# US 11,111,521 B2 · Compositions and methods for analyte detection

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- Number: US 11,111,521 B2
- Document ID: US11111521B2
- Status: Granted
- Kind: B2
- Application number: 17/122,168
- Filed: 2020-12-15
- Published: 2021-04-08
- Granted: 2021-09-07
- Earliest claimed: 2011-12-22
- Assignees: President and Fellows of Harvard College
- Applicants: President and Fellows of Harvard College
- Inventors: George M. Church; Je-hyuk Lee; Richard C. Terry; Evan R. Daugharthy
- Web page: https://patentgrep.com/patent/US11111521B2

## CPC

- C12Q 1/6806: Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence)
- C12P 19/34: Polynucleotides, e.g. nucleic acids, oligoribonucleotides
- C12Q 1/6804: Nucleic acid analysis using immunogens (immunoassay G01N33/53)
- C12Q 1/6816: characterised by the detection means (C12Q1/6804 takes precedence)
- C12Q 1/6837: using probe arrays or probe chips (C12Q1/6874 takes precedence)
- C12Q 1/6844: Nucleic acid amplification reactions
- C12Q 1/6869: Methods for sequencing
- C12Q 1/6874: involving nucleic acid arrays, e.g. sequencing by hybridisation
- G01N 33/53: Immunoassay; Biospecific binding assay; Materials therefor
- G01N 2458/10: Oligonucleotides as tagging agents for labelling antibodies
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12P: FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE {(brewing of beer C12C; producing vinegar C12J; producing specific peptides or proteins C07K; producing enzymes C12N9/00; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; measuring or testing processes involving enzymes or microorganisms C12Q; measuring or testing processes involving nucleic acid amplification reactions C12Q1/6844; fermentation processes to form a food composition, A21 or A23; compounds in general, see the relevant compound class, e.g. C01, C07)}
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)

## Abstract

Methods of making a three-dimensional matrix of nucleic acids within a cell is provided.

## Claims

### Claim 1

A method for biological analysis, comprising:
(a) providing a cell or tissue sample, wherein said cell or tissue sample comprises an analyte at a spatial location in said cell or tissue sample;
(b) binding a detection reagent to said analyte at said spatial location, wherein said detection reagent comprises (i) a probe that binds to said analyte and (ii) one or more pre-determined subsequences;
(c) performing a plurality of detection steps at said spatial location to obtain a time series of signal signatures corresponding to said analyte, wherein said plurality of detection steps comprises (i) detecting a first detectable label associated with said one or more pre-determined subsequences to obtain a first signal signature at said spatial location at a first time point of said time series; (ii) removing said first detectable label from said one or more pre-determined subsequences; and (iii) detecting a second detectable label associated with said one or more pre-determined subsequences to obtain a second signal signature at said spatial location at a second time point of said time series, wherein said second time point of said time series is subsequent to said first time point of said time series; and
(d) using said time series of signal signatures to identify said analyte at said spatial location in said cell or tissue sample.

### Claim 2 (depends on 1)

The method of claim 1, wherein said analyte is a cellular nucleic acid molecule, wherein said detection reagent is a nucleic acid molecule comprising said probe and said one or more pre-determined subsequences, and wherein said probe comprises a sequence that binds said cellular nucleic acid molecule.

### Claim 3 (depends on 2)

The method of claim 2, wherein said cellular nucleic acid molecule is a cellular deoxyribonucleic acid (DNA) molecule.

### Claim 4 (depends on 2)

The method of claim 2, wherein said cellular nucleic acid molecule is a cellular ribonucleic acid (RNA) molecule.

### Claim 5 (depends on 4)

The method of claim 4, wherein said cellular RNA molecule is a messenger RNA molecule.

### Claim 6 (depends on 1)

The method of claim 1, wherein said probe comprises an antibody or an antigen-binding fragment of an antibody.

### Claim 7 (depends on 6)

The method of claim 6, wherein said analyte is a protein.

### Claim 8 (depends on 1)

The method of claim 1, wherein said one or more pre-determined subsequences comprises a first pre-determined subsequence and a second pre-determined subsequence; wherein said first detectable label is associated with said first pre-determined subsequence; and wherein said second detectable label is associated with said second pre-determined subsequence.

### Claim 9 (depends on 8)

The method of claim 8, further comprising:
using a first decoder probe complementary to said first predetermined subsequence to associate said first detectable label with said first pre-determined subsequence; and
using a second decoder probe complementary to said second predetermined subsequence to associate said second detectable label with said second pre-determined subsequence.

### Claim 10 (depends on 9)

The method of claim 9, wherein said first detectable label is covalently attached to said first decoder probe and wherein said second detectable label is covalently attached to said second decoder probe.

### Claim 11 (depends on 9)

The method of claim 9, wherein said first detectable label is attached to said first decoder probe via a first linker and wherein said second detectable label is attached to said second decoder probe via a second linker.

### Claim 12 (depends on 9)

The method of claim 9, wherein said first detectable label is indirectly attached to said first decoder probe and wherein said second detectable label is indirectly attached to said second decoder probe.

### Claim 13 (depends on 9)

The method of claim 9, wherein said first detectable label is attached to a first molecule that binds said first decoder probe and wherein said second detectable label is attached to a second molecule that binds said second decoder probe.

### Claim 14 (depends on 9)

The method of claim 9, wherein said first detectable label is a first optical label and wherein said second detectable label is a second optical label.

### Claim 15 (depends on 14)

The method of claim 14, wherein said first optical label is a first fluorescent molecule and said second optical label is a second fluorescent molecule.

### Claim 16 (depends on 15)

The method of claim 15, wherein said first fluorescent molecule and said second fluorescent molecule have the same fluorescent color.

### Claim 17 (depends on 15)

The method of claim 15, wherein said first fluorescent molecule and said second fluorescent molecule have different fluorescent colors.

### Claim 18 (depends on 8)

The method of claim 8, wherein said first pre-determined subsequence is 5-30 nucleotides in length and said second pre-determined subsequence is 5-30 nucleotides in length.

### Claim 19 (depends on 1)

The method of claim 1, wherein said cell or tissue sample is immobilized on a solid support.

### Claim 20 (depends on 19)

The method of claim 19, wherein said solid support does not comprise discrete sites configured to attach to beads.

### Claim 21 (depends on 19)

The method of claim 19, wherein said solid support comprises discrete sites configured to attach to or associate with beads.

### Claim 22 (depends on 1)

The method of claim 1, wherein said cell or tissue sample is a tissue section on a slide.

### Claim 23 (depends on 22)

The method of claim 22, wherein (b) comprises contacting said tissue section with said detection reagent.

### Claim 24 (depends on 22)

The method of claim 22, further comprising, prior to (b), permeabilizing tissue of said tissue section.

### Claim 25

A method for biological analysis, comprising:
(a) binding a detection reagent to a target nucleic acid molecule at a spatial location in a cell or tissue sample, wherein said detection reagent comprises a nucleic acid molecule comprising (i) a probe sequence that binds to said target nucleic acid molecule and (ii) a label that identifies said probe, wherein said label comprises a first predetermined subsequence and a second predetermined subsequence;
(b) performing a plurality of detection steps at said spatial location to obtain a time series of signal signatures corresponding to said label, wherein said plurality of detection steps comprises (i) using a first decoder probe complementary to said first predetermined subsequence to associate a first detectable label with said first pre-determined subsequence; (ii) detecting said first detectable label in said cell or tissue sample to obtain a first signal signature at said spatial location at a first time point of said time series of signal signatures; (iii) removing said first detectable label from said cell or tissue sample; and (iv) using a second decoder probe complementary to said second predetermined subsequence to associate a second detectable label with said second pre-determined subsequence; and (v) detecting said second detectable label in said cell or tissue sample to obtain a second signal signature at said spatial location at a second time point of said time series of signal signatures, wherein said second time point of said time series is subsequent to said first time point of said time series; and
(c) using said time series of signal signatures to identify said analyte at said spatial location in said cell or tissue sample.

### Claim 26 (depends on 25)

The method of claim 25, wherein said cell or tissue sample is immobilized on a solid support.

### Claim 27 (depends on 25)

The method of claim 25, wherein said cell or tissue sample is a tissue section on a slide.

### Claim 28 (depends on 25)

The method of claim 25, wherein said first detectable label is covalently attached to said first decoder probe and wherein said second detectable label is covalently attached to said second decoder probe.

### Claim 29 (depends on 25)

The method of claim 25, wherein said first detectable label is attached to said first decoder probe via a first linker and wherein said second detectable label is attached to said second decoder probe via a second linker.

### Claim 30 (depends on 25)

The method of claim 25, wherein said first detectable label is indirectly attached to said first decoder probe and wherein said second detectable label is indirectly attached to said second decoder probe.

### Claim 31 (depends on 1)

The method of claim 1, wherein said probe comprises an aptamer.

### Claim 32 (depends on 12)

The method of claim 12, wherein said first detectable label is indirectly attached to said first decoder probe via a first binding molecule that hybridizes to said first decoder probe, and wherein said second detectable label is indirectly attached to said second decoder probe via a second binding molecule that hybridizes to said second decoder probe.

### Claim 33 (depends on 32)

The method of claim 32, wherein said first binding molecule is directly attached to said first detectable label and wherein said second binding molecule is directly attached to said second detectable label.

### Claim 34 (depends on 14)

The method of claim 14, wherein removing said first detectable label from said one or more pre-determined subsequences comprises photo-bleaching, quenching, chemically degrading, bleaching, or oxidizing said first optical label.

### Claim 35 (depends on 9)

The method of claim 9, wherein removing said first detectable label from said one or more pre-determined subsequences comprises washing or displacing said first decoder probe from said first predetermined sequence.

### Claim 36 (depends on 35)

The method of claim 35, wherein washing or displacing said first decoder probe from said first predetermined sequence further comprises use of heat, varying a salt concentration, applying a detergent, applying a denaturant, or any combinations thereof.

### Claim 37 (depends on 9)

The method of claim 9, wherein removing said first detectable label from said one or more pre-determined subsequences comprises cleaving or enzymatically digesting said first decoder probe.

### Claim 38 (depends on 9)

The method of claim 9, wherein said first decoder probe comprises a cleavable linker and wherein removing said first detectable label from said one or more pre-determined subsequences comprises cleaving said cleavable linker.

### Claim 39 (depends on 1)

The method of claim 1, wherein said detection reagent is a linear nucleic acid molecule comprising said probe and said one or more pre-determined subsequences.

### Claim 40 (depends on 39)

The method of claim 39, further comprising, prior to (c), circularizing said detection reagent and performing rolling circle amplification to generate an amplified detection reagent.

### Claim 41 (depends on 40)

The method of claim 40, wherein circularizing said detection reagent comprises use of a ligase.

### Claim 42 (depends on 19)

The method of claim 19, wherein said solid support is a planar substrate.

### Claim 43 (depends on 22)

The method of claim 22, wherein said tissue section is derived from a fixed tissue.

### Claim 44 (depends on 1)

The method of claim 1, wherein said one or more pre-determined subsequences comprises a first predetermined sequence, wherein (c)(i) comprises using a first decoder probe complementary to said first predetermined subsequence to associate said first detectable label with said first pre-determined subsequence, and wherein (c)(iii) comprises using a second decoder probe complementary to said first predetermined subsequence to associate said second detectable label with said first pre-determined subsequence.

### Claim 45 (depends on 1)

The method of claim 1, wherein each of said one or more pre-determined subsequences is 3-50 nucleotides in length.

### Claim 46 (depends on 1)

The method of claim 1, wherein (b) comprises binding an additional detection reagent to said analyte at said spatial location, wherein said additional detection reagent comprises (i) an additional probe that binds to said analyte and (ii) an additional one or more pre-determined subsequences; wherein said time series of signal signatures comprises signal signatures associated with said one or more pre-determined subsequences and said additional one or more pre-determined subsequences; and wherein said plurality of detection steps further comprises detecting a third detectable label associated with said additional one or more pre-determined subsequences to obtain a third signal signature at said spatial location at a third time point of said time series.

### Claim 47 (depends on 25)

The method of claim 25, wherein said target nucleic acid molecule is a cellular deoxyribonucleic acid (DNA) molecule.

### Claim 48 (depends on 25)

The method of claim 25, wherein said target nucleic acid molecule is a cellular ribonucleic acid (RNA) molecule.

### Claim 49 (depends on 25)

The method of claim 25, wherein said first detectable label is attached to a first molecule that binds said first decoder probe and wherein said second detectable label is attached to a second molecule that binds said second decoder probe.

### Claim 50 (depends on 25)

The method of claim 25, wherein said first detectable label is indirectly attached to said first decoder probe via a first binding molecule that hybridizes to said first decoder probe, and wherein said second detectable label is indirectly attached to said second decoder probe via a second binding molecule that hybridizes to said second decoder probe.

### Claim 51 (depends on 50)

The method of claim 50, wherein said first binding molecule is directly attached to said first detectable label and wherein said second binding molecule is directly attached to said second detectable label.

### Claim 52 (depends on 25)

The method of claim 25, wherein said first pre-determined subsequence is 5-30 nucleotides in length and said second pre-determined subsequence is 5-30 nucleotides in length.

### Claim 53 (depends on 26)

The method of claim 26, wherein said solid support does not comprise discrete sites configured to attach to beads.

### Claim 54 (depends on 26)

The method of claim 26, wherein said solid support comprises discrete sites configured to attach to or associate with beads.

### Claim 55 (depends on 26)

The method of claim 26, wherein said solid support is a planar substrate.

### Claim 56 (depends on 27)

The method of claim 27, wherein said tissue section is derived from a fixed tissue.

### Claim 57 (depends on 26)

The method of claim 26, further comprising, prior to (a), permeabilizing said tissue section.

### Claim 58 (depends on 25)

The method of claim 25, wherein said first detectable label is a first optical label and wherein said second detectable label is a second optical label.

### Claim 59 (depends on 58)

The method of claim 58, wherein said first optical label is a first fluorescent molecule and said second optical label is a second fluorescent molecule.

### Claim 60 (depends on 59)

The method of claim 59, wherein said first fluorescent molecule and said second fluorescent molecule have the same fluorescent color.

### Claim 61 (depends on 59)

The method of claim 59, wherein said first fluorescent molecule and said second fluorescent molecule have different fluorescent colors.

### Claim 62 (depends on 58)

The method of claim 58, wherein removing said first detectable label from said from said cell or tissue sample comprises photo-bleaching, quenching, chemically degrading, bleaching, or oxidizing said first optical label.

### Claim 63 (depends on 25)

The method of claim 25, wherein removing said first detectable label from said cell or tissue sample comprises washing or displacing said first decoder probe from said first predetermined sequence.

### Claim 64 (depends on 63)

The method of claim 63, wherein washing or displacing said first decoder probe from said cell or tissue sample further comprises heating said cell or tissue sample, varying a salt concentration of said cell or tissue sample, applying a detergent to said cell or tissue sample, applying a denaturant to said cell or tissue sample, or any combinations thereof.

### Claim 65 (depends on 25)

The method of claim 25, wherein removing said first detectable label from said cell or tissue sample comprises cleaving or enzymatically digesting said first decoder probe.

### Claim 66 (depends on 25)

The method of claim 25, wherein said first decoder probe comprises a cleavable linker and wherein removing said first detectable label from said cell or tissue sample comprises cleaving said cleavable linker.

### Claim 67 (depends on 25)

The method of claim 25, wherein said detection reagent is a linear nucleic acid molecule comprising said probe sequence and said label.

### Claim 68 (depends on 67)

The method of claim 67, further comprising, prior to (b), circularizing said detection reagent and performing rolling circle amplification to generate an amplified detection reagent.

### Claim 69 (depends on 68)

The method of claim 68, wherein circularizing said detection reagent comprises use of a ligase.

### Claim 70 (depends on 25)

The method of claim 25, wherein (a) comprises binding an additional detection reagent to said target nucleic acid molecule at said spatial location, wherein said additional detection reagent comprises (i) an additional probe sequence that binds to said analyte and (ii) an additional label that identifies said additional probe, wherein said additional label comprises a third pre-determined subsequence; wherein said time series of signal signatures comprises signal signatures associated with said label and said additional label; and wherein said plurality of detection steps further comprises (vi) removing said second detectable label from said cell or tissue sample; (vii) using a third decoder probe complementary to said third predetermined subsequence to associate a third detectable label with said third pre-determined subsequence; and (viii) detecting said third detectable label in said cell or tissue sample to obtain a third signal signature at said spatial location at a third time point of said time series of signal signatures.

### Claim 71 (depends on 1)

The method of claim 1, wherein said cell or tissue sample is paraffin-embedded.

### Claim 72 (depends on 25)

The method of claim 25, wherein said cell or tissue sample is paraffin-embedded.