Abstract
ABSTRACT
Cell‐surface receptors are essential biomarkers and therapeutic targets, yet detecting them in native biological environments remains challenging. Turn‐on, ligand‐directed fluorescent probes have advanced the study of these receptors by enabling low‐background imaging and the sensing of interactions with binding partners. However, their broader utility is hindered by the difficulty of tuning specificity, binding affinity, and emission properties for each new target. Here, we introduce a highly modular molecular beacon (MB)‐based platform that overcomes these limitations. Building on the spatial proximity of many cell‐surface receptors and the conformational switching of MBs, we designed multivalent, ligand‐appended hairpin probes that strongly engage these receptors while undergoing stem opening and fluorescence activation at the cell surface. This design, together with the structural programmability of these single‐stranded DNA constructs, enables tuning of emission wavelength, receptor selectivity, and binding strength through simple exchange of fluorophores and ligands, and modulation of valency. For rapidly endocytosed receptors, we further demonstrate an alternative long‐stem MB design that exploits intracellular probe cleavage to trigger fluorescence. By applying these MBs to image distinct receptors, distinguish cellular states, and detect receptor–inhibitor interactions under wash‐free conditions, we demonstrate a versatile and generalizable strategy for probing cell‐surface receptors in living cells.