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AssigneesAlbert Einstein College of Medicine, University of Helsinki
InventorsVladislav V. Verkhusha, Anna V. Leopold
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This disclosure provides a generalized approach for engineering receptor tyrosine kinases (RTKs) optically controlled with far-red light, named eDrRTKs, by targeting a bacterial phytochrome (e.g., DrBphP) to the cell surface and allowing its light-induced conformational changes to be transmitted across the plasma membrane via transmembrane helices to intracellular RTK domains. The ability to activate eDrRTKs with far-red light enabled cross-talk free spectral multiplexing with fluorescent probes operating in a shorter spectral range, allowing for all-optical assays, including non-invasive stimulation in the brain of a live animal. The disclosed engineering approach can be applied beyond RTKs to any membrane receptors, channels, surface antigens, or membrane antibodies that share high similarity with RTKs in mechanisms of their activation.
A polynucleotide encoding a chimeric polypeptide comprising:
The polynucleotide of any one of the preceding claims, wherein the light-responsive polypeptide comprises an N-terminal photosensory core module (PCM) of Deinococcus radiodurance bacteriophytochrome (DrBphP-PCM) or a variant thereof.
The polynucleotide any one of the preceding claims, wherein the light-responsive polypeptide comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:
The polynucleotide of any one of the preceding claims, wherein the receptor is a receptor tyrosine kinase (RTK).
The polynucleotide of any one of the preceding claims, wherein the transmembrane domain further comprises one or more repeats of Tyrosine (Y)-Phenylalanine (F).
The polynucleotide of any one of the preceding claims, wherein the transmembrane domain comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of any one of SEQ ID NOs:
The polynucleotide of any one of the preceding claims, wherein the intracellular domain is a tyrosine kinase domain of a second receptor tyrosine kinase.
The polynucleotide of any one of the preceding claims, wherein the intracellular domain comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of any one of SEQ ID NOs:
The polynucleotide of any one of the preceding claims, wherein the chimeric polypeptide further comprises a signaling peptide linked to the N-terminus of the light-responsive polypeptide.
The polynucleotide of any one of the preceding claims, wherein the chimeric polypeptide further comprises a Golgi-export peptide linked to the C-terminus of the intracellular domain.
The polynucleotide of any one of the preceding claims, wherein:
The polynucleotide of any one of the preceding claims, wherein the chimeric polypeptide comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of any one of SEQ ID NOs:
The polynucleotide of any one of the preceding claims, wherein the wavelength is in far-red or near-infrared spectrum.
The polynucleotide of any one of the preceding claims, wherein the wavelength is from about 650 nm to about 900 nm.
A polypeptide encoded by the polynucleotide of any one of the preceding claims.
The method of 41, wherein the receptor is a receptor tyrosine kinase (RTK).
Dependencies are inferred from the claim text.