Electrodes linked via conductive oligomers to nucleic acids

• US 20030003473A1
• Filed: 02/20/2002
• Published: 01/02/2003
• Est. Priority Date: 11/05/1996
• Status: Active Grant

• Current Assignee: Osmetech Technology Inc. (Roche Holding AG)
• Original Assignee: Clinical Micro Sensors Inc. (Roche Holding AG)
• Inventors: Kayyem, Jon Faiz, Meade, Thomas J., Yu, Changjun, Gozin, Michael, OʼConnor, Stephen D.
• Granted Patent: US 6,977,151 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• 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: C12Q 1/6825 Nucleic acid detection involving sensors

Abstract

The invention relates to nucleic acids covalently coupled to electrodes via conductive oligomers. More particularly, the invention is directed to the site-selective modification of nucleic acids with electron transfer moieties and electrodes to produce a new class of biomaterials, and to methods of making and using them.

Claims

1. A composition comprising:

a) an electrode;

b) at least one nucleoside; and

c) a conductive oligomer covalently attached to both said electrode and said nucleoside, wherein said conductive oligomer has the formula;

 

wherein

Y is an aromatic group;

n is an integer from 1 to 50;

g is either 1 or zero;

e is an integer from zero to 10; and

m is zero or 1;

wherein when g is 1, B-D is a conjugated bond; and

wherein when g is zero, e is 1 and D is preferably carbonyl, or a heteroatom moiety, wherein the heteroatom is selected from oxygen, sulfur, nitrogen or phosphorus.

3. A composition according to claim 1 or 2 wherein said nucleoside is part of a nucleic acid.

4. A composition according to claim 3 further comprising a plurality of conductive oligomers each covalently attached to a nucleic acid.

5. A composition according to claim 4 wherein said nucleic acids are all the same.

6. A composition according to claim 4 wherein at least one of said nucleic acids is different.

7. A composition according to claim 1 or 2 wherein said covalent attachment of said conductive oligomer to said nucleoside is to the ribose or phosphate of said nucleoside.

8. A composition according to claim 1 or 2 wherein said covalent attachment of said conductive oligomer to said nucleoside is to the base of said nucleoside.

9. A composition according to claim 1 or 2 wherein said electrode further comprises at least one passavation agent.

10. A composition according to claim 1 or 2 wherein said electrode further comprises a monolayer of passavation agents.

11. A composition according to claim 10 further comprising a hybridization indicator.

13. A composition according to claim 12 wherein said second electron transfer moiety comprises a transition metal complex.

14. A composition according to claim 12 wherein said second electron transfer moiety comprises an organic electron transfer moiety.

15. A composition according to claim 12 wherein said covalent attachment of said second electron transfer moiety is to the ribose-phosphate backbone of said nucleic acid.

16. A composition according to claim 12 wherein said covalent attachment of said second electron transfer moiety is to a base of said nucleic acid.

17. A composition according to claim 12 wherein said electrode further comprises at least one passavation agent.

18. A composition according to claim 12 wherein said electrode further comprises a monolayer of passavation agents.

19. A composition according to claim 18 further comprising a hybridization indicator.

20. A composition according to claim 12 wherein said conductive oligomer has the structure:

21. A composition according to claim 1 wherein said conductive oligomer has the structure:

34. A method of making a composition according to claim 1, 2 or 12 comprising:

a) providing a conductive oligomer covalently attached to a nucleoside; and

b) attaching said conductive oligomer to said electrode.

35. A method of making a composition according to claim 1, 2 or 12 comprising:

a) attaching a conductive oligomer to an electrode; and

b) attaching at least one nucleotide to said conductive oligomer.

2. A composition comprising:

a) an electrode;

b) at least one nucleoside; and

c) a conductive oligomer covalently attached to both said electrode and said nucleoside, wherein said conductive oligomer has the formula;

 

wherein

n is an integer from 1 to 50;

m is 0 or 1;

C is carbon;

J is carbonyl or a heteroatom moeity, wherein the heteroatom is selected from the group consisting of nitrogen, silicon, phosphorus, sulfur; and

G is a bond selected from alkane, alkene or acetylene.

12. A composition comprising:

a) a first electron transfer moiety comprising an electrode;

b) a nucleic acid;

c) a second electron transfer moiety covalently attached to said nucleic acid; and

d) a conductive oligomer covalently attached to both said electrode and said nucleic acid.

22. A method of detecting a target sequence in a nucleic acid sample comprising

a) applying an input signal to a hybridization complex comprising:

i) a probe nucleic acid comprising a conductive oligomer covalently attached to a first electron transfer moiety comprising an electrode and to a single stranded nucleic acid capable of hybridizing to said target sequence, said single stranded nucleic acid comprising a covalently attached second electron transfer moiety, wherein said conductive oligomer has the formula;

 

wherein

Y is an aromatic group;

n is an integer from 1 to 50;

g is either 1 or zero;

e is an integer from zero to 10; and

m is zero or 1;

wherein when g is 1, B-D is a conjugated bond; and

wherein when g is zero, e is 1 and D is preferably carbonyl, or a heteroatom moiety, wherein the heteroatom is selected from oxygen, sulfur, nitrogen or phosphorus; and

ii) a target nucleic acid hybridized to said probe sequence to form said hybridization complex; and

b) detecting electron transfer between said electrode and said second electron transfer moiety, if present, as an indicator of the present or absence of said target sequence.

24. A method according to claim 22 or 23 further comprising the step of hybridizing said probe nucleic acid to said target nucleic acid prior to said applying step.

25. A method according to claim 22 or 23 wherein said input signal includes the use of a co-redoxant.

29. A method according to claim 28, further comprising adding additional subunits to form said conductive oligomer.

30. A method according to claim 29, further comprising adding at least first nucleoside to said conductive oligomer.

31. A method according to claim 30, further comprising adding additional nucleosides to said first nucleoside to form a nucleic acid.

32. A method according to claim 29 or 31, further comprising attaching said conductive oligomer to said gold electrode.

23. A method of detecting a target sequence in a nucleic acid sample comprising

a) applying an input signal to a hybridization complex comprising:

i) a probe nucleic acid comprising a conductive oligomer covalently attached to a first electron transfer moiety comprising an electrode and to a single stranded nucleic acid capable of hybridizing to said target sequence, said single stranded nucleic acid comprising a covalently attached second electron transfer moiety, wherein said conductive oligomer has the formula;

 

wherein

n is an integer from 1 to 50;

m is 0 or 1;

C is carbon;

J is carbonyl or a heteroatom moeity, wherein the heteroatom is selected from the group consisting of nitrogen, silicon, phosphorus, sulfur; and

G is a bond selected from alkane, alkene or acetylene; and

ii) a target nucleic acid hybridized to said probe sequence to form said hybridization complex; and

b) detecting electron transfer between said electrode and said second electron transfer moiety, if present, as an indicator of the present or absence of said target sequence.

26. A method of detecting a target sequence in a nucleic acid wherein said target sequence comprises a first target domain and a second target domain, said method comprising:

a) hybridizing a first probe nucleic acid to said first target domain, if present, to form a hybridization complex, wherein said first probe nucleic acid comprises;

i) a conductive oligomer covalently attached to a first electron transfer moiety comprising an electrode and to a single stranded nucleic acid capable of hybridizing to said target sequence, wherein said conductive oligomer has the formula;

 

wherein

Y is an aromatic group;

n is an integer from 1 to 50;

g is either 1 or zero;

e is an integer from zero to 10; and

m is zero or 1;

wherein when g is 1, B-D is a conjugated bond; and

wherein when g is zero, e is 1 and D is preferably carbonyl, or a heteroatom moiety, wherein the heteroatom is selected from oxygen, sulfur, nitrogen or phosphorus;

b) hybridizing a second single stranded nucleic acid comprising a covalently attached electron transfer moiety to said second target domain; and

c) detecting electron transfer between said electrode and said second electron transfer moiety, if present, as an indicator of the present or absence of said target sequence.

27. A method of detecting a target sequence in a nucleic acid wherein said target sequence comprises a first target domain and a second target domain, said method comprising:

a) hybridizing a first probe nucleic acid to said first target domain, if present, to form a hybridization complex, wherein said first probe nucleic acid comprises;

i) a conductive oligomer covalently attached to a first electron transfer moiety comprising an electrode and to a single stranded nucleic acid capable of hybridizing to said target sequence, wherein said conductive oligomer has the formula;

 

wherein

n is an integer from 1 to 50;

m is 0 or 1;

C is carbon;

J is carbonyl or a heteroatom moeity, wherein the heteroatom is selected from the group consisting of nitrogen, silicon, phosphorus, sulfur; and

G is a bond selected from alkane, alkene or acetylene; and

b) hybridizing a second single stranded nucleic acid comprising a covalently attached electron transfer moiety to said second target domain; and

c) detecting electron transfer between said electrode and said second electron transfer moiety, if present, as an indicator of the present or absence of said target sequence.

28. A method for attaching a conductive oligomer to a gold electrode comprising

a) adding an ethyl pyridine protecting group to a sulfur atom attached to a first subunit of said conductive oligomer.

33. A conductive oligomer with a ethyl-pyridine protected sulfur atom.

38. A composition according to claim 36 or 37 further comprising a hybridization indicator.

36. A composition comprising a conductive oligomer covalently attached to a nucleoside, wherein said conductive oligomer has the formula:

37. A composition comprising a conductive oligomer covalently attached to a nucleoside, wherein said conductive oligomer has the formula:

39. A composition comprising a conductive oligomer covalently attached to a phosphoramidite nucleoside, wherein said conductive oligomer has the formula:

40. A composition comprising a conductive oligomer covalently attached to a phosphoramidite nucleoside, wherein said conductive oligomer has the formula:

41. A composition comprising a conductive oligomer covalently attached to a CPG-nucleoside.

42. A composition comprising a nucleoside covalently linked to a metallocene.

43. A composition according to claim 42 wherein said metallocene is ferrocene or substituted ferrocene.

44. A composition according to claim 42 wherein said metallocene is covalently attached to the base of said nucleoside.

45. A composition comprising:

a) an electrode;

b) at least one metallocene; and

c) a conductive oligomer covalently attached to both said electrode and said metallocene, wherein said conductive oligomer has the formula;

 

wherein

n is an integer from 1 to 50;

m is 0 or 1;

C is carbon;

J is carbonyl or a heteroatom moeity, wherein the heteroatom is selected from the group consisting of nitrogen, silicon, phosphorus, sulfur; and

G is a bond selected from alkane, alkene or acetylene

46. A composition comprising:

a) an electrode;

b) at least one metallocene; and

c) a conductive oligomer covalently attached to both said electrode and said metallocene, wherein said conductive oligomer has the formula;

 

wherein

Y is an aromatic group;

n is an integer from 1 to 50;

g is either 1 or zero;

e is an integer from zero to 10; and

m is zero or 1;

wherein when g is 1, B-D is a conjugated bond; and

wherein when g is zero, e is 1 and D is preferably carbonyl, or a heteroatom moiety, wherein the heteroatom is selected from oxygen, sulfur, nitrogen or phosphorus.