Chimeric Ligase Enzymes for Stable RNA-DNA Hybrid Binding
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Solution Overview
Problem
Current RNA-templated DNA ligation reactions suffer from inefficiency, lack of specificity, and poor thermostability due to the premature release of ligases from RNA-DNA hybrids, leading to incomplete ligation reactions.
Innovation Solution
Development of chimeric ligase enzymes by fusing a ligase polypeptide domain with a catalytically inactive ribonuclease H (RNAseH) polypeptide domain, which enhances binding to RNA-DNA hybrids, preventing premature release and improving ligation efficiency and fidelity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If DNA ligases are used for RNA-templated DNA ligation, then ligation can occur, but the ligases are prematurely released from RNA-DNA hybrids resulting in low efficiency and incomplete reactions
Solution Approach 1:
The patent combines a DNA ligase domain with an RNAseH domain to create a chimeric enzyme. The RNAseH domain specifically binds to RNA-DNA hybrid structures, while the ligase domain performs the ligation reaction. This merging ensures the enzyme remains stably associated with the RNA-DNA hybrid substrate throughout the reaction, preventing premature release and improving both ligation efficiency and completeness
Solution Approach 2:
The RNAseH domain acts as an intermediary that mediates the interaction between the ligase and the RNA-DNA hybrid substrate. By providing a specific binding interface for the RNA-DNA hybrid through the RNAseH domain, the ligase can maintain stable association with the substrate, ensuring complete ligation reactions
2Measurement precision
If standard ligases are used for RNA-templated DNA ligation, then ligation reactions can proceed, but they lack specificity and produce high background noise
Solution Approach 1:
The chimeric enzyme merges the ligase function with the RNAseH binding function, creating a single enzyme that performs both substrate recognition and ligation. This ensures that only substrates properly bound by the RNAseH domain can be ligated, improving specificity and reducing background noise from non-specific ligation events
3Speed
If DNA ligases are used at elevated temperatures, then reaction speed increases, but the ligases lose stability and activity
Solution Approach 1:
The RNAseH domain in the chimeric enzyme provides enhanced thermal stability, allowing the ligase to maintain activity at elevated temperatures. The stable RNAseH-RNA-DNA hybrid interaction at higher temperatures anchors the ligase to the substrate, preventing thermal denaturation and maintaining both reaction speed and enzyme 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
The chimeric ligases exhibit enhanced RNA-templated DNA ligation activity, increased thermostability, and reduced ligation junction bias, enabling more efficient and specific detection of RNA sequences in biological samples.
Implementation Method 1
a catalytically inactive RNAseH polypeptide domain... enhances binding to RNA-DNA hybrids
Implementation Method 2
RNA-DNA hybrid duplex binding domains
Implementation Method 3
ligases, which are enzymes that catalyze the joining of two nucleic acid molecules by forming a new chemical bond
Implementation Method 4
forming a new chemical bond... catalyze the joining of two nucleic acid molecules
Data Source
AI summary
The present disclosure provides chimeric ligase polypeptides comprising a ligase polypeptide domain operably linked to a catalytically inactive ribonuclease H (RNAseH) polypeptide domain; compositions, isolated nucleic acid, and vectors comprising the chimeric ligase polypeptides; and multiplexed methods for detecting nuclei acids in a biological sample using hybridization-based in situ sequencing assays.