Methods and systems for determining spatial patterns of biological targets in a sample

Patent metadata

Field	Value
Publication number	US-9879313-B2
Application number	US-201414900604-A
Country	US
Kind code	B2
Filing date	Jun 25, 2014
Priority date	Jun 25, 2013
Publication date	Jan 30, 2018
Grant date	Jan 30, 2018

Abstract

The present disclosure provides methods and assay systems for use in spatially encoded biological assays, including assays to determine a spatial pattern of abundance, expression, and/or activity of one or more biological targets across multiple sites in a sample. In particular, the biological targets comprise proteins, and the methods and assay systems do not depend on imaging techniques for the spatial information of the targets. The present disclosure provides methods and assay systems capable of high levels of multiplexing where reagents are provided to a biological sample in order to address tag the sites to which reagents are delivered; instrumentation capable of controlled delivery of reagents; and a decoding scheme providing a readout that is digital in nature.

First claim

We claim:

1. A method, comprising:
delivering a probe for a target protein to multiple sites in a sample, wherein the probe comprises: (1) a target-binding moiety capable of binding to the target protein; (2) a first address tag that identifies each of the multiple sites to which the probe is delivered; and (3) an identity tag that identifies the target protein or the target-binding moiety;
allowing the probe to bind to the target protein in the sample;
analyzing the probe bound to the target protein, the analysis comprising: (1) determining abundance, expression, and/or activity of the target protein by assessing the amount of the probe bound to the target protein; and (2) determining the identities of the identity tag and the first address tag of the probe for the target protein; and
determining a spatial pattern of abundance, expression, and/or activity of the target protein across the multiple sites in the sample based on the analysis,
the method further comprising:
delivering a probe for a target polynucleotide to each of the multiple sites in the sample, wherein the probe for the target polynucleotide comprises: (1) a sequence that hybridizes to and identifies the target polynucleotide; (2) a second address tag that identifies each of the multiple sites to which the probe for the target polynucleotide is delivered;
allowing the probe for the target polynucleotide to bind to the target polynucleotide in the sample;
analyzing the probe bound to the target polynucleotide, the analysis comprising: (1) determining abundance, expression, and/or activity of the target polynucleotide by assessing the amount of the probe bound to the target polynucleotide; and (2) determining the identities of the sequence that hybridizes to and identifies the target polynucleotide and the second address tag of the probe for the target polynucleotide; and
determining a spatial pattern of abundance, expression, and/or activity of the target polynucleotide across the multiple sites in the sample based on the analysis of the probe bound to the target polynucleotide at each of the multiple sites in the sample.

2. The method of claim 1 , wherein the analyzing step is carried out by sequencing, wherein the amount of a sequencing product indicates abundance, expression, and/or activity of the target protein, the sequencing product comprising all or a portion of the first address tag sequence and all or a portion of the identity tag sequence.

3. A method, comprising:
delivering a probe for a target protein to multiple sites in a sample, wherein the probe comprises: (1) a target-binding moiety capable of binding to the target protein; and (2) an identity tag that identifies the target protein or the protein-binding moiety;
allowing the probe to bind to the target protein in the sample;
delivering a first address tag to each of the multiple sites in the sample, wherein the first address tag is to be coupled to the probe bound to the target protein and identifies the site to which it is delivered;
analyzing the probe/first address tag conjugate bound to the target protein, the analysis comprising: (1) determining abundance, expression, and/or activity of the target protein by assessing the amount of the probe/first address tag conjugate bound to the target protein; and (2) determining the identities of the identity tag and the first address tag of the probe/first address tag conjugate; and
determining a spatial pattern of abundance, expression, and/or activity of the target protein across the multiple sites in the sample based on the analysis,
the method further comprising:
delivering a probe for a target polynucleotide to each of the multiple sites in the sample, wherein the probe for the target polynucleotide comprises a sequence that hybridizes to and identifies the target polynucleotide;
allowing the probe for the target polynucleotide to bind to the target polynucleotide in the sample;
delivering a second address tag to each of the multiple sites in the sample, wherein the second address tag is to be coupled to the probe bound to the target polynucleotide and identifies the site to which it is delivered;
analyzing the probe/second address tag conjugate bound to the target polynucleotide, the analysis comprising: (1) determining abundance, expression, and/or activity of the target polynucleotide by assessing the amount of the probe/second address tag conjugate bound to the target polynucleotide; and (2) determining the identities of the sequence that hybridizes to and identifies the target polynucleotide and the second address tag of the probe/second address tag conjugate; and
determining a spatial pattern of abundance, expression, and/or activity of the target polynucleotide across the multiple sites in the sample based on the analysis of the probe/second address tag conjugate bound to the target polynucleotide at each of the multiple sites in the sample.

4. The method of claim 3 , wherein the target polynucleotide or the complement thereof encodes all or a portion of the target protein.

5. The method of claim 3 , wherein the step of analyzing the probe or probe/first address tag conjugate bound to the target protein and the step of analyzing the probe or probe/second address tag conjugate bound to the target polynucleotide are performed in parallel in the same reaction run.

6. The method of claim 3 , wherein the first address tag and the second address tag are the same for a given site of the multiple sites in the sample.

7. The method of claim 3 , wherein the first address tag and the second address tag are different for a given site of the multiple sites in the sample.

8. The method of claim 3 , further comprising associating abundance, expression, and/or activity of the target protein to abundance, expression, and/or activity of the target polynucleotide at each of the multiple sites in the sample.

9. The method of claim 3 , wherein the target protein comprises an enzyme activity.

10. The method of claim 3 , wherein the target-binding moiety of the probe comprises a moiety selected from the group consisting of an antibody or an antigen binding fragment thereof, an aptamer, a small molecule, an enzyme substrate, a putative enzyme substrate, an affinity capture agent, and a combination thereof.

11. The method of claim 3 , wherein the target-binding moiety is conjugated to a polynucleotide comprising the identity tag.

12. The method of claim 3 , wherein the target-binding moiety is conjugated to a polynucleotide capable of specifically hybridizing to a polynucleotide comprising the identity tag.

13. The method of claim 3 , wherein the probe comprises a multiplicity of target-binding moieties capable of binding to the same domain or different domains of the target, or capable of binding to different targets.

14. The method of claim 3 , wherein there are two address tags that identify each of the multiple sites in the sample.

15. The method of claim 3 , wherein two probes for each target are delivered to the sample.

16. The method of claim 3 , wherein the sample is a biological sample selected from the group consisting of a freshly isolated sample, a fixed sample, a frozen sample, an embedded sample, a processed sample, or a combination thereof.

17. The method of claim 3 , wherein the address tag comprises an oligonucleotide.

18. The method of claim 3 , wherein the identity tag comprises an oligonucleotide.

19. The method of claim 3 , wherein the analyzing step is performed by nucleic acid sequencing or high-throughput sequencing.

Assignees

• Prognosys Biosciences Inc

Inventors

• Chee Mark S
• Routenberg David A

Classifications

• C12Q1/6853 Primary

  using modified primers or templates · CPC title

• C12Q1/6841

  In situ hybridisation · CPC title

• B01L3/5027

  by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip · CPC title

• C12Q1/6809 Primary

  Methods for determination or identification of nucleic acids involving differential detection · CPC title

• C12Q2565/629

  being a microfluidic device · CPC title