# US 8,569,002 B2 · Multiplexed luciferase reporter assay systems

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- Number: US 8,569,002 B2
- Document ID: US8569002B2
- Status: Granted
- Kind: B2
- Application number: 12/908,754
- Filed: 2010-10-20
- Published: 2012-04-26
- Granted: 2013-10-29
- Earliest claimed: 2010-10-20
- Assignees: Board of Regents, The University of Texas System
- Applicants: Lawrence Lum; Ozlem Kulak
- Inventors: Lawrence Lum; Ozlem Kulak
- Web page: https://patentgrep.com/patent/US8569002B2

## CPC

- C12Q 1/66: involving luciferase
- 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

Inhibitors of luciferase enzymes are disclosed and find use in multiplexed assays using multiple luciferases and multiple inhibitors, in both in vitro and in vivo embodiments.

## Claims

### Claim 1

A method of detecting a plurality of distinct luciferase molecules comprising:
(a) providing a plurality of distinct luciferase molecules in combination with substrates therefor, wherein said plurality of distinct luciferase molecules includes at least renilla luciferase and firefly luciferase;
(b) providing a plurality of distinct luciferase inhibitors, at least one of which selectively inhibits renilla luciferase and one of which selectively inhibits firefly luciferase molecules;
(c) sequentially measuring chemiluminescence from said plurality of distinct luciferase molecules prior to and after combination of said plurality of distinct luciferase inhibitors with said plurality of distinct luciferase molecules,
whereby the addition of said renilla luciferase inhibitor permits the detection of chemiluminescence from non-renilla luciferase molecules, and the addition of said firefly luciferase inhibitor permits the detection of chemiluminescence from non-firefly luciferase molecules.

### Claim 2 (depends on 1)

The method of claim 1, wherein said plurality of distinct luciferase inhibitors are provided sequentially.

### Claim 3 (depends on 1)

The method of claim 1, wherein said plurality of distinct luciferase inhibitors are provided simultaneously.

### Claim 4 (depends on 1)

The method of claim 1, wherein said plurality of distinct luciferase molecules comprises 2, 3, 4, 5 or 6 distinct luciferase molecules,

### Claim 5 (depends on 1)

The method of claim 1, wherein said plurality of distinct luciferase inhibitors comprises 2, 3, or 4 distinct luciferase inhibitors.

### Claim 6 (depends on 1)

The method of claim 1, wherein said plurality of distinct luciferase molecules further comprises one or more of gaussia luciferase, cypridina luciferase, gaussia luciferase lacking a secretion signal sequence or having an endoplasmic retention sequence, or cypridina luciferase lacking a secretion signal sequence or having an endoplasmic retention sequence.

### Claim 7 (depends on 1)

The method of claim 1, wherein said plurality of luciferases are expressed from cells in culture.

### Claim 8 (depends on 7)

The method of claim 7, further comprising separating said cells from cell medium.

### Claim 9 (depends on 8)

The method of claim 8, further comprising disrupting said cells to form a cell lysate.

### Claim 10 (depends on 1)

The method of claim 1, wherein said substrates comprise two or all three of D-luciferin, oxyluciferin, and coelenterazine.

### Claim 11 (depends on 1)

The method of claim 1. wherein an inhibitor is selective or specific for renilla luciferase.

### Claim 12 (depends on 11)

The method of claim 11, wherein said inhibitor selective or specific for renilla luciferase has the formula:
wherein a is substituted or unsubstituted alkyl (C1-C10), substituted or unsubstituted alkenyl (C1-C10), substituted or unsubstituted alkynyl (C1-C10), substituted or unsubstituted alkyoxy (C1-C10), amido or halide, b is H, substituted or unsubstituted alkyl (C1-C10), substituted or unsubstituted alkyoxy (C1-C10), substituted or unsubstituted alkenyl (C1-C10), substituted or unsubstituted alkynyl (C1-C10), amido or halide, and c is H, substituted or unsubstituted alkyl (C1-C10), substituted or unsubstituted alkyoxy (C1-C10), substituted or unsubstituted alkenyl (C1-C10), substituted or unsubstituted alkynyl (C1-C10), amido or halide.

### Claim 13 (depends on 1)

The method of claim 1, wherein an inhibitor is cross-reactive with renilla luciferase and firefly luciferase.

### Claim 14 (depends on 1)

The method of claim 1, wherein an inhibitor is selective or specific for firefly luciferase.

### Claim 15 (depends on 14)

The method of claim 14, wherein said inhibitor selective or specific for firefly luciferase has the formula:
wherein a is H, substituted or unsubstituted alkyl (C1-C10), substituted or unsubstituted alkyoxy (C1-C10), substituted or unsubstituted alkenyl (C1-C10), substituted or unsubstituted alkynyl (C1-C10), amido or halide.

### Claim 16 (depends on 1)

The method of claim 1, wherein said method comprises measuring firefly lueiferase, renilla luciferase, and one or more of gaussia luciferase, cypridina luciferase, gaussia luciferase lacking a secretion signal sequence or having an endoplasmic retention sequence, or cypridina luciferase lacking a secretion signal sequence or having an endoplasmic retention sequence.

### Claim 17 (depends on 1)

The method of claim 1, wherein said method is peformed in an intact cell.

### Claim 18 (depends on 1)

The method of claim 1, wherein said method is peformed in a cell-free system.