Antidote-Mediated Aptamer Staining for Native-State Cell Sorting
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Existing cell purification methods using antibodies for selective labeling are irreversible, compromising the utility of isolated cells in downstream applications and disrupting native signaling pathways, making them unsuitable for clinical and basic research.
Innovation Solution
The use of aptamers and antidotes for reversible cell staining, allowing for the identification and sorting of live cells by binding to surface molecular targets and subsequently removing the aptamer to restore the cells to their native state.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If antibodies are used for selective cell labeling, then cell identification and sorting capability is improved, but the labeling becomes irreversible and cell function is compromised
Solution Approach 1:
The patent introduces an intermediary system consisting of aptamer-cell surface target binding followed by antidote-aptamer binding. The antidote acts as a mediator that displaces the aptamer from the cell surface target, enabling reversible labeling. This two-step intermediary mechanism resolves the contradiction by providing both specific identification (through aptamer binding) and functional restoration (through antidote-mediated displacement), unlike direct antibody labeling which is irreversible.
Solution Approach 2:
The patent exploits parameter changes in binding affinity through the introduction of antidotes. By changing the binding parameters (adding complementary oligonucleotides that bind to the aptamer with high affinity), the aptamer is displaced from the cell surface target. This dynamic parameter change enables reversible labeling, allowing cells to be identified during sorting while maintaining functionality for downstream applications after the antidote is added.
2Quantity of substance
If antibodies are used for cell purification, then selective labeling is achieved, but native signaling pathways are disrupted
Solution Approach 1:
The aptamer serves as an intermediary binding molecule that temporarily labels cell surface targets for purification purposes. Unlike antibodies that permanently block receptors, the aptamer can be displaced by antidotes, allowing the cell surface targets to resume their native signaling functions after purification. This intermediary approach maintains purification selectivity while avoiding permanent disruption of cellular pathways.
Solution Approach 2:
The patent applies preliminary anti-action by pre-binding aptamers to cell surface targets for purification, then subsequently applying antidotes to reverse the binding and restore native function. This two-stage approach first achieves selective purification, then proactively counteracts any potential functional disruption by displacing the aptamer, preventing harmful effects before they can manifest in downstream applications.
3Productivity
If antibody labeling is used for cell sorting, then cell separation efficiency is improved, but the labeled cells are unsuitable for clinical therapeutic use due to immunogenicity
Solution Approach 1:
The patent substitutes the antibody-based labeling mechanism with an aptamer-based mechanism. Aptamers are nucleic acid-based molecules that can be synthesized without immunogenic protein components. This substitution maintains the ability to selectively label and sort cells while eliminating the immunogenicity problem, making the labeled cells suitable for clinical therapeutic applications where patient safety is critical.
Solution Approach 2:
The aptamer molecules serve as temporary, disposable labeling agents that can be easily removed by antidotes. Unlike antibodies that remain bound to cells and pose immunogenic risks, the short-lived aptamer labels are designed to be transient and removable, ensuring that no foreign immunogenic material remains in the purified cells for clinical use.
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
Enables the positive sorting and identification of cells within heterogeneous populations while maintaining their viability and functional integrity, overcoming the limitations of irreversible antibody labeling.
Implementation Method 1
contacting the surface-bound molecular target with a sorting aptamer having a binding affinity for the molecular target
Implementation Method 2
removing the sorting aptamer from the surface-bound molecular target may comprise contacting the sorting aptamer with an oligonucleotide antidote, the oligonucleotide antidote comprising a sequence at least partially complementary to a portion of the sorting aptamer
Data Source
AI summary
Provided herein are compositions and methods for sorting and/or identifying live cells. The compositions and methods provide for staining of live cells with aptamer so particular cells can be identified within or sorted from a heterogeneous population of live cells and subsequent reversal of the staining to prepare sorted and/or identified cells in their native state.