ToxN Endoribonuclease Cleavage for Specific Single-Stranded RNA Analysis
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Solution Overview
Problem
Current endoribonucleases face challenges in efficiently and specifically cleaving single-stranded RNA molecules, particularly for RNA analysis and synthesis, due to high fragmentation, unspecific cleavage, and reliance on hybridization or costly and unstable catalytic nucleic acids, leading to poor resolution in analytical methods.
Innovation Solution
The use of a sequence-specific ToxN endoribonuclease from Type III toxin-antitoxin systems, which operates optimally at specific monovalent salt concentrations and is inhibited by divalent metal cations, allowing for precise cleavage of single-stranded RNA molecules without the need for DNA probes or RNA guides, thus enhancing RNA analysis and synthesis methods.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If RNase I is used for RNA cleavage, then high cutting frequency is achieved, but high degree of fragmentation occurs
Solution Approach 1:
The patent applies local quality by using sequence-specific endoribonucleases that cleave RNA at specific recognition sites rather than along the entire molecule. This localized cleavage approach maintains high productivity at target sites while preventing unwanted fragmentation elsewhere in the RNA structure.
Solution Approach 2:
The patent changes the specificity parameter of the endoribonuclease from non-specific (RNase I) to sequence-specific (ToxN, RNase H, Csy4). This parameter change enables controlled cleavage at defined locations, resolving the contradiction between high cutting frequency and fragmentation control.
2Measurement precision
If RNase H is used for RNA cleavage, then sequence-specific cleavage is achieved, but unspecific and incomplete cleavage occurs
Solution Approach 1:
The patent optimizes the cleavage parameters of RNase H by adjusting DNA probe concentration, hybridization conditions, and enzyme incubation parameters. These parameter changes enable complete and specific digestion of the RNA target while minimizing unspecific cleavage.
Solution Approach 2:
The patent employs feedback mechanisms through optimization of hybridization conditions and probe design to ensure complete and specific cleavage. The system adjusts conditions based on cleavage efficiency to achieve reliable results.
3Measurement precision
If Csy4 endoribonuclease is used for RNA cleavage, then guide RNA-dependent specific cleavage is achieved, but complex guide RNA synthesis is required
Solution Approach 1:
The patent extracts the guide RNA component from the Csy4 system and replaces it with a simpler DNA probe-based approach using RNase H. This extraction eliminates the complex guide RNA synthesis requirement while maintaining sequence-specific cleavage capability.
Solution Approach 2:
The patent introduces a DNA probe as an intermediary element that mediates between the sequence-specific cleavage requirement and the simplified system design. The DNA probe serves as a stable, easily synthesized alternative to guide RNA, enabling specific cleavage without complex RNA synthesis.
4Measurement precision
If ribozymes are used for RNA cleavage, then sequence-specific cleavage is achieved, but high production cost and low stability occur
Solution Approach 1:
The patent uses a DNA probe as an intermediary that mediates the cleavage reaction, replacing the ribozyme catalytic RNA. This intermediary approach maintains sequence-specific cleavage while using more stable and easier-to-produce DNA molecules, reducing production costs and improving stability.
Solution Approach 2:
The patent substitutes the ribozyme catalytic mechanism with a DNA probe-based RNase H system. This replacement transitions from RNA-based catalysis to a DNA-protein hybrid system that is more stable and cost-effective to produce while maintaining specific cleavage function.
5Productivity
If DNAzymes are used for RNA cleavage, then catalytic activity is achieved, but lack of stability occurs
Solution Approach 1:
The patent introduces a DNA probe as an intermediary that mediates the catalytic cleavage reaction. This DNA-based intermediary is more stable than RNA-based catalysts while maintaining catalytic activity, thus resolving the contradiction between productivity and 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
ToxN endoribonuclease provides efficient and stable RNA cleavage, reducing unspecific activity and enabling accurate analysis of RNA modifications, such as 5'capping efficiency and poly(A) tail generation, with improved resolution in analytical techniques like gel electrophoresis and mass spectrometry.
Implementation Method 1
Endoribonucleases are a group of enzymes that cleaves internal phosphodiester bonds between adjacent nucleotides of RNA
Implementation Method 2
ToxN endoribonucleases recognises and cleaves single stranded ribonucleic acid (RNA) molecules
Data Source
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AI summary
The present disclosure provides compositions comprising sequence specific endoribonuclease and methods of their use in RNA analysis, RNA synthesis and fingerprinting of RNA molecules. In particular the present disclosure relates to compositions and samples comprising ToxN endoribonucleases that recognises and cleaves single stranded RNA and optimal conditions for obtaining cleavage.