# US 8,569,468 B2 · Nanoplasmonic molecular ruler for nuclease activity and DNA footprinting

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- Number: US 8,569,468 B2
- Document ID: US8569468B2
- Status: Granted
- Kind: B2
- Application number: 12/440,579
- Filed: 2007-09-14
- Published: 2010-12-23
- Granted: 2013-10-29
- Earliest claimed: 2006-09-14
- Assignees: The Regents of the University of California
- Applicants: Fanqing Frank Chen; Gang L. Liu; Luke P. Lee
- Inventors: Fanqing Frank Chen; Gang L. Liu; Luke P. Lee
- Web page: https://patentgrep.com/patent/US8569468B2

## CPC

- C12Q 1/683: involving restriction enzymes, e.g. restriction fragment length polymorphism [RFLP]
- C12Q 1/6825: Nucleic acid detection involving sensors
- C12Q 1/6827: for detection of mutation or polymorphism
- C12Q 1/6834: Enzymatic or biochemical coupling of nucleic acids to a solid phase
- C12Q 1/6876: Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- G01N 21/554: {detecting the surface plasmon resonance of nanostructured metals, e.g. localised surface plasmon resonance}
- 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

This invention provides a nanoplasmonic molecular ruler, which can perform label-free and real-time monitoring of nucleic acid (e.g., DNA) length changes and perform nucleic acid footprinting. In various embodiments the ruler comprises a nucleic acid attached to a nanoparticle, such that changes in the nucleic acid length are detectable using surface plasmon resonance. The nanoplasmonic ruler provides a fast and convenient platform for mapping nucleic acid-protein interactions, for nuclease activity monitoring, and for other footprinting related methods.

## Claims

### Claim 1

A nanoplasmon resonance ruler for calibrating nucleic acid length changes in a plasmon resonance system, said ruler comprising:
a nanoparticle having a nucleic acid attached thereto to form a nanoparticle-nucleic acid conjugate, where said nucleic acid comprises at least a first restriction site and a second restriction site positioned such that the conjugates cleaved at the first restriction site provide plasmon resonance signatures that are distinguishable from the plasmon resonance signatures produced by the nucleic acid conjugates cleaved at second restriction site, and/or the intact conjugate, and wherein the nanoparticle surface is functionalized with a phosphine layer.

### Claim 2

A nanoplasmon resonance ruler for calibrating nucleic acid length changes in a plasmon resonance system, said ruler comprising:
a nanoparticle having a nucleic acid attached thereto to form a nanoparticle-nucleic acid conjugate, where said nucleic acid comprises at least a first restriction site and a second restriction site positioned such that the conjugates cleaved at the first restriction site provide plasmon resonance signatures that are distinguishable from the plasmon resonance signatures produced by the nucleic acid conjugates cleaved at second restriction site, and/or the intact conjugate, and wherein the nanoparticle surface is silica coated and functionalized using amino-silane molecules.

### Claim 3

A nanoplasmon resonance ruler for calibrating nucleic acid length changes in a plasmon resonance system, said ruler comprising:
a nanoparticle having a nucleic acid attached thereto to form a nanoparticle-nucleic acid conjugate, where said nucleic acid comprises at least a first restriction site and a second restriction site positioned such that the conjugates cleaved at the first restriction site provide plasmon resonance signatures that are distinguishable from the plasmon resonance signatures produced by the nucleic acid conjugates cleaved at second restriction site, and/or the intact conjugate, and wherein the conjugate comprises from about 10 to about 10,000 nucleic acid molecules per nanoparticle.

### Claim 4

A nanoplasmon resonance ruler for calibrating nucleic acid length changes in a plasmon resonance system, said ruler comprising:
a nanoparticle having a nucleic acid attached thereto to form a nanoparticle-nucleic acid conjugate, where said nucleic acid comprises at least a first restriction site and a second restriction site positioned such that the conjugates cleaved at the first restriction site provide plasmon resonance signatures that are distinguishable from the plasmon resonance signatures produced by the nucleic acid conjugates cleaved at second restriction site, and/or the intact conjugate, and wherein the conjugate is immobilized on a substrate appropriate for surface plasmon resonance.

### Claim 5

A nanoplasmon resonance ruler for calibrating nucleic acid length changes in a plasmon resonance system, said ruler comprising:
a nanoparticle having a nucleic acid attached thereto to form a nanoparticle-nucleic acid conjugate, where said nucleic acid comprises at least a first restriction site and a second restriction site positioned such that the conjugates at the first restriction site provide plasmon resonance signatures that are distinguishable from the plasmon resonance signatures produced by the nucleic acid conjugates cleaved at second restriction site, and/or the intact conjugate, and wherein the conjugate is electrostatically immobilized on a glass, quartz, or ceramic surface.

### Claim 6 (depends on 1, 2, 3, 4)

The nanoplasmon resonance ruler according to any one of claims 1, 2, 3, 4, and 5, wherein said nucleic acid is a double-stranded DNA, or a double-stranded RNA.

### Claim 7 (depends on 6)

The nanoplasmon ruler of claim 6, wherein said nucleic acid is a double-stranded nucleic acid comprising a first strand attached to said nanoparticle and a second strand hybridized to said first strand, but not otherwise attached to said nanoparticle.

### Claim 8 (depends on 1, 2, 3, 4)

The nanoplasmon resonance ruler according to any one of claims 1, 2, 3, 4, and 5, wherein said nucleic acid comprises at least three different restriction sites.

### Claim 9 (depends on 1, 2, 3, 4)

The nanoplasmon resonance ruler according to any one of claims 1, 2, 3, 4, and 5, wherein said nucleic acid further comprises a protein binding site.

### Claim 10 (depends on 1, 2, 3, 4)

The nanoplasmon resonance ruler according to any one of claims 1, 2, 3, 4, and 5, wherein said nucleic acid ranges in length from 3 nucleotides to about 500 nucleotides.

### Claim 11 (depends on 1, 2, 3, 4)

The nanoplasmon resonance ruler according to any one of claims 1, 2, 3, 4, and 5, wherein the nanoparticle is selected from the group consisting of a nanosphere, a nanocrescent, a nanowire, a nanohorn, a nanotube, a nanopyramid, a nanorod, a nanotetrepod, a single- or multi-layered nanodisk, and a nanohorn.

### Claim 12 (depends on 1, 2, 3, 4)

The nanoplasmon resonance ruler according to any one of claims 1, 2, 3, 4, and 5, wherein the nanoparticle comprises a metal or semiconductor material.

### Claim 13 (depends on 1, 2, 3, 4)

The nanoplasmon resonance ruler according to any one of claims 1, 2, 3, 4, and 5, wherein the nanoparticle comprises a material selected from the group consisting of a noble metal, a noble metal alloy, a noble metal composite.

### Claim 14 (depends on 1, 2, 3, 4)

The nanoplasmon resonance ruler according to any one of claims 1, 2, 3, 4, and 5, wherein the nanoparticle comprises a material selected from the group consisting of gold, gold alloy, silver, silver alloy, copper, copper alloy, platinum, platinum alloy, CdSe semiconductor, CdS semiconductor, CdSe coated with ZnS, magnetic colloidal materials, ZnS, ZnO, TiO2, AgI, AgBr, HgI2, PbS, PbSe, ZnTe, CdTe, In2S3, In2Se3, Cd3P2, Cd3As2, InAs, and GaAs.

### Claim 15 (depends on 1, 2, 3, 4)

The nanoplasmon resonance ruler according to any one of claims 1, 2, 3, 4, and 5, wherein the nanoparticle material comprises silver.

### Claim 16 (depends on 1, 2, 3, 4)

The nanoplasmon resonance ruler according to any one of claims 1, 2, 3, 4, and 5, wherein the nanoparticle material comprises gold.

### Claim 17 (depends on 1, 2, 3, 4)

The nanoplasmon resonance ruler according to any one of claims 1, 2, 3, 4, and 5, wherein the nanoparticle size ranges from about 5 nm to about 150 nm.