Very large scale immobilized polymer synthesis

• US 6,566,495 B1
• Filed: 12/17/1999
• Issued: 05/20/2003
• Est. Priority Date: 06/07/1989
• Status: Expired due to Fees

• Current Assignee: Affymetrix, Inc. (Thermo Fisher Scientific Incorporated)
• Original Assignee: Affymetrix, Inc. (Thermo Fisher Scientific Incorporated)
• Inventors: Pirrung, Michael C., Fodor, Stephen P. A., Stryer, Lubert, Read, J. Leighton
• Primary Examiner(s): ZITOMER, STEPHANIE W
• Application Number:
  US09/465,126
• Time in Patent Office: 1,250 Days
• Field of Search: 435/6, 435/7.1, 536/25.3, 536/25.31, 536/24.3, 530/300, 530/334, 530/335, 530/336, 530/339, 530/337, 530/350, 530/387.1
• US Class Current: 506/15
• 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 2219/00315 Microtiter plates
• CPC Class Codes: B01J 2219/00432 Photolithographic masks
• CPC Class Codes: B01J 2219/00434 Liquid crystal masks
• CPC Class Codes: B01J 2219/00436 Maskless processes
• CPC Class Codes: B01J 2219/00459 Beads
• CPC Class Codes: B01J 2219/00468 by manipulation of individual beads
• CPC Class Codes: B01J 2219/00475 Sheets
• CPC Class Codes: B01J 2219/005 Beads
• CPC Class Codes: B01J 2219/00527 Sheets
• CPC Class Codes: B01J 2219/00529 DNA chips
• CPC Class Codes: B01J 2219/00531 essentially square
• CPC Class Codes: B01J 2219/00585 Parallel processes
• 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 2219/00608 DNA chips
• CPC Class Codes: B01J 2219/00612 the surface being inorganic
• CPC Class Codes: B01J 2219/00617 by chemical means
• CPC Class Codes: B01J 2219/00626 Covalent
• CPC Class Codes: B01J 2219/00637 : by coating it with another layer
• CPC Class Codes: B01J 2219/00648 : by the use of solid beads
• CPC Class Codes: B01J 2219/00659 : Two-dimensional arrays
• CPC Class Codes: B01J 2219/00689 : using computers
• CPC Class Codes: B01J 2219/00695 : Synthesis control routines, e.g. using computer programs
• CPC Class Codes: B01J 2219/00711 : Light-directed synthesis
• CPC Class Codes: B01J 2219/00722 : Nucleotides
• CPC Class Codes: B01J 2219/00725 : Peptides
• CPC Class Codes: B82Y 10/00 : Nanotechnology for information processing, storage or transmission, e.g. quantum computing or single electron logic
• CPC Class Codes: B82Y 30/00 : Nanotechnology for materials or surface science, e.g. nanocomposites
• CPC Class Codes: C07B 2200/11 : Compounds covalently bound to a solid support
• CPC Class Codes: C07C 229/14 : to carbon atoms of carbon skeletons containing rings
• CPC Class Codes: C07C 229/16 : to carbon atoms of hydrocarbon radicals substituted by amino or carboxyl groups, e.g. ethylenediamine-tetra-acetic acid, iminodiacetic acids
• CPC Class Codes: C07D 263/44 : Two oxygen atoms
• CPC Class Codes: C07D 317/62 : with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to atoms of the carbocyclic ring
• CPC Class Codes: C07H 19/073 : with 2-deoxyribosyl as the saccharide radical
• CPC Class Codes: C07H 19/173 : with 2-deoxyribosyl as the saccharide radical
• CPC Class Codes: C07H 21/04 : with deoxyribosyl as saccharide radical
• CPC Class Codes: C07K 1/042 : characterised by the nature of the carrier
• CPC Class Codes: C07K 1/045 : using devices to improve synthesis, e.g. reactors, special vessels
• CPC Class Codes: C07K 1/047 : Simultaneous synthesis of different peptide species; Peptide libraries
• CPC Class Codes: C07K 1/062 : for alpha- or omega-carboxy functions
• CPC Class Codes: C07K 17/06 : attached to the carrier via a bridging agent
• CPC Class Codes: C07K 17/14 : Peptides being immobilised on, or in, an inorganic carrier
• CPC Class Codes: C07K 7/06 : having 5 to 11 amino acids
• CPC Class Codes: C12Q 1/6809 : Methods for determination or identification of nucleic acids involving differential detection
• CPC Class Codes: C12Q 1/6816 : characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6837 : using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 1/6874 : involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C40B 40/06 : Libraries containing nucleotides or polynucleotides, or derivatives thereof
• CPC Class Codes: C40B 40/10 : Libraries containing peptides or polypeptides, or derivatives thereof
• CPC Class Codes: C40B 60/14 : for creating libraries
• CPC Class Codes: C40B 80/00 : Linkers or spacers specially adapted for combinatorial chemistry or libraries, e.g. traceless linkers or safety-catch linkers
• CPC Class Codes: G01N 15/1433 : using image recognition
• CPC Class Codes: G01N 21/253 : for batch operation, i.e. multisample apparatus analytical automats G01N35/00
• CPC Class Codes: G01N 21/6428 : Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" in vivo A61B5/00; immunoassay G01N33/53
• CPC Class Codes: G01N 21/6452 : Individual samples arranged in a regular 2D-array, e.g. multiwell plates
• CPC Class Codes: G01N 21/6458 : Fluorescence microscopy fluorescence microscopes per se G02B21/0076 and G02B21/16
• CPC Class Codes: G03F 7/00 : Photomechanical, e.g. photolithographic, production of textured or patterned surfaces, e.g. printing surfaces; Materials therefor, e.g. comprising photoresists; Apparatus specially adapted therefor using photoresist structures for special production processes, see the relevant places, e.g. B44C, H01L, e.g. H01L21/00, H05K
• CPC Class Codes: G03F 7/0045 : with organic non-macromolecular light-sensitive compounds not otherwise provided for, e.g. dissolution inhibitors
• CPC Class Codes: G03F 7/265 : Selective reaction with inorganic or organometallic reagents after image-wise exposure, e.g. silylation
• CPC Class Codes: G03F 7/38 : Treatment before imagewise removal, e.g. prebaking G03F7/265 takes precedence
• CPC Class Codes: G11C 13/0014 : comprising cells based on organic memory material
• CPC Class Codes: G11C 13/0019 : comprising bio-molecules
• CPC Class Codes: Y02P 20/55 : Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
• CPC Class Codes: Y10S 435/969 : Multiple layering of reactants
• CPC Class Codes: Y10S 435/973 : Simultaneous determination of more than one analyte
• CPC Class Codes: Y10S 436/807 : Apparatus included in process claim, e.g. physical support structures
• CPC Class Codes: Y10S 436/809 : Multifield plates or multicontainer arrays

Abstract

A synthetic strategy for the creation of large scale chemical diversity. Solid-phase chemistry, photolabile protecting groups, and photolithography are used to achieve light-directed spatially-addressable parallel chemical synthesis. Binary masking techniques are utilized in one embodiment. A reactor system, photoremovable protective groups, and improved data collection and handling techniques are also disclosed. A technique for screening linker molecules is also provided.

Claims

1. A method of electrically directed synthesis of a plurality of biological polymers on a surface of a substrate, comprising:

a) providing a substrate having a surface with a plurality of localized areas, wherein each localized area comprises a molecule having a protected functional group is attached thereto;

b) electrically activating a first set of one or more localized areas by applying an electric field or current to the first set of localized areas, thereby generating one or more activated groups in the first set of localized areas;

c) selectively binding a first monomer having a protected functional group to an activated functional group in the first set of localized areas by reacting the first monomer with the molecule attached to the first set of localized areas;

d) electrically activating a second set of one or more localized areas, said second set of localized areas containing the same, different or a combination of the same and different localized areas as in the first set of localized areas, by applying an electric field or current to the second set of localized areas, thereby generating one or more activated functional groups in the second set of localized areas;

e) selectively binding a second monomer having a protected functional group to an activated functional group in the second set of localized areas by reacting the second monomer with the molecule attached to the second set of localized areas; and

f) repeating (b)-(e) to synthesize a plurality of biological polymers on the surface of the substrate.

2. The method of claim 1, wherein at least one of the plurality of biological polymers is an oligonucleotide.

3. The method of claim 1, wherein at least one of the plurality of biological polymers is a nucleic acid.

4. The method of claim 2, wherein the oligonucleotide is a deoxyribonucleic acid (DNA).

5. The method of claim 1, further comprising exposing the substrate bearing the plurality of biological polymers to a receptor, and determining which biological polymers bind to the receptor.

6. The method of claim 5, wherein the receptor is a nucleic acid.

7. The method of claim 1, wherein at least one of the plurality of biological polymers is a peptide.

26. The method of claim 1, wherein at least one of the plurality of biological polymers is a polynucleotide.

27. The method of claim 1, wherein the first monomer is a nucleotide.

28. The method of claim 1, wherein the second monomer is a nucleotide.

29. The method of claim 1, wherein the first monomer and the second monomer are each a nucleotide.

30. The method of claim 1, wherein the first monomer is an amino acid.

31. The method of claim 1, wherein the second monomer is an amino acid.

32. The method of claim 1, wherein the first monomer and the second monomer are each an amino acid.

8. A method of electrically directed synthesis of a plurality of biological polymers at a plurality of localized areas on a surface of a substrate, comprising:

a) providing a substrate having a surface comprising a plurality of localized areas, wherein each localized area comprises a molecule having a protected functional group attached thereto;

b) selectively activating a first set of one or more localized areas by applying an electric current or field using a microelectrode to the first set of localized areas, thereby generating one or more activated functional groups in the first set of localized areas;

c) binding a first monomer having a protected functional group to an activated functional group in the first set of localized areas by reacting the first monomer with the molecule attached to the first set of localized areas;

d) selectively activating a second set of one or more localized areas, said second set of localized areas containing the same, different or a combination of the same and different localized areas as in the first set of localized areas by applying an electric current or field using a microelectrode to the second set of localized areas, thereby generating one or more activated functional groups in the second set of localized areas;

e) binding a second monomer having a protected functional group to an activated functional group in the second set of localized areas by reacting the second monomer with the molecule attached to the second set of localized areas; and

f) repeating (b)-(e) to synthesize a plurality of biological polymers at a plurality of localized areas on the surface of the substrate.

9. The method of claim 8, wherein at least one of the plurality of biological polymers is an oligonucleotide.

10. The method of claim 9, wherein the oligonucleotide comprises a deoxyribonucleic acid (DNA).

11. The method of claim 9, wherein at least one of the plurality of biological polymers is a nucleic acid.

33. The method of claim 8, wherein at least one of the plurality of biological polymers is a polynucleotide.

34. The method of claim 8, wherein at least one of the plurality of biological polymers is a peptide.

35. The method of claim 8, wherein the first monomer is a nucleotide.

36. The method of claim 8, wherein the second monomer is a nucleotide.

37. The method of claim 8, wherein the first monomer and the second monomer are each a nucleotide.

38. The method of claim 8, wherein the first monomer is an amino acid.

39. The method of claim 8, wherein the second monomer is an amino acid.

40. The method of claim 8, wherein the first monomer and the second monomer are each independently an amino acid.

12. A method for coupling a biological monomer to a localized area of a solid support, comprising:

(a) applying an electric current or field to the localized area of the solid support, wherein the localized area comprises one or more molecules each having a protected functional group attached thereto, thereby generating one or more activated molecules in the localized area;

(b) adding a biological monomer having a protected functional group to the localized area of the solid support; and

(c) attaching the biological monomer to an activated molecule in the localized area.

13. A method for forming a biological polymer on a localized area of a solid support, comprising the steps of:

a) applying an electric current or field to the localized area of the solid support, wherein the localized area comprises one or more molecules each having a protected functional group attached thereto, thereby generating one or more activated molecules in the localized area;

b) adding a monomer having a protected functional group to the localized area of the solid support;

c) attaching the monomer to an activated molecule in the localized area, thereby generating an attached monomer;

d) applying an electric current or field to the localized area of the solid support;

e) adding an additional monomer having a protected functional group to the localized area;

f) attaching the additional monomer to the attached monomer, wherein the additional monomer is the same or different from the attached monomer; and

g) repeating steps d)-f), thereby forming a biological polymer bound to the localized area of the solid support.

14. The method of claim 13, wherein the biological polymer is a nucleic acid.

15. The method of claim 13, wherein the biological polymer is a peptide.

16. The method of claim 13, wherein the biological polymer is a protein.

17. The method of claim 13, further comprising repeating said steps a)-f) to form a biological polymer bound to said solid support at a second localized area.

18. The method of claim 13, wherein the biological polymer is a heteropolymer, said heteropolymer comprising a drug bound to a nucleic acid, polysaccharide, phospholipid, or peptide.

41. The method of claim 13, wherein the biological polymer is an oligonucleotide.

42. The method of claim 13, wherein the biological polymer is a polynucleotide.

43. The method of claim 13, wherein the monomer and the additional monomer are each a nucleotide.

44. The method of claim 13, wherein the monomer and the additional monomer are each an amino acid.

19. A method for creating an array of biological molecules bound to a solid support, comprising:

(a) applying an electric current or field to localized areas of the solid support, wherein each localized area comprises one or more biological molecules each having a protected functional group attached thereto, thereby generating one or more activated biological molecules in the localized area;

(b) adding additional biological molecules to the localized areas of the solid support; and

(c) attaching the additional biological molecules to the activated biological molecules in the localized areas, thereby creating an array of biological molecules bound to the solid support.

20. The method of claim 19, wherein the biological molecules comprise an oligonucleotide.

21. The method of claim 19, wherein the biological molecules comprise a polynucleotide.

22. The method of claim 19, wherein the biological molecules comprise a nucleic acid.

23. The method of claim 19, wherein the biological molecules comprise a peptide.

24. The method of claim 19, wherein the biological molecules comprise a protein.

25. The method of claim 19, wherein the biological molecules comprise a heteropolymer, said heteropolymer comprising a drug bound to a nucleic acid, polysaccharide, phospholipid, or peptide.