DNA-Based Proximity Labeling for Live-Cell Metabolite Mapping
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Solution Overview
Problem
Current methods for identifying protein-metabolite and protein-metal ion interactions in live cells are limited by a lack of generalizable and discovery-based approaches, as existing tools struggle with the transient and low-affinity nature of these interactions and often require cell lysates, losing spatial context.
Innovation Solution
A conformationally gated sensor, such as aptamers or DNAzymes, is administered to cells to interact with metabolites or metal ions, undergoing a conformational change to expose reactive electrophiles that label nearby macromolecules, followed by extraction and identification using quantitative mass spectrometry.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If conventional protein interaction detection tools are used, then protein-protein interactions can be identified, but they fail to detect protein-metabolite and protein-metal ion interactions due to transient and low-affinity nature
Solution Approach 1:
The patent introduces a proximity labeling reagent as an intermediary that mediates between the target protein and the detection system. The reagent contains a reactive group that covalently labels nearby proteins, enabling indirect detection of transient interactions that conventional direct binding methods cannot capture
Solution Approach 2:
The patent changes the detection parameter from direct binding affinity to spatial proximity through covalent labeling. By using a reactive electrophile that forms covalent bonds with nearby nucleophiles, the method transforms transient weak interactions into stable detectable labels, resolving the reliability issue
2Ease of operation
If cell lysates are used for interaction detection, then interactions can be studied in controlled conditions, but spatial context and intracellular distribution information are lost
Solution Approach 1:
The patent performs preliminary action by introducing the proximity labeling reagent into live cells before lysis. The reagent labels proteins in their native spatial context within intact cells, preserving location information that would be lost if lysis were performed first
Solution Approach 2:
The patent replaces the mechanical disruption of cell lysis with a chemical labeling approach that works in situ. The electrophilic labeling reagent chemically modifies proteins in living cells without requiring mechanical breakdown, thereby preserving spatial information
3Measurement precision
If known metabolite binding pockets are used for labeling, then specific metabolites can be targeted, but discovery of novel interactions is limited
Solution Approach 1:
The patent extracts the requirement for pre-known binding pockets by using a general electrophilic labeling reagent that reacts with any nearby protein containing nucleophilic residues. This removes the constraint of needing defined binding sites, enabling discovery of novel interactions while maintaining spatial precision through the proximity-based mechanism
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 detection of macromolecule interactors in the vicinity of metabolite or metal ion pools within live cells, providing spatially relevant interaction data.
Implementation Method 1
the sensor to interact with the metabolite or metal ion, thereby undergoing a conformational change upon binding the metabolite or metal ion, thereby exposing the reactive (e.g., protein-reactive) electrophile
Implementation Method 2
a conformationally gated sensor that has affinity for the metabolite or metal ion
Implementation Method 3
the exposed reactive (e.g., protein-reactive) electrophile can label nearby macromolecules (e.g., proteins or nucleic acids)
Implementation Method 4
extracting and identifying the proximal macromolecules (e.g., proteins or nucleic acids) that have been labeled by the sensor's reactive (e.g., protein-reactive) electrophile
Data Source
AI summary
Provided herein are methods of detecting macromolecule (e.g., proteins or nucleic acids) interactors in the vicinity of a metabolite or metal ion pool in a cell. The methods may comprise DNAzyme-and aptamer-based proximity labeling identification (DAP-ID) to identify macromolecules (e.g., proteins or nucleic acids) in the vicinity of intracellular metabolites or metal ions. DAP-ID leverages metabolite-selective aptamers and metal ion-selective DNAzymes that undergo target-induced conformational changes to expose reactive electrophiles (e.g., sulfonyl fluoride electrophiles) for covalent macromolecule (e.g., protein or nucleic acid) tagging.