Monomeric NIR Fluorescent Proteins Brightness Engineering
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Solution Overview
Problem
Current monomeric near-infrared (NIR) fluorescent proteins derived from bacterial phytochromes face limitations in brightness, stability, and oligomerization, hindering their use in deep-tissue imaging and biosensing applications, particularly due to dimerization issues and reliance on external BV supply.
Innovation Solution
Development of bright, spectrally distinct monomeric NIR fluorescent proteins (miRFPs) that rely on endogenous BV for fluorescence, with specific amino acid mutations to prevent dimerization and enhance brightness, allowing for use in protein fusions, biosensors, and imaging across various scales.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Illumination intensity
If BphP-based NIR FPs are used for deep-tissue imaging, then the imaging depth is improved, but the brightness and stability are insufficient
Solution Approach 1:
The patent applies parameter changes by systematically optimizing amino acid sequences at specific positions (particularly in the C-terminal region) to transform dimeric BphP-based FPs into monomeric variants. This involves changing the oligomeric state parameter and spectral properties parameters to achieve both brightness enhancement and monomeric stability, resolving the contradiction between imaging depth requirements and protein reliability
Solution Approach 2:
The patent creates multiple copy variants of the monomeric NIR FP (including red-shifted variants) with optimized sequences that replicate and improve upon the parental protein's function. These copied and optimized variants maintain the desired monomeric state while enhancing brightness and stability for deep-tissue imaging applications
2Stability of the object's composition
If monomeric NIR FPs are engineered from BphPs, then the oligomerization is reduced, but the brightness is limited
Solution Approach 1:
The patent employs parameter changes by optimizing amino acid sequences at specific positions to simultaneously achieve monomeric stability and enhanced brightness. The engineered variants exhibit both stable monomeric states and improved quantum yields/extinction coefficients, resolving the contradiction between oligomerization control and brightness enhancement
Solution Approach 2:
The patent creates composite fluorescent protein variants by combining optimized amino acid sequences from different BphP sources and engineering specific structural features. These composite monomeric FPs integrate beneficial properties from parental proteins while achieving superior brightness and stable monomeric configuration
3Adaptability or versatility
If spectral distinctness is achieved in NIR FPs, then the multicolor labeling capability is improved, but the brightness and stability are compromised
Solution Approach 1:
The patent applies parameter changes by systematically optimizing amino acid sequences in spectrally distinct variants to maintain or improve brightness and stability despite spectral shifts. The engineered monomeric FPs show that spectral optimization and performance enhancement can be achieved simultaneously through sequence engineering, resolving the contradiction between spectral versatility and protein reliability
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 miRFPs provide high brightness and stability, enabling effective deep-tissue imaging and biosensing without the need for external BV, and can be used in various applications from microscopy to whole-body imaging, while maintaining spectral distinctness for multicolor labeling and biosensor development.
Implementation Method 1
Bright monomeric near-infrared fluorescent proteins engineered from bacterial phytochromes
Implementation Method 2
light absorption results in BV isomerization and conformational changes
Implementation Method 3
light absorption results in BV isomerization and conformational changes of the protein backbone
Data Source
AI summary
Nucleic acid molecules encoding monomeric near-infrared fluorescent proteins, variants and derivatives thereof are provided, as well as proteins and peptides encoded by these nucleic acids. Also provided are proteins that are substantially similar to, or derivatives, homologues, or mutants of, the above-referenced specific proteins. Also provided are fragments of the nucleic acids and the peptides encoded thereby, specifically split fluorescent proteins. In addition, host-cells, stable cell lines and transgenic organisms comprising above-referenced nucleic acid molecules are provided. The invention also refers to methods of making and using monomeric fluorescent proteins derived from bacterial phytochromes. The subject protein and nucleic acid compositions find use in a variety of different applications and methods, particularly for labeling of biomolecules, cells or cell organelles, and for detecting protein-protein interactions. Finally, kits for use in such methods and applications are provided.