A structural reorganization-based catalytic hairpin assembly enabling small-molecule monitoring in living cells

Abstract

Small-molecule drugs, constituting over 60% of FDA-approved therapeutics (2017–2022), present unresolved challenges relating to elucidating their intracellular mechanisms. We present a dual-strategy platform integrating “in silico aptamer affinity maturation” (ISAAM) and “structural reorganization-catalytic hairpin assembly” (SR-CHA). ISAAM computationally designs high-affinity aptamers, while SR-CHA eliminates undesired signals via energy-minimized conformational control, achieving a signal-to-background improvement over conventional CHA. This system enables ultrasensitive small-molecule monitoring in live cells, resolving traditional challenges of false positives and inefficiency. Demonstrated through intracellular imaging and kinetic studies, SR-CHA offers a robust tool for probing small-molecule interactions in biological systems, advancing drug discovery and diagnostic applications.