Single-Component NIR Optogenetic System for Gene Regulation
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Solution Overview
Problem
Current NIR optogenetic systems require co-expression of large protein components and exhibit high background noise, limiting their efficiency and practicality for gene transcription control.
Innovation Solution
Development of a single-component IsPadC-PCM-based optogenetic system that utilizes a chimeric polypeptide with a light-responsive domain linked to a DNA binding domain, capable of switching between different oligomeric states in response to far-red and near-infrared light, allowing for precise control of gene transcription without the need for multiple protein components.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If two-component heterodimerization systems (e.g., PhyB-PIF6 or RpBphP1-RpPpsR2) are used for NIR light control, then light-induced gene transcription control is achieved, but device complexity increases and background noise increases
Solution Approach 1:
The patent merges the light-sensing phytochrome domain and the DNA-binding/transcriptional control domain into a single chimeric protein (IsPadC-PCM). This single-component system eliminates the need for separate partner proteins required in traditional heterodimerization systems, thereby reducing device complexity while maintaining NIR light-induced gene transcription control functionality
Solution Approach 2:
The IsPadC-PCM protein performs multiple functions within a single component: it senses NIR light through its phytochrome chromophore, undergoes photoconversion between Pr and Pfr states, and directly binds to DNA through its DNA-binding domain. This multi-functionality replaces the need for multiple specialized proteins in traditional systems
2Reliability
If two-component heterodimerization systems are used, then NIR light control is achieved, but background noise increases
Solution Approach 1:
The patent extracts and eliminates the source of background noise by removing one of the two required protein components. The traditional two-component systems (e.g., PhyB-PIF6) generate background noise through spontaneous dimerization or non-specific interactions in the absence of light. By using a single self-sufficient IsPadC-PCM component with an intrinsically inactive state in the Pr form, the system eliminates this background noise while maintaining specific light-induced activation
3Reliability
If full-length phytochrome proteins are used, then light sensing is achieved, but protein size increases making AAV packaging difficult
Solution Approach 1:
The patent segments the full-length phytochrome protein into its functional core module (PCM) containing the essential phytochrome chromophore and light-sensing domains. This segmented IsPadC-PCM retains the necessary light-sensing capability while significantly reducing protein size compared to full-length phytochromes, making it suitable for AAV packaging and cellular delivery
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
The IsPadC-PCM system achieves high efficiency in gene transcription regulation in mammalian cells, neurons, and mouse tissue, with reduced background noise and improved spectral multiplexing capabilities.
Implementation Method 1
BphPs exist in two interconvertible states, Pr (absorbs at 660-700 nm) and Pfr (absorbs at 740-780 nm). Upon NIR illumination, BphP-bound BV isomerizes
Implementation Method 2
Upon NIR illumination, BphP-bound BV isomerizes via the fourth D-ring rotation around its 15-16 double bond. This Z-E isomerization results in the subsequent structural changes in an N-terminal photosensory core module (PCM)
Data Source
AI summary
This disclosure provides the single-component near-infrared light-controlled IsPadC-PCM-based optogenetic systems for prokaryotic and eukaryotic cells, consisting of an evolved photosensory core module of the Idiomarina sp. bacterial phytochrome, named iLight. The iLight systems are smaller and packable in an adeno-associated virus, as compared to the other near-infrared optogenetic systems based on phytochromes. This disclosure demonstrates the high light-activation efficiency of the developed iLight systems in gene transcription regulation in bacteria, cultured mammalian cells, primary isolated neurons, and living mouse tissue in vivo. The iLight systems also enable crosstalk-free spectral multiplexing with optogenetic systems and fluorescent probes activated or excited by light of the visible spectral range.