Selective attachment of nucleic acid molecules to patterned self-assembled surfaces

• US 5,688,642 A
• Filed: 12/01/1994
• Issued: 11/18/1997
• Est. Priority Date: 12/01/1994
• Status: Expired due to Term

• Current Assignee: the united states of america as represented by the secretary of the navy
• Original Assignee: the united states of america as represented by the secretary of the navy
• Inventors: Chrisey, Linda A., Dressick, Walter J., Calvert, Jeffrey M.
• Primary Examiner(s): Jones, W. Gary
• Assistant Examiner(s): REES, DIANE
• Application Number:
  US08/352,126
• Time in Patent Office: 1,083 Days
• Field of Search: 435/6, 435/5, 435/91.1, 435/91.2, 435/176, 204/403, 356/445, 536/24.3, 536/24.33, 536/25.3
• US Class Current: 435/6.11
• CPC Class Codes: B01J 2219/00432 Photolithographic masks
• CPC Class Codes: B01J 2219/00527 Sheets
• CPC Class Codes: B01J 2219/00585 Parallel processes
• CPC Class Codes: B01J 2219/00596 Solid-phase processes
• CPC Class Codes: B01J 2219/00605 the compounds being directly bound or immobilised to solid supports
• CPC Class Codes: B01J 2219/0061 The surface being organic
• 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 2219/00659 Two-dimensional arrays
• CPC Class Codes: B01J 2219/00711 Light-directed synthesis
• CPC Class Codes: B01J 2219/00722 Nucleotides
• CPC Class Codes: B82Y 30/00 Nanotechnology for materials or surface science, e.g. nanocomposites
• CPC Class Codes: B82Y 5/00 Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
• CPC Class Codes: C12Q 1/6834 Enzymatic or biochemical coupling of nucleic acids to a solid phase
• CPC Class Codes: C40B 40/06 Libraries containing nucleotides or polynucleotides, or derivatives thereof
• CPC Class Codes: C40B 60/14 for creating libraries

Abstract

Patterns of pre-formed hybridizable nucleic acid oligomers are formed upon a substrate. The substrate is coated with molecules, such as aminosilanes, whose reactivity with nucleic acid molecules can be transformed by irradiation. The coated substrate exposed to patterned irradiation then contacted with pre-formed nucleic acid oligomers. The binding of the preformed nucleic acid oligomers to the coating molecules may be covalent or non-covalent (for example, ionic bonding or hydrogen bonding). If desired, a heterobifunctional crosslinker may be employed, before or after irradiation, with the coating to promote covalent binding of the nucleic acid oligomers to the coating molecules. Also, the irradiation step may be performed with the assistance of a positive-tone or negative-tone photoresist.

Claims

1. A method of attaching pre-formed hybridizable synthetic nucleic acid oligomers, modified to include a thiol group, to selected portions along a surface of a substrate to form a pattern of synthetic hybridizable nucleic acid oligomers thereon, comprising the steps of:

covalently binding an organosilane coating to said surface of said substrate, the organosilane molecules of said covalently-bound organosilane coating having, at a site distal from a site of attachment of said organosilane molecule to said surface, at least one active site incapable of binding to said surface and incapable of binding to the other organosilane molecules of said organosilane coating, but available as a covalent attachment site for binding to a heterobifunctional crosslinker, said heterobifunctional crosslinker including a first reactive site that covalently binds to said at least one active site of said organosilane molecule of said coating, a second reactive site available for directly covalently binding to said thiol group of said pre-formed synthetic hybridizable nucleic acid oligomers, thus forming an organosilane-coated assembly;

reacting said organosilane coating with said heterobifunctional crosslinker;

defining, after said reacting step, a pattern within said organosilane coating including a first region, said first region including said organosilane molecules, and said organosilane molecules in said first region retaining said at least one active site available for binding said heterobifunctional crosslinker, and a second region that lacks organosilane molecules having an available active site for binding said heterobifunctional crosslinker, by exposing said organosilane-coated assembly to a pattern of radiation thereby forming a patterned surface;

exposing said patterned surface to said modified pre-formed synthetic nucleic acid oligomers to selectively and covalently attach said thiol group of said modified pre-formed synthetic nucleic acid oligomers to said first region and to non-specifically adsorb said modified pre-formed synthetic nucleic acid oligomers to said patterned surface;

removing said non-specifically adsorbed pre-formed synthetic nucleic acid oligomers from said patterned surface, thereby forming a pattern of covalently attached hybridizable synthetic nucleic acid oligomers upon said substrate.

2. The method of claim 1, wherein said radiation is ultraviolet radiation.

3. The method of claim 2, wherein said organosilane is an aminosilane.

4. The method of claim 2, wherein said organosilane is an aminosilane and said at least one active site is a terminal amino group.

5. The method of claim 4, wherein said terminal amino group is a primary amino group.

6. The method of claim 4, wherein said aminosilane is an amino-terminated aminosilane also having a hydroxyl-reactive terminus selected from the group consisting of a trichloro group, a trialkoxy group, a dichloro group, a monochloro group, a dialkoxy group and a monoalkoxy group.

7. The method of claim 6, wherein said aminosilane is selected from the group consisting of N-(2-aminoethyl)-3-aminopropyltrimethoxysilane, trimethoxysilylpropyldiethylenetriamine, and (aminoethylaminomethyl)-phenyethyltrimethoxysilane.

8. The method of claim 1, wherein said organosilane-coated assembly is exposed to said patterned radiation by employing ultraviolet light and a lithographic mask.

9. The method of claim 1, wherein said first reactive site of said heterobifunctional crosslinker is an N-succinimide ester group, an isothiocyanate group, an imidoester group, or a nitroaryl halide group, and said second reactive site is a maleimide group, an iodoacetyl group, or a disulfide group.

10. The method of claim 12, wherein said heterobifunctional crosslinker is succinimidyl 4-(p-maleimidophenyl)butyrate.

11. The method of claim 1, further comprising the step of coating said heterobifunctional crosslinker-bound organosilane layer with a photoresist material before said step of defining said pattern, so that said second reactive sites of said underlying heterobifunctional crosslinker are rendered unavailable for covalently bonding with nucleic acid molecule.

12. The method of claim 11, wherein said step of defining said pattern includes the steps of:

exposing part of said photoresist to said pattern of ultraviolet radiation, thus forming a corresponding pattern including an exposed region of said photoresist and an unexposed region of said photoresist;

selectively removing said exposed or said unexposed region of said photoresist to expose said heterobifunctional crosslinker-bound organosilane layer underlying said selectively removed region of said photoresist, so that said second reactive sites of said heterobifunctional crosslinkers underlying said removed region of said photoresist are made available for covalently bonding with nucleic acid molecules.

13. The method of claim 1, wherein said nucleic acid oligomers have from 4 to about 400 bases.

14. The method of claim 13, wherein said nucleic acid oligomers have from about 20 to about 100 bases.

18. The method of claim 1, wherein said non-specifically adsorbed pre-formed nucleic acid oligomers are removed from said patterned surface by exposing said patterned surface to a salt of sufficiently high concentration to desorb said non-specifically adsorbed pre-formed nucleic acid oligomers from said patterned surface.

15. A method of attaching pre-formed, hybridizable, synthetic nucleic acid oligomers to selected portions along a surface of a substrate to form a pattern of hybridizable nucleic acid oligomers thereon, comprising the steps of:

covalently binding a coating of non-DNA-binding organosilane molecules to said surface of said substrate, thus forming an organosilane-coated assembly;

defining a pattern including a first region, said first region including said non-DNA-binding organosilane molecules, and a second region in which said non-DNA-binding organosilane molecules have been removed from the surface by exposing said organosilane-coated assembly to a pattern of ultraviolet radiation, thereby forming a patterned surface;

covalently binding nucleic acid-binding organosilane molecules to said patterned surface at said second region, said nucleic acid-binding organosilane molecules having, at a site distal from a site of attachment of said nucleic acid-binding organosilane molecule to said surface of said second region, at least one active site incapable of binding to said surface, incapable of binding to the non-DNA-binding organosilane molecules of said coating, and incapable of binding to said nucleic acid-binding organosilanes, but available as an attachment site for binding a heterobifunctional crosslinker, said heterobifunctional crosslinker including a first reactive site that covalently binds to said at least one active site of said organosilane molecule of said coating, and a second reactive site available for directly and covalently binding to said thiol group of said modified synthetic nucleic acid molecules;

reacting said organosilane coating with said heterobifunctional crosslinker;

exposing, after said reacting step, said patterned surface having said nucleic acid-binding organosilanes therein to said modified pre-formed synthetic nucleic acid oligomers to selectively and covalently attach said thiol group of said pre-formed, synthetic nucleic acid oligomers to said second region and to non-specifically adsorb said modified pre-formed nucleic acid oligomers to said patterned surface; and

removing said non-specifically adsorbed pre-formed synthetic nucleic acid oligomers from said patterned surface, thereby forming a pattern of covalently attached hybridizable synthetic nucleic acid oligomers upon said substrate.

16. The method of claim 15, wherein said organosilane is an aminosilane and said at least one of active site is a terminal amino group.

17. A method of attaching pre-formed synthetic hybridizable nucleic acid oligomers, modified to include a thiol group, to selected portions along a surface of a substrate to form a pattern of said modified hybridizable synthetic nucleic acid oligomers thereon, comprising the steps

covalently binding an organosilane coating to said surface of said substrate, the molecules of said covalently-bound organosilane coating having, at a site distal from a site of attachment of said coating molecule to said surface, at least one active site incapable of binding to said surface and incapable of binding to the other molecules of said coating, the ability of which to covalently bind a heterobifunctional crosslinker is reversed by exposure to irradiation, thus forming an organosilane-coated assembly;

defining a pattern in said organosilane coating, said pattern including a first region, said first region including said coating molecules, and said at least one active site of said coating molecules in said first region having the ability to bind said heterobifunctional crosslinker, and a second region that lacks coating molecules with the ability to bind said heterobifunctional crosslinker, by exposing said coated assembly to a pattern of radiation thereby forming a patterned surface;

reacting said organosilane coating with said heterobifunctional crosslinker, said heterobifunctional crosslinker including a first reactive site that covalently binds to said at least one active site of said coating molecules in said first region, and a second reactive site available for directly and covalently binding to said thiol group of said modified synthetic nucleic acid oligomers;

exposing said patterned surface having said nucleic acid-binding organosilanes therein to said modified pre-formed synthetic nucleic acid oligomers to selectively and covalently bind said thiol group of said modified synthetic nucleic acid oligomers to said first region and to non-specifically adsorb said modified preformed synthetic nucleic acid oligomers to said patterned surface; and

removing said non-specifically adsorbed pre-formed synthetic nucleic acid oligomers from said patterned surface, thereby forming a pattern of covalently attached hybridizable synthetic nucleic acid oligomers upon said substrate.