Modified Restriction Enzyme Selectivity for 5βghmC
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Solution Overview
Problem
Current enzymes used for mapping genomic 5-hydroxymethylcytosine (5 hmC) have limited selectivity between 5β-glucosylhydroxymethylcytosine (5βghmC) and 5-methylcytosine (5 mC), leading to high background digestion and reduced efficiency in modification determination.
Innovation Solution
Development of non-natural variants of wild-type restriction enzymes with specific amino acid substitutions, such as at positions V72, T152, or R282, which exhibit a 2-fold increase in cleavage specificity for 5βghmC compared to 5 mC, enhancing discrimination between the two modifications.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If wild type restriction enzyme is used, then DNA cleavage activity is maintained, but selectivity between 5βghmC and 5 mC is insufficient leading to high background digestion
Solution Approach 1:
The patent applies parameter changes by modifying amino acid residues at specific positions (V72, T152, R282) in the restriction enzyme sequence. These parameter changes in the enzyme's primary structure alter its substrate binding properties, enhancing selectivity for 5βghmC over 5 mC by at least 2-fold compared to wild type, thereby reducing background digestion while maintaining cleavage activity.
2Measurement precision
If amino acid substitutions are introduced to enhance discrimination, then selectivity for 5βghmC increases, but enzyme activity may be affected
Solution Approach 1:
The patent systematically changes amino acid parameters at positions V72, T152, and R282 to enhance discrimination between 5βghmC and 5 mC/C while monitoring and maintaining enzyme activity. The selected substitutions (excluding F, Y, I, V at R282) are designed to optimize substrate recognition without compromising the catalytic function of the enzyme.
Solution Approach 2:
The patent employs feedback by assaying variant enzymes for both cleavage activity and selectivity, then selecting variants that achieve at least 2-fold increase in selectivity while maintaining functional activity. This iterative optimization process ensures that improvements in discrimination do not come at the cost of enzyme reliability.
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 modified enzymes demonstrate significantly improved selectivity for 5βghmC over 5 mC, reducing background digestion and enabling more accurate determination of 5 hmC modifications in genomic DNA, as shown by assays and sequence alignments.
Implementation Method 1
a non-natural variant of a wild type restriction enzyme is provided wherein the wild type restriction enzyme is defined by SEQ ID NO: 20, and wherein the variant has at least 90% sequence identity to the wild type enzyme and has at least a 2 fold increase in cleavage at 5βghmC compared with 5 mC relative to the wild type enzyme
Implementation Method 2
reacting the DNA with β glucosyltransferase (βGT) prior to reacting the variant enzyme with the DNA, thereby converting any hydroxymethylcytosines in the DNA to 5-β glucosylhydroxymethylcytosines
Data Source
AI summary
Provided herein in some embodiments is a non-naturally occurring variant of a wild type restriction enzyme defined by SEQ ID NO: 20, wherein the variant has at least a 2 fold increase in cleavage at 5-β glucosylhydroxymethylcytosine (5βghmC) compared with methylcytosine relative to the wild type enzyme. Methods for examining hydroxymethylation of a DNA sample using the variant enzyme are also provided.