Methylcytosine-Selective Deaminases for Epigenetic Sequencing
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Solution Overview
Problem
Current DNA sequencing methods for distinguishing cytosine (C) from its methylated forms, such as 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC), are complex, require additional processing steps, and often result in higher DNA input amounts and processing time.
Innovation Solution
A sequencing method involving the use of a methylcytosine-selective deaminase that preferentially converts 5mC and 5hmC to thymidine (T) and hydroxymethyluridine (hmU), respectively, while leaving cytosine (C) substantially unaltered, thereby simplifying the sequencing workflow.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If bisulfite deamination is used to distinguish C from 5mC, then C can be converted to U for sequencing differentiation, but DNA samples undergo chemical degradation and require additional protection steps
Solution Approach 1:
Instead of converting C to U (standard approach), the invention inverts the approach by using a deaminase that selectively converts 5mC to T while leaving C unchanged. This inversion eliminates the need for protective pretreatments and simplifies the workflow while maintaining differentiation accuracy.
Solution Approach 2:
The invention extracts and removes the problematic protective pretreatment steps (oxidase and glucosidase treatments) from the workflow by using a deaminase that inherently selectively targets 5mC without requiring prior protection of methylated cytosines.
2Measurement precision
If enzymatic deamination with protective pretreatments is used, then C to U conversion can be achieved, but DNA input amount requirements increase and processing time extends
Solution Approach 1:
The invention inverts the conventional approach by using a deaminase that selectively converts 5mC to T rather than converting C to U. This eliminates the need for multiple protective pretreatment steps, thereby reducing processing time and DNA input requirements while maintaining accurate differentiation.
3Reliability
If multiple enzymatic pretreatments are applied to protect methylcytosine, then selective deamination can be achieved, but the number of processing steps and failure points increases
Solution Approach 1:
The invention extracts and eliminates the complex sequence of protective pretreatments (oxidase treatment, glucosidase treatment, and their combinations) by using a single deaminase enzyme that inherently provides the necessary selectivity without requiring multiple protective steps.
Solution Approach 2:
Instead of protecting 5mC from deamination and then differentiating based on protection status, the invention inverts the approach by using a deaminase that naturally selectively targets 5mC, eliminating the need for protective pretreatments and reducing the number of processing steps.
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 approach allows for more direct and efficient epigenetic sequencing by reducing the complexity of the sequencing workflow, minimizing DNA input requirements, and reducing processing time, while maintaining high accuracy in distinguishing methylated cytosines.
Implementation Method 1
a methylcytosine-selective deaminase that preferentially converts 5mC and 5hmC to thymidine (T) and hydroxymethyluridine (hmU), respectively
Data Source
AI summary
Provided are methylcytosine-selective deaminases, compositions, kits, and methods employing them, including a sequencing method involving (a) contacting a DNA substrate comprising cytosine (C) and at least one methylcytosine nucleobase selected from 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) or comprising C and both 5mC and 5hmC, with a methylcytosine-selective deaminase to produce a deamination product, wherein the methylcytosine-selective deaminase (i) is capable of deaminating 5mC to thymidine (T) and/or 5hmC to hydroxymethyluridine (hmU) and (ii) preferentially deaminates the at least one methylcytosine nucleobase relative to cytosine (C); and (b) sequencing the deamination product, or amplifying the deamination product to produce an amplification product, and sequencing the amplification product, in each case, to produce sequence reads, wherein positions of Cs and position of the at least one methylcytosine nucleobase in the DNA substrate is determined based on the sequence reads.