# US 8,785,615 B2 · Single molecule sensitive probes for detecting RNA

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- Number: US 8,785,615 B2
- Document ID: US8785615B2
- Status: Granted
- Kind: B2
- Application number: 13/258,830
- Filed: 2010-03-25
- Published: 2012-05-03
- Granted: 2014-07-22
- Earliest claimed: 2009-03-25
- Assignees: Georgia Tech Research Corporation
- Applicants: Philip J. Santangelo
- Inventors: Philip J. Santangelo
- Web page: https://patentgrep.com/patent/US8785615B2

## CPC

- C07H 21/00: Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
- C12Q 1/682: Signal amplification
- C12Q 1/6841: In situ hybridisation
- C07H 21/02: with ribosyl as saccharide radical
- C07H 21/04: with deoxyribosyl as saccharide radical
- C07H: SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS (derivatives of aldonic or saccharic acids C07C, C07D; aldonic acids, saccharic acids C07C59/105, C07C59/285; cyanohydrins C07C255/16; glycals C07D; compounds of unknown constitution C07G; polysaccharides, derivatives thereof C08B; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; sugar industry C13)
- 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

## Abstract

The various embodiments of the present disclosure relate generally to single molecule sensitive probes for detecting RNA, and more particularly to multivalent fluorescent probes for detecting a single molecule of RNA in a cell. The present invention includes a RNA imaging probe comprising: a multivalent core comprising a plurality of attachment sites; a plurality of RNA/DNA chimeric oligonucleotides having a specificity for a target RNA, wherein a RNA/DNA chimeric oligonucleotide is bound to an attachment site of the multivalent core; and a plurality of fluorophores, wherein a fluorophore is bound to the RNA/DNA chimeric oligonucleotide.

## Claims

### Claim 1

A biomolecule probe comprising:
a multivalent core comprising a plurality of attachment sites;
a plurality of RNA/DNA chimeric linear oligonucleotides having specificity for a target, wherein the plurality of RNA/DNA chimeric linear oligonucleotides are each bound to an attachment site of the multivalent core; and
a plurality of reporter molecules, wherein each reporter molecule is bound to the RNA/DNA chimeric linear oligonucleotide.

### Claim 2 (depends on 1)

The biomolecule probe of claim 1, wherein a ratio of the plurality of RNA/DNA chimeric linear oligonucleotides to the plurality of attachment sites on the multivalent core is equal to or less than about 1.

### Claim 3 (depends on 1)

The biomolecule probe of claim 1, wherein a ratio of reporter molecule to plurality of RNA/DNA chimeric linear oligonucleotides is greater than or equal to about 1.

### Claim 4 (depends on 3)

The biomolecule probe of claim 3, wherein the ratio of reporter molecule to the plurality of RNA/DNA chimeric linear oligonucleotides is about 3.

### Claim 5 (depends on 4)

The biomolecule probe of claim 4, wherein the target is a RNA.

### Claim 6 (depends on 4)

The biomolecule probe of claim 4, wherein the reporter molecule comprises a fluorophore.

### Claim 7 (depends on 3)

The biomolecule probe of claim 3, wherein the nucleic acid comprises an attachment region, a spacer region, and a hybridization region.

### Claim 8 (depends on 3)

The biomolecule probe of claim 3, wherein multivalent core comprises avidin or a derivative thereof.

### Claim 9 (depends on 3)

The biomolecule probe of claim 3, wherein multivalent core comprises a multivalent polyethylene glycol core.

### Claim 10 (depends on 9)

The biomolecule probe of claim 9, further comprising a peptide, wherein the peptide is bound to an attachment site of the multivalent polyethylene glycol core.

### Claim 11 (depends on 10)

The biomolecule probe of claim 10, wherein the peptide comprises a targeting moiety.

### Claim 12 (depends on 10)

The biomolecule probe of claim 10, wherein the peptide comprises an epitope moiety.

### Claim 13

A RNA imaging probe comprising:
a multivalent core comprising a plurality of attachment sites;
a plurality of RNA/DNA chimeric linear oligonucleotides having a specificity for a plurality of sites on a target RNA, wherein a RNA/DNA chimeric linear oligonucleotide is bound to an attachment site of the multivalent core; and
a plurality of fluorophores, wherein a fluorophore is bound to the RNA/DNA chimeric linear oligonucleotide.

### Claim 14 (depends on 13)

The RNA imaging probe of claim 13, wherein a ratio of fluorophore to RNA/DNA chimeric linear oligonucleotide is greater than 1.

### Claim 15 (depends on 14)

The RNA imaging probe of claim 14, wherein a ratio of fluorophore to RNA/DNA chimeric linear oligonucleotide is greater than or equal to about 3.

### Claim 16 (depends on 13)

The RNA imaging probe of claim 13, wherein the RNA/DNA chimeric linear oligonucleotide comprises an attachment region, a spacer region, and a hybridization region.

### Claim 17 (depends on 16)

The RNA imaging probe of claim 16, wherein multivalent core comprises a tetravalent strepavidin core, and wherein the attachment region of the RNA/DNA chimeric linear oligonucleotide comprises biotin.

### Claim 18 (depends on 16)

The RNA imaging probe of claim 16, wherein multivalent core comprises a multivalent polyethylene glycol core, and wherein the attachment group comprises a 5′ thiol modified 2′-O-methyl uridine.

### Claim 19 (depends on 16)

The RNA imaging probe of claim 16, wherein the spacer region comprises a plurality of deoxythymidines, a plurality of deoxyadenonsines, or combinations thereof.

### Claim 20 (depends on 16)

The RNA imaging probe of claim 16, wherein the RNA/DNA chimeric linear oligonucleotide further comprises a plurality of amino-modified deoxythymidines.

### Claim 21 (depends on 13)

The RNA imaging probe of claim 13 further comprising a targeting moiety.

### Claim 22 (depends on 13)

The RNA imaging probe of claim 13 further comprising an epitope moiety.

### Claim 23 (depends on 13)

A method for detecting a single molecule of RNA using the RNA imaging probe of claim 13 comprising:
delivering an effective amount of the RNA imaging probe to a cell; and
detecting a molecule of a target RNA in a cell.

### Claim 24 (depends on 23)

The method for detecting a single molecule of RNA of claim 23, wherein the effective amount of an RNA imaging probe has a concentration of less than about 1 micromolar (μM).

### Claim 25 (depends on 24)

The method for detecting a single molecule of RNA of claim 24, wherein the effective amount of an effective amount of an RNA imaging probe has a concentration ranging from about 5 nanomolar (nM) to about 30 nanomolar (nM).

### Claim 26 (depends on 23)

The method for detecting a single molecule of RNA of claim 23, wherein the delivering an effective amount of an RNA imaging probe to a cell comprises permeabilizing the cell with streptolysin 0.

### Claim 27 (depends on 23)

The method for detecting a single molecule of RNA of claim 23, wherein the delivering an effective amount of an RNA imaging probe to a cell comprises microinjecting the RNA imaging probe into a cell.

### Claim 28 (depends on 23)

The method for detecting a single molecule of RNA of claim 23, wherein the detecting a molecule of a RNA in a cell comprises visualizing the association of the RNA imaging probe with the molecule of a target RNA in a cell through microscopy.

### Claim 29 (depends on 23)

The method for detecting a single molecule of RNA of claim 23, wherein the RNA is a native, non-engineered RNA.

### Claim 30 (depends on 23)

The method for detecting a single molecule of RNA of claim 23, wherein the RNA is a cellular RNA.

### Claim 31 (depends on 30)

The method for detecting a single molecule of RNA of claim 30, further comprising detecting a protein that is associated with the target RNA.

### Claim 32 (depends on 23)

The method for detecting a single molecule of RNA of claim 23, wherein the RNA is a viral RNA.