Biosensors for chloride ions

• US 10,640,766 B2
• Filed: 04/10/2018
• Issued: 05/05/2020
• Est. Priority Date: 04/12/2017
• Status: Active Grant

• Current Assignee: Tempo Biosciences Inc.
• Original Assignee: Tempo Biosciences Inc.
• Inventors: Huang, Angela
• Primary Examiner(s): Landsman, Robert S
• Application Number:
  US15/949,694
• Publication Number:
  US 20190112597A1
• Time in Patent Office: 756 Days
• CPC Class Codes: C07K 14/705 Receptors; Cell surface antigens; Cell surface determinants tumour specific antigens C07K14/4748
• CPC Class Codes: C07K 2319/03 containing a transmembrane segment
• CPC Class Codes: C07K 2319/033 containing a motif for targeting to the internal surface of the plasma membrane, e.g. containing a myristoylation motif
• CPC Class Codes: C07K 2319/05 containing a GOLGI retention signal
• CPC Class Codes: C07K 2319/60 containing spectroscopic/fluorescent detection, e.g. green fluorescent protein [GFP]
• CPC Class Codes: C12N 15/1086 Preparation or screening of expression libraries, e.g. reporter assays
• CPC Class Codes: C12N 15/52 Genes encoding for enzymes or proenzymes
• CPC Class Codes: C12N 15/85 for animal cells
• CPC Class Codes: C12N 2015/859 Animal models comprising reporter system for screening tests
• CPC Class Codes: C12N 5/0606 Pluripotent embryonic cells, e.g. embryonic stem cells [ES] embryonic germ cells C12N5/0611, induced pluripotent stem cells C12N5/0696
• CPC Class Codes: C12N 5/0647 Haematopoietic stem cells; Uncommitted or multipotent progenitors
• CPC Class Codes: C12N 5/0656 Adult fibroblasts
• CPC Class Codes: C12N 5/0696 Artificially induced pluripotent stem cells, e.g. iPS
• CPC Class Codes: G01N 2333/705 Assays involving receptors, cell surface antigens or cell surface determinants
• CPC Class Codes: G01N 33/52 Use of compounds or compositions for colorimetric, spectrophotometric or fluorometric investigation, e.g. use of reagent paper and including single- and multilayer analytical elements immunological elements G01N33/54386; involving labelled immunochemicals G01N33/58; for haemoglobin or occult blood G01N33/72
• CPC Class Codes: G01N 33/68 involving proteins, peptides or amino acids involving lipoproteins G01N33/92
• CPC Class Codes: G01N 33/84 involving inorganic compounds or pH

Abstract

This invention provides biosensors, cell models, and methods of their use for monitoring chloride ion, where the biosensors can include targeting domains, sensing domains and reporting domains. Biosensors can be introduced into cells reprogrammed to represent experimental or pathologic cells of interest, including as detectors of chloride ions, as TempoChloro™ accomplishes.

Claims

1. A chloride ion sensor peptide construct comprising:

a chloride ion sensing domain having a sequence at least 95% identical to SEQ ID NO;

1; and

, a fluorescent reporter domain, wherein the fluorescent reporter domain is adapted to change fluorescent emissions characteristics in response to chloride ion concentration; and

wherein the fluorescent reporter domain has a fluorescent emission range of 500 nm to 700 nm and a fluorescent excitation range of 400 nm to 700 nm.

2. The chloride ion sensor peptide construct of claim 1 wherein the fluorescent reporter comprises a sequence at least 95% identical to SEQ ID NO:

3.

3. The chloride ion sensor peptide construct of claim 1 wherein the chloride ion sensing domain binds to chloride ion.

4. The chloride ion sensor peptide construct of claim 1 further including one or more targeting peptide segments with affinity for a cellular target.

5. The chloride ion sensor peptide construct of claim 4 wherein the targeting peptide segments are one or more of:

an NLS tag, an ER tag, an endosome tag, a human endosome sequence tag, a ciliary tag, a human synapsin tag, and a human synaptic vesicle tag.

6. A chloride ion sensor peptide construct comprising:

a chloride ion sensing domain having a sequence at least 95% identical to SEQ ID NO;

1; and

, a fluorescent reporter domain;

wherein the fluorescent reporter domain is adapted to change fluorescent emissions characteristics in response to chloride ion concentration; and

wherein the fluorescent reporter domain has a fluorescent emission range of 500 nm to 700 nm and a fluorescent excitation range of 400 nm to 700 nm.

7. The chloride ion sensor peptide construct of claim 6 wherein the fluorescent reporter comprises a sequence at least 95% identical to SEQ ID NO:

3.

8. The chloride ion sensor peptide construct of claim 6 wherein the chloride ion sensing domain binds to chloride ion.

9. The chloride ion sensor peptide construct of claim 6 further including one or more targeting peptide segments with affinity for a cellular target.

10. The chloride ion sensor peptide construct of claim 6 wherein the targeting peptide segments are one or more of:

an NLS tag, an ER tag, an endosome tag, a human endosome sequence tag, a ciliary tag, a human synapsin tag, and a human synaptic vesicle tag.