Thermostable Nucleic Acid Ligase Mutations for Single-Stranded Ligation
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Solution Overview
Problem
There is a need for thermostable single-stranded nucleic acid ligases with higher ligation efficiency, as existing enzymes like HyperLigase suffer from insufficient efficiency in catalyzing ligation reactions between single-stranded DNAs and RNAs, particularly at higher temperatures.
Innovation Solution
Genetic mutations at specific positions (79, 281, 370, and 372) in the HyperLigase enzyme, such as substituting Arg with Ala or Lys with other amino acids, enhance the enzymatic and catalytic activities of the ligase, resulting in improved ligation efficiency.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Temperature
If thermostable ligases are used for single-stranded DNA ligation at higher temperatures, then thermal stability is improved, but ligation efficiency is insufficient
Solution Approach 1:
The patent applies parameter changes by mutating specific amino acid residues in the thermostable ligase structure to optimize its catalytic parameters. The mutations at positions 79, 281, 370, and 372 modify the enzyme's active site characteristics, enabling it to maintain both high thermal stability and improved ligation efficiency for single-stranded DNA substrates at elevated temperatures.
Solution Approach 2:
The patent applies local quality by introducing specific mutations at key positions (79, 281, 370, 372) within the ligase structure while leaving the rest of the thermostable framework intact. This localized modification approach allows the enzyme to gain enhanced single-stranded DNA ligation capability without compromising its overall thermal stability.
2Temperature
If existing thermostable ligases like TS2126 are used, then thermal stability is improved, but substrate specificity causes selective ligation bias
Solution Approach 1:
The patent applies universality by engineering the ligase to accept multiple substrate types including single-stranded DNA, double-stranded DNA, RNA, and pre-adenylated substrates. The mutated enzyme maintains thermal stability while gaining broad substrate acceptance, eliminating the selective ligation bias present in earlier thermostable ligases.
3Productivity
If Taq DNA ligase is used for single-stranded DNA ligation, then ligation efficiency is improved, but requirement for complementary template strand increases complexity
Solution Approach 1:
The patent applies taking out by removing the requirement for a complementary template strand from the ligation reaction system. The mutated thermostable ligase directly ligates single-stranded DNA substrates without needing Taq polymerase or template DNA, thereby maintaining high ligation efficiency while significantly simplifying the reaction system.
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 mutated HyperLigase enzymes demonstrate significantly higher catalytic activities and ligation efficiency compared to wild-type and commercially available enzymes like CircLigase, particularly in single-stranded DNA and RNA ligation reactions at elevated temperatures.
Implementation Method 1
Nucleic acid ligases are metal ion-dependent enzymes that catalyze the formation of a phosphodiester bond between the adjacent 3′ end and 5′ end of DNA or RNA molecules
Implementation Method 2
The mutated HyperLigase enzymes demonstrate significantly higher catalytic activities and ligation efficiency compared to wild-type and commercially available enzymes
Data Source
AI summary
Provided is a nucleic acid ligase, which comprises an amino acid sequence having a mutation at one or more positions selected from positions 79, 281, 370 and 372 compared to the amino acid sequence of Hyperligase of the prior art (SEQ ID NO: 1). Also provided are a nucleic acid molecule encoding the enzyme, a vector comprising the nucleic acid molecule, and a recombinant cell comprising the nucleic acid molecule or the vector. Also provided are a composition containing the enzyme and a use of the enzyme.