Modified DNA Methyltransferase for CC Dinucleotide Recognition
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Solution Overview
Problem
Existing DNA methyltransferases (MTases) have recognition sequences that overlap with CG dinucleotide sequences, leading to interference with endogenous methylation in mammalian cells, and their longer recognition sequences limit fine epigenetic mapping resolution.
Innovation Solution
A novel DNA methyltransferase (MTase) is developed that specifically recognizes and methylates CC dinucleotide sequences, avoiding overlap with CG dinucleotides and utilizing a shorter recognition sequence for improved epigenetic mapping resolution.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If existing DNA methyltransferases are used, then methylation can be introduced, but the recognition sequences overlap with CG dinucleotide sequences causing interference with endogenous methylation
Solution Approach 1:
The patent modifies the recognition sequence specificity of DNA methyltransferase to target CC dinucleotides instead of CG dinucleotides. This local change in substrate specificity allows the enzyme to perform methylation without interfering with endogenous CG methylation processes, resolving the contradiction between introducing methylation and avoiding interference with endogenous methylation.
2Measurement precision
If existing DNA methyltransferases with longer recognition sequences are used, then stable enzyme-DNA binding is achieved, but the resolution for fine epigenetic mapping is limited
Solution Approach 1:
The patent changes the recognition sequence parameter from longer sequences (e.g., 4-6 bases) to a shorter CC dinucleotide sequence (2 bases). This parameter change in recognition sequence length enables higher resolution epigenetic mapping while the enzyme maintains sufficient binding stability through optimized catalytic activity on the shorter sequence.
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 novel MTase enables distinguishable methylation from endogenous methylation, allowing for simultaneous profiling of endogenous methylation and other epigenomes with enhanced resolution, facilitating advanced epigenome analysis.
Implementation Method 1
DNA methyltransferases (MTases) are enzymes inducing methylation, which is one of the representative epigenetic modifications of DNA
Implementation Method 2
a protein modified to delete some amino acid residues including the N-terminal end of DNA methyltransferase M.CviQIX or M.CviPII and thereby acquire the ability to specifically recognize a CC dinucleotide sequence and 5-methylate the 5'-position cytosine residue in the sequence
Data Source
Figure 1
Figure 2A~2C
Figure 3A~3E
AI summary
The present invention provides a DNA methyltransferase that is capable of recognizing and methylating a short context other than but equivalent to a CG dinucleotide sequence where endogenous methylation occurs, etc. The present invention relates to a protein modified to delete some amino acid residues including the N-terminal end of DNA methyltransferase M.CviQIX or M.CviPII and thereby acquire the ability to specifically recognize a CC dinucleotide sequence and 5-methylate the 5'-position cytosine residue in the sequence, etc.