# US 11,299,767 B2 · Method for generating a three-dimensional nucleic acid containing matrix

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- Number: US 11,299,767 B2
- Document ID: US11299767B2
- Status: Granted
- Kind: B2
- Application number: 17/363,097
- Filed: 2021-06-30
- Published: 2021-10-21
- Granted: 2022-04-12
- Earliest claimed: 2013-03-12
- 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/US11299767B2

## 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 1/6844: Nucleic acid amplification reactions
- C12Q 1/6869: Methods for sequencing
- C12Q 1/6874: involving nucleic acid arrays, e.g. sequencing by hybridisation
- 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)}

## Abstract

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

## Claims

### Claim 1

A method of analyzing a biological sample, comprising:
(a) permeabilizing said biological sample, wherein said biological sample comprises a plurality of cells, wherein a cell of said plurality of cells comprises cellular nucleic acid molecules having a relative three-dimensional (3D) spatial orientation within said cell;
(b) generating a 3D matrix comprising said cellular nucleic acid molecules attached thereto, wherein said 3D matrix preserves said relative 3D spatial orientation;
(c) contacting said 3D matrix with reagents to selectively remove a non-nucleic acid component from said biological sample; and
(d) using 3D fluorescence imaging to identify said cellular nucleic acid molecules and said relative 3D spatial orientation of said cellular nucleic acid molecules within said cell.

### Claim 2 (depends on 1)

The method of claim 1, wherein (d) comprises hybridizing fluorescently labeled oligonucleotides to said cellular nucleic acid molecules or derivatives thereof and imaging said 3D matrix to detect said fluorescently labeled oligonucleotides.

### Claim 3 (depends on 2)

The method of claim 2, further comprising, (i) stripping said fluorescently labeled oligonucleotides from said cellular nucleic acid molecules or derivatives thereof; (ii) hybridizing additional fluorescently labeled oligonucleotides to said cellular nucleic acid molecules or derivatives thereof; and (iii) imaging said 3D matrix to detect said additional fluorescently labeled oligonucleotides.

### Claim 4 (depends on 2)

The method of claim 2, further comprising, prior to (d), contacting said cellular nucleic acid molecules with a plurality of nucleic acid probes and wherein (d) comprises hybridizing said fluorescently labeled oligonucleotides to said plurality of nucleic acid probes.

### Claim 5 (depends on 4)

The method of claim 4, further comprising, (i) stripping said fluorescently labeled oligonucleotides from said plurality of nucleic acid probes; (ii) hybridizing additional fluorescently labeled oligonucleotides to said plurality of nucleic acid probes; and (iii) imaging said 3D matrix to detect said additional fluorescently labeled oligonucleotides.

### Claim 6 (depends on 1)

The method of claim 1, further comprising, prior to (d), contacting said cellular nucleic acid molecules with a plurality of nucleic acid probes and wherein (d) comprises sequencing at least a portion of said plurality of nucleic acid probes.

### Claim 7 (depends on 6)

The method of claim 6, wherein said sequencing is sequencing-by-ligation.

### Claim 8 (depends on 6)

The method of claim 6, wherein said sequencing is sequencing-by-synthesis.

### Claim 9 (depends on 6)

The method of claim 6, wherein said sequencing is sequencing-by-hybridization.

### Claim 10 (depends on 1)

The method of claim 1, wherein (d) comprises sequencing said cellular nucleic acid molecules or derivatives thereof.

### Claim 11 (depends on 1)

The method of claim 1, wherein, prior to (b), said cellular nucleic acid molecules comprise a functional moiety and wherein said functional moiety is attached to said 3D matrix.

### Claim 12 (depends on 11)

The method of claim 11, wherein said functional moiety comprises acrydite, wherein (b) comprises contacting said biological sample with a matrix forming material and polymerizing said matrix forming material to generate said 3D matrix, and wherein said acrydite is polymerized into said 3D matrix thereby attaching said cellular nucleic acid molecules to said 3D matrix.

### Claim 13 (depends on 1)

The method of claim 1, wherein said cellular nucleic acid molecules are attached to said 3D matrix through a non-covalent interaction.

### Claim 14 (depends on 13)

The method of claim 13, wherein said non-covalent interaction is hydrogen bonding.

### Claim 15 (depends on 1)

The method of claim 1, wherein said cellular nucleic acid molecules are attached to said 3D matrix through a ligand-ligand binding pair, wherein a ligand of said ligand-ligand binding pair comprises a functional moiety that is attached to said 3D matrix.

### Claim 16 (depends on 15)

The method of claim 15, wherein said functional moiety comprises acrydite, wherein (b) comprises contacting said biological sample with a matrix forming material and polymerizing said matrix forming material to generate said 3D matrix, and wherein said acrydite is polymerized into said 3D matrix.

### Claim 17 (depends on 1)

The method of claim 1, wherein said reagents comprise a protease, an organic solvent or a denaturant.

### Claim 18 (depends on 1)

The method of claim 1, wherein said reagents comprise a detergent.

### Claim 19 (depends on 18)

The method of claim 18, wherein said reagents further comprise a protease.

### Claim 20 (depends on 1)

The method of claim 1, wherein said biological sample is a formalin-fixed, wax embedded tissue sample on a glass slide.

### Claim 21 (depends on 1)

The method of claim 1, further comprising, subsequent to (d), performing a 3D reconstruction of said cellular nucleic acid molecules within said cell.