Engineered RNA-Binding Proteins for In Vivo Protein Tethering
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Solution Overview
Problem
Current techniques lack effective methods for determining the function of RNA-binding proteins in vivo, particularly in plants, due to the absence of reliable protein-RNA artificial tethering tools.
Innovation Solution
Development of engineered proteins that can tether a protein of interest to a target RNA molecule using an RNA-binding polypeptide with specific binding domains, allowing for the modulation and study of post-transcriptional regulation of RNAs.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If in vitro assays with heterologous expression and purification are used to determine RNA-binding protein function, then protein function can be studied, but the complexity and laborious nature of the process increases and in vivo relevance is lost
Solution Approach 1:
The patent introduces engineered tethering proteins as intermediary molecules that bridge the protein of interest and the target RNA. These tethering proteins contain RNA-binding domains that specifically bind to the target RNA, thereby mediating the interaction between the protein of interest and RNA in a controlled manner, simplifying the assay process while maintaining in vivo relevance
Solution Approach 2:
The patent segments the functional elements into separate components: the protein of interest, the tethering protein with RNA-binding domain, and the target RNA with recognition sequence. This segmentation allows independent optimization and expression of each component, reducing the overall complexity of the assay system
2Measurement precision
If mutation of the protein of interest is used to determine function, then functional analysis is possible, but the lack of alternative methods limits the ability to study RNA-binding protein mechanisms
Solution Approach 1:
The tethering protein acts as an intermediary that enables functional analysis without requiring protein mutation. The RNA-binding domain of the tethering protein binds to a specific recognition sequence on the target RNA, thereby mediating the interaction and allowing functional study through controlled tethering rather than genetic mutation
Solution Approach 2:
The patent changes the experimental parameter from protein mutation to controlled protein-RNA tethering. By using engineered tethering proteins with specific RNA-binding domains that recognize specific sequences, the method allows functional analysis through parameter control (tethering strength, specificity) rather than genetic modification
3Reliability
If reliable protein-RNA artificial tethering tools are developed, then in vivo function determination becomes possible, but the development and validation of such tools remains challenging
Solution Approach 1:
The patent designs tethering proteins with universal features: a standardized protein of interest fusion, a conserved RNA-binding domain (such as RRM, KH, or zinc finger), and compatibility with various target RNAs containing recognition sequences. This universality allows the same tethering approach to be applied to different proteins and RNAs, simplifying tool development and validation
Solution Approach 2:
The patent uses engineered tethering proteins that copy the essential function of natural RNA-binding proteins (specific RNA binding) while adding the capability for controlled tethering. The tethering proteins replicate the RNA-binding function of natural proteins but with enhanced specificity and controllability for experimental purposes
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 the investigation of protein functions in RNA-protein interactions within living cells, facilitating the understanding of RNA sorting pathways and gene expression regulation.
Implementation Method 1
the RNA-binding domain recognizes and specifically binds an RNA recognition sequence in the target RNA molecule
Data Source
AI summary
The instant disclosure provides engineered proteins, constructs for expressing the engineered proteins, and methods of using the engineered proteins and constructs for tethering a protein of interest to an RNA molecule.