Large scale photolithographic solid phase synthesis of polypeptides and receptor binding screening thereof

• US 5,143,854 A
• Filed: 03/07/1990
• Issued: 09/01/1992
• Est. Priority Date: 06/07/1989
• Status: Expired due to Term

• Current Assignee: Affymetrix, Inc. (Thermo Fisher Scientific Incorporated)
• Original Assignee: Affymax Incorporated
• Inventors: Pirrung, Michael C., Fodor, Stephen P. A., Stryer, Lubert, Read, J. Leighton
• Primary Examiner(s): Kepplinger, Esther L.
• Assistant Examiner(s): Spiegel, Carol A.
• Application Number:
  US07/492,462
• Time in Patent Office: 909 Days
• Field of Search: 436/518, 436/527, 436/807, 436/528, 530/333-335, 530/336-337, 435/7.92, 435/7.94, 435/7.95, 435/961, 435/968, 435/973
• 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 2219/00315 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 2219/0061 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 2219/00619 : using hydrophilic or hydrophobic regions
• 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 2219/00639 : the compounds being trapped in or bound to a porous medium
• 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: B01L 7/52 : with provision for submitting samples to a predetermined sequence of different temperatures, e.g. for treating nucleic acid samples amplification or hybridisation processes per se C12Q1/68; controlling sequential reactions for synthesis B01J19/0046
• 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 19/04 : Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
• CPC Class Codes: C07H 19/10 : with the saccharide radical esterified by phosphoric or polyphosphoric acids
• 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 1/04 : on carriers C07K1/003, C07K1/006 take precedence
• 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: C08G 69/00 : Macromolecular compounds obtained by reactions forming a carboxylic amide link in the main chain of the macromolecule products obtained from isocyanates or isothiocyanates C08G18/00; polyamide-imides C08G73/14
• 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: 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 7/26 : Processing photosensitive materials; Apparatus therefor G03F7/12 - G03F7/24 take precedence
• 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: H01L 25/50 : Multistep manufacturing processes of assemblies consisting of devices, each device being of a type provided for in group H01L27/00 or H01L29/00 H01L21/50 takes precedence
• CPC Class Codes: H01L 2924/00 : Indexing scheme for arrangements or methods for connecting or disconnecting semiconductor or solid-state bodies as covered by H01L24/00
• CPC Class Codes: H01L 2924/0002 : Not covered by any one of groups H01L24/00, H01L24/00 and H01L2224/00
• CPC Class Codes: Y02P 20/55 : Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
• CPC Class Codes: Y10S 435/961 : including a step of forming, releasing, or exposing the antigen or forming the hapten-immunogenic carrier complex or the antigen per se
• CPC Class Codes: Y10S 435/968 : High energy substrates, e.g. fluorescent, chemiluminescent, radioactive
• 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
• CPC Class Codes: Y10T 428/31971 : Of carbohydrate
• CPC Class Codes: Y10T 436/2575 : Volumetric liquid transfer

Abstract

Polypeptide arrays can be synthesized on a substrate by attaching photoremovable groups to the surface of a substrate, exposing selected regions of the substrate to light to activate those regions, attaching an amino acid monomer with a photoremovable group to the activated regions, and repeating the steps of activation and attachment until polypeptides of the desired length and sequences are synthesized. The resulting array can be used to determine which peptides on the array can bind to a receptor.

Claims

1. A method of identifying at least one polypeptide for binding with a receptor comprising the sequential steps of:

a) generating a pattern of light and dark areas by selectively irradiating at least a first area of a surface of a substrate, said surface comprising polypeptides immobilized on said surface, said polypeptides having a photoremovable protective group, without irradiating at least a second area of said surface, to remove said protective group from said polypeptides in said first area;

b) simultaneously contacting said first area and said second area of said surface with a first amino acid to couple said first amino acid to said polypeptides in said first area, and not in said second area, said first amino acid having said photoremovable protective group;

c) generating another generating another pattern of light and dark areas by selectively irradiating with light at least a part of said first area of said surface and at least a part of said second area to remove said protective group in said at least a part of said first area and said at least a part of said second area;

d) simultaneously contacting said first area and said second area of said surface with a second amino acid to couple said second amino acid to said polypeptides in said at least a part of said first area and said at least a part of said second area;

e) performing additional irradiating and amino acid contacting and coupling steps so that a matrix array of at least 100 different polypeptides is formed on said surface, each different polypeptide synthesized in an area of less than 0.1 cm², whereby said different polypeptides have amino acid sequences and locations on said surface defined by the patterns of light and dark areas formed during the irradiating steps and the amino acids coupled in said contacting steps; and

f) contacting said surface with a receptor and identifying which polypeptides on said surface specifically bind to said receptor.

2. The method as recited in claim 1 wherein said amino acids are selected from the group consisting of L-amino acids, D-amino acids, and synthetic amino acids.

3. The method as recited in claim 1 wherein said receptor is an antibody.

4. The method as recited in claim 1 wherein said substrate is selected from the group consisting of Langmuir Blodgett film, glass, germanium, silicon, (poly)tetrafluorethylene, polystyrene, gallium arsenide, gallium phosphide, silicon oxide, silicon nitride, and combinations thereof.

5. The method as recited in claim 1 wherein said protective group is selected from the group consisting of 6-nitroveratryloxycarbonyl, 2-nitrobenzyloxycarbonyl, dimethoxybenzyloxy carbonyl, 5-bromo-7-nitroindolinyl, o-hydroxy-alpha-methyl cinnamoyl, 2-oxymethylene anthriquinone, and mixtures thereof.

6. The method as recited in claim 1 wherein each of said different polypeptides is contained within an area of between about 1 μ

m² and 10,000 μ

m².

7. The method as recited in claim 1 wherein said steps of irradiating are carried out while said substrate is in contact with a wash solution.

8. The method as recited in claim 1 wherein said receptor further comprises a marker selected from the group consisting of radioactive markers and fluorescent markers and wherein said identifying step is a step of detecting the location on said substrate of said marker.

9. The method as recited in claim 1 wherein the irradiating step c) further comprises steps of:

i) placing a mask adjacent to said substrate, said mask having substantially transparent regions and substantially opaque regions at a wavelength of light; and

ii) illuminating said mask with a light source, said light source producing at least said wavelength of light, said mask permitting illumination of half of said substrate which was illuminated and half of said substrate which was not illuminated in said step a).

10. The method as recited in claim 1 wherein said additional steps are performed so as to synthesize 10³ different peptides in 10³ respective preselected regions on said substrate.

11. The method as recited in claim 1 wherein said additional steps are performed so as to synthesize 10⁶ different peptides in 10⁶ respective preselected regions on said substrate.

12. The method as recited in claim 1 wherein the step of identifying further comprises the step of contacting said receptor with a second receptor having a marker wherein said second receptor specifically binds to said receptor bound to said polypeptide.

13. The method of claim 1 wherein said additional steps are performed so that at least 1,000 different polypeptides are synthesized on said surface, and each different polypeptide is contained within an area less than about 1×

10^-3 cm².