Hyper-thermostable Lysine-Mutant Ligases for High-Temperature Nucleic Acid Ligation
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Solution Overview
Problem
Current DNA and RNA ligases are limited in their ability to efficiently ligate single-stranded nucleic acid sequences at high temperatures, particularly above 65°C, which restricts their application in next-generation sequencing and other molecular biology protocols.
Innovation Solution
Development of hyper-thermostable lysine-mutant ssDNA/RNA ligases with both ssRNA and ssDNA ligase activity, capable of operating at temperatures of at least 75°C, achieved by mutating the catalytic lysine in the Motif I of precursor hyper-thermostable ssRNA ligases, allowing for efficient ligation of 5' adenylated ends to 3' ends of single-stranded nucleic acid sequences.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Temperature
If conventional DNA/RNA ligases are used for ligation reactions, then the enzymes can catalyze phosphodiester bond formation, but they cannot efficiently ligate single-stranded nucleic acid sequences at temperatures above 65°C
Solution Approach 1:
The patent introduces a lysine mutation in the Motif I region of hyper-thermostable ssRNA ligases to create variants with enhanced thermostability and dual ssDNA/ssRNA ligation activity. This amino acid substitution changes the enzyme's properties to maintain catalytic function at temperatures of 75°C or higher, resolving the contradiction between temperature and ligation efficiency for single-stranded substrates
2Temperature
If precursor hyper-thermostable ssRNA ligases are used, then the enzymes can operate at high temperatures, but they lack ssDNA ligase activity
Solution Approach 1:
The patent engineers lysine-mutant variants of hyper-thermostable ssRNA ligases that acquire dual functionality to catalyze both ssRNA and ssDNA ligation reactions. The Motif I lysine mutation enables these enzymes to process both RNA and DNA substrates at high temperatures, eliminating the need for separate enzymes and expanding substrate versatility while maintaining thermal stability
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
Enables efficient ligation of nucleic acid sequences at elevated temperatures, enhancing the efficiency of next-generation sequencing and other molecular biology applications by allowing for higher temperature reactions, thus improving the robustness and accuracy of library preparation and sequencing processes.
Implementation Method 1
hyper-thermostable lysine-mutant ssDNA/RNA ligases that possesses both ssRNA ligase and ssDNA ligase activity... ligate a first single-strand nucleic acid sequence with a 5' adenylated end to a second single-strand nucleic acid sequence
Data Source
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AI summary
Provided herein are compositions, systems, and methods employing hyper-thermostable lysine-mutant ssDNA/RNA ligases that possesses both ssRNA ligase and ssDNA ligase activity. In certain embodiments, such hyper-thermostable lysine-mutant ssDNA/RNA ligases are used to ligate an first single stranded nucleic acid sequence with a 5' adenylated end to a second single stranded nucleic acid sequence (e.g., at a temperature of at least 75°C) to form a ligated nucleic acid sequence. In further embodiments, the ligated nucleic acid sequence is sequenced.