Combinatorial strategies for polymer synthesis

• US 5,677,195 A
• Filed: 11/20/1992
• Issued: 10/14/1997
• Est. Priority Date: 11/22/1991
• Status: Expired due to Term

• Current Assignee: Affymetrix, Inc. (Thermo Fisher Scientific Incorporated)
• Original Assignee: Affymax Incorporated
• Inventors: Modlin, Douglas N., Winkler, James L., Ross, Debra A., Aldwin, Lois, Fodor, Stephen P. A., Buchko, Christopher J.
• Primary Examiner(s): Spiegel, Carol A.
• Application Number:
  US07/980,523
• Time in Patent Office: 1,789 Days
• Field of Search: 436/89, 436/116, 436/518, 436/527-528, 436/807, 436/809, 530/333-337, 435/6, 435/7.1, 435/7.4, 435/7.92, 435/961, 435/968-970, 435/973, 435/975, 422/131, 422/134, 422/149, 935/88, 536/25.3, 536/25.31, 536/25.32
• US Class Current: 436/518
• 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 2219/00364 Pipettes
• CPC Class Codes: B01J 2219/00367 capillary
• CPC Class Codes: B01J 2219/00369 in multiple or parallel arrangements
• CPC Class Codes: B01J 2219/00378 Piezoelectric or ink jet dispensers
• CPC Class Codes: B01J 2219/00389 Feeding through valves
• CPC Class Codes: B01J 2219/00416 Vacuum
• CPC Class Codes: B01J 2219/0043 for direct application of reagents, e.g. through openings in a shutter
• CPC Class Codes: B01J 2219/00432 Photolithographic masks
• 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 2219/00536 in the shape of disks
• 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 2219/0061 : The surface being organic
• CPC Class Codes: B01J 2219/00612 : the surface being inorganic
• CPC Class Codes: B01J 2219/00617 : by chemical means
• CPC Class Codes: B01J 2219/00621 : by physical means, e.g. trenches, raised areas
• 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 2219/00675 : In-situ synthesis on the substrate
• CPC Class Codes: B01J 2219/00686 : Automatic
• CPC Class Codes: B01J 2219/00689 : using computers
• CPC Class Codes: B01J 2219/00702 : Processes involving means for analysing and characterising the products
• 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 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: 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 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 5/1008 : the side chain containing 0 or 1 carbon atoms, i.e. Gly, Ala
• CPC Class Codes: C07K 5/1016 : and aromatic or cycloaliphatic
• 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 50/14 : Solid phase synthesis, i.e. wherein one or more library building blocks are bound to a solid support during library creation; Particular methods of cleavage from the solid support
• CPC Class Codes: C40B 60/14 : for creating libraries
• CPC Class Codes: G01N 35/1072 : with provision for selective pipetting of individual channels
• CPC Class Codes: Y02P 20/55 : Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
• CPC Class Codes: Y10S 435/97 : Test strip or test slide
• CPC Class Codes: Y10S 435/973 : Simultaneous determination of more than one analyte
• CPC Class Codes: Y10T 436/143333 : Saccharide [e.g., DNA, etc.]
• CPC Class Codes: Y10T 436/2575 : Volumetric liquid transfer

Abstract

A method and device for forming large arrays of polymers on a substrate (401). According to a preferred aspect of the invention, the substrate is contacted by a channel block (407) having channels (409) therein. Selected reagents are delivered through the channels, the substrate is rotated by a rotating stage (403), and the process is repeated to form arrays of polymers on the substrate. The method may be combined with light-directed methodolgies.

Claims

1. A method of forming oligonucleotides or peptides having diverse sequences on a single substrate, said substrate comprising a surface with a plurality of selected regions, said method comprising the steps of:

a) forming a plurality of flow channels adjacent said surface, each of said flow channels at least partially comprising a first wall thereof defined by a first portion of said selected regions and second and third walls which form substantially fluid-tight seals with said surface, and a flow inlet and a flow outlet; and

said first portion of said selected regions comprising oligonucleotides or peptides with a first known sequence of nucleic acids or amino acids different from oligonucleotides or peptides with a second known sequence of nucleic acids or amino acids bound to at least one other of said selected regions;

b) flowing selected nucleic acids or amino acids through said flow channels and binding covalently said selected nucleic acids or amino acids to said oligonucleotides or peptides at said first portion of said selected regions; and

c) repeating steps a) and b) wherein additional flow channels are formed along subsequent portions of said selected regions by altering the relative positions of the substrate surface and the second and third walls until said oligonucleotides or peptides having diverse sequences are formed.

2. The method as recited in claim 1 wherein said step of forming a plurality of flow channels comprises placing a channel block adjacent said surface, said channel block having a plurality of grooves therein, the walls of said grooves and said surface at least partially defining said flow channels.

3. The method as recited in claim 1 wherein the step of flowing said selected nucleic acids or amino acids in said flow channels comprises the steps of:

removing a first protective group from an active site in at least a first flow channel;

flowing a first selected nucleic acid or amino acid through said first flow channel, said first selected nucleic acid or amino acid comprising a second protective group thereon, and binding covalently said first selected nucleic acid or amino acid to said active site in said first flow channel;

removing a third protective group from said active site contacted by at least a second flow channel, wherein at least a portion of said second flow channel overlaps a portion of said substrate contacted by said first flow channel; and

flowing a second selected nucleic acid or amino acid through said second flow channel, and binding covalently said second selected nucleic acid or amino acid to said active site in said second flow channel.

4. The method as recited in claim 3 wherein the steps of removing and flowing further comprise the steps of:

contacting a channel block containing flow channels therein with said surface in a first orientation and flowing the first selected nucleic acid or amino acid through at least one flow channel in said channel block;

rotating said channel block relative to said substrate; and

contacting said channel block with said surface in a second orientation and flowing a second selected nucleic acid or amino acid through at least one flow channel in said channel block.

5. The method as recited in claim 1 further comprising a step of screening said oligonucleotides or peptides having diverse sequences against a receptor to determine the binding affinity of said oligonucleotides or peptides having diverse sequences with respect to said receptor.

6. The method as recited in claim 1 wherein at least 10 different oligonucleotides or peptides having diverse sequences are formed on said surface.

7. The method as recited in claim 1 wherein at least 10,000 different oligonucleotides or peptides having diverse sequences are formed on said surface.

8. The method as recited in claim 1 wherein at least 100,000 different oligonucleotides or peptides having diverse sequences are formed on said surface.

9. The method as recited in claim 1 wherein said selected regions each have an area of less than about 10,000 microns².

10. The method as recited in claim 1 wherein each step of flowing selected nucleic acids or amino acids through said flow channels comprises:

placing a pipet in fluid communication with said flow channels; and

injecting said selected nucleic acids or amino acids through said flow channels.

11. The method as recited in claim 10 wherein said step of placing a pipet in fluid communication with said flow channels comprises placing said pipet in contact with an orifice on a side of said substrate opposite said selected regions.

12. The method as recited in claim 10 wherein said step of placing a pipet in fluid communication with said flow channel is a step of placing a plurality of pipets in communication with a plurality of said flow channels and flowing different reagents through at least two of said flow channels, said reagents selected from the group consisting of nucleic acids, amino acids, wash solutions and protective group removal agents.

13. The method as recited in claim 1, wherein said substrate comprises photoremovable groups and said method is preceded by the step of irradiating selected portions of said substrate with light whereby said photoremovable groups are removed from said substrate to form active sites thereon.

14. The method as recited in claim 13, wherein said selected irraditation portions are in the form of stripes, and wherein said step of forming said flow channels comprises forming said flow channels along a path of said stripes, and said selected nucleic acids or amino acids are flowed in at least a portion of said flow channels.

15. A method of forming oligonucleotides or peptides having diverse sequences on a single substrate, said substrate comprising a surface with a plurality of selected regions, said method comprising the steps of:

a) forming a plurality of valves and flow channels adjacent said surface whereby fluid may be directed to said selected regions on said surface, each of said flow channels at least partially comprising a first wall thereof defined by a first portion of said selected regions and second and third walls which form substantially fluid-tight seals with said surface, and a flow inlet and a flow outlet; and

said first portion of said selected regions comprising oligonucleotides or peptides with a known sequence of nucleic acids or amino acids different from oligonucleotides or peptides with a second known sequence of nucleic acids or amino acids bound to at least one other of said selected regions;

b) selectively operating said valves and flowing said selected nucleic acids or amino acids through said flow channels and binding covalently said selected nucleic acids or amino acids to said oligonucleotides or peptides at said first portion of said selected regions; and

c) repeating steps a) and b) wherein additional flow channels are formed along subsequent portions of said selected regions by altering the relative positions of the substrate surface and the second and third walls until said oligonucleotides or peptides having diverse sequences are formed.

16. A method of forming a plurality of oligonucleotide sequences on a surface of a single substrate comprising the steps of:

a) contacting said substrate with a channel block in a first orientation, said channel block comprising a substantially planar member with a plurality of recessed regions and raised walls on a surface thereof, said recessed regions forming channels, said raised walls effective to produce substantially fluid-tight seals between said raised walls and said substrate upon said contacting to define thereby a first plurality of flow channels between said channel block and said substrate, said first plurality of flow channels dividing said substrate surface into at least a first portion and a second portion;

b) flowing at least a first nucleic acid through at least one of said first plurality of flow channels, and covalently coupling said first nucleic acid to said first portion of said substrate surface, said first nucleic acid comprising a reactive group protected by a protecting group;

c) flowing at least a second nucleic acid through at least one of said first plurality of flow channels, and covalently coupling said second nucleic acid to said second portion of said substrate surface, said second nucleic acid comprising a reactive group protected by a protecting group;

d) translating said channel block relative to said substrate and contacting said substrate with said channel block in a second orientation to produce substantially fluid-tight seals between said raised walls and said substrate upon said contacting to define thereby a second plurality of flow channels between said channel block and said substrate, said second flow channels dividing said substrate surface into at least a third portion and a fourth portion, said third and fourth portions each having at least one intersection with each of said first and second portions of said substrate surface;

e) removing said protective group from at least a portion of said first or second nucleic acids to form a first set of deprotected first or second nucleic acids and flowing a third nucleic acid through at least one of said second plurality of flow channels, covalently coupling said third nucleic acid to said third portion of said substrate surface and covalently coupling said third nucleic acid to at least a portion of said first set of deprotected first or second nucleic acids through a oligonucleotide bond therewith where said first and second portions of said substrate surface intersect with said third portion of said substrate surface to form first and second oligonucleotide sequences; and

f) removing said protective group from at least a portion of said first and second nucleic acids to form a second set of deprotected first or second nucleic acids and flowing a fourth nucleic acid through at least one of said second plurality of flow channels, covalently coupling said fourth nucleic acid to said fourth portion of said substrate surface and covalently coupling said fourth nucleic acid to at least a portion of said second set of deprotected first or second nucleic acids through a oligonucleotide bond therewith where said fourth portion of said substrate surface intersects with said first and second portions of said substrate surface to form third and fourth oligonucleotide sequences.

17. The method as recited in claim 16 wherein at least 10 different oligonucleotide sequences are formed on said surface.

18. The method as recited in claim 16 wherein at least 100 different oligonucleotide sequences are formed on said surface.

19. The method as recited in claim 16 wherein at least 1,000 different oligonucleotide sequences are formed on said surface.

20. The method as recited in claim 16 wherein at least 10,000 different oligonucleotide sequences are formed on said surface.

21. The method as recited in claim 16 wherein at least 100,000 different oligonucleotide sequences are formed on said surface.

22. The method as recited in claim 16 wherein each different oligonucleotide sequence is in a region having an area of less than about 1 cm².

23. The method as recited in claim 16 wherein each different oligonucleotide sequence is in a region having an area of less than about 1 mm².

24. The method as recited in claim 16 wherein each different oligonucleotide sequence is in a region having an area of less than about 10,000 microns².

25. The method as recited in claim 16 wherein the surface of said substrate comprises active groups capable of forming a covalent bond with nucleic acids, said active groups protected with photoremovable groups, and wherein the step of forming said first plurality of flow channels is preceded by a step of irradiating portions of said substrate with a light source shone through a mask whereby said photoremovable groups are removed from at least some of the active groups on said substrate surface in order for at least said first nucleic acid to bind covalently thereto.

26. The method as recited in claim 25 wherein said irradiated portions are in the form of stripes, and wherein said step of forming said first plurality of flow channels comprises forming said first flow channels along a path of said stripes, and wherein different reagents are placed in at least a portion of said first flow channels, said reagents selected from the group consisting of nucleic acids, wash solutions and protective group removal agents.

27. The method as recited in claim 16 wherein each said step of flowing said first, second, third or fourth nucleic acid through its respective said first or second plurality of flow channels comprises:

placing a pipet in fluid communication with at least one of said first or second plurality of flow channels; and

injecting said first, second, third or fourth nucleic acid from said pipet through its respective said at least one of said first or second plurality of flow channels.

28. The method as recited in claim 27 wherein said step of placing the pipet in fluid communication with said at least one of said first or second plurality of flow channels comprises placing said pipet in contact with an orifice on a side of said substrate.

29. The method as recited in claim 27 wherein said step of placing the pipet in fluid communication with said at least one of said first or second plurality of flow channels is a step of placing a plurality of pipets in fluid communication with a plurality of said first and second plurality of flow channels and flowing reagents selected from the group consisting of said first, second, third and fourth nucleic acids, wash solutions and protective group removal agents through at least two of said first or second plurality of flow channels.

30. A method of forming oligonucleotides or peptides having diverse sequences on a single substrate, said substrate comprising a surface with a plurality of selected regions, said method comprising the steps of:

a) on said surface providing a photoresist;

b) selectively removing said photoresist to expose a portion of said plurality of selected regions;

c) removing protecting groups on said surface by exposing said surface to a protecting group removal agent;

d) contacting and covalently binding selected nucleic acids or amino acids with said surface at said portion of said plurality of selected regions; and

e) repeating steps a), b), c) and d) to form oligonucleotides or peptides having diverse sequences at the selected regions.

31. The method of claim 30 wherein said removing step comprises the steps of:

a) exposing said portion of said plurality of selected regions to radiation to make said photoresist located therein capable of dissolution; and

b) etching said portion of said plurality of selected regions to remove said photoresist.

32. The method as recited in claim 30, wherein diverse sequences of oligonucleotides are formed on said substrate.

33. The method as recited in claim 32, wherein at least 10 different oligonucleotide sequences are formed on said surface.

34. The method as recited in claim 32, wherein at least 100 different oligonucleotide sequences are formed on said surface.

35. The method as recited in claim 32, wherein at least 1000 different oligonucleotide sequences are formed on said surface.

37. The method as recited in claim 32, wherein at least 10,000 different oligonucleotide sequences are formed on said surface.

38. The method as recited in claim 32, wherein at least 100,000 different oligonucleotide sequences are formed on said surface.

39. The method as recited in claim 32, wherein each different oligonucleotide sequence is in a region having an area of less than about 1 cm².

40. The method as recited in claim 32, wherein each different oligonucleotide sequence is in a region having an area of less than about 1 mm².

41. The method as recited in claim 32, wherein each different oligonucleotide sequence is in a region having an area of less than about 10,000 μ

m².

36. A method of forming oligonucleotides or peptides having diverse sequences on a single substrate, said substrate comprising a surface with a plurality of selected regions with acid-labile protecting groups protecting activated sites capable of covalently binding nucleic acids or amino acids, said method comprising the steps of:

a) providing on said surface a photoresist wherein said photoresist is an acid generating polymer;

b) selectively irradiating a portion of said plurality of selected regions whereby acid is generated thereby removing said acid-labile protecting groups to provide said activated sites in said selectively irradiated portion;

c) removing said photoresist in said selectively irradiated portion of said plurality of selected regions;

d) contacting and covalently binding selected nucleic acids or amino acids to said activated sites in said selectively irradiated portion;

e) sequentially contacting and covalently bonding one or more selected nucleic acids or amino acids to the substrate of step d) to form said oligonucleotides or peptides having diverse sequences at the selectively irradiated portions.

42. The method as recited in claim 36 wherein diverse sequences of oligonucleotides are formed on said substrate.

43. The method as recited in claim 36, wherein at least 10 different oligonucleotide sequences are formed on said surface.

44. The method as recited in claim 36, wherein at least 100 different oligonucleotide sequences are formed on said surface.

45. The method as recited in claim 36, wherein at least 1000 different oligonucleotide sequences are formed on said surface.

46. The method as recited in claim 36, wherein at least 10,000 different oligonucleotide sequences are formed on said surface.

47. The method as recited in claim 36, wherein at least 100,000 different oligonucleotide sequences are formed on said surface.

48. The method as recited in claim 36, wherein each different oligonucleotide sequence is in a region having an area of less than about 1 cm².

49. The method as recited in claim 36, wherein each different oligonucleotide sequence is in a region having an area of less than about 1 mm².

50. The method as recited in claim 36, wherein each different oligonucleotide sequence is in a region having an area of less than about 10,000 μ

m².