Engineered Ligases for Thermostable RNA-Templated DNA Ligation
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Solution Overview
Problem
Existing ligases, such as PCBV-1 DNA ligase, suffer from low thermostability and propensity to aggregate, limiting their utility in ligation of single-stranded DNA polynucleotides hybridized onto complementary RNA molecules.
Innovation Solution
Development of ligases with amino acid substitutions and attachment of hydrophilic polymers, such as PEG, to enhance thermostability and prevent aggregation, along with kits including reaction buffers and cofactors for optimized ligation.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If PCBV-1 DNA ligase is used to ligate single-stranded DNA polynucleotides hybridized onto RNA molecules, then ligation activity is achieved, but thermostability is low and aggregation occurs
Solution Approach 1:
The patent applies parameter changes by introducing specific amino acid substitutions at positions 22, 298, and 302 of the PCBV-1 DNA ligase sequence. These substitutions (such as C22S, C298S, K302R) modify the enzyme's physical and chemical properties to enhance thermostability and prevent aggregation, allowing the ligase to maintain activity at elevated temperatures up to 55°C while reducing harmful aggregation behavior
Solution Approach 2:
The patent creates composite enzyme structures by fusing the PCBV-1 DNA ligase with polynucleotide-binding domains from proteins such as SSB, RPA, or T7 gp4. These fusion proteins combine the ligation activity of PCBV-1 ligase with the RNA/DNA binding and stabilizing properties of the fused domains, resulting in a composite enzyme that exhibits enhanced thermostability and reduced aggregation while maintaining catalytic function
2Productivity
If PCBV-1 DNA ligase operates at elevated temperatures to improve ligation efficiency, then productivity increases, but thermostability decreases and aggregation increases
Solution Approach 1:
The amino acid substitutions fundamentally change the thermal stability parameters of the ligase, enabling it to withstand elevated temperatures (37-55°C) that increase ligation efficiency for RNA-hybridized substrates without undergoing denaturation or aggregation. The modified ligase maintains catalytic activity across a broader temperature range
Solution Approach 2:
The patent creates dynamically adaptable ligase variants that can function optimally across varying temperature conditions. The engineered ligases adjust their conformational dynamics to maintain stability at higher temperatures while preserving the flexibility needed for catalytic activity, allowing the enzyme to adapt its behavior based on operational conditions
3Reliability
If amino acid substitutions are introduced to enhance thermostability, then reliability improves, but manufacturing complexity increases
Solution Approach 1:
The patent applies local quality changes by introducing targeted amino acid substitutions at specific positions (22, 298, 302) rather than globally modifying the entire enzyme. These localized changes at critical residues provide the necessary thermostability improvement with minimal impact on overall enzyme structure and function, simplifying the engineering process compared to comprehensive redesign
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 modified ligases exhibit increased thermostability, improved ligation efficiency, and resistance to aggregation, enabling effective ligation of polynucleotides even at elevated temperatures.
Implementation Method 1
Ligases catalyze the joining of the 5′-phosphorus-terminated strand with the 3′-hydroxyl-terminated strand, specifically within double-stranded polynucleotides
Implementation Method 2
the ligase includes one or more hydrophilic polymers (e.g., PEG)
Implementation Method 3
the ligase includes an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO:1 attached (e.g., covalently attached) to a polynucleotide-binding polypeptide
Data Source
AI summary
Disclosed herein, inter alia, are ligases, kits, and methods of use thereof.