Non-covalent Genome Targeting via Segmented Protein Assembly
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Solution Overview
Problem
Current methods for site-directed genome modification, such as fusion proteins, often result in incorrect protein folding, compromised function, and limited targeting specificity due to covalent linkages, and are restricted by viral delivery capacity for large fusion proteins.
Innovation Solution
The use of non-covalent linkages between DNA localization components and effector molecules, such as antibody fragments or protein binding domains, allows for temporary and specific interactions that enable precise genome modification without the limitations of covalent fusion.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Stability of the object's composition
If covalent fusion of DNA binding domain and effector molecule is used, then stable linkage is achieved, but protein folding and function are compromised
Solution Approach 1:
The system divides the fusion protein into two separate components: a DNA-binding component and an effector component. These components are delivered separately and assemble non-covalently at the target site, avoiding the folding and functional issues caused by covalent fusion while maintaining stable interaction at the target location.
Solution Approach 2:
The patent introduces a non-covalent interaction interface as an intermediary between the DNA-binding component and effector component. This intermediary allows stable association at the target site without the permanent constraints of covalent bonding, enabling proper folding and function of both components.
2Adaptability or versatility
If fusion protein strategy is used, then DNA binding and effector function are combined, but steric hindrance blocks protein function
Solution Approach 1:
By separating the DNA-binding component and effector component into distinct molecules that assemble non-covalently, the system eliminates steric hindrance caused by covalent fusion. Each component can fold and function independently while maintaining spatial proximity at the target site through non-covalent interaction.
3Adaptability or versatility
If large fusion protein is created, then comprehensive function is achieved, but viral delivery capacity is exceeded
Solution Approach 1:
The system divides the functional elements into separate DNA components that can be delivered independently via viral vectors. This segmentation reduces the size of each individual DNA construct to fit within viral delivery capacity while maintaining the comprehensive functional capability through non-covalent assembly of the separate components at the target site.
Data Source
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Figure 2A~2B
AI summary
Disclosed are compositions and methods for directing proteins to specific loci in the genome and uses thereof. In one aspect, the disclosed methods allow for directing proteins to specific loci in the genome of an organism, including the steps of providing a DNA localization component and an effector molecule, wherein the DNA localization component and the effector molecule are capable of being operatively linked via a non-covalent linkage.