Zinc Finger Nucleic Acid Cleaving Agent for Site-Specific DNA Manipulation

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Solution Overview

Problem

Current restriction enzymes are limited in their ability to selectively cleave specific nucleotide sequences in large DNA molecules, leading to impractical fragmentation and a need for tools that can efficiently manipulate large DNA for gene recombination and therapy.

Innovation Solution

A nucleic acid cleaving agent is designed with at least two independent zinc finger proteins bound to a nucleic acid cleaving moiety, allowing specific binding upstream and downstream of the target cleavage site, enabling site-specific cleavage and catalytic turnover, thereby overcoming the limitations of existing ZFNs.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Measurement precision

If traditional restriction enzymes are used to cleave large DNA molecules, then the DNA is cleaved at multiple sites, but this results in fragmentation that makes desired gene recombination procedures impracticable

Engineering Contradiction:

Improvesite-specificity of cleavageVSAvoidpracticality of gene recombination

Core Design Contradiction:

Measurement precisionVSProductivity

Solution Approach 1:

The invention divides the DNA binding function into separate zinc finger domains that can independently bind to specific sequences upstream and downstream of the target site, allowing precise control over cleavage location in large DNA molecules

Inventive Principle:

Principle #1Segmentation

Solution Approach 2:

The invention introduces a DNA-binding protein component that acts as an intermediary between the cleaving enzyme and the target DNA sequence, enabling site-specific cleavage in large DNA molecules without random fragmentation

Inventive Principle:

Principle #24Intermediary (Mediator)

2Reliability

If zinc finger proteins are bound to the end of a nucleic acid cleaving moiety (ZFN structure), then the molecule can bind to target sites, but it fails to catalytically exert cleavage action by repeating binding and cleavage cycles

Engineering Contradiction:

Improvebinding affinity to target siteVSAvoidcatalytic cleavage activity

Core Design Contradiction:

ReliabilityVSProductivity

Solution Approach 1:

The invention segments the protein structure into independent zinc finger domains that can dissociate after cleavage, enabling catalytic turnover. Each zinc finger domain binds independently to specific sequences, allowing the complex to release and rebind to multiple target sites

Inventive Principle:

Principle #1Segmentation

Solution Approach 2:

The invention introduces dynamic binding characteristics where zinc finger proteins can associate and dissociate from DNA target sites. This dynamic behavior enables the cleaving agent to perform multiple cleavage cycles on different DNA molecules, achieving catalytic activity

Inventive Principle:

Principle #15Dynamics

3Reliability

If two ZFN molecules are needed to act cooperatively for each cleavage site, then homologous recombination can occur, but this increases the complexity and reagent requirements for gene therapy applications

Engineering Contradiction:

Improvehomologous recombination capabilityVSAvoidnumber of molecules required per cleavage site

Core Design Contradiction:

ReliabilityVSDevice complexity

Solution Approach 1:

The invention merges the DNA binding function and nucleic acid cleaving function into a single integrated molecule. The zinc finger domains and cleaving moiety are covalently linked, allowing one molecule to perform both binding and cleavage functions that previously required two separate ZFN molecules

Inventive Principle:

Principle #5Merging (Combining)

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 agent efficiently cleaves large DNA molecules at specific sites with high activity and low affinity post-cleavage, allowing for repeated turnover and enhanced efficiency compared to traditional ZFNs, facilitating gene manipulation and therapy.

Implementation Method 1

at least one of the zinc finger proteins is capable of specifically binding to a nucleotide sequence located upstream from the target cleavage site, and at least one of the remaining zinc finger proteins is capable of specifically binding to a nucleotide sequence located downstream from the target cleavage site

Methodology Applied

Scientific EffectSequence-specific binding:

Implementation Method 2

a nucleic acid cleaving moiety... enabling site-specific cleavage and catalytic turnover

Methodology Applied

Scientific EffectCatalysis: Catalysis

Implementation Method 3

allows easy dissociation of the agent from the nucleic acid after the cleavage. This configuration results in substantial decrease in the affinity of the agent for the nucleic acid compared to the affinity prior to the cleavage

Methodology Applied

Scientific EffectDissociation:

Data Source

PatentUS8318155B2Nucleic acid cleaving agent

Publication Date: 2012.11.27 SERA TAKASHI

AI summary

A nucleic acid cleaving agent having a cleaving activity specific to a desired cleavage site in a nucleic acid such as large DNA, which comprises (1) a nucleic acid cleaving moiety, and (2) at least two zinc finger proteins bound to the nucleic acid cleaving moiety, wherein at least one of the zinc finger proteins can specifically bind to a nucleotide sequence located upstream from the target cleavage site, and at least one of the remaining zinc finger proteins can specifically bind to a nucleotide sequence located downstream from the target cleavage site.