Engineered MBD Variants for oxi-mC Configuration Resolution
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Solution Overview
Problem
Current technologies are unable to resolve individual oxidized 5-methylcytosine (oxi-mC) configurations in DNA molecules due to binary read-out methods and lack of suitable binders that cannot discriminate neighboring modifications, hindering the understanding of their biological functions in mammalian genomes.
Innovation Solution
Engineered Methyl-CpG binding domains (MBD) with specific amino acid substitutions that provide differential interactions with different mC and oxi-mC configurations, enabling the creation of molecular probes for precise configuration resolution across DNA double-strands, suitable for affinity enrichment, imaging, and high-throughput sequencing.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of operation
If binary read-out methods are used for sequencing, then the sequencing process is simple, but individual oxi-mC configurations cannot be resolved
Solution Approach 1:
The patent segments the binary read-out process into multiple sequential affinity enrichment steps, each targeting a specific oxi-mC configuration. By dividing the complex measurement into discrete stages with specialized binders, the method resolves individual configurations while maintaining operational feasibility through systematic processing of DNA pools.
Solution Approach 2:
The patent introduces engineered MBD proteins as intermediary binders that specifically recognize and bind to different oxi-mC configurations. These intermediary molecules bridge the gap between the DNA sample and the detection system, enabling precise configuration resolution without requiring direct observation methods.
2Adaptability or versatility
If generic binders are used for DNA binding, then binder availability is high, but discrimination of neighboring modifications is impossible
Solution Approach 1:
The patent applies local quality by engineering specific amino acid substitutions in the MBD proteins at positions that directly contact the DNA bases. These localized modifications create distinct binding specificities for different oxi-mC configurations, enabling discrimination of neighboring modifications while maintaining the overall binder structure and availability.
Solution Approach 2:
The patent changes the chemical parameters of the binders by introducing specific amino acid substitutions that alter the binding interface properties. These parameter changes in the binder structure enable selective recognition of different oxidation states and configurations, transforming generic binders into configuration-specific probes.
3Measurement precision
If engineered MBD variants with specific amino acid substitutions are used, then configuration-specific binding is achieved, but protein design complexity increases
Solution Approach 1:
The patent employs dynamics by using library screening approaches that allow the binding properties of MBD variants to be dynamically optimized. Through iterative selection and characterization of variants with different amino acid substitutions, the method identifies proteins with desired specificities without requiring de novo design of entire protein structures, thus managing complexity through evolutionary optimization.
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
These molecular probes allow for the precise determination and enrichment of DNA molecules with defined oxi-mC configurations, enabling genome-wide mapping and correlation with epigenetic features to reveal biological functions.
Implementation Method 1
an isolated Methyl-CpG binding domain (MBD) variant... comprising at least one amino acid substitution relative to the corresponding wildtype MBD... for the determination of the methylation state of cytosine residues and/or oxidation state of 5-methylated cytosine residues
Data Source
AI summary
An isolated Methyl-CpG binding domain (MBD) variant may include an MBD core domain having at least 60% sequence homology relative to any one of SEQ ID Nos. 1-45 and comprising at least one amino acid substitution relative to the corresponding wildtype MBD in various positions. The isolated MBD variant or the conjugate may be used for determining the methylation state of cytosine residues and/or oxidation state of 5-methylated cytosine residues in a CpG dinucleotide of interest and its complement in a DNA molecule or for the enrichment of DNA molecules comprising a CpG dinucleotide of interest and its complement. At least one cytosine nucleobase in the CpG dinucleotide may be modified to be 5-methylcytosine (mC), 5-hydroxymethylcytosine (hmC), 5-formylcytosine (fC), or 5-carboxylcytosine (caC).