Ligase Mutants Enhance Nucleic Acid Linking Activity and Stability
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current techniques for T4 RNA ligase 2 do not provide ligase mutants with improved properties for nucleic acid linking activities, such as enhanced efficiency and stability.
Innovation Solution
Development of three types of ligase mutants (Mut1 to 3) with specific amino acid sequence identities to T4 RNA ligase 2, exhibiting improved nucleic acid-linking activities and stability, which can efficiently link various nucleic acid materials including RNA, DNA, and modified nucleic acids.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If T4 RNA ligase 2 is used for nucleic acid linking, then the enzyme can form phosphodiester bonds to link nucleic acids, but the linking activity and stability are insufficient
Solution Approach 1:
The patent applies parameter changes by mutating specific amino acid residues in the T4 RNA ligase 2 enzyme sequence. Three specific mutants are provided (Mut1, Mut2, Mut3) with different amino acid sequences compared to the wild-type T4 RNA ligase 2. These mutations modify the enzyme's kinetic parameters and stability properties, resulting in enhanced nucleic acid linking activity and improved thermal stability while maintaining the phosphodiester bond formation function.
2Reliability
If wild-type T4 RNA ligase 2 is used, then the enzyme maintains natural functionality, but it lacks enhanced efficiency and stability for industrial applications
Solution Approach 1:
The patent applies local quality by making specific targeted mutations at particular positions in the enzyme sequence rather than random mutagenesis. Each mutant (Mut1, Mut2, Mut3) has specific amino acid changes at defined locations that locally optimize the enzyme's active site or structural stability. This localized modification approach allows enhancement of specific functions (linking efficiency or stability) while preserving the overall enzyme structure and natural 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 ligase mutants demonstrate increased activity and stability, enabling efficient production of nucleic acid products like siRNA and heteroduplex nucleic acids, with activities up to 4.2 times higher than T4 RNA ligase 2 and maintaining stability under elevated temperatures.
Implementation Method 1
T4 RNA ligase 2 has an ability of forming a phosphodiester bond to link a phosphate group (donor) at the 5′ end of a nucleic acid to a hydroxy group (acceptor) at the 3′ end thereof
Implementation Method 2
T4 RNA ligase 2 is an enzyme that is one type of RNA ligase (EC6.5.1.3) having an ability of linking ribonucleotides in the presence of ATP
Data Source
AI summary
Ligase mutants of the following (1), (2), or (3):(1) a ligase mutant comprising an amino acid sequence showing 95% or more identity to the amino acid sequence of SEQ ID NO: 1, and having a nucleic acid-linking activity;(2) a ligase mutant comprising an amino acid sequence showing 90% or more identity to the amino acid sequence of SEQ ID NO: 2, and having a nucleic acid-linking activity; or(3) a ligase mutant comprising an amino acid sequence showing 97% or more identity to the amino acid sequence of SEQ ID NO: 3, and having a nucleic acid-linking activity, have excellent properties.