Methods and devices based upon a novel form of nucleic acid duplex on a surface

• US 20030134299A1
• Filed: 07/11/2002
• Published: 07/17/2003
• Est. Priority Date: 07/11/2001
• Status: Active Grant

• Current Assignee: Baylor College of Medicine (Baylor College), Genomics USA, Inc.
• Original Assignee: Genomics USA, Inc.
• Inventors: Belosludtsev, Yuri, Hogan, Michael, Mitra, Rahul, Powdrill, Tom, Lemeshko, Sergy
• Granted Patent: US 7,354,710 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: B01J 19/0046 Sequential or parallel reactions, e.g. for the synthesis of polypeptides or polynucleotides; Apparatus and devices for combinatorial chemistry or for making molecular arrays synthesis methods per se C40B50/00
• CPC Class Codes: B01J 2219/00376 in multiple or parallel arrangements
• CPC Class Codes: B01J 2219/00387 Applications using probes
• CPC Class Codes: B01J 2219/00497 Features relating to the solid phase supports
• CPC Class Codes: B01J 2219/005 Beads
• CPC Class Codes: B01J 2219/00529 DNA chips
• CPC Class Codes: B01J 2219/00576 fluorophore
• CPC Class Codes: B01J 2219/00605 the compounds being directly bound or immobilised to solid supports
• CPC Class Codes: B01J 2219/00608 DNA chips
• CPC Class Codes: B01J 2219/00612 the surface being inorganic
• CPC Class Codes: B01J 2219/00626 Covalent
• CPC Class Codes: B01J 2219/00637 by coating it with another layer
• 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 2219/00677 Ex-situ synthesis followed by deposition on the substrate
• CPC Class Codes: B01J 2219/00691 using robots
• CPC Class Codes: B01J 2219/00722 Nucleotides
• CPC Class Codes: C12Q 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 2523/308 Adsorption or desorption
• CPC Class Codes: C40B 60/14 for creating libraries

Abstract

The present invention relates to simple method to fabricate DNA hybridization devices based upon adsorptive attachment of oligonucleotides to a positively charged surface. Such adsorbed oligonucleotide probes form a densely packed monolayer, which retains capacity for base-pair specific hybridization with a solution state nucleic acid target strand to form the duplex. However, both strand dissociation kinetics and the rate of DNase digestion suggest on symmetry grounds that solution-state nucleic acid binds to such adsorbed oligonucleotides to form a highly asymmetric and unwound duplex, with structural details that are substantially different from that known for the Watson-Crick DNA duplex. This novel nucleic acid duplex form can serve as the basis for a new class of hybridization device and methods for their use. It is also disclosed that new methods of nucleic acid duplex detection can be developed which are based upon the interaction of enzymes and dye labels with the unique structural characteristics of the non-helical duplex described herein. Preferred implementations of the invention include DNA microarrays, bead-based nucleic acid analysis, microelectronic devices to detect nucleic acid hybridization and more traditional methods of laboratory analysis, including hybridization on membranous and other solid supports.

Claims

1. A biomolecular hybridization device comprising a surface and a first nucleic acid adsorbed thereto, wherein the surface is substantially saturated with positive charge groups and wherein said first nucleic acid is linker-free and noncovalently attached to the surface.

2. The biomolecular hybridization device of claim 1 wherein the density of said charge groups is about 1 charge group per 5 Å

on center.

3. The biomolecular hybridization device of claim 1 wherein said charge groups are uniformly distributed.

4. The biomolecular hybridization device of claim 1 wherein the nucleic acid contains about 1 nucleotide to about 100 nucleotides.

5. The biomolecular hybridization device of claim 4 wherein the nucleic acid contains about 12 nucleotide to about 60 nucleotides.

6. The biomolecular hybridization device of claim 1 wherein the nucleic acid is DNA.

7. The biomolecular hybridization device of claim 1 wherein the nucleic acid is RNA.

8. The biomolecular hybridization device of claim 1 wherein the nucleic acid is single-stranded.

9. The biomolecular hybridization device of claim 1 wherein the nucleic acid is double-stranded.

10. The biomolecular hybridization device of claim 1 wherein said surface is substantially saturated with said nucleic acid.

11. The biomolecular hybridization device of claim 8 wherein said surface is substantially saturated with said nucleic acid.

12. The biomolecular hybridization device of claim 8 wherein said nucleic acid forms a monolayer.

13. The biomolecular hybridization device of claim 1 wherein said device further comprises one or more additional surfaces wherein each additional surface is substantially saturated with positive charge groups.

14. The biomolecular hybridization device of claim 13 wherein each additional surface is substantially saturated with a noncovalently attached additional nucleic acid.

15. The biomolecular hybridization device of claim 14 wherein the sequence of each additional nucleic acid is identical to said first nucleic acid.

16. The biomolecular hybridization device of claim 14 wherein the sequence of each additional nucleic acid is not identical to said first nucleic acid.

17. The biomolecular hybridization device of claim 14 wherein said surfaces comprise a microarray format.

18. The biomolecular hybridization device of claim 1 wherein the device further comprises a support.

19. The biomolecular hybridization device of claim 18 wherein said support comprises a material selected from the group consisting of a ceramic material, a silicon dioxide material, and a metal.

20. The biomolecular hybridization device of claim 18 wherein the form of said device is selected from the group consisting of a slide and a bead.

28. The biomolecular hybridization device of claim 20 wherein said surface comprises an inorganic material.

29. The biomolecular hybridization device of claim 20 wherein the device further comprises a support.

30. The biomolecular hybridization device of claim 29 wherein said support comprises a material selected from the group consisting of a ceramic material, a silicon dioxide material, and a metal.

31. The biomolecular hybridization device of claim 29 wherein said device is a bead or a slide.

21. A biomolecular hybridization device comprising a surface and a first nucleic acid adsorbed thereto, wherein the surface is substantially saturated with with functional groups and wherein said first nucleic acid is linker-free and noncovalently attached to the surface.

22. The biomolecular hybridization device of claim 21 wherein said noncovalent attachment comprises electrostatic interactions, hydrogen bonding, Van der Waals interactions, London interactions, hydrophobic interactions or combinations thereof between said functional groups and said nucleic acid.

23. The biomolecular hybridization device of claim 21 wherein said surface is substantially saturated with said functional groups.

24. The biomolecular hybridization device of claim 21 wherein said surface is hydrophilic.

25. The biomolecular hybridization device of claim 21 wherein said surface is hydrophobic.

26. The biomolecular hybridization device of claim 21 wherein said surface comprises an organic material.

27. The biomolecular hybridization device of claim 26 wherein said organic material is selected from the group consisting of amino acids, lipids, nucleotides, carbohydrates, hydrocarbons, and isoprenoids.

32. The biomolecular hybridization device of claim 21 wherein the nucleic acid contains about 1 nucleotide to about 100 nucleotides.

33. The biomolecular hybridization device of claim 21 wherein the nucleic acid contains about 12 nucleotide to about 60 nucleotides.

34. The biomolecular hybridization device of claim 21 wherein the nucleic acid is DNA.

35. The biomolecular hybridization device of claim 21 wherein the nucleic acid is RNA.

36. The biomolecular hybridization device of claim 21 wherein the nucleic acid is single-stranded.

37. The biomolecular hybridization device of claim 21 wherein the nucleic acid is double-stranded.

38. The biomolecular hybridization device of claim 21 wherein said surface is substantially saturated with said nucleic acid.

39. The biomolecular hybridization device of claim 36 wherein said surface is substantially saturated with said nucleic acid.

40. The biomolecular hybridization device of claim 36 wherein said nucleic acid forms a monolayer.

41. A method for preparing a biomolecular hybridization device comprising a surface and a nucleic acid probe adsorbed thereto comprising:

contacting the surface with a saturating amount of a nucleic acid probe under conditions that permit noncovalent adsorption of the probe onto the surface

wherein the surface is substantially saturated with functional groups that mediate noncovalent interaction between the nucleic acid and the surface.

42. The method of claim 41 wherein the functional groups are cationic charge groups.

43. The method of claim 41 wherein the functional groups are zwitterionic charge groups.

44. The method of claim 41 wherein the nucleic acid is single stranded.

45. The method of claim 41 wherein the nucleic acid is DNA.

46. The method of claim 41 wherein the nucleic acid is RNA.

47. The method of claim 41 wherein said contacting comprises depositing said nucleic acid probe on said surface in solution state.

48. The method of claim 41 wherein said contacting comprises vapor deposition of said nucleic acid probe on said surface.

49. The method of claim 41 further comprising fabricating said surface by covalently modifying a substrate to form said surface.

50. The method of claim 49 wherein said covalent modification is aminosilanization.

51. The method of claim 49 wherein said substrate is a glass slide.

52. The method of claim 41 further comprising capping said surface.

53. The method of claim 52 wherein said capping comprises vapor deposition of the capping material.

54. The method of claim 52 wherein said capping comprises deposition of a surfactant.

55. A method for hybridizing solution-state target nucleic acids to probe nucleic acids comprising:

exposing a biomolecular hybridization device comprising a surface and a nucleic acid probe adsorbed thereto to at least one solution-state nucleic acid target under conditions that permit hybridization;

wherein probe-target hybrids are formed.

56. The method of claim 55 wherein the functional groups are cationic charge groups.

57. The method of claim 55 wherein the functional groups are zwitterionic charge groups.

58. The method of claim 55 wherein the nucleic acid is single stranded.

59. The method of claim 55 wherein the nucleic acid is DNA.

60. The method of claim 55 wherein the nucleic acid is RNA.

61. A method for identifying a nucleotide sequence to which a nucleotide-binding protein binds comprising:

contacting a biomolecular hybridization device comprising a surface and a nucleic acid probe-target duplex with a nucleotide-binding protein under conditions that permit binding;

eluting duplex-protein complex(es) formed from the surface; and

sequencing at lest one strand of said eluted duplex.

wherein said nucleic acid probe is noncovalently attached to said surface and said target nucleic acid is hybridized to said nucleic acid probe.

62. The method of claim 61 wherein said eluting comprises contacting at least one surface having a duplex-protein complex with a salt solution.

63. The method of claim 62 wherein said salt solution has the ionic strength of an aqueous solution of from about 1 M to about 5 M NaCl.