# US 11,078,520 B2 · Method for generating a three-dimensional nucleic acid containing matrix

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- Number: US 11,078,520 B2
- Document ID: US11078520B2
- Status: Granted
- Kind: B2
- Application number: 16/157,243
- Filed: 2018-10-11
- Published: 2019-01-24
- Granted: 2021-08-03
- 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/US11078520B2

## 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 for analysis, comprising:
(a) providing a biological sample comprising a plurality of cells, wherein a cell of said plurality of cells comprises a plurality of cellular nucleic acids;
(b) generating a three-dimensional matrix comprising cellular nucleic acids of said plurality of cellular nucleic acids or derivatives of said cellular nucleic acids, wherein said cellular nucleic acids or derivatives of said cellular nucleic acids have a three-dimensional position in said three-dimensional matrix; and
(c) detecting signals from said cellular nucleic acids or derivatives of said cellular nucleic acids to identify said three-dimensional position in said three-dimensional matrix.

### Claim 2 (depends on 1)

The method of claim 1, further comprising, prior to (b), permeabilizing said cell.

### Claim 3 (depends on 2)

The method of claim 2, wherein (b) comprises contacting said cell with a matrix-forming material and using said matrix-forming material to form said three-dimensional matrix.

### Claim 4 (depends on 3)

The method of claim 3, wherein said three-dimensional matrix is a cross-linked or polymer matrix.

### Claim 5 (depends on 3)

The method of claim 3, wherein said three-dimensional matrix comprises polyacrylamide, cellulose, alginate, polyamide, cross-linked agarose, cross-linked dextran, or cross-linked polyethylene glycol.

### Claim 6 (depends on 3)

The method of claim 3, wherein (b) further comprises exposing said matrix-forming material to a polymerization inducing catalyst, ultraviolet (UV) light, or a functional cross-linker.

### Claim 7 (depends on 3)

The method of claim 3, wherein said matrix-forming material comprises a polymer, wherein said three-dimensional polymer matrix is generated at least in part by crosslinking said polymer.

### Claim 8 (depends on 3)

The method of claim 3, wherein said matrix-forming material comprises a plurality of monomers, and wherein said three-dimensional matrix is generated at least in part by polymerizing said plurality of monomers.

### Claim 9 (depends on 1)

The method of claim 1, further comprising, prior to (b), generating a plurality of amplicons from said cellular nucleic acids.

### Claim 10 (depends on 9)

The method of claim 9, wherein said derivatives of said cellular nucleic acids comprise amplicons of said plurality of amplicons and (b) comprises generating said three-dimensional matrix comprising said amplicons, wherein said amplicons have said three-dimensional position in said three-dimensional matrix.

### Claim 11 (depends on 10)

The method of claim 10, wherein (b) comprises covalently attaching said amplicons to said three-dimensional matrix thereby substantially retaining said three-dimensional position of said amplicons in said three-dimensional matrix.

### Claim 12 (depends on 10)

The method of claim 10, wherein said amplicons comprise a functional moiety and wherein said amplicons are covalently attached to said three-dimensional matrix via said functional moiety.

### Claim 13 (depends on 12)

The method of claim 12, wherein said functional moiety comprises an amine, an acrydite, an alkyne, a biotin, an azide, or a thiol.

### Claim 14 (depends on 12)

The method of claim 12, wherein said amplicons comprising said functional moiety are generated by performing a nucleic acid amplification reaction in the presence of a modified nucleotide comprising said functional moiety such that said modified nucleotide is incorporated into said amplicons.

### Claim 15 (depends on 14)

The method of claim 14, wherein said modified nucleotide comprises uracil.

### Claim 16 (depends on 12)

The method of claim 12, wherein said functional moiety is covalently cross-linked to said three-dimensional matrix to covalently attach said amplicons to said three-dimensional matrix.

### Claim 17 (depends on 16)

The method of claim 16, further comprising using a cross-linker to covalently attach said amplicons to said three-dimensional matrix, wherein said cross-linker comprises a reactive group that reacts with said functional moiety.

### Claim 18 (depends on 17)

The method of claim 17, wherein said reactive group comprises an imidoester, a succinimide ester, a maleimide, a carbodiimide, or a phenyl azide.

### Claim 19 (depends on 17)

The method of claim 17, wherein said cross-linker further comprises a spacer.

### Claim 20 (depends on 19)

The method of claim 19, wherein said spacer comprises polyethylene glycol, a carbon backbone, or a photo-cleavable moiety.

### Claim 21 (depends on 12)

The method of claim 12, wherein said functional moiety is co-polymerized with matrix-forming material to covalently attach said amplicons to said three-dimensional matrix.

### Claim 22 (depends on 21)

The method of claim 21, wherein said functional moiety comprises acrydite and wherein said matrix forming material comprises an acrylamide.

### Claim 23 (depends on 10)

The method of claim 10, wherein said amplicons are labelled with detectable labels, and wherein (c) comprises detecting signals from said detectable labels.

### Claim 24 (depends on 23)

The method of claim 23, wherein said detectable labels are fluorescent labels.

### Claim 25 (depends on 23)

The method of claim 23, wherein detecting signals from said detectable labels comprises imaging.

### Claim 26 (depends on 23)

The method of claim 23, wherein said signals are detected in a plurality of optical sections to identify said three-dimensional position in said three-dimensional matrix.

### Claim 27 (depends on 23)

The method of claim 23, further comprising generating a visualization comprising a three-dimensional reconstruction of said three-dimensional position in said three-dimensional matrix.

### Claim 28 (depends on 2)

The method of claim 2, further comprising, prior to permeabilizing said cell, using a fixative to substantially fix said cellular nucleic acids.

### Claim 29 (depends on 10)

The method of claim 10, wherein (c) comprises sequencing said amplicons.

### Claim 30

A method for analysis, comprising:
(a) providing a biological sample comprising a plurality of cells, wherein a cell of said plurality of cells comprises a plurality of cellular nucleic acids;
(b) generating a plurality of amplicons from said plurality of cellular nucleic acids;
(c) generating a three-dimensional matrix comprising amplicons of said plurality of amplicons, wherein said amplicons are covalently attached to said three-dimensional matrix, and wherein said amplicons have a three-dimensional position in said three-dimensional matrix; and
(d) hybridizing fluorescently labeled oligonucleotides to said amplicons and imaging said amplicons to identify said three-dimensional position in said three-dimensional matrix.

### Claim 31 (depends on 29)

The method of claim 29, wherein said amplicons are generated through rolling circle amplification of circularized probes.