miRNA Isolation Using Anti-Binding Proteins and Protease
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Solution Overview
Problem
Current methods for isolating microRNAs from biofluids are complex, not readily automatable, and inefficient, particularly for low-abundance miRNAs, and often require toxic compounds, making it challenging to diagnose and monitor diseases effectively.
Innovation Solution
A method involving the use of a surface active agent and an anti-miRNA-binding protein reagent to form immunoprecipitated miRNA complexes, followed by protease treatment at room temperature to release miRNAs without purification, allowing for the simultaneous isolation of both vesicle-associated and non-vesicle associated miRNAs in high yield.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Extent of automation
If spin columns are used for miRNA isolation, then isolation can be performed, but the method is not readily automatable and cannot be scaled up
Solution Approach 1:
The patent extracts the miRNA from complex biological matrices by utilizing the specific binding interaction between anti-miRNA-binding protein reagents and miRNA-binding proteins. This extraction approach eliminates the need for complex spin column procedures, enabling straightforward automation and scaling while maintaining isolation effectiveness.
Solution Approach 2:
The patent introduces an intermediary anti-miRNA-binding protein reagent that mediates the isolation process. This reagent acts as a bridge between the miRNA target and the isolation system, simplifying the overall procedure and making it amenable to automation without requiring complex mechanical separation devices.
2Ease of manufacture
If current isolation methods are used, then miRNA can be isolated, but the methods involve toxic compounds and are complicated
Solution Approach 1:
The patent converts the naturally occurring miRNA-binding proteins into beneficial tools for isolation. Instead of using toxic chemical reagents, the method utilizes the specific binding properties of endogenous proteins, turning a potential complicating factor into an advantageous feature that simplifies the procedure and eliminates toxic compounds.
Solution Approach 2:
The patent employs disposable anti-miRNA-binding protein reagents that can be used in a single isolation cycle and then discarded. This approach eliminates the need for complex cleanup procedures and toxic solvent systems required by traditional methods, making the process simpler and safer while maintaining high isolation efficiency.
3Measurement precision
If current methods are used for low-abundance miRNAs, then isolation is attempted, but detection is challenging due to low abundance
Solution Approach 1:
The patent segments the isolation process into specific binding and release steps, allowing for the enrichment of low-abundance miRNAs through high-affinity binding. This segmented approach enables the concentration of trace miRNA molecules from large volumes of biofluid, significantly improving detection precision even when starting quantities are extremely low.
Solution Approach 2:
The patent performs preliminary enrichment of miRNAs using anti-miRNA-binding protein reagents before detection. This preliminary action concentrates the low-abundance target molecules, making them detectable by subsequent analytical methods and overcoming the limitation of low starting quantities in biofluid samples.
4Adaptability or versatility
If vesicular or non-vesicular miRNAs are specifically isolated, then one type is obtained, but the other type is excluded
Solution Approach 1:
The patent employs a universal anti-miRNA-binding protein reagent that can bind to miRNAs regardless of whether they are vesicular or non-vesicular. This multi-functional approach allows simultaneous isolation of both miRNA populations, expanding the scope of the method and maximizing total miRNA recovery from biological samples.
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
This method enables efficient, scalable, and automated isolation of pure miRNAs from dilute extracellular fluids, reducing contamination and improving the detection of miRNAs for diagnostic purposes.
Implementation Method 1
contacting the biological sample with a surface active agent and an anti-miRNA-binding protein reagent, wherein the surface active agent dissociates sample components
Implementation Method 2
releasing miRNA from the immunoprecipitated miRNA complexes without purification by contacting, at room temperature, the immunoprecipitated miRNA complexes with a protease
Data Source
Figure 1A~1B
Figure 2A~2C
Figure 3A~3C
AI summary
The present disclosure provides methods and kits for isolating miRNAs from biological fluids.