CpG Binding Protein Mapping of Modified Cytosines Without DNA Damage
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current methods for mapping modified DNA cytosines, such as 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC), suffer from DNA degradation, loss of sample complexity, multi-step processes, and limited resolution, making it difficult to determine their exact genomic locations.
Innovation Solution
A one-step enzymatic method using a CpG binding protein and altered cytidine deaminases that selectively convert modified cytosines to thymidine or uracil, avoiding harsh chemical treatments and enabling single-base resolution mapping of 5mC and 5hmC.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If bisulfite treatment is used to map 5hmC, then detection capability is improved, but DNA integrity is degraded and sample complexity is lost
Solution Approach 1:
The patent replaces harsh chemical bisulfite treatment with an enzymatic system using TET proteins and CpG binding proteins. The TET enzyme catalyzes oxidation of 5hmC to formable cytosine, which is then detected through enzymatic conversion rather than chemical degradation. This substitution of chemical mechanism with enzymatic mechanism achieves detection while preserving DNA integrity and avoiding the formation of uracil that causes complexity loss.
Solution Approach 2:
The patent introduces CpG binding proteins as intermediaries that selectively bind to unmethylated CpG sites, preventing TET enzyme activity at these locations. This intermediary mechanism allows selective detection of 5hmC at methylated sites while avoiding non-specific effects. The intermediary protein complex enables precise spatial control of the enzymatic conversion process, improving detection accuracy without degrading DNA.
2Measurement precision
If multiple enzymatic steps are used to map modified cytosines, then detection accuracy is improved, but process complexity increases
Solution Approach 1:
The patent merges the functions of 5hmC detection and CpG site mapping into a single integrated enzymatic process. The TET protein performs oxidation of 5hmC while the CpG binding protein simultaneously provides spatial guidance and prevents off-target activity. This consolidation of multiple functional steps into one coordinated enzymatic reaction reduces process complexity while maintaining or improving detection accuracy through the synergistic action of the protein complex.
3Measurement precision
If antibody-based detection is used for 5mC, then detection capability is improved, but resolution is limited to approximately 150 bp
Solution Approach 1:
The patent replaces antibody-based detection with an enzymatic conversion approach using TET proteins. Instead of relying on physical proximity detection that is limited to ~150 bp resolution, the enzymatic method converts 5hmC to formable cytosine at the exact site of modification. This chemical conversion creates a permanent, detectable change at the precise location of the original modification, enabling single-base resolution mapping that overcomes the spatial limitation of antibody-based methods.
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
This method preserves DNA integrity, reduces sample complexity, and achieves single-base resolution mapping of modified cytosines without the need for multi-step processes, enhancing the accuracy of methylation status determination.
Implementation Method 1
a cytidine deaminase that selectively converts modified cytosines to thymidine or uracil
Implementation Method 2
a CpG binding protein for binding to CpG sites in a DNA sample
Data Source
AI summary
Provided herein are methods, compositions, and kits related to using a CpG binding protein. In one embodiment, the present disclosure includes methods, compositions, and kits related to using a CpG binding protein with a cytidine deaminase protein to identify methylated cytosine nucleotides. The cytidine deaminase can be an altered cytidine deaminase that includes an amino acid substitution mutation at a position functionally equivalent to (Tyr/Phe)130 in a wild-type APOBEC3A protein. In another embodiment, the present disclosure includes methods, compositions, and kits related to using a CpG binding protein with a ten-eleven translocase (TET) protein to identify methylated cytosine nucleotides.