Structural Reorganization-based Catalytic Hairpin Assembly Enable Small Molecule Monitoring in Living Cell

Abstract

Small-molecule drugs, constituting over 60% of FDA-approved therapeutics (2017–2022), face unresolved challenges in elucidating intracellular mechanism. 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.