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US-12624347-B2 - Wireless activation of channelrhodopsin via in situ self-assembly of semiconductor quantum dots at the plasma membrane

US12624347B2US 12624347 B2US12624347 B2US 12624347B2US-12624347-B2

Abstract

A quantum dot (QD)-rhodopsin bioconjugate system uses Förster resonance energy transfer (FRET)-mediated induction of cellular membrane depolarization via optical activation of ion channel proteins channelrhodopsin (ChR).

Inventors

  • Okhil K. Nag
  • Megan E. Muroski
  • Michael H. Stewart
  • Alexander Efros
  • Scott Walper
  • James Delehanty
  • Eunkeu Oh

Assignees

  • THE GOVERNMENT OF THE UNITED STATES OF AMERICA, AS REPRESENTED BY THE SECRETARY OF THE NAVY

Dates

Publication Date
20260512
Application Date
20220916

Claims (6)

  1. 1 . A method of cell depolarization comprising: providing a cell expressing a channelrhodopsin protein comprising a polyhistidine domain on an extracellular portion thereof; contacting the cell with a quantum dot configured as a FRET donor to the channelrhodopsin and allowing the quantum dot to self-assemble via metal affinity coordination to the polyhistidine domain; and then stimulating the quantum dot to emit light, thereby optically activating the channelrhodopsin via a FRET process to depolarize the cell.
  2. 2 . The method of claim 1 , wherein said channelrhodopsin protein comprises SEQ ID NO: 1.
  3. 3 . The method of claim 1 , wherein said stimulating comprises two-photon illumination.
  4. 4 . The method of claim 1 , wherein said quantum dot comprises a colloidally stable CdSe/ZnS core/shell.
  5. 5 . The method of claim 4 , wherein said quantum dot comprises a zwitterionic ligand.
  6. 6 . The method of claim 1 , wherein the polyhistidine domain is positioned at an N-terminus of the protein and a far red fluorescent protein (iRFP682) is positioned at a C-terminus of the protein.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 63/246,111 filed on Sep. 20, 2021, incorporated herein by reference in its entirety. FEDERALLY-SPONSORED RESEARCH AND DEVELOPMENT The United States Government has ownership rights in this invention. Licensing inquiries may be directed to Office of Technology Transfer, US Naval Research Laboratory, Code 1004, Washington, D.C. 20375, USA; +1.202.767.7230; techtran@nrl.navy.mil, referencing NC 114119. INCORPORATION BY REFERENCE This Application incorporates by reference the Sequence Listing XML file submitted via the patent office electronic filing system having the file name “114119-US2.XML” and created on Sep. 16, 2022 with a file size of 2,910 bytes. STATEMENT REGARDING PRIOR DISCLOSURES BY THE INVENTOR(S) A prior disclosure, Nag, 0. K., Muroski, M. E., Field, L. D., Stewart, M. H., Oh, E., Susumu, K., Spangler, J. R., Walper, S. A., Delehanty, J. B., “In Situ Self-Assembly of Quantum Dots at the Plasma Membrane Mediates Energy Transfer-Based Activation of Channelrhodopsin,” Part. Part. Syst. Charact. 2021, 2100053, was made by one or more of the inventors with other named authors. Those other authors who are not named as inventors of this patent application were working under the direction and supervision of at least one of the inventors. BACKGROUND The therapeutic modulation of membrane potential of electrically excitable cells has gained significant interest in recent years as membrane potential regulates proliferation, migration, and communication among neuronal cells and contraction in muscle cells. Furthermore, normal cells exhibit resting membrane potentials between −60 to −100 mV as compared to cancer cells, which have elevated resting membrane potentials between −55 mV to +5 mV. Exploiting the large differences in membrane potential provides a unique opportunity for researchers to target therapeutics specifically to cancer cells. However, at the single cell level, the functional control of ionic currents and ion channel activity using nanoscale devices has yet to be elucidated. Optically activatable ion channel proteins in the cellular plasma membrane, such as channelrhodopsin (ChR), play critical rules in maintaining the membrane potential of excitable cells. ChRs are highly conserved seven transmembrane domain proteins containing an 11-cis-retinal chromophore that undergoes light-activated photoisomerization (typically with blue light) to open the channel and allow the passage of ions (Na+, K+, Ca2+) into the cell. Recently, the development of genetically-encoded ChRs have opened up the possibility of light-activated control of membrane potential (optogenetics), but this requires cellular transfection with exogenous nucleic acids which poses challenges for their use in vivo. Further, genetically-encoded ChRs suffer from bleaching/inactivation, short lifetime in the activated state, and small two-photon absorption cross sections (˜102 GM units) which limits their ability to be excited with longer wavelength light in the optical tissue transparency window (˜700-1100 nm) where water and hemoglobin have minimal absorbance. A need exists to overcome these limitations of ChRs. BRIEF SUMMARY In one embodiment, method of cell depolarization includes providing a cell expressing a channelrhodopsin protein comprising a polyhistidine domain on an extracellular portion thereof; contacting the cell with a quantum dot configured as a FRET donor to the channelrhodopsin and allowing the quantum dot to self-assemble via metal affinity coordination to the polyhistidine domain; and then stimulating the quantum dot to emit light, thereby optically activating the channelrhodopsin via a FRET process to depolarize the cell. In one aspect, the channelrhodopsin protein comprises SEQ ID NO: 1. BRIEF DESCRIPTION OF THE DRAWINGS The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee. FIG. 1 schematically depicts the photoactivation of channelrhodopsin acceptor using a membrane-tethered quantum dot (QD) as a donor. A hydrophilic QD (as Förster resonance energy transfer donor) is self-assembled to the exofacial side of channelrhodopsin C1V1 (His6-ChR-C1V1) via an N-terminal polyhistidine tract (red) that mediates metal affinity coordination to the ZnS shell of the QD. The QD donor self-assembles specifically to the ChR-C1V1 and is in close proximity to the 11-cis-retinal acceptor (orange dot). The QD acts as a light harvesting transducer to photoactivate the retinal moiety which undergoes photoisomerization, resulting in opening of the channel and the influx of Na+, K+, and Ca2+ ions. Depolarization of membrane potential is iteratively controlled by excitation of the QD. In all experiments herein, His6-ChR-C1V1 was expressed as a fusion wit