Exosome-like Vesicle Production for Noninvasive MicroRNA Acquisition
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Solution Overview
Problem
The challenge lies in noninvasively acquiring sufficient microRNAs (miRNAs) from cells, as they are present in low abundance in the cytoplasm and their encapsulation in exosomes results in extremely low concentrations, making accurate diagnosis and drug development difficult.
Innovation Solution
A method involving the de novo design of a miRNA binding protein and a vesicle-forming protein, where the miRNA binding protein includes an MID domain and a PIWI domain of an Argonaute protein combined with a virus protein R, and the vesicle-forming protein contains palmitoylation or myristoylation signals, self-assembling domains, and a Gag p6 domain, is introduced into cells to produce exosome-like vesicles encapsulating miRNAs, which are then collected and extracted from extracellular fluid.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If exosomes are formed by incorporating cytoplasm to encapsulate miRNAs, then miRNAs can be noninvasively acquired from cells, but the concentration of miRNAs remains extremely low
Solution Approach 1:
The patent introduces a designer protein as an intermediary substance that specifically binds to miRNAs. This protein is engineered with a nanocage domain that facilitates encapsulation into exosomes. The protein-miRNA complex serves as a mediator that increases the effective concentration and detectability of miRNAs in exosomes, resolving the contradiction between noninvasive acquisition and measurement precision
Solution Approach 2:
The patent changes the concentration parameter of miRNAs in exosomes by introducing exogenous designer proteins that bind to miRNAs. The binding protein acts as a carrier that increases the apparent concentration of miRNAs in the exosomal compartment, thereby improving measurement precision while maintaining noninvasive acquisition methodology
2Ease of manufacture
If exosome-like vesicles are produced by incorporating cytoplasm, then vesicles can be formed to encapsulate target proteins, but miRNAs present in low abundance in cytoplasm cannot be acquired
Solution Approach 1:
The designer protein serves as an intermediary that bridges the gap between low-abundance cytoplasmic miRNAs and exosome encapsulation. The protein's nanocage domain facilitates efficient packaging, while its miRNA-binding domain concentrates scarce miRNAs, thereby improving yield without complicating the manufacturing process
Solution Approach 2:
The patent applies preliminary action by pre-loading the designer protein with miRNAs in the cytoplasm before exosome formation. This pre-concentration step ensures that even low-abundance miRNAs are captured and packaged into exosomes at sufficient levels, improving yield without requiring changes to the overall manufacturing ease
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 approach significantly increases the concentration of miRNAs in extracellular fluid, enabling more accurate and sensitive noninvasive analysis and facilitating the development of nucleic acid medicine by providing a higher yield of miRNAs.
Implementation Method 1
the microRNA-binding protein comprises a first portion consisting of an MID domain and a PIWI domain of an Argonaute protein
Implementation Method 2
the vesicle-forming protein comprises: a palmitoylation or myristoylation signal or a pleckstrin homology domain; a self-assembling domain
Implementation Method 3
thereby an exosome-like vesicle including a microRNA is produced, (2) collecting an extracellular fluid of the cell
Data Source
AI summary
A method for noninvasively acquiring a microRNA from a cell, the method comprising (1) introducing a nucleic acid coding a microRNA-binding protein and a nucleic acid coding a vesicle-forming protein into the cell, wherein the microRNA-binding protein comprises a first portion consisting of an MID domain and a PIWI domain of an Argonaute protein and a second portion consisting of a virus protein R, and the vesicle-forming protein comprises: a palmitoylation or myristoylation signal or a PH domain; a self-assembling domain; an ESCRT or ESCRT-related factor-binding domain; and a Gag p6 domain; thereby producing an exosome-like vesicle comprising a micro-RNA, (2) collecting an extracellular fluid of the cell, and (3) extracting the micro-RNA from the extracellular fluid.