WO 2013/016693 A2 - Near-infrared Light-activated Proteins - The Lens

Near-infrared Light-activated Proteins

Published: Jan 31, 2013
Earliest Priority: Jul 27 2011
Family: 8
Cited Works: 66
Cited by: 1
Cites: 2
Sequences: 13
Additional Info: Cited Works Full text Published Sequence

Abstract

Methods and constructs are provided for controlling processes in live animal, plant or microbial organisms via genetically engineered near-infrared light-activated or light- inactivated proteins. In embodiments, these proteins are chimeras comprised of the photosensory modules of bacteriohytochromes and output modules that possess enzymatic activity and/or ability to bind to DNA, RNA, protein. or small molecules. DNA encoding these proteins can be introduced as genes into live animals, plants or microbes, where their activities can be turned on by near-infrared light, controlled by the intensity of light, and turned off by near-infrared light or a different wavelength than the activating light. By using light-inducible proteins, one can regulate diverse cellular processes with high spatial and temporal precision, in a nontoxic manner, often using external light sources. For example, near-infrared light-activated proteins possessing nucleotidyl cyclase, protein kinase, protease, DNA-binding and RNA-binding activities can be used to control signal transduction, cell apoptosis, proliferation, adhesion, differentiation and other cell processes.

Claims

A method for producing photoactive fusion proteins having a desired activity controllable by far-red and/or near-infrared (NIR) light, said method comprising the steps: a. designing one or more homodimeric fusion proteins, each comprising a
photoreceptor protein module and a heterologous. output module, wherein: i. said homodimeric fusion proteins comprise two monomers that each comprise:
(1 ) a photoreceptor module of a bacteriophytochrome; and
(2) a heterologous output module capable of being activated upon homodimerization to perform said desired activity; and ii. said monomers are not active when separated, but are capable of combining to form homodimers that are controllable by NIR light;
wherein designing said fusion proteins comprises identifying candidate output domains based on 3D structures or models, identifying candidate protein fusion sites and estimating lengths of D-helices linking said output modules to said photosensor/ modules;
b. producing a plurality of DNA molecules, each encodings said monomer of a said homodimeric fusion protein that has at least one unique fusion site; c. screening said DNA molecules for their ability to produce homodimeric
photoactive fusion proteins capable of performing said desired activity by a method comprising:
i. transforming a designed test organism with a plurality of different said DNA molecules such that different said fusion proteins are expressed in each test organism;
ii. allowing the expressed fusion proteins to bind bacteriophytochrome chromophore and form homodimeric proteins; and iii. applying selected wavelengths of NI light to said transformed
organisms and determining the level of said desired activity of said
45 fusion proteins in said organisms in the presence and absence of said selected wavelengths of light;
wherein the level of said desired activity of said fusion proteins is controllable by NIR light when the level of said desired activity is changed by the presence and/or absence of NIR light having said selected wavelengths.
The method of claim 1 for producing fusion proteins with enhanced controllability by NIR light, wherein such enhanced controllability exists when said fusion proteins have high ratios of activity in the light versus dark or vice versa.
The method of claim 1 also comprising transforming said test organisms with DNA encoding a heme oxygenase gene capable of being expressed in said test organisms to produce a biliverdin IXD chromophore.
The method of claim 3 wherein said test organisms do not comprise an endogenous chromophore.
The method of claim 1 wherein said test organisms comprise, an endogenous;
chromophore.
The method of claim 1 also comprising modifying the design of said fusion proteins that are controllable by NIR light to produce additional candidate fusion proteins by designing additional fusion sites and linkers for said fusion proteins and repeating the steps of producing DNA encoding the additional fusion proteins, transforming suitable organisms with this DNA, expressing the DNA, and screening the resultant fusion proteins for additional fusion proteins controllable by NIR light.
The method of claim 6 wherein said linker lengths are increased or decreased by the length of one or more helical turns to produce said additional candidate fusion proteins.
The method of claim 7 wherein said linker lengths are increased or decreased by three or four amino acids.
The method of claim 1 wherein said fusion proteins controllable by NIR light, or additional fusion proteins controllable by NIR light produced by increasing or decreasing their linker lengths, are mutagenized to create further candidate fusion proteins controllable
46 by NIR light, and said screen steps are repeated to screen for further photoactivated fusion proteins.
The method of claim 1 wherein said bacteriophytochrome photorece tor module is from the BphG1 protein from Rhodobacter sphaeroides.
The method of claim 1 wherein said suitable organism for expression of said fusion protein is a cultured organism selected from the group consisting of E. coli, yeast plant, or mammalian cells selected or modified so as to detectiably exhibit the level of activity of said expressed fusion protein controllable by the presence or absence of NIR light.
The method of claim 1 wherein said fusion protein is a light-responsive nucleotidyl cyclase or light-responsive uncleavable procaspase-3.
The method of claim 1 wherein said desired activity is increased by the application of NIR light of a selected wavelength.
The method of claim 1 wherein said desired activity is decreased by the application of NIR light of a selected wavelength.
The method of claim 1 wherein said desired activity is gradually decreased or gradually increased by ceasing to apply NIR light of a selected wavelength.
The method of claim 1 wherein said desired activity is immediately increased or decreased by the application of NIR light of a selected wavelength.
A homodimeric fusion protein controllable by NIR light, said fusion protein comprising a photoreceptor module comprising a bacteriophytochrome and a heterologous output module capable of producing a desired activity.
The homodimeric fusion protein of claim 17 which is a lights-activated nucleotidyl cyclase or light-activated uncleavable procaspase-3.
A recombinant DNA molecule encoding the homodimeric fusion protein of claim 17.
A method for controlling an in vivo process in a host which is a living cell or organism comprising:
a. introducing into the cell or organism a DNA sequence encoding a
homodimeric fusion protein comprising a photoreceptor module comprising a
47 bacteriophytochrome and a heterologous output module capable of modulating said process;
b. allowing said fusion protein to be expressed in said host; and
c. applying NIR light of a selected wavelength to the host or preventing NIR light of a selected wavelength from contacting the host; thereby modulating the process under control of NIR light.
The method of claim 20 wherein said process is selected from the group consisting of metabolic processes, signal transduction, cell apoptosis, cell proliferation, cell adhesion, and cell differentiation.
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