Modified DNA Cleaving Enzyme with Beta-Bridge Mutation

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Solution Overview

Problem

The toxicity of enzymes like T7 Endonuclease I, which are used in molecular biology, hinders their overproduction and specific activity, due to their broad substrate specificity and potential harm to host cells.

Innovation Solution

A modified DNA cleaving enzyme with at least 35% amino acid sequence identity to T7 Endo I, featuring a mutation in the β-bridge, reduces toxicity and alters cleavage activity, allowing for specific recognition and cleavage of DNA structures such as cruciforms, mismatch base pairs, and pre-existing nick sites, with enhanced activity in manganese-containing buffers.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Adaptability or versatility

If T7 Endonuclease I is used for DNA cleavage, then broad substrate specificity is achieved, but toxicity to host cells increases

Engineering Contradiction:

Improvesubstrate specificityVSAvoidtoxicity to host cells

Core Design Contradiction:

Adaptability or versatilityVSObject-affected harmful factors

Solution Approach 1:

The patent applies local quality by making a specific localized change to the enzyme structure - mutating amino acids in the β-bridge region (specifically positions 46-47) - to alter the enzyme's properties locally without changing its overall structure or function. This localized modification reduces toxicity while preserving DNA cleavage capability.

Inventive Principle:

Principle #3Local quality

2Productivity

If T7 Endonuclease I is overproduced in host cells, then enzyme quantity increases, but toxicity to host cells increases

Engineering Contradiction:

Improveenzyme production quantityVSAvoidtoxicity to host cells

Core Design Contradiction:

ProductivityVSObject-affected harmful factors

Solution Approach 1:

The patent applies parameter changes by modifying the amino acid sequence parameters of the enzyme - specifically changing residues in the β-bridge region - to alter the enzyme's biological properties. This parameter modification allows the enzyme to be overproduced without the same toxic effects, enabling higher productivity.

Inventive Principle:

Principle #35Parameter changes

3Object-affected harmful factors

If the enzyme structure is modified to reduce toxicity, then host cell compatibility improves, but cleavage activity may be altered

Engineering Contradiction:

Improvetoxicity reductionVSAvoidcleavage activity consistency

Core Design Contradiction:

Object-affected harmful factorsVSReliability

Solution Approach 1:

The patent applies segmentation by separating the enzyme into functional regions - the catalytic domains that perform DNA cleavage and the β-bridge region that connects them. By modifying only the β-bridge segment (positions 46-47) and leaving the catalytic domains intact, the invention reduces toxicity while preserving the essential cleavage function.

Inventive Principle:

Principle #1Segmentation

Data Source

PatentUS7851192B2Modified DNA cleavage enzymes and methods for use

Publication Date: 2010.12.14 NEW ENGLAND BIOLABS INC

AI summary

Compositions and methods are provided that relate to a modified DNA cleaving enzyme having at least 35% amino acid sequence identity with T7 Endo I. The modified enzyme includes two catalytic centers separated by a β-bridge where the β-bridge contains at least one mutation having an effect of altering enzyme cleavage activity compared to the unmodified enzyme. Activities associated with the modified DNA cleaving enzyme that can be modulated in different reaction conditions include at least one of: (a) non-sequence specific nicking activity; (b) cleaving the second strand of a duplex DNA at a preexisting nick site to produce a linear duplex with a single strand overhang; (c) non-sequence specific DNA cleavage; (d) cleaving DNA flanking a mismatch; and (e) cleavage at a cruciform structure in a DNA duplex.