Recombinant TET3 Enzyme Oxidation for Accurate mdC Sequencing
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Solution Overview
Problem
Current methods for sequencing 5-methylcytosine (mdC) in nucleic acids, such as bisulfite treatment and 3rd generation sequencing, face challenges like DNA fragmentation, cumbersome protocols, and low signal differentiation, making them unsuitable for limited input samples and requiring resource-intensive data analysis.
Innovation Solution
A recombinant Ten Eleven Translocation 3 (TET3) enzyme is developed, comprising specific domains and a heterologous connecting sequence, which selectively oxidizes mdC to 5-carboxycytidine (cadC) for enhanced signal differentiation, enabling accurate sequencing without bisulfite addition.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If bisulfite treatment is used for mdC sequencing, then sequencing accuracy is improved, but DNA fragmentation increases and input DNA amount is reduced
Solution Approach 1:
The patent extracts and removes the harsh bisulfite treatment step from the sequencing protocol, replacing it with a milder oxidation method using TET3 enzyme that converts mdC to cadC without causing extensive DNA fragmentation, thus preserving input DNA amount while maintaining sequencing accuracy
Solution Approach 2:
The patent changes the chemical method parameter from bisulfite deamination to TET3-mediated oxidation, which operates under milder conditions and converts mdC to cadC with different chemical properties that enable sequencing while preserving DNA integrity and input material
2Device complexity
If APOBEC3A deamination is used for mdC sequencing, then sequencing is simplified, but genome complexity is reduced making assembly challenging
Solution Approach 1:
The patent applies local quality modification by selectively oxidizing only the mdC bases to cadC while leaving other genomic sequences unchanged, thereby maintaining full genome complexity and four-letter code integrity while simplifying the sequencing protocol through enzymatic conversion
3Measurement precision
If formic acid treatment is used for mdC detection, then mdC concentration determination is achieved, but mdC groups are hydrolysed
Solution Approach 1:
The patent converts the harmful effect of strong acid treatment into a beneficial enzymatic oxidation process using TET3 enzyme, which selectively transforms mdC to cadC under mild conditions, achieving accurate detection while preserving mdC groups from hydrolysis
4Measurement precision
If cadC sequencing is used instead of mdC sequencing, then signal differentiation is improved, but proper oxidizing enzyme and data analysis algorithms are not available
Solution Approach 1:
The patent employs the TET3 enzyme that naturally occurs in the system and can be recombinantly produced, eliminating the need for external oxidizing enzymes, while the enzyme itself performs the oxidation function required for signal differentiation without requiring additional complex components
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 TET3 enzyme provides highly selective and quantitative oxidation of mdC, facilitating non-invasive, time- and cost-effective early tumor diagnosis by enhancing signal differentiation and reducing data complexity.
Implementation Method 1
A recombinant Ten Eleven Translocation 3 (TET3) enzyme is developed, comprising specific domains and a heterologous connecting sequence, which selectively oxidizes mdC to 5-carboxycytidine (cadC) for enhanced signal differentiation
Data Source
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AI summary
The present invention refers to a recombinant Ten Eleven Translocation 3 (TET3) enzyme, a nucleic acid molecule encoding the TET3-enzyme, a vector comprising the nucleic acid molecule and a host cell transformed or transfected with the vector, the use of the recombinant TET3-enzyme for sequencing of a nucleic acid substrate, a method for sequencing a nucleic acid substrate and a reagent for sequencing a nucleic acid substrate.