PEG-Mediated Delivery of LNA-Modified Nucleobases for Plant Mutagenesis
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Solution Overview
Problem
Current methods for targeted mutagenesis in plant cells are not precise and have low efficiency due to lack of control over genomic insertion positions and high degradation of mutagenic nucleobases by nucleases, leading to variable and often undetectable mutation frequencies.
Innovation Solution
The use of polyethylene glycol (PEG) mediated transformation to introduce single-stranded mutagenic nucleobases into plant protoplasts, which are modified with locked nucleic acids (LNAs) and propynyl modifications to enhance nuclease resistance and binding affinity, allowing for precise nucleotide alterations.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If exogenous DNA fragments are added to the genome of a cell, then new genetic properties are conferred to the cell, but the precision of genomic insertion position control is lost
Solution Approach 1:
The patent applies preliminary action by pre-modifying the mutagenic nucleobase with locked nucleic acid (LNA) chemistry before introduction into the cell. This pre-modification ensures nuclease resistance and precise base-pairing capability are established in advance, allowing the nucleobase to reach its target genomic position intact and function with high precision at the intended insertion site.
Solution Approach 2:
The patent uses polyethylene glycol (PEG) as an intermediary delivery vehicle to transport the mutagenic nucleobase into the cell nucleus. PEG mediates the introduction process, protecting the nucleobase from degradation and facilitating its delivery to the target genomic location, thereby enabling precise insertion without random integration.
2Manufacturing precision
If mutagenic nucleobases are introduced into plant cells, then targeted nucleotide conversion is achieved, but the efficiency is low due to nuclease degradation
Solution Approach 1:
The patent applies beforehand cushioning by chemically modifying the mutagenic nucleobase with locked nucleic acid (LNA) structure prior to introduction. This pre-established chemical protection shields the nucleobase from nuclease degradation that would otherwise occur after cellular introduction, thereby maintaining high mutagenesis efficiency while preserving targeted nucleotide conversion precision.
Solution Approach 2:
The patent changes the chemical parameters of the mutagenic nucleobase by incorporating LNA modifications and propynyl groups. These parameter changes increase the nucleobase's resistance to enzymatic degradation and enhance its binding affinity, directly improving mutagenesis efficiency while maintaining the precision of targeted nucleotide conversion at the intended genomic locus.
3Ease of manufacture
If unmodified mutagenic nucleobases are used, then the process is simpler, but the frequency of targeted mutagenesis is low and variable
Solution Approach 1:
The patent modifies key chemical parameters of the mutagenic nucleobase, specifically incorporating locked nucleic acid (LNA) structures and propynyl modifications. These parameter changes dramatically increase nuclease resistance and binding affinity, transforming the unreliable and variable mutagenesis process into a highly reliable method with consistent, detectable mutation frequencies across experiments.
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
PEG-mediated transformation significantly increases the efficiency of targeted mutagenesis in plant cells, with higher protoplast survival rates and successful nucleotide conversions, compared to electroporation and biolistics, enabling more reliable and precise genetic modifications.
Implementation Method 1
The use of polyethylene glycol (PEG) mediated transformation to introduce single-stranded mutagenic nucleobases into plant protoplasts
Implementation Method 2
modified with locked nucleic acids (LNAs) and propynyl modifications to enhance nuclease resistance and binding affinity
Implementation Method 3
molecules consisting of short stretches of nucleotide-like moieties that resemble DNA in their Watson-Crick basepairing properties
Data Source
AI summary
Method for targeted alteration of a duplex acceptor DNA sequence in a plant cell protoplast, comprising combining the duplex acceptor DNA sequence with a donor mutagenic nucleobase, wherein the duplex acceptor DNA sequence contains a first DNA sequence and a second DNA sequence which is the complement of the first DNA sequence and wherein the donor mutagenic nucleobase comprises at least one mismatch with respect to the duplex acceptor DNA sequence to be altered, preferably with respect to the first DNA sequence, wherein the method further comprises a step of introducing the donor mutagenic nucleobase into the cell protoplasts using polyethylene glycol (PEG) mediated transformation and the use of PEG protoplast transformation for enhancing the rate of targeted mutagenesis.