Engineered DNA Deaminases for Methylation Analysis
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current methods for identifying modified cytosines in DNA require a denaturation step, which is undesirable, and existing deaminases are limited in their ability to act on double-stranded DNA substrates without denaturing them.
Innovation Solution
Development of double-stranded DNA deaminases that can deaminate cytosines in both double-stranded and single-stranded DNA substrates without denaturing the DNA, allowing for the identification of modified cytosines such as 5mC and 5hmC without the need for denaturation steps, using enzymes like TET methylcytosine dioxygenase and DNA beta-glucosyltransferase to modify substrates and enhance specificity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of operation
If current deaminase methods are used to identify modified cytosines, then cytosine deamination can be achieved, but a denaturation step is required which complicates the workflow
Solution Approach 1:
The patent changes the substrate binding parameters of deaminases through protein engineering, enabling them to recognize and bind to double-stranded DNA structures that natural deaminases cannot process. This allows deamination to occur on dsDNA without requiring denaturation, eliminating the extra workflow step while maintaining deamination functionality
Solution Approach 2:
The engineered deaminases gain the ability to process both double-stranded and single-stranded DNA substrates, making them universally applicable to different DNA forms. This multi-functionality eliminates the need for separate denaturation and deamination steps, simplifying the overall workflow for identifying modified cytosines
2Adaptability or versatility
If existing deaminases are used, then cytosine deamination is possible, but they cannot act on double-stranded DNA without denaturing it
Solution Approach 1:
The patent modifies key parameters of deaminase proteins including their substrate binding pockets and structural conformations to enable recognition of double-stranded DNA. This allows the enzymes to reliably process dsDNA substrates that were previously inaccessible, expanding substrate type flexibility while maintaining deamination reliability
Solution Approach 2:
The patent introduces intermediary structural elements or modifications to the deaminase proteins that facilitate their interaction with double-stranded DNA. These intermediaries act as mediators between the enzyme and the dsDNA substrate, enabling the enzyme to access and process cytosines in the context of double-stranded DNA without requiring denaturation
3Measurement precision
If denaturation step is included in the workflow, then modified cytosines can be identified, but the workflow complexity increases
Solution Approach 1:
The patent extracts and eliminates the denaturation step from the workflow by engineering deaminases that can directly process double-stranded DNA. This removal of the unnecessary intermediate step simplifies the workflow while preserving the ability to accurately identify modified cytosines through deamination and sequencing
Solution Approach 2:
The patent enables continuous deamination action on double-stranded DNA without interruption for denaturation. The engineered deaminases can process dsDNA substrates continuously, maintaining measurement precision for modified cytosine identification while reducing workflow complexity by eliminating the break in the process
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
Enables efficient identification of modified cytosines in DNA without denaturing the DNA, simplifying workflows and improving the accuracy of methylation analysis by allowing sequencing of double-stranded DNA substrates, reducing the complexity of current methods and enhancing the detection of methylation profiles.
Implementation Method 1
cytosine deaminases that are active on DNA substrates... deaminate cytosines in a double-stranded DNA substrate
Implementation Method 2
one or more enzymes that alter the deamination susceptibility of one or more modified cytosines (e.g., a TET methylcytosine dioxygenase)
Implementation Method 3
a TET methylcytosine dioxygenase and/or a DNA beta-glucosyltransferase
Data Source
AI summary
Provided herein, among other things, is a method for deaminating a double-stranded nucleic acid. In some embodiments, the method may comprise contacting a double-stranded DNA substrate that comprises cytosines and a double-stranded DNA deaminase having an amino acid sequence that is at least 80% identical to any of SEQ ID NOS: 21, 40, 47, 49, 50, 55, 58, 59, 62, 63, 65, 67, 70, 71, 76, 106, 107, 110, 112, 114, 117, 163 and/or 164 to produce a deamination product that comprises deaminated cytosines. Enzymes and kits for performing the method are also provided.