PolX DNA Polymerase Variants for Modified Nucleotide Synthesis
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Solution Overview
Problem
Current DNA polymerases of the polX family are limited in their ability to synthesize nucleic acid molecules without a template strand, especially when using modified nucleotides, as they lose catalytic activity due to steric hindrance and are not suitable for industrial-scale production.
Innovation Solution
Development of DNA polymerase variants with mutations in specific amino acid positions to enlarge the catalytic pocket and increase accessibility, allowing for the incorporation of modified nucleotides with greater steric hindrance, such as those with 3′-OH modifications.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If natural DNA polymerases are used for synthesizing nucleic acid without template strand, then the synthesis can be performed, but the catalytic activity is lost when modified nucleotides with greater steric hindrance are used
Solution Approach 1:
The patent applies parameter changes by mutating specific amino acid residues (such as F346A, F334A, W450A) in the DNA polymerase structure to alter the physical parameters of the catalytic pocket, specifically increasing its volume and reducing steric constraints. This enables the enzyme to accommodate modified nucleotides with greater steric hindrance while maintaining catalytic activity.
Solution Approach 2:
The patent applies local quality by making targeted mutations only in specific regions of the DNA polymerase molecule, particularly in the catalytic pocket and nucleotide binding site, while leaving the rest of the enzyme structure unchanged. This localized modification allows the enzyme to gain the ability to process modified nucleotides without losing overall catalytic function.
2Adaptability or versatility
If DNA polymerase variants with enlarged catalytic pocket are created to accommodate modified nucleotides, then the ability to incorporate modified nucleotides is improved, but the enzyme structure becomes more complex
Solution Approach 1:
The patent applies local quality by making targeted mutations only in specific regions of the DNA polymerase molecule, particularly in the catalytic pocket and nucleotide binding site, while leaving the rest of the enzyme structure unchanged. This localized modification allows the enzyme to gain the ability to process modified nucleotides without losing overall catalytic function.
Solution Approach 2:
The patent applies parameter changes by mutating specific amino acid residues (such as F346A, F334A, W450A) in the DNA polymerase structure to alter the physical parameters of the catalytic pocket, specifically increasing its volume and reducing steric constraints. This enables the enzyme to accommodate modified nucleotides with greater steric hindrance while maintaining catalytic activity.
3Adaptability or versatility
If currently available DNA polymerase variants are used, then some ability to incorporate modified nucleotides is achieved, but the activity is low and only suitable for laboratory scale
Solution Approach 1:
The patent applies parameter changes by mutating specific amino acid residues (such as F346A, F334A, W450A) in the DNA polymerase structure to alter the physical parameters of the catalytic pocket, specifically increasing its volume and reducing steric constraints. This enables the enzyme to accommodate modified nucleotides with greater steric hindrance while maintaining catalytic activity.
Solution Approach 2:
The patent applies universality by creating DNA polymerase variants that can function with both natural and modified nucleotides, making the enzyme applicable to a broader range of substrates. The mutated enzymes maintain their ability to process natural nucleotides while gaining enhanced capability to incorporate modified nucleotides, thus achieving multi-functionality.
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 variants exhibit enhanced catalytic activity and flexibility, enabling efficient synthesis of nucleic acid molecules using modified nucleotides, suitable for both laboratory and industrial scales, with improved incorporation kinetics and specificity.
Implementation Method 1
The present invention relates to a variant of a DNA polymerase of the polX family capable of synthesizing a nucleic acid molecule without a template strand
Data Source
AI summary
The invention relates to variants of a DNA polymerase of the polX family capable of synthesizing a nucleic acid molecule without a template strand, or of a functional fragment of such a polymerase, comprising at least one mutation of a residue in at least one specific position, and to uses of said variants, in particular for the synthesis of nucleic acid molecules comprising 3′-OH modified nucleotides.