Logic-Gated Protein Actuators for Antigen-Proximity Targeting
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Solution Overview
Problem
Existing technologies lack efficient and specific methods for targeting and manipulating proteins within cells based on Boolean logic operations, limiting precise control over protein activity and function.
Innovation Solution
A logic-gated protein actuator system utilizing caged split inteins and antigen-binding domains that undergo protein trans-splicing when specific surface antigens are proximal, allowing for the formation of desired proteins only under defined conditions, enabling Boolean logic gates such as AND, OR, NOT, and combinations thereof.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If traditional protein targeting methods are used, then protein delivery to cells is achieved, but specificity and control over protein activity are insufficient
Solution Approach 1:
The protein actuator system is divided into multiple independent components: caged split intein fragments (N-terminal and C-terminal), antigen-binding domains, and payload proteins. These segments are expressed separately and only assemble into functional actuators when all components are present and properly localized, enabling precise spatial and temporal control while maintaining high specificity for target cell populations.
Solution Approach 2:
The caged split intein fragments are pre-loaded with payload proteins and targeted to specific cell surfaces via antigen-binding domains before activation. The intein fragments are prepared in an inactive caged state and only become catalytically active upon encountering the correct combination of surface antigens, ensuring that protein formation occurs only at the intended location and time.
2Adaptability or versatility
If caged split intein fragments are used for protein trans-splicing, then conditional protein formation is achieved, but the system requires multiple components increasing complexity
Solution Approach 1:
The caged split intein fragments serve multiple functions simultaneously: they act as protein tags for purification, as catalytic domains for trans-splicing, and as structural scaffolds for assembling the complete actuator complex. This multi-functionality reduces the need for separate specialized components, thereby reducing overall system complexity while maintaining versatile conditional protein formation capabilities.
Solution Approach 2:
The invention merges the antigen-binding function, the catalytic activation function, and the payload delivery function into a single integrated actuator complex. The caged split intein fragments are co-expressed with antigen-binding domains and payload proteins, creating a unified system that performs multiple operations sequentially without requiring separate molecular machines for each function.
3Manufacturing precision
If protein trans-splicing is activated by antigen proximity, then precise spatial control is achieved, but activation conditions become more restrictive
Solution Approach 1:
The actuator system dynamically responds to the local cellular environment by monitoring the presence and proximity of specific surface antigens. The caged split intein fragments remain inactive until they encounter the correct antigen combination, at which point they undergo conformational changes that activate catalysis. This dynamic activation mechanism allows precise spatial control while adapting to varying antigen expression levels and combinations across different cell types.
Solution Approach 2:
The system changes its activation parameters by recognizing different combinations of surface antigens with varying affinities and stoichiometries. By adjusting which antigens are required for activation and their relative abundances, the system can be tuned to target specific cell populations with high precision while maintaining flexibility to adapt to different biological contexts and disease states.
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 precise and conditional protein formation and activity within cells, allowing for targeted protein functions like toxin delivery or imaging, with high specificity and minimal off-target effects.
Implementation Method 1
The first caged split intein fragment and the second caged split intein fragment can undergo protein trans-splicing to form a protein of interest from the first protein-of-interest fragment and the second protein-of-interest fragment
Data Source
AI summary
A logic-gated protein device in which proximity-gated protein trans-splicing governs formation of an active protein from two otherwise inactive fragments.