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

• US 20090011949A1
• Filed: 04/04/2008
• Published: 01/08/2009
• Est. Priority Date: 07/11/2001
• Status: Active Grant

• Current Assignee: Genomics USA, Inc., Gmsbiotech Incorporated
• Original Assignee: Genomics USA, Inc.
• Inventors: Eggers, Frederick H., Powdrill, Thomas F., Lemeshko, Sergy, Utermohlen, Joseph G., Hogan, Michael E., Belosludtsev, Yuri, Mitra, Rahul
• Granted Patent: US 10,272,409 B2
• Time in Patent Office: Days
• US Class Current: 506/9
• 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 2219/00497 Features relating to the solid phase supports
• CPC Class Codes: B01J 2219/005 Beads
• 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 2219/00653 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

Abstract

Provided herein are biomolecular hybridization devices comprising a substrate with a permanently and covalently attached surface of functional groups and an adsorbed monolayer of unmodified, single-stranded oligonucleotides all of which are 10 to about 24 bases in length as a saturated film of constrained oligonucleotides on the surface via direct non-covalent phosphate-surface adsorptive contact of substantially all phosphate groups of each oligonucleotide. The constrained oligonucleotides are effective to dissociably hybridize to a complementary single-stranded nucleic acid with asymmetric, non-helical base pairing and without oligonucleotide dissociation from the surface of the device. Also, provided are methods for hybridizing solution-state target nucleic acids to probe nucleic acids and for identifying a nucleotide sequence to which a nucleotide-binding protein binds using the biomolecular hybridization devices.

Claims

1. A biomolecular hybridization device comprising:

a substrate having a surface of functional groups permanently and covalently attached thereto; and

an adsorbed monolayer of unmodified, single-stranded oligonucleotides all of which are 10 to about 24 bases in length as a saturated film of constrained oligonucleotides on the surface via direct non-covalent phosphate-surface adsorptive contact of substantially all phosphate groups of each oligonucleotide;

wherein each constrained oligonucleotide base plane is presented from the surface in a manner effective to dissociably hybridize to a complementary single-stranded nucleic acid with asymmetric, non-helical base pairing without alteration of the oligonucleotide base plane presentation and without oligonucleotide phosphate group dissociation from the surface.

2. The biomolecular hybridization device of claim 1, further comprising a non-hybridizing poly-T sequence at one or both of the 5′

or 3′

end of the oligonucleotides such that the adsorbed oligonucleotides are each about 30 bases in length.

3. The biomolecular hybridization device of claim 1, further comprising a capping material disposed on the surface.

4. The biomolecular hybridization device of claim 3, wherein the capping material is a surfactant or an acid anhydride.

5. The biomolecular hybridization device of claim 1, further comprising a single-stranded nucleic acid reversibly hybridized to the oligonucleotide as a non-helical duplex of 10 to about 24 base pairs long adsorbed to the surface.

6. The biomolecular hybridization device of claim 5, wherein the nucleic acid is DNA or RNA.

7. The biomolecular hybridization device of claim 1, wherein the phosphate groups adsorb to the surface at a density of one phosphate group per about 0.5 square nanometers to less than about 1 square nanometer of surface.

8. The biomolecular hybridization device of claim 1, wherein the functional groups form a positively charged, polar or negatively charged hydrophilic surface or form a hydrophobic surface.

9. The biomolecular hybridization device of claim 8, wherein the positively charged surface is an aminosilane.

10. The biomolecular hybridization device of claim 8, wherein the surface comprises an organic material or an inorganic material.

11. The biomolecular hybridization device of claim 10, wherein the organic material is amino acids, lipids, nucleotides, carbohydrates, hydrocarbons, or isoprenoids.

12. The biomolecular hybridization device of claim 10, wherein the inorganic material is a hydrogen bond donor metal oxide.

13. The biomolecular hybridization device of claim 12, wherein the metal oxide is ferrite, aluminum dioxide or titanium dioxide or ferrite coated with an oxide of aluminum, titanium, zirconium, barium, calcium, cadmium, cobalt, iron, magnesium, nickel, or zinc.

14. The biomolecular hybridization device of claim 1, wherein the form of the substrate is a slide or a bead.

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

contacting the biomolecular hybridization device of claim 1 with at least one solution-state nucleic acid target under conditions that permit hybridization thereto such that non-helical, asymmetrical probe-target duplexes are formed.

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

contacting the biomolecular hybridization device of claim 5 with a nucleotide-binding protein under conditions that permit binding;

eluting formed duplex-protein complex(es) from the surface with a salt solution with an ionic strength of an aqueous solution of from about 1 M to about 5 M NaCl; and

sequencing at least one strand of the eluted nucleic acid duplex.

17. A biomolecular hybridization device comprising:

a substrate having a surface of functional groups permanently and covalently attached thereto; and

an adsorbed monolayer of unmodified, single-stranded oligonucleotides all of which are about 30 bases in length as a saturated film of constrained oligonucleotides on the surface via direct non-covalent phosphate-surface adsorptive contact of substantially all phosphate groups of each oligonucleotide;

wherein the constrained oligonucleotides each have a targeted, hybridizing domain of 10 to about 24 bases and a non-targeted, non-hybridizing domain of bases flanking one or both of the 5′

or 3′

ends thereof such that each constrained oligonucleotide base plane within the targeted domain is presented from the surface in a manner effective to dissociably hybridize to a complementary single-stranded nucleic acid target with asymmetric, non-helical base pairing without alteration of the oligonucleotide base plane presentation and without oligonucleotide phosphate group dissociation from the surface.

18. The biomolecular hybridization device of claim 17, further comprising a capping material disposed on the surface.

19. The biomolecular hybridization device of claim 18, wherein the capping material is a surfactant or an acid anhydride.

20. The biomolecular hybridization device of claim 17, further comprising a single-stranded nucleic acid reversibly hybridized to the oligonucleotide as a non-helical duplex of 10 to about 24 base pairs long adsorbed to the surface.

21. The biomolecular hybridization device of claim 20, wherein the nucleic acid is DNA or RNA.

22. The biomolecular hybridization device of claim 17, wherein the non-targeted, non-hybridizing domain bases are a poly-T sequence or other inert sequence of bases.

23. The biomolecular hybridization device of claim 17, wherein the adsorbed monolayer is formed from an about twice saturating amount of unmodified single-stranded oligonucleotides.

24. The biomolecular hybridization device of claim 17, wherein the phosphate groups adsorb to the surface at a density of one phosphate group per about 0.5 square nanometers to less than about 1 square nanometer of surface.

25. The biomolecular hybridization device of claim 17, wherein the functional groups form a positively charged, polar or negatively charged hydrophilic surface or form a hydrophobic surface.

26. The biomolecular hybridization device of claim 25, wherein the positively charged surface is an aminosilane.

27. The biomolecular hybridization device of claim 25, wherein the surface comprises an organic material or an inorganic material.

28. The biomolecular hybridization device of claim 27, wherein the organic material is amino acids, lipids, nucleotides, carbohydrates, hydrocarbons, or isoprenoids.

29. The biomolecular hybridization device of claim 27, wherein the inorganic material is a hydrogen bond donor metal oxide.

30. The biomolecular hybridization device of claim 29, wherein the metal oxide is ferrite, aluminum dioxide or titanium dioxide or ferrite coated with an oxide of aluminum, titanium, zirconium, barium, calcium, cadmium, cobalt, iron, magnesium, nickel, or zinc.

31. The biomolecular hybridization device of claim 17, wherein the form of the substrate is a slide or a bead.

32. A biomolecular hybridization device comprising:

a substrate having an aminosilanized surface permanently and covalently attached thereto; and

an adsorbed monolayer of unmodified single-stranded oligonucleotides all of which are 10 to 24 bases in length adsorbed to the aminosilanized surface as a saturated film of constrained oligonucleotides on the surface via direct non-covalent phosphate-surface adsorptive contact of substantially all phosphate groups of each oligonucleotide at a density of one phosphate group per about 0.5 square nanometers to less than about 1 square nanometer of surface, wherein each constrained oligonucleotide base plane is presented from the surface in a manner effective to dissociably hybridize to a complementary single-stranded nucleic acid with asymmetric, non-helical base pairing without alteration of the oligonucleotide base plane presentation and without oligonucleotide phosphate group dissociation from the surface.

33. The biomolecular hybridization device of claim 32, further comprising a non-hybridizable poly-T sequence at one or both of the 5′

or 3′

end of the oligonucleotides such that the adsorbed oligonucleotides are each about 30 bases in length.

34. The biomolecular hybridization device of claim 32, further comprising a capping material disposed on the surface.

35. The biomolecular hybridization device of claim 34, wherein the capping material is a surfactant or an acid anhydride.

37. The biomolecular hybridization device of claim 33, further comprising a single-stranded DNA or RNA reversibly hybridized to the oligonucleotide as a non-helical duplex of 10 to 24 base pairs long adsorbed to the surface.

38. The biomolecular hybridization device of claim 33, wherein the form of the substrate is a slide or a bead.