Nanoparticles having oligonucleotides attached thereto and uses therefor

• US 6,730,269 B2
• Filed: 10/12/2001
• Issued: 05/04/2004
• Est. Priority Date: 07/29/1996
• Status: Expired due to Term

• Current Assignee: Nanosphere Incorporated (DiaSorin S.p.A.)
• Original Assignee: Nanosphere Incorporated (DiaSorin S.p.A.)
• Inventors: Mucic, Robert C., Mirkin, Chad A., Storhoff, James J., Elghanian, Robert, Taton, Thomas A., Letsinger, Robert L.
• Primary Examiner(s): Riley, Jezia
• Application Number:
  US09/976,617
• Publication Number:
  US 20020137072A1
• Time in Patent Office: 935 Days
• Field of Search: 435/6, 536/231, 536/24.3, 536/25.3
• US Class Current: 422/68.1
• CPC Class Codes: B01J 2219/00274 Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
• CPC Class Codes: B01J 2219/005 Beads
• CPC Class Codes: B01J 2219/00585 Parallel processes
• CPC Class Codes: B01J 2219/00596 Solid-phase processes
• CPC Class Codes: B01J 2219/00603 Making arrays on substantially continuous surfaces
• CPC Class Codes: B01J 2219/00648 by the use of solid beads
• CPC Class Codes: B01J 2219/00653 the compounds being bound to electrodes embedded in or on the solid supports
• CPC Class Codes: B01J 2219/00659 Two-dimensional arrays
• CPC Class Codes: B01J 2219/00702 Processes involving means for analysing and characterising the products
• CPC Class Codes: B01J 2219/00722 Nucleotides
• CPC Class Codes: B82B 1/00 Nanostructures formed by manipulation of individual atoms or molecules, or limited collections of atoms or molecules as discrete units
• CPC Class Codes: B82Y 15/00 Nanotechnology for interacting, sensing or actuating, e.g. quantum dots as markers in protein assays or molecular motors
• CPC Class Codes: B82Y 30/00 Nanotechnology for materials or surface science, e.g. nanocomposites
• CPC Class Codes: C07B 2200/11 Compounds covalently bound to a solid support
• 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 1/6813 Hybridisation assays
• CPC Class Codes: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• 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 1/6827 for detection of mutation or polymorphism
• 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 2525/161 : incorporating target specific and non-target specific sites
• CPC Class Codes: C12Q 2525/197 : incorporating a spacer/coupling moiety
• CPC Class Codes: C12Q 2537/125 : Sandwich assay format
• CPC Class Codes: C12Q 2537/143 : Multiplexing, i.e. use of multiple primers or probes in a single reaction, usually for simultaneously analyse of multiple analysis
• CPC Class Codes: C12Q 2563/143 : Magnetism, e.g. magnetic label
• CPC Class Codes: C12Q 2563/155 : Particles of a defined size, e.g. nanoparticles
• CPC Class Codes: C12Q 2565/625 : being a nucleic acid test strip device, e.g. dipsticks, strips, tapes, CD plates
• CPC Class Codes: C40B 40/06 : Libraries containing nucleotides or polynucleotides, or derivatives thereof
• CPC Class Codes: Y02A 50/30 : Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
• CPC Class Codes: Y10S 977/924 : using nanostructure as support of dna analysis

Abstract

The invention provides methods of detecting a nucleic acid. The methods comprise contacting the nucleic acid with one or more types of particles having oligonucleotides attached thereto. In one embodiment of the method, the oligonucleotides are attached to nanoparticles and have sequences complementary to portions of the sequence of the nucleic acid. A detectable change (preferably a color change) is brought about as a result of the hybridization of the oligonucleotides on the nanoparticles to the nucleic acid. The invention also provides compositions and kits comprising particles. The invention further provides methods of synthesizing unique nanoparticle-oligonucleotide conjugates, the conjugates produced by the methods, and methods of using the conjugates. In addition, the invention provides nanomaterials and nanostructures comprising nanoparticles and methods of nanofabrication utilizing nanoparticles. Finally, the invention provides a method of separating a selected nucleic acid from other nucleic acids.

Claims

1. A method of nanofabrication comprising

providing at least one type of linking oligonucleotide having a selected sequence, the sequence of each type of linking oligonucleotide having at least two portions;

providing one or more types of nanoparticles having oligonucleotides bound thereto, wherein the oligonucleotides are present on a surface of the nanoparticles at a surface density of at least 10 picomoles/cm², at least some of the oligonucleotides on each type of nanoparticles comprise at least one type of recognition oligonucleotides, each type of recognition oligonucleotides comprises a spacer portion and a recognition portion, the spacer portion being designed so that it is bound to the nanoparticles, the recognition portion comprises a sequence complementary to at least a portion of the sequence of a specific type of linking oligonucleotide; and

contacting the linking oligonucleotides and nanoparticles under conditions effective to allow hybridization of at least a portion of the oligonucleotides on the nanoparticles to the linking oligonucleotides so that a desired nanomaterial or nanostructure is formed.

2. The method of claim 1 wherein the linking oligonucleotide is single-stranded.

3. The method of claim 1 wherein the linking oligonucleotide is double-stranded and has overhanging ends.

4. The method of claim 1 wherein the linking oligonucleotide is a triple-stranded oligonucleotide connector.

5. The method of claim 1 wherein the linking oligonucleotide comprises a peptide nucleic acid chain.

7. The method of any one of claims 1 or 6 wherein the oligonucleotides are attached to the nanoparticles in a stepwise ageing process comprising (i) contacting the oligonucleotides with the nanoparticles in a first aqueous solution for a period of time sufficient to allow some of the oligonucleotides to bind to the nanoparticles;

(ii) adding at least one salt to the first aqueous solution to create a second aqueous solution; and

(iii) contacting the oligonucleotides and nanoparticles in the second aqueous solution for an additional period of time to enable additional oligonucleotides to bind to the nanoparticles.

8. The method of claim 7 wherein the second aqueous solution has an ionic strength sufficient to overcome at least partially the electrostatic attraction or repulsion of the oligonucleotides for the nanoparticles and the electrostatic repulsion of the oligonucleotides to each other.

9. The method of claim 7 wherein the oligonucleotides and nanoparticles are contacted in aqueous solution for about 12 to about 24 hours.

10. The method of claim 7 wherein salt is added to the aqueous solution to form the aqueous salt solution which is buffered at pH 7.0 and which contains about 0.1 M NaCl.

11. The method of claim 7 wherein the oligonucleotides and nanoparticles are contacted in the aqueous salt solution for an additional 40 hours to increase the density of oligonucleotides bound to the nanoparticles.

12. The method of claim 10 wherein the salt is added to the first aqueous solution in a single addition.

13. The method of claim 10 wherein the salt is added gradually to the first aqueous solution over time.

14. The method of claim 10 wherein the salt is selected from the group consisting of consisting of sodium chloride, magnesium chloride, potassium chloride, ammonium chloride, sodium acetate, ammonium acetate, a combination of two or more of these salts, one of these salts in a phosphate buffer, and a combination of two or more these salts in a phosphate buffer.

15. The method of claim 14 wherein the salt is sodium chloride in a phosphate buffer.

16. The method of claim 1 or 6 wherein the oligonucleotides are present on the surface of the nanoparticles at a surface density of at least 15 picomoles/cm².

17. The method of claim 16 wherein the oligonucleotides are present on the surface of the nanoparticles at a surface density from about 15 picomoles/cm² to about 40 picomoles/cm².

18. The method of any one of claim 1 or 6 where the nanoparticles are gold metal nanoparticles or semiconductor nanoparticles.

19. The method of claim 18 wherein the nanoparticles are gold nanoparticles.

20. The method of claim 18 wherein the semiconductor nanoparticles are made of CdSe/ZnS (core/shell).

21. The method of any one of claim 1 or 6 wherein the spacer portion has a moiety covalently bound to it, the moiety comprising a functional group through which the spacer portion is bound to the nanoparticles.

22. The method of any one of claim 1 or 6 wherein the spacer portion comprises at least 10 nucleotides.

23. The method of claim 22 wherein the spacer portion comprises from about 10 to about 30 nucleotides.

24. The method of claim 22 wherein the bases of the nucleotides of the spacer portion arc all adenines, all thymines, all cytosines, all uracils or all guanines.

25. The method of any one of claim 1 or 6 wherein at least some the oligonucleotides bound to the nanoparticles comprise a type of diluent oligonucleotides.

26. The method of claim 25 wherein the diluent oligonucleotides contain about the same number of nucleotides as are contained in the spacer portions of the recognition oligonucleotides.

27. The method of claim 26 wherein the sequence of the diluent oligonucleotides is the same as hat of the spacer portions of the recognition oligonucleotides.

28. The method of any one of claim 1 or 6 wherein the oligonucleotides are bound to the nanoparticles through sulfur linkages.

29. A nanomaterial or nanostructure prepared in accordance with any one of claims 1-28.

6. A method of nanofabrication comprising:

providing at least two types of nanoparticles having oligonucleotides bound thereto wherein the oligonucleotides are present on a surface of the nanoparticles at a surface density of at least 10 picomoles/cm², wherein at least some of the oligonucleotides on each type of nanoparticles comprise one or more types of recognition oligonucleotides, each type of recognition oligonucleotides comprises a spacer portion and a recognition portion, the spacer portion being designed so that it is bound to the nanoparticles, at least one type of recognition oligonucleotides on a first type of nanoparticles comprises a recognition portion having a sequence complementary to at least a portion of the oligonucleotides on a second type of nanoparticles and at least one type of recognition oligonucleotides on the second type of nanoparticles comprises a recognition portion having a sequence complementary to at least a portion of the oligonucleotides on the first type of nanoparticles; and

contacting the first and second types of nanoparticles under conditions effective to allow hybridization of at least a portion of the oligonucleotides on the nanoparticles to each other so that a desired nanomaterial or nanostructure is formed.

30. A kit comprising

(i) one or more types of linking oligonucleotide having a selected sequence, the sequence of each type of linking oligonucleotide having at least two portions;

and

(ii) one or more types of nanoparticles having oligonucleotides bound thereto wherein the oligonucleotides are present on a surface of the nanoparticles at a surface density of at least 10 picomoles/cm², at least some of the oligonucleotides on each type of nanoparticles comprise at least one type of recognition oligonucleotides, each type of recognition oligonucleotides comprises a spacer portion and a recognition portion, the spacer portion being designed so that it is bound to the nanoparticles, and the recognition portion having a sequence complementary to at least a portion of the sequence of a specific type of linking oligonucleotide.

31. The kit of claim 30 wherein the linking oligonucleotide is single-stranded.

32. The kit of claim 30 wherein the linking oligonucleotide is double-stranded and has overhanging ends.

33. The kit of claim 30 wherein the linking oligonucleotide is a triple-stranded oligonucleotide connector.

34. The kit of claim 30 wherein the linking oligonucleotide comprises a peptide nucleic acid chain.

35. The kit of claim 30 wherein the oligonucleotides are attached to the nanoparticles in a stepwise ageing process comprising (i) contacting the oligonucleotides with the nanoparticles in a first aqueous solution for a period of time sufficient to allow some of the oligonucleotides to bind to the nanoparticles;

(ii) adding at least one salt to the first aqueous solution to create a second aqueous solution; and

(iii) contacting the oligonucleotides and nanoparticles in the second aqueous solution for an additional period of time to enable additional oligonucleotides to bind to the nanoparticles.

36. The kit of claim 35 wherein the second aqueous solution has an ionic strength sufficient to overcome at least partially the electrostatic attraction or repulsion of the oligonucleotides for the nanoparticles and the electrostatic repulsion of the oligonucleotides to each other.

37. The kit of claim 35 wherein the oligonucleotides and nanoparticles are contacted in aqueous solution for about 12 to about 24 hours.

38. The kit of claim 35 wherein salt is added to the first aqueous solution to form the second aqueous solution which is buffered at pH 7.0 and which contains about 0.1 M NaCl.

39. The kit of claim 35 wherein the oligonucleotides and nanoparticles are contacted in the second aqueous solution for an additional 40 hours to increase the density of oligonucleotides bound to the nanoparticles.

40. The kit of claim 38 wherein the salt is added to the first aqueous solution in a single addition.

41. The kit of claim 38 wherein the salt is added gradually to the first aqueous solution over time.

42. The kit of claim 38 wherein the salt is selected from the group consisting of consisting of sodium chloride, magnesium chloride, potassium chloride, ammonium chloride, sodium acetate, ammonium acetate, a combination of two or more of these salts, one of these salts in a phosphate buffer, and a combination of two or more these salts in a phosphate buffer.

43. The kit of claim 42 wherein the salt is sodium chloride in a phosphate buffer.

44. The kit of claim 30 wherein the oligonucleotides are present on the surface of the nanoparticles at a surface density of at least 15 picomoles/cm².

45. The kit of claim 44 wherein the oligonucleotides are present on the surface of the nanoparticles at a surface density from about 15 picomoles/cm to about 40 picomoles/cm².

46. The kit of claim 30 wherein the nanoparticles are metal nanoparticles or semiconductor nanoparticles.

47. The kit of claim 46 wherein the nanoparticles are gold nanoparticles.

48. The kit of claim 47 wherein the semiconductor nanoparticles are made of CdSe/ZnS (core/shell).

49. The kit of claim 30 wherein the spacer portion has a moiety covalently bound to it, the moiety comprising a functional group through which the spacer portion is bound to the nanoparticles.

50. The kit of claim 30 wherein the spacer portion comprises at least 10 nuclcotides.

51. The kit of claim 50 wherein the spacer portion comprises from about 10 to about 30 nucleotides.

52. The kit of claim 50 wherein the bases of the nucleotides of the spacer portion are all adenines, all thymines, all cytosines, all uracils or all guanines.

53. The kit of claim 50 wherein the bases of the nucleotides of the spacer portion are all adenines, all thymines, all cytosines, all uracils or all guanines.

54. The kit of claim 30 wherein at least some the oligonucleotides bound to the nanoparticles comprise a type of diluent oligonucleotides.

55. The kit of claim 54 wherein the diluent oligonucleotides contain about the same number of nucleotides as are contained in the spacer portions of the recognition oligonucleotides.

56. The kit of claim 54 wherein the sequence of the diluent oligonucleotides is the same as that of the spacer portions of the recognition oligonucleotides.

57. The kit of claim 30 wherein the oligonucleotides are bound to the nanoparticles through sulfur linkages.