# US 2025/0154221 A1 · Voltage indicators for two-photon imaging > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 2025/0154221 A1 - Document ID: US20250154221A1 - Status: Published application - Kind: A1 - Application number: 18/943,722 - Filed: 2024-11-11 - Published: 2025-05-15 - Earliest claimed: 2023-11-10 - Applicants: Howard Hughes Medical Institute - Inventors: Ahmed Abdelfattah; Rosario Valenti; Eric R. Schreiter - Web page: https://patentgrep.com/patent/US20250154221A1 ## CPC - C07K 14/705: Receptors; Cell surface antigens; Cell surface determinants {(tumour specific antigens C07K14/4748)} - 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)} - G01N 21/6458: {Fluorescence microscopy (fluorescence microscopes per se G02B21/0076 and G02B21/16)} - C07K 2319/60: containing spectroscopic/fluorescent detection, e.g. green fluorescent protein [GFP] - G01N 2021/6439: {with indicators, stains, dyes, tags, labels, marks} - 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) - 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 A voltage indicator is described, including a voltage-sensitive rhodopsin domain having mutations to replace canonical counterion charge residues with neutral residues, and a capture protein that binds a fluorescent element, wherein the capture protein is provided together with the voltage-sensitive rhodopsin domain in a fusion protein. ## Claims ### Claim 1 A voltage indicator, comprising: a voltage-sensitive rhodopsin domain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1-26, including a mutation to replace a charged canonical amino acid residue with a neutral residue at one, two, or three residues within a pump or channel core motif of the rhodopsin domain; and a capture protein that covalently or noncovalently binds a fluorescent element that is a fluorescent protein, or a fluorescent dye; wherein the capture protein is provided together with the voltage-sensitive rhodopsin domain in a fusion protein. ### Claim 2 (depends on 1) The voltage indicator of claim 1, wherein the rhodopsin domain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 31-74. ### Claim 3 (depends on 1) The voltage indicator of claim 1, wherein the rhodopsin domain comprises the amino acid sequence of SEQ ID NO: 1, including one or two mutations to replace charged canonical amino acid residues at counterion positions with neutral residues. ### Claim 4 (depends on 3) The voltage indicator of claim 3, wherein the one or two mutations are selected from the group consisting of: D89X, D100X, and E206X, where X is a neutral residue. ### Claim 5 (depends on 1) The voltage indicator of claim 1, wherein the rhodopsin domain comprises the amino acid sequence of SEQ ID NO: 31. ### Claim 6 (depends on 1) The voltage indicator of claim 1, wherein the rhodopsin domain comprises the amino acid sequence of SEQ ID NO: 2, including one or two mutations to replace charged canonical amino acid residues at counterion positions with neutral residues. ### Claim 7 (depends on 6) The voltage indicator of claim 6, wherein the mutation is selected from the group consisting of: D136X, D147X, and E254X, where X is a neutral residue. ### Claim 8 (depends on 1) The voltage indicator of claim 1, wherein the rhodopsin domain comprises the amino acid sequence of SEQ ID NO: 32. ### Claim 9 (depends on 1) The voltage indicator of claim 1, wherein the capture protein is selected from the group consisting of a self-labeling protein and avidin. ### Claim 10 (depends on 1) The voltage indicator of claim 1, wherein the capture protein is a self-labeling protein. ### Claim 11 (depends on 10) The voltage indicator of claim 10, wherein the fluorescent element is the fluorescent dye, and the self-labeling protein binds the fluorescent dye via a self-labeling protein ligand. ### Claim 12 (depends on 1) The voltage indicator of claim 1, further comprising a 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid linker between the voltage-sensitive rhodopsin domain and the capture protein. ### Claim 13 (depends on 1) The voltage indicator of claim 1, and further comprising a fluorescent element. ### Claim 14 (depends on 1) The voltage indicator of claim 1, further comprising a targeting sequence and/or an endoplasmic reticulum (ER) export sequence. ### Claim 15 A nucleotide molecule, comprising a nucleotide encoding an amino acid sequence selected from the group consisting of SEQ ID NO: 1-26, including a mutation to replace a charged canonical amino acid residue with a neutral residue at a residue within a pump or channel core motif of the rhodopsin domain. ### Claim 16 (depends on 15) The nucleotide molecule of claim 15, comprising a nucleotide encoding an amino acid selected from the group consisting of SEQ ID NO: 31-74. ### Claim 17 (depends on 15) The nucleotide molecule of claim 15, and further comprising a nucleotide encoding an amino acid sequence for a capture protein. ### Claim 18 (depends on 17) A vector, comprising the nucleotide of claim 17. ### Claim 19 A method of measuring voltage, the method comprising: (i) delivering to a cell a nucleotide encoding an amino acid molecule comprising a voltage-sensitive rhodopsin domain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1-26, including a mutation to replace a charged canonical amino acid residue with a neutral residue at a residue within a pump or channel core motif of the rhodopsin domain; and a capture protein that covalently or noncovalently binds a fluorescent element that is a fluorescent protein, or a fluorescent dye; wherein the capture protein is provided together with the voltage-sensitive rhodopsin domain in a fusion protein; and (ii) delivering to the cell a fluorescent element that is a fluorescent protein or a fluorescent dye, wherein when the fluorescent element is a fluorescent protein, it is provided together with the capture protein and the voltage-sensitive rhodopsin domain in a fusion protein; and when the fluorescent element is a fluorescent dye, it is attached to a ligand of the capture protein; and (iii) determining changes in fluorescence of the fluorescent element, as an measurement of voltage changes in the cell, wherein an increase in membrane potential lead to an increase in fluorescence. ### Claim 20 (depends on 19) The method of claim 19, wherein the microscope is a two-photon microscope.