# US 12,601,016 B2 · Genetically encoded fluorescent indicators under optogenetic control and uses thereof

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- Number: US 12,601,016 B2
- Document ID: US12601016B2
- Status: Granted
- Kind: B2
- Application number: 17/289,946
- Filed: 2019-10-31
- Published: 2022-01-20
- Granted: 2026-04-14
- Earliest claimed: 2018-10-31
- Assignees: LumiSTAR Biotechnology, Inc.; The Governors of the University of Alberta
- Applicants: Lumistar Biotechnology, Inc.; The Governors of the University of Alberta
- Inventors: Yu-fen Chang; Yi Shen; Yong Qian; Min-wen Chung; Robert Campbell
- Web page: https://patentgrep.com/patent/US12601016B2

## CPC

- C07K 14/415: from plants
- C12Q 1/6897: involving reporter genes operably linked to promoters
- G01N 21/6428: {Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" (in vivo A61B5/00; immunoassay G01N33/53)}
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)
- 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

Disclosed are methods and compositions for real-time monitoring cellular calcium ion dynamics by red-shifted genetically encoded indicators under optogenetic control, in which millisecond-timescale of temporal control of optical activation or inactivation and signal recording can be achieved. The methods include artifact-free functional imaging in conjunction with optogenetic tools for studying cellular physiology, signal transduction and neuronal activity. Thus, all-optical and non-invasive approaches for drug screening, toxicity testing and assessment of cell functions may be provided.

## Claims

### Claim 1

A method for examining a function of a cell in a sample, the method comprising:
(a) expressing a red-shifted genetically encoded Ca2+ indicator (GECI) comprising the amino acid sequence of SEQ ID NO: 5 in the cell;
(b) obtaining an optical signal from the red-shifted GECI in response to a stimulation; and
(c) characterizing the sample by analyzing the optical signal responses in order to determine functional properties of the sample.

### Claim 2 (depends on 1)

The method of claim 1, wherein the sample is a two- or three-dimensional (2D or 3D) cell culture, cellular organelles, stem cells or a mammalian blood plasma.

### Claim 3 (depends on 1)

The method of claim 1, wherein the sample characterization step (c) comprises determining cell response to exposure to an agent.

### Claim 4 (depends on 1)

The method of claim 1, wherein analyzing the optical signal responses comprises characterization of calcium influx of the sample.

### Claim 5 (depends on 1)

The method of claim 1, wherein the cell is transformed or transfected with a nucleic acid encoding the red-shifted GECI.

### Claim 6 (depends on 5)

The method of claim 5, wherein the nucleic acid has the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 2.