Surface-bound, unimolecular, double-stranded DNA

• US 5,770,722 A
• Filed: 06/13/1996
• Issued: 06/23/1998
• Est. Priority Date: 10/24/1994
• Status: Expired due to Term

• Current Assignee: Affymetrix, Inc. (Thermo Fisher Scientific Incorporated)
• Original Assignee: Affymetrix, Inc. (Thermo Fisher Scientific Incorporated)
• Inventors: Vetter, Dirk, Lockhart, David J., Diggelmann, Martin
• Primary Examiner(s): Chambers, Jasemine C.
• Assistant Examiner(s): Priebe, Scott D.
• Application Number:
  US08/664,093
• Time in Patent Office: 740 Days
• Field of Search: 536/23.1, 536/25.3, 435/6
• US Class Current: 536/25.3
• 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 2219/00529 DNA chips
• CPC Class Codes: B01J 2219/00608 DNA chips
• CPC Class Codes: B01J 2219/0061 The surface being organic
• CPC Class Codes: B01J 2219/00612 the surface being inorganic
• CPC Class Codes: B01J 2219/00619 using hydrophilic or hydrophobic regions
• CPC Class Codes: B01J 2219/00626 Covalent
• CPC Class Codes: B01J 2219/00635 by reactive plasma treatment
• 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 2219/00722 Nucleotides
• 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: C07K 7/06 having 5 to 11 amino acids
• CPC Class Codes: C12N 15/1093 General methods of preparing gene libraries, not provided for in other subgroups
• CPC Class Codes: C12Q 1/6811 Selection methods for production or design of target specific oligonucleotides or binding molecules
• CPC Class Codes: C12Q 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 2525/205 Aptamer
• CPC Class Codes: C12Q 2565/525 characterised by the capture oligonucleotide being double stranded
• CPC Class Codes: C40B 40/00 Libraries per se, e.g. arrays, mixtures
• CPC Class Codes: C40B 40/06 : Libraries containing nucleotides or polynucleotides, or derivatives thereof
• CPC Class Codes: G01N 33/5308 : for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites
• CPC Class Codes: G01N 33/551 : the carrier being inorganic

Abstract

Libraries of unimolecular, double-stranded oligonucleotides on a solid support. These libraries are useful in pharmaceutical discovery for the screening of numerous biological samples for specific interactions between the double-stranded oligonucleotides, and peptides, proteins, drugs and RNA. In a related aspect, the present invention provides libraries of conformationally restricted probes on a solid support. The probes are restricted in their movement and flexibility using double-stranded oligonucleotides as scaffolding. The probes are also useful in various screening procedures associated with drug discovery and diagnosis. The present invention further provides methods for the preparation and screening of the above libraries.

Claims

1. A method of forming a plurality of diverse unimolecular, double-stranded oligonucleotides on a solid support, said support comprising a surface with a plurality of preselected regions, said method comprising:

(a) forming on each of said preselected regions a different first oligonucleotide, each of said first oligonucleotides comprising from 6 to 30 monomers;

(b) attaching to the distal end of each of said first oligonucleotides of step (a) a linking group; and

(c) forming on the distal end of each of said linking groups a second oligonucleotide, wherein each of said second oligonucleotides is complementary to said first oligonucleotide which is attached within the same preselected region, and wherein said linking groups have sufficient length such that said first and second oligonucleotides form a unimolecular, double-stranded oligonucleotide.

2. The method of claim 1, wherein said method of construction of step (a) and step (b) is by light-directed synthesis.

3. The method of claim 1, wherein said linking group comprises a member selected from the group consisting of a polyethyleneglycol group and a polynucleotide.

4. The method of claim 3, wherein said linking group comprises a polyethyleneglycol group.

5. The method of claim 1, wherein said plurality is from 2 up to 100 diverse unimolecular, double-stranded oligonucleotides.

6. The method of claim 1, wherein said plurality is from 100 to 10000 diverse unimolecular, double-stranded oligonucleotides.

7. The method of claim 1, wherein said plurality is from 10000 to 1000000 diverse unimolecular, double-stranded oligonucleotides.

8. The method of claim 1, wherein said plurality of diverse unimolecular, double-stranded oligonucleotides is present on said solid support at a density of more than 100 members per cm².

9. The method of claim 1, wherein said plurality of diverse unimolecular, double-stranded oligonucleotides is present on said solid support at a density of more than 1000 members per cm².

10. The method of claim 1, wherein said plurality of diverse unimolecular, double-stranded oligonucleotides is present on said solid support at a density of more than 10000 members per cm².

11. The method of claim 10, wherein said solid support has a spacer attached thereto at each of said plurality of preselected regions, and wherein said step (a) of forming a different first oligonucleotide comprises attaching each of said different first oligonucleotides to the distal end of each of said spacers at each of said plurality of preselected regions.

12. The method of claim 11, wherein each of said spacers comprise a surface attaching portion which is a member selected from the group consisting of aminoalkylsilane and hydroxyalkylsilane.

13. The method of claim 12, wherein said surface attaching portion is selected from the group consisting of bis(2-hydroxyethyl)-aminopropyltriethoxysilane, 2-hydroxyethylaminopropyltriethoxysilane, aminopropyltriethoxysilane and hydroxypropyltriethoxysilane.

14. The method of claim 11, wherein each of said spacers comprises a longer chain portion which is a member selected from the group consisting of a polyethyleneglycol and a polynucleotide.

15. The method of claim 11, wherein said solid support is a silica support, said spacer comprises a polyethyleneglycol conjugated to an aminoalkylsilane, and said linking group is a polyethyleneglycol group.