Methods for discovering ligands

• US 6,080,585 A
• Filed: 10/02/1996
• Issued: 06/27/2000
• Est. Priority Date: 02/01/1994
• Status: Expired due to Fees

• Current Assignee: Oxford Gene Technology Limited (Sysmex Corporation)
• Original Assignee: Oxford Gene Technology Limited (Sysmex Corporation)
• Inventors: Mir, Kalim Ullah, Southern, Edwin Mellor, Case-Green, Stephen Charles
• Primary Examiner(s): Sisson, Bradley L.
• Application Number:
  US08/676,140
• Time in Patent Office: 1,364 Days
• Field of Search: 435/4, 435/6, 435/7.1, 435/183, 436/501, 436/94, 536/24.3, 536/23.1, 536/24.5, 935/77, 935/78
• US Class Current: 436/94
• 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 2219/0043 for direct application of reagents, e.g. through openings in a shutter
• CPC Class Codes: B01J 2219/00527 Sheets
• CPC Class Codes: B01J 2219/0059 Sequential 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 2219/00612 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 2219/00722 Nucleotides
• 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: C12N 15/1048 SELEX
• 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: C40B 40/06 Libraries containing nucleotides or polynucleotides, or derivatives thereof
• CPC Class Codes: C40B 50/06 Biochemical methods, e.g. using enzymes or whole viable microorganisms
• CPC Class Codes: C40B 60/14 for creating libraries
• CPC Class Codes: Y10T 436/143333 Saccharide [e.g., DNA, etc.]

Abstract

A method of identifying one or a combination of ligands, e.g. oligonucleotides or analogues, that interact specifically with a target, e.g. a DNA or an RNA molecule having a secondary or tertiary structure. One ligand may be pre-reacted to open up the target for interaction with other ligands forming an array on a solid surface.

Claims

1. A method of investigating the intramolecular structure of a polynucleotide, which method comprises:

providing a polynucleotide, said polynucleotide having a known sequence and having an intramolecular structure, and providing a plurality of different oligonucleotides in the form of an array on a solid surface, all of said different oligonucleotides being fully complementary to different segments of the polynucleotide;

applying the polynucleotide in solution to the array of oligonucleotides under hybridisation conditions which preserve the intramolecular structure of the polynucleotide; and

observing quantitative differences between interactions of the polynucleotide with different oligonucleotides of the array, said differences between interactions being indicative of the intramolecular structure of the polynucleotide.

2. The method according to claim 1, wherein the polynucleotide is an RNA.

3. The method according to claim 1, wherein the polynucleotide is a DNA.

4. The method according to claim 1, which further comprises an additional step of designing an antisense oligonucleotide based upon said quantitative differences.

5. A method of identifying oligonucleotides which specifically bind to a polynucleotide, which method comprises:

a) binding at least one oligonucleotide to the polynucleotide to form a polynucleotide-oligonucleotide complex,

b) applying the polynucleotide-oligonucleotide complex to an array comprising a plurality of different oligonucleotides immobilized on a solid surface under hybridisation conditions which allow for interaction between the array of immobilized oligonucleotides and the polynucleotide-oligonucleotide complex, and

c) identifying an immobilized oligonucleotide on the array which specifically binds to the polynucleotide-oligonucleotide complex.

6. The method as claimed in claim 5, wherein one oligonucleotide is bound to the polynucleotide in step a) to form the polynucleotide-oligonucleotide complex, and then steps b) and c) are conducted.

7. The method as claimed in claim 5, wherein two or more oligonucleotides are bound to the polynucleotide in step a) to form the polynucleotide-oligonucleotide complex, and then steps b) and c) are conducted.

8. The method as claimed in claim 5, wherein the at least one oligonucleotide to be bound to the polynucleotide to form the polynucleotide-oligonucleotide complex in step a) is chosen by mixing the polynucleotide with a library of oligonucleotides and choosing from the library at least one oligonucleotide that binds to the polynucleotide.

9. The method as claimed in claim 5, wherein the polynucleotide is an RNA.

10. The method as claimed in claim 5, wherein the polynucleotide is a DNA.

11. The method as claimed in claim 5, wherein the polynucleotide has a secondary or tertiary structure, and wherein the polynucleotide-oligonucleotide complex is caused to interact with the array of oligonucleotides under hybridisation conditions, whereby the secondary or tertiary structure of the polynucleotide is retained during hybridisation.

12. The method as claimed in claim 5, wherein the polynucleotide has a known sequence.

13. The method as claimed in claim 5, which further includes a step of designing an antisense oligonucleotide based upon the identification of the immobilized oligonucleotide.