer
• CPC Class Codes: C12Q 1/6825 Nucleic acid detection involving sensors
• CPC Class Codes: C12Q 2563/113 the label being electroactive, e.g. redox labels
• CPC Class Codes: C12Q 2563/137 Metal/ion, e.g. metal label
• CPC Class Codes: C12Q 2565/101 Interaction between at least two labels
• CPC Class Codes: C12Q 2565/1015 labels being on the same oligonucleotide
• CPC Class Codes: C12Q 2565/401 Signal amplification by chemical polymerisation
• CPC Class Codes: C12Q 2565/607 being a sensor, e.g. electrode

Abstract

The present invention is directed to solid supports having metallic surfaces comprising blocking moieties and modified nucleic acids, which exhibit excellent characteristics in hybridization assays, in a stable, reproducible, rapid manner. In an additional aspect, the invention provides methods utilizing the solid supports to hybridize probe nucleic acid to target nucleic acid and methods for detecting the hybridization complex.

Claims

1. A method of detecting at least one hybridization complex comprising a target nucleic acid, said method comprising:

a) adding a target nucleic acid to an array to form at least a first hybridization complex, said array comprising a solid support having a plurality of regions,

each region comprising an electrode and a self-assembled mixed monolayer comprising

i) blocking moieties, having a first end attached to said electrode, wherein said blocking moieties shield nucleic acids from said electrode; and

ii) at least one modified nucleic acid comprising a nucleic acid and a linker moiety having a first and second end;

wherein said first end of said linker is attached to said electrode and said second end is covalently attached to said nucleic acid;

wherein at least two different regions comprise different probe nucleic acids;

b) adding an agent that distinguishes between single and double stranded nucleic acids; and

c) detecting the presence of said first hybridization complex.

2. A method according to claim 1, wherein said first end of said blocking moieties is attached to said electrode via a sulfur linkage.

3. A method according to claim 2, wherein said first end of said linker is attached to said electrode via a sulfur linkage.

4. A method according to claim 1, 2, or 3, wherein said electrode comprises gold.

5. A method according to claim 1, wherein said blocking moieties have the formula:

wherein

SCM is a sulfur-containing moiety, wherein said sulfur containing moiety is attached to said electrode;

R₁ and R₂ are independently selected from the group consisting of hydrogen and substituent groups;

n is an integer from 3 to 50; and

X is a terminal group.

6. A method according to claim 5, wherein R₁ and R₂ are hydrogen.

7. A method according to claim 6, wherein said blocking moieties comprise alkyl groups.

8. A method according to claim 5, 6, or 7, wherein n is ≧

6.

9. A method according to claim 1, wherein said blocking moiety is a branched molecule.

10. A method according to claim 9, wherein said blocking moiety is a straight chain alkyl group.

11. A method according to claim 10, wherein said alkyl ranges from 1 to 20 carbon atoms.

12. A method according to claim 1, wherein said array comprises a plurality of different blocking moieties.

13. A method according to claim 12, wherein at least one of said blocking moieties is a branched molecule.

14. A method according to claim 12 or 13, wherein at least one of said blocking moieties is an alkyl group.

15. A method according to claim 5, wherein for said blocking moiety,

SCM is a thiol containing moiety;

R₁ and R₂ are hydrogen;

n is 16; and

X is hydroxyl.

16. A method according to claim 1, wherein said linker moiety is a straight chain alkyl group.

17. A method according to claim 16, wherein said alkyl group ranges from 1 to 20 carbon atoms.

18. A method according to claim 1, wherein said linker moiety has the formula

wherein

SCM is a thiol containing moiety;

R₁ and R₂ are hydrogen;

n is 16; and

Y is oxygen.

19. A method according to claim 5, wherein n is ≧

6.

20. A method according to claim 1, wherein said blocking moiety comprises a phosphorus-containing moiety.

21. A method according to claim 1, wherein said nucleic acid is attached to said linker at a 2′

position of a ribose.

22. A method according to claim 1, wherein said nucleic acid is attached to said linker at a 3′

position of a ribose.

23. A method according to claim 1, wherein said nucleic acid is attached to said linker at a base of said nucleic acid.

24. A method according to claim 1, wherein said nucleic acid is attached to said linker at a phosphate linkage of said nucleic acid.

25. A method according to claim 1, wherein said agent is an intercalating agent.

26. A method of detecting at least one hybridization complex comprising a target nucleic acid, said method comprising:

a) adding a target nucleic acid to an array to form at least a first hybridization complex, said array comprising a solid support having a plurality of regions, each region comprising an electrode and a self-assembled mixed monolayer comprising

i) blocking moieties, having a first end attached to said electrode, wherein said blocking moieties shield nucleic acids from said electrode; and

ii) at least one modified nucleic acid comprising a nucleic acid and a linker moiety having a first and second end;

wherein said first end of said linker is attached to said electrode and said second end is covalently attached to said nucleic acid; and

wherein said modified nucleic acid the formula;

wherein;

SCM is a sulfur-containing moiety, wherein said sulfur containing moiety is attached to said electrode;

R₁ and R₂ are independently selected from the group consisting of hydrogen and substituent groups; and

n is an integer from 3 to 50; and

wherein at least two different regions comprise different probe nucleic acids;

b) adding an agent that distinguishes between single and double stranded nucleic acids; and

c) detecting the presence of said first hybridization complex.

28. A method according to claim 26 or 27 wherein said first end of said blocking moieties is attached to said electrode via a sulfur linkage.

29. A method according to claim 26 or 27 wherein said blocking moieties have the formula:

wherein

SCM is a sulfur-containing moiety, wherein said sulfur containing moiety is attached to said electrode;

R₁ and R₂ are independently selected from the group consisting of hydrogen and substituent groups;

n is an integer from 3 to 50; and

X is a terminal group.

30. A method according to claim 26, wherein said blocking moiety is a branched molecule.

31. A method according to claim 26 or 27, wherein said array comprises a plurality of different blocking moieties.

32. A method according to claim 26 or 27, wherein for said linker moiety,

SCM is a thiol containing moiety;

R₁ and R₂ are hydrogen;

n is 16; and

Y is oxygen.

33. A method according to claim 26 or 27, wherein n is ≧

6.

34. A method according to claim 26 or 27, wherein said blocking moiety comprises a phosphorus-containing moiety.

35. A method according to claim 26 or 27, wherein said nucleic acid is attached to said linker at a 2′

position of a ribose.

36. A method according to claim 26 or 27, wherein said nucleic acid is attached to said linker at a 3′

position of a ribose.

37. A method according to claim 26 or 27, wherein said nucleic acid is attached to said linker at a base of said nucleic acid.

38. A method according to claim 26 or 27, wherein said agent is an intercalating agent.

27. A method of detecting at least one hybridization complex comprising a target nucleic acid, said method comprising:

a) adding a target nucleic acid to an array to form at least a first hybridization complex, said array comprising a solid support having a plurality of regions, each region comprising an electrode and a self-assembled mixed monolayer comprising

i) branched molecule blocking moieties, having a first end attached to said electrode, wherein said blocking moieties shield nucleic acids from said electrode; and

ii) at least one modified nucleic acid comprising a nucleic acid and a linker moiety having a first and second end;

wherein said first end of said linker is attached to said electrode and said second end is covalently attached to said nucleic acid; and

wherein said modified nucleic acid the formula;

wherein;

SCM is a sulfur-containing moiety, wherein said sulfur containing moiety is attached to said electrode;

R₁ and R₂ are independently selected from the group consisting of hydrogen and substituent groups; and

n is an integer from 3 to 50; and

wherein at least two different regions comprise different probe nucleic acids;

b) adding an agent that distinguishes between single and double stranded nucleic acids; and

c) detecting the presence of said first hybridization complex.