Split Peroxidase EM Reporter for Fixed Cell Imaging
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Solution Overview
Problem
Current genetically encoded reporters for electron microscopy, such as Horse Radish Peroxidase (HRP) and miniSOG, are limited in their ability to image cellular compartments beyond the secretory pathway and are prone to inactivation due to strong fixation methods, restricting their application in electron microscopy.
Innovation Solution
Development of split peroxidases, which are enzymatically active in various subcellular compartments and remain active after membrane-preserving fixation, allowing for the conversion of substrates into detectable signal-releasing products, enabling imaging across multiple cellular compartments.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of operation
If HRP is used as a reporter for EM, then it is easy to use and robust, but it is inactive in cellular compartments other than the secretory pathway due to disruption of four disulfide bonds
Solution Approach 1:
The peroxidase enzyme is divided into two separate fragments that can independently function in different cellular compartments. When these fragments are brought together through protein-protein interaction, they reconstitute the active enzyme. This segmentation allows the reporter to be active in compartments where the full enzyme would be inactive, thereby resolving the contradiction between ease of use and cellular compartment adaptability.
Solution Approach 2:
A linker peptide acts as an intermediary that connects the two peroxidase fragments and mediates their interaction. The linker contains a protein-protein interaction motif that brings the fragments into proximity, enabling enzymatic activity. This intermediary mechanism allows the split peroxidase to function in diverse cellular compartments while maintaining robustness and ease of use.
2Measurement precision
If miniSOG is used as an EM reporter, then it can image small fields of view, but it requires laser and blown oxygen making it limited to small fields of view
Solution Approach 1:
The invention changes the biochemical parameters of the imaging system by using a peroxidase-based chemistry that does not require laser excitation or high oxygen concentrations. This parameter change enables the reporter to function in larger fields of view and under physiological conditions, resolving the contradiction between imaging precision and field of view adaptability.
3Measurement precision
If strong fixation is employed in EM, then spatial resolution is improved, but reporters are prone to inactivation
Solution Approach 1:
The split peroxidase system is designed to be resilient to fixation conditions. The enzyme is reconstituted in fixed cells, and the fragments are engineered to remain stable and functional after fixation. This beforehand cushioning approach ensures that the reporter remains active even after strong fixation, resolving the contradiction between spatial resolution and reporter reliability.
4Power
If a whole peroxidase is used, then enzymatic activity is high, but it cannot be targeted to specific subcellular compartments due to disulfide bond disruption
Solution Approach 1:
The peroxidase is segmented into two fragments that can be independently targeted to different subcellular compartments using localization signals. When the fragments co-localize and interact, they reconstitute the active enzyme in situ. This segmentation strategy maintains high enzymatic activity while enabling precise subcellular compartment targeting that would be impossible with the whole enzyme.
Solution Approach 2:
Different fragments of the peroxidase can be equipped with different localization signals that direct them to specific subcellular compartments. This creates local quality differences in the fragments, allowing them to be selectively targeted and reconstituted in specific compartments, thereby resolving the contradiction between enzymatic activity and compartmental adaptability.
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
Enables effective imaging and detection of proteins in a wide range of cellular compartments, including the cytosol and mitochondria, with enhanced sensitivity and robustness, overcoming the limitations of existing reporters.
Implementation Method 1
contacting the sample with a peroxidase substrate to allow conversion of the peroxidase substrate into a product via an enzymatic reaction catalyzed by a reconstituted peroxidase
Implementation Method 2
the product releases a detectable signal
Data Source
AI summary
An imaging method utilizing a split peroxidase is described herein. Imaging methods involve contacting a cell with a split peroxidase and a substrate thereof to allow conversion of a substrate into a product via an enzymatic reaction catalyzed by the reconstitute split peroxidase. Also disclosed herein are split peroxidases, related products and kits.