# US 10,501,779 B2 · Oligonucleotide trapping

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- Number: US 10,501,779 B2
- Document ID: US10501779B2
- Status: Granted
- Kind: B2
- Application number: 13/469,242
- Filed: 2012-05-11
- Published: 2013-06-06
- Granted: 2019-12-10
- Earliest claimed: 2011-05-12
- Assignees: President and Fellows of Harvard College
- Applicants: Chao-ting Wu; Brian Beliveau
- Inventors: Chao-ting Wu; Brian Beliveau
- Web page: https://patentgrep.com/patent/US10501779B2

## CPC

- C12Q 1/6841: In situ hybridisation
- G01N 33/50: Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing (measuring or testing processes involving enzymes or microorganisms, compositions or test papers therefor; processes for forming such compositions, condition responsive control in microbiological or enzymological processes C12Q)
- G01N 33/53: Immunoassay; Biospecific binding assay; Materials therefor
- G01N 33/58: involving labelled substances (G01N33/53 takes precedence)
- 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
- 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

Novel methods and compositions for identifying one or more factors associated with a nucleic acid sequence (e.g., DNA and/or RNA) of interest are provided.

## Claims

### Claim 1

A method for identifying one or more factors bound to one or more targeting moieties of a plurality of oligonucleotide paints in a biological sample by in situ hybridization, comprising the steps of:
hybridizing the plurality of oligonucleotide paints to different chromosomal regions of a chromosome in the biological sample by in situ hybridization, thereby forming hybridized oligonucleotide paints, wherein each of the oligonucleotide paints comprises a 5′ primer sequence, a nucleotide sequence capable of hybridizing to a specific region of the chromosome, 3′ primer sequence, and one or more targeting moieties, and the nucleotide sequence capable of hybridizing to the specific region of the chromosome is located between the 5′ primer sequence and the 3′ primer sequence and is different in each of the oligonucleotide paints and the 5′ primer sequence and the 3′ primer sequence comprises either nucleotide sequences of a universal primer or nucleotide sequences fully complementary to the universal primer;
removing the oligonucleotide paints unbound to the different chromosomal regions of the chromosome from the hybridized oligonucleotide paints;
covalently attaching the targeting moieties of the hybridized oligonucleotide paints to the one or more factors in the biological sample; and
identifying the one or more factors bound to the one or more targeting moieties of the plurality of oligonucleotide paints in the biological sample by identifying the factors attached to the targeting moieties of the hybridized oligonucleotide paints.

### Claim 2 (depends on 1)

The method of claim 1, wherein the one or more factors are selected from the group consisting of a protein, a peptide, a carbohydrate, a lipid, a chemical moiety and any combination thereof.

### Claim 3 (depends on 1)

The method of claim 1, wherein the oligonucleotide paints are between about 20 base pairs and about 32 base pairs in length.

### Claim 4 (depends on 1)

The method of claim 1, wherein the nucleotide sequence capable of hybridizing to the specific region of the chromosome is between about 32 base pairs and about 40 base pairs in length.

### Claim 5 (depends on 1)

The method of claim 1, further comprising the step of retrieving the hybridized oligonucleotide paints after the attaching step, wherein the oligonucleotide paints further include a retrievable moiety.

### Claim 6 (depends on 5)

The method of claim 5, wherein the retrievable moiety is one component of a binding pair.

### Claim 7 (depends on 5)

The method of claim 5, wherein the retrievable moiety is biotin.

### Claim 8 (depends on 5)

The method of claim 5, wherein the hybridized oligonucleotide paints are retrieved by binding the retrievable moiety.

### Claim 9 (depends on 1)

The method of claim 1, further comprising activation of the targeting moieties of the hybridized oligonucleotide paints before the attaching step.

### Claim 10 (depends on 9)

The method of claim 9, wherein the activation of the targeting moieties of the hybridized oligonucleotide paints is performed by heat activation of the targeting moieties, light activation of the targeting moieties, or chemical activation of the targeting moieties or any combination thereof.

### Claim 11 (depends on 1)

The method of claim 1, wherein the one or more targeting moieties are covalently attached to a protein, a peptide, a carbohydrate, a lipid, or a chemical moiety or any combination thereof.

### Claim 12 (depends on 11)

The method of claim 11, wherein the chemical moiety is a methyl group or an acetyl group.

### Claim 13 (depends on 1)

The method of claim 1, wherein the one or more factors are selected from the group consisting of histone proteins, scaffold proteins, transcription factors, DNA binding proteins, DNA repair factors, and nucleases or any combination thereof.

### Claim 14 (depends on 1)

The method of claim 1, wherein the chromosome is within a cell from the biological sample.

### Claim 15 (depends on 1)

The method of claim 1, wherein the oligonucleotide paints are ligated oligonucleotide paints.

### Claim 16 (depends on 1)

The method of claim 1, wherein the oligonucleotide paints are hapten-conjugated oligonucleotide paints.

### Claim 17 (depends on 1)

The method of claim 1, wherein the oligonucleotide paints unbound to the different chromosomal regions of the chromosome are removed by washing a support having the biological sample immobilized on the support.

### Claim 18 (depends on 17)

The method of claim 17, wherein the different chromosomal regions are present in single or low copy numbers.

### Claim 19 (depends on 1)

The method of claim 1 wherein one of the one or more targeting moieties is a protein capture moiety, a lipid capture moiety, a sugar capture moiety or a nucleic acid capture moiety.

### Claim 20 (depends on 1)

The method of claim 1 wherein one of the one or more targeting moieties is

### Claim 21 (depends on 1)

The method of claim 1 wherein the plurality of oligonucleotide paints comprises a set of oligonucleotide paints bound to a plurality of sub-chromosomal regions.

### Claim 22 (depends on 21)

The method of claim 21 wherein the plurality of sub-chromosomal regions comprises at least 50 regions.

### Claim 23

A method for identifying one or more factors bound to one or more targeting moieties of a plurality of oligonucleotide paints in a biological sample by in situ hybridization, comprising the steps of:
hybridizing the plurality of oligonucleotide paints to different regions of genomic DNA or mitochondrial DNA in the biological sample by in situ hybridization, thereby forming hybridized oligonucleotide paints, wherein each of the oligonucleotide paints comprises a 5′ primer sequence, a nucleotide sequence capable of hybridizing to a specific region of the genomic DNA or the mitochondrial DNA, 3′ primer sequence, and one or more targeting moieties, and the nucleotide sequence capable of hybridizing to the specific region of the genomic DNA or the mitochondrial DNA is located between the 5′ primer sequence and the 3′ primer sequence and is different in each of the oligonucleotide paints and the 5′ primer sequence and the 3′ primer sequence comprises either nucleotide sequences of a universal primer or nucleotide sequences fully complementary to the universal primer;
removing the oligonucleotide paints unbound to the different regions of the genomic DNA or the mitochondrial DNA from the hybridized oligonucleotide paints;
covalently attaching the targeting moieties of the hybridized oligonucleotide paints to the one or more factors in the biological sample; and
identifying the one or more factors bound to the one or more targeting moieties of the plurality of oligonucleotide paints in the biological sample by identifying the factors attached to the targeting moieties of the hybridized oligonucleotide paints.

### Claim 24 (depends on 23)

The method of claim 23, wherein the one or more factors are selected from the group consisting of a protein, a peptide, a carbohydrate, a lipid, and a chemical moiety or any combination thereof.

### Claim 25

A method for identifying one or more factors bound to one or more targeting moieties of a plurality of oligonucleotide paints in a biological sample by in situ hybridization, comprising the steps of:
hybridizing the plurality of oligonucleotide paints to different regions of genomic DNA or mitochondrial DNA in the biological sample by in situ hybridization, thereby forming hybridized oligonucleotide paints, wherein the plurality of oligonucleotide paints has retrievable moieties, and wherein each of the oligonucleotide paints comprises a 5′ primer sequence, a nucleotide sequence capable of hybridizing to a specific region of the genomic DNA or the mitochondrial DNA, 3′ primer sequence, and one or more targeting moieties, and the nucleotide sequence capable of hybridizing to the specific region of the genomic DNA or the mitochondrial DNA is located between the 5′ primer sequence and the 3′ primer sequence and is different in each of the oligonucleotide paints and the 5′ primer sequence and the 3′ primer sequence comprises either nucleotide sequences of a universal primer or nucleotide sequences fully complementary to the universal primer;
removing the oligonucleotide paints unbound to the different regions of the genomic DNA or mitochondrial DNA from the hybridized oligonucleotide paints;
covalently attaching the targeting moieties of the hybridized oligonucleotide paints to the one or more factors in the biological sample; and
identifying the one or more factors bound to the one or more targeting moieties of the plurality of oligonucleotide paints in the biological sample by identifying the factors attached to the targeting moieties of the hybridized oligonucleotide paints.

### Claim 26 (depends on 25)

The method of claim 25, wherein the one or more factors are selected from the group consisting of a protein, a peptide, a carbohydrate, a lipid, and a chemical moiety or any combination thereof.