SPATIALLY ENCODED BIOLOGICAL ASSAYS

• US 20160298180A1
• Filed: 06/20/2016
• Published: 10/13/2016
• Est. Priority Date: 04/05/2010
• Status: Active Grant

• Current Assignee: Prognosys Biosciences Inc.
• Original Assignee: Prognosys Biosciences Inc.
• Inventors: CHEE, Mark S.
• Granted Patent: US 10,308,982 B2
• Time in Patent Office: Days
• US Class Current: 1/1
• CPC Class Codes: B01L 3/502715 characterised by interfacing components, e.g. fluidic, electrical, optical or mechanical interfaces
• CPC Class Codes: C12Q 1/6804 Nucleic acid analysis using immunogens immunoassay G01N33/53
• CPC Class Codes: C12Q 1/6809 Methods for determination or identification of nucleic acids involving differential detection
• CPC Class Codes: C12Q 1/6834 Enzymatic or biochemical coupling of nucleic acids to a solid phase
• CPC Class Codes: C12Q 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 1/6841 In situ hybridisation
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2535/122 Massive parallel sequencing
• CPC Class Codes: C12Q 2543/10 the purpose being "in situ" analysis
• CPC Class Codes: C12Q 2563/179 the label being a nucleic acid
• CPC Class Codes: C12Q 2565/515 characterised by the interaction between or sequential use of two or more arrays
• CPC Class Codes: C40B 30/04 by measuring the ability to specifically bind a target molecule, e.g. antibody-antigen binding, receptor-ligand binding
• CPC Class Codes: C40B 60/04 Integrated apparatus specially adapted for both screening libraries and identifying library members
• CPC Class Codes: G01N 2458/10 Oligonucleotides as tagging agents for labelling antibodies
• CPC Class Codes: G01N 33/5308 for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites
• CPC Class Codes: G01N 33/543 with an insoluble carrier for immobilising immunochemicals
• CPC Class Codes: G01N 33/6845 Methods of identifying protein-protein interactions in protein mixtures

Abstract

The present invention provides assays and assay systems for use in spatially encoded biological assays. The invention provides an assay system comprising an assay capable of high levels of multiplexing where reagents are provided to a biological sample in defined spatial patterns; instrumentation capable of controlled delivery of reagents according to the spatial patterns; and a decoding scheme providing a readout that is digital in nature.

Claims

1. An assay system to determine spatial patterns of abundance or activity or both of multiple biological targets at multiple sites in a sample, wherein the assay system performs the following steps:

providing a sample affixed to a support;

delivering encoded probes for the multiple biological targets to the multiple sites in the sample in a known spatial pattern, wherein each encoded probe comprises a probe region that may interact with the biological targets and a coding tag that identifies a location of the site to which the encoded probe was delivered;

allowing the encoded probes to interact with the biological targets;

separating encoded probes that interact with the biological targets from encoded probes that do not interact with the biological targets;

determining all or a portion of a sequence of the encoded probes, and

associating the abundance or activity or both of the multiple biological targets to the locations of the sites in the sample.

2. The assay system of claim 1, wherein the biological targets are nucleic acids and the encoded probes are oligonucleotides.

3. The assay system of claim 2, where there are two encoded probes for each of the multiple nucleic acid targets.

4. The assay system of claim 1, wherein the multiple biological targets are proteins, the probe regions of the encoding probes are proteins and the coding tags comprise oligonucleotides.

5. The assay system of claim 4, wherein the multiple biological targets comprise enzymes.

6. The assay system of claim 1, wherein probe regions of the encoded probes comprise antibodies.

7. The assay system of claim 1, wherein the probe regions of the encoded probes are aptamers.

8. The assay system of claim 1, wherein the probe regions of the encoded probes are small molecules.

9. The assay system of claim 1, further comprising an amplification step between the separating step and the determining step.

10. The assay system of claim 1, wherein the determining step is performed by nucleic acid sequencing.

11. The assay system of claim 10, wherein the sequencing is high-throughput digital nucleic acid sequencing.

12. The assay system of claim 1, wherein the product of the multiple biological targets being assayed and the multiple sites in the sample is greater than 20.

13. The assay system of claim 12, wherein the product of the multiple biological targets being assayed and the multiple sites in the sample is greater than 50.

14. The assay system of claim 13, wherein the product of the multiple biological targets being assayed and the multiple sites in the sample is greater than 75.

15. The assay system of claim 14, wherein the product of the multiple biological targets being assayed and the multiple sites in the sample is greater than 100.

16. The assay system of claim 15, wherein the product of the multiple biological targets being assayed and the multiple sites in the sample is greater than 1,000.

17. The assay system of claim 16, wherein the product of the multiple biological targets being assayed and the multiple sites in the sample is greater than 10,000.

18. The assay system of claim 17, wherein the product of the multiple biological targets being assayed and the multiple sites in the sample is greater than 100,000.

19. The assay system of claim 18, wherein the product of the multiple biological targets being assayed and the multiple sites in the sample is greater than 1,000,000.

20. The assay system of claim 11, wherein the sequence of at least one hundred thousand encoding probes are determined in parallel.

21. The assay system of claim 20, wherein the sequence of at least five hundred thousand encoding probes are determined in parallel.

22. The assay system of claim 21, wherein the sequence of at least one million encoding probes are determined in parallel.

23. The assay system of claim 1, wherein the known spatial pattern is determined by histological features of the sample.

24. The assay system of claim 1, wherein software programmed hardware performs at least two steps of the delivering step, the separation step, the determining step and the associating step.

25. The assay system of claim 1, wherein the probe regions of the encoded probes are proteins and the separating step is accomplished by encoded probes that interact with the biological targets being captured by an affinity capture agent.

26. The assay system of claim 1, wherein the probe regions of the encoding probes are nucleic acids and the separating step is accomplished by a washing of the sample.

27. An assay system to determine spatial patterns of abundance or activity or both of multiple nucleic acid targets at multiple sites in a sample, wherein the assay system performs the following steps:

providing a sample affixed to a support;

delivering oligonucleotide probes for multiple nucleic acid targets to the multiple sites in the sample in a known spatial pattern;

allowing the oligonucleotide probes to hybridize with the nucleic acid targets;

washing unhybridized encoded oligonucleotide probes from the sample;

delivering one or more encoding agents to locations of the multiple sites in the sample according to a known spatial pattern, wherein the combination of encoding agents delivered to each site is different;

coupling the encoding agents and the oligonucleotide probes to form encoded probes;

determining all or a portion of a sequence of the encoded probes using high-throughput sequencing, and

associating the abundance or activity or both of multiple biological targets to the locations of multiple sites in the sample.

28. The assay system of claim 27, wherein the product of the multiple biological targets being assayed and the multiple sites in the sample is greater than 20.

29. The assay system of claim 28, wherein the product of the multiple biological targets being assayed and the multiple sites in the sample is greater than 50.

30. The assay system of claim 29, wherein the product of the multiple biological targets being assayed and the multiple sites in the sample is greater than 75.

31. The assay system of claim 30, wherein the product of the multiple biological targets being assayed and the multiple sites in the sample is greater than 10,000.

32. The assay system of claim 31, wherein the product of the multiple biological targets being assayed and the multiple sites in the sample is greater than 100,000.

33. The assay system of claim 32, wherein the product of the multiple biological targets being assayed and the multiple sites in the sample is greater than 1,000,000.

34. The assay system of claim 33, wherein the sequence of at least one hundred thousand encoding probes are determined in parallel.

35. The assay system of claim 34, wherein the sequence of at least one million encoding probes are determined in parallel.

36. The assay system of claim 27, where two oligonucleotide probes are delivered for each of the multiple nucleic acid targets.

37. The assay system of claim 27, wherein the coupling step is performed by ligation.

38. The assay system of claim 27, wherein the coupling step is performed by extension followed by ligation.

39. The assay system of claim 27, further comprising an amplification step between the coupling step and the determining step.

56. The assay system of claim 36, wherein the coding tags are oligonucleotides.

40. An assay system to determine spatial patterns of abundance or activity or both of multiple protein targets at multiple sites in a sample, wherein the assay system performs the following steps:

providing a sample affixed to a support;

delivering encoded probes for the multiple protein targets to the multiple sites in the sample in a known spatial pattern, wherein each encoded probe comprises a protein probe region that may interact with the protein targets and a coding tag that identifies a location of the site to which the encoded probe was delivered and the protein probe region of the encoding probe of which the coding tag is part;

allowing the encoded probes to interact with the protein targets;

separating encoded probes that interact with the protein targets from encoded probes that do not interact with the protein targets;

determining all or a portion of a sequence of the encoded probes by high throughput sequencing, and

associating the abundance or activity or both of the multiple protein targets to the locations of the multiple sites in the sample.

41. The assay system of claim 40, wherein the product of the multiple biological targets being assayed and the multiple sites in the sample is greater than 20.

42. The assay system of claim 41, wherein the product of the multiple biological targets being assayed and the multiple sites in the sample is greater than 50.

43. The assay system of claim 42, wherein the product of the multiple biological targets being assayed and the multiple sites in the sample is greater than 75.

44. The assay system of claim 43, wherein the product of the multiple biological targets being assayed and the multiple sites in the sample is greater than 100.

45. The assay system of claim 44, wherein the product of the multiple biological targets being assayed and the multiple sites in the sample is greater than 500.

46. The assay system of claim 45, wherein the product of the multiple biological targets being assayed and the multiple sites in the sample is greater than 1000.

47. The assay system of claim 46, wherein the sequence of at least ten thousand encoding probes are determined in parallel.

48. The assay system of claim 40, wherein the sequence of at least one hundred thousand encoding probes are determined in parallel.

49. The assay system of claim 48, wherein the sequence of at least one million encoding probes are determined in parallel.

50. The assay system of claim 40, further comprising an amplification step between the coupling step and the determining step.

51. The assay system of claim 40, wherein the protein targets are enzymes, and the probe regions of the encoded probes are substrates, putative substrates of both for the enzymes.

52. The assay system of claim 40, wherein the probe regions of the encoded probes are affinity capture agents.

53. The assay system of claim 52, wherein the probe regions of the encoded probes are antibodies.

54. The assay system of claim 52, wherein the probe regions of the encoded probes are aptamers.

55. The assay system of claim 40, wherein separating encoded probes that interact with the protein targets from encoded probes that do not interact with the protein targets is accomplished by an affinity capture agent that distinguishes encoded probes that interact with the protein targets from encoded probes that do not interact with the protein targets.

57. An assay system to determine spatial patterns of abundance or activity or both of multiple biological targets at multiple sites in a sample, wherein the assay system performs the following steps:

providing a sample affixed to a support;

delivering encoded probes for the multiple biological targets to the multiple sites in the sample in a known spatial pattern, wherein each encoded probe comprises a probe region that may interact with the biological targets and a coding tag that identifies a location of the site to which the encoded probe was delivered and identifies the biological target;

allowing the encoded probes to interact with the biological targets;

determining all or a portion of a sequence of the encoded probes, and

associating the abundance or activity or both of the multiple biological targets to the locations of the sites in the sample.