Understanding how metabolites and metal ions interact with proteins inside living cells is essential for decoding cellular signaling, metabolic regulation, and disease mechanisms. These small molecules function as cofactors, second messengers, and allosteric regulators, orchestrating critical pathways that govern cell behavior. However, traditional biochemical methods often rely on lysate-based assays that disrupt native spatial organization and lose transient or low-affinity interactions, creating significant gaps in our knowledge of dynamic cellular processes.
Existing interactome mapping approaches—such as affinity purification-mass spectrometry and enzyme-mediated proximity labeling—face notable limitations. Lysate-based workflows destroy subcellular localization and underrepresent fast or labile interactions. Genetically encoded tags used in enzyme-based methods are bulky and difficult to apply in primary cells or tissues, and small-molecule probes frequently suffer from off-target effects and inefficient intracellular delivery. Moreover, slow reaction kinetics limit the capture of transient biological events, making it difficult to fully map metabolite and metal ion signaling under physiological conditions.
This technology introduces functional DNA-based sensors that use aptamers or DNAzymes engineered to carry a sulfonyl fluoride (SuFEx) electrophile at the 2′ sugar position. In their resting state, the electrophile is sequestered within a stable DNA duplex, preventing premature activation. Upon binding a specific intracellular metabolite or metal ion, a conformational change exposes the SuFEx group, triggering proximity-based covalent labeling of proteins near the target pool.
The labeled proteins are subsequently identified and quantified via quantitative proteomics, all without requiring cell lysis or disrupting cellular organization. Demonstrated for ATP and sodium ions (Na⁺), the platform provides high spatial and temporal resolution of dynamic interactomes in living cells. Its modular architecture allows rapid substitution of DNA sensors to adapt to different biological targets, and ongoing improvements in delivery and reaction kinetics further enhance its utility for diverse biological systems.