Triblock Copolymer Cell Fixation for RNA Retention
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Solution Overview
Problem
Existing methods for stabilizing biomolecules in single cells result in poor RNA retention and high ambient RNA noise in sequencing data, leading to data loss and reduced accuracy.
Innovation Solution
A triblock copolymer system with hydrophobic and hydrophilic regions is used to stabilize cells by forming an internal polymer network, retaining biomolecules within the cell membrane, allowing for improved compatibility with downstream assays like single-cell RNA sequencing.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Stability of the object's composition
If conventional fixation methods (paraformaldehyde, methanol) are used to stabilize biomolecules in bulk cells, then biomolecule stability is improved, but RNA retention deteriorates leading to high ambient RNA noise in sequencing data
Solution Approach 1:
The invention divides the stabilization function into two distinct components: (1) conventional fixation agents that stabilize biomolecules, and (2) a novel polymer system (polyethylene glycol-polypropylene oxide-polyethylene glycol triblock copolymer) that specifically retains RNA and prevents ambient noise. This segmentation allows each component to perform its specialized function without interfering with the other, resolving the contradiction between stabilization and retention.
Solution Approach 2:
The invention uses a composite material system combining traditional fixation chemicals with a specially designed triblock copolymer. The copolymer contains hydrophobic polypropylene oxide regions that interact with cell membranes and hydrophilic polyethylene glycol regions that interact with RNA, creating a composite stabilization-retention system that overcomes the limitations of conventional single-function fixation methods.
2Duration of action of stationary object
If fixation methods are used to preserve biomolecules, then sample stability during transportation and storage is improved, but compatibility with downstream single-cell assays deteriorates
Solution Approach 1:
The triblock copolymer acts as an intermediary substance between the fixed cell and downstream sequencing assays. It provides long-term stabilization during storage while being chemically inert and compatible with subsequent single-cell lysis and RNA extraction protocols, thus bridging the gap between long-term stability and assay compatibility.
Solution Approach 2:
The invention changes the physical-chemical parameters of the fixation system by using a polymer-based approach rather than traditional crosslinking agents. This allows for adjustable parameters such as polymer concentration, molecular weight, and block ratios to be optimized for both long-term stability and downstream assay compatibility, providing versatility across different single-cell applications.
3Loss of information
If conventional fixation is applied to single cells, then biomolecule preservation is improved, but data quality and yield deteriorate due to poor RNA retention
Solution Approach 1:
The invention converts the potential harm of RNA degradation and loss into a benefit by using the triblock copolymer to actively retain RNA within fixed cells. The polymer's amphiphilic structure creates a protective environment that not only prevents degradation but also enriches RNA retention, turning the challenge of maintaining RNA integrity into an advantage for improving data quality and yield.
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 triblock copolymer system enhances biomolecule retention, reducing data loss and improving signal-to-noise ratio, thereby increasing the flexibility and accuracy of sample collection, transportation, and storage for single-cell analysis.
Implementation Method 1
the hydrophobic region is capable of stable insertion into and retention within a cell membrane
Implementation Method 2
the first hydrophilic region, and optionally the second hydrophilic region, comprises a bioorthogonal reactive moiety
Data Source
AI summary
Systems and methods are provided for stabilization of cells and retention of biomolecules therein for further analysis. Block copolymers are inserted into membranes of biological particles, cell membranes or organelles (e.g., organelle membranes) and polymerized to form an intra-biological particle (e.g., intracellular) polymer network.