TALE RVDs for Epigenetic Base Recognition
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Solution Overview
Problem
Current techniques for gene regulation and editing lack specificity in recognizing and targeting epigenetic modifications such as 5-methylcytosine (5mC), 5-hydroxymethylcytosine (5hmC), and N6-methyladenine (6mA), which are crucial for precise manipulation of gene expression and epigenetic modifications.
Innovation Solution
Development of DNA binding polypeptides and fusion proteins containing specific Repeat-Variable DiResidues (RVDs) that recognize and bind to 5mC, 5hmC, and 6mA, enabling targeted gene activation, editing, and detection by integrating these proteins with functional domains for transcription regulation, genome editing, or epigenetic modification.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional TALE RVDs (NI, NG, HD, NN) are used for DNA recognition, then binding to canonical bases (A, T, C, G) is achieved, but specificity for modified bases (5mC, 5hmC, 6mA) is lost
Solution Approach 1:
The patent applies local quality by modifying specific positions (12 and 13) within the TALE repeat structure to create specialized RVDs that recognize modified bases. Each RVD is locally optimized with specific amino acid combinations (e.g., HA for 5mC, FS for 5hmC, NP for 6mA) while maintaining the overall TALE framework, enabling selective recognition of epigenetic modifications without compromising overall binding capability.
Solution Approach 2:
The patent employs parameter changes by systematically varying the amino acid composition at positions 12 and 13 of the TALE repeats to create different RVDs with distinct recognition specificities. This includes using hydrophobic amino acids (Ala, Val, Leu, Ile) at position 12 combined with various amino acids at position 13 to recognize different modified bases, thereby tuning the recognition parameters to achieve high specificity for 5mC, 5hmC, and 6mA.
2Adaptability or versatility
If TALE proteins are fused with functional domains for gene regulation and editing, then versatility of the tool is improved, but device complexity increases
Solution Approach 1:
The patent applies universality by creating a modular TALE platform that can be fused with various functional domains (transcriptional activators, repressors, nucleases, epigenetic modifiers) to achieve multiple functions. The standardized TALE-RVD recognition module serves as a universal binding platform that can be combined with different effector domains to perform gene activation, repression, editing, or epigenetic modification, thereby achieving multi-functionality through a common framework.
Solution Approach 2:
The patent employs segmentation by dividing the TALE protein into distinct functional modules: the N-terminal and C-terminal non-repeat regions, the central repeat region containing RVDs for base recognition, and fused functional domains for specific activities. This modular segmentation allows independent optimization of each component and facilitates combinatorial assembly to create customized tools for different applications.
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 use of these RVDs allows for precise recognition and manipulation of epigenetic marks, facilitating methylation-dependent gene activation, efficient genome editing, and targeted detection of 5hmC and 6mA, thereby enhancing the specificity and effectiveness of gene regulation and editing processes.
Implementation Method 1
RVDs contact with DNA bases in a direct, sequence-specific manner. The full potential of RVDs in recognizing 5mC, 5hmC and 6mA remains to be further explored.
Implementation Method 2
5-methylcytosine (5mC) known as the fifth DNA base, is an important epigenetic marker that regulates gene expression. N6-methyladenine (6mA), plays an important role in prokaryotic cells as a covalent modification of adenine in DNA
Data Source
AI summary
RVDs with recognition preferences for 5mC, 5hmC and 6 mA and different binding properties to these epigenetic modifications are identified in this present invention. Methylation-dependent gene activation, efficient genome editing, targeted detection of 5hmC and other applications can be achieved by using these RVDs. The present invention therefore provides an isolated DNA binding polypeptide containing TALEs, a fusion protein, a polynucleotide, a vector comprising the polynucleotide and a host cell, and the use of the protein comprising TALE repeats domain in the preparation of a reagent for detecting a methylated base in a target sequence of a gene of interest, as well as a method for targeting and binding to a target sequence of a gene of interest in a cell.