Aptamer and Thiol Co‐Regulated Color‐Shifting Fluorophores via Dynamic Through‐Bond/Space Conjugation for Constructing Ratiometric RNA Sensor

Abstract Fluorophores with color‐shifting characteristics have attracted enormous research interest in the quantitative application of RNA sensors. It reports here a simple synthesis, luminescent properties, and co‐transcription ability of de‐conjugated triphenylmethane leucomalachite green (LMG). This novel clusteroluminescence fluorophore is rapidly synthesized from malachite green (MG) in reductive transcription system containing dithiothreitol, emitting fluorescence in the UV region through space conjugation. The co‐transcribed MG RNA aptamer (MGA) bound to the ligand, resulting in red fluorescence from the through‐bond conjugation. Given the equilibrated color‐shifting fluorophores, they are rationally employed in a 3WJ‐based rolling circle transcription switch, with the target‐aptamer acting as an activator to achieve steric allosterism. This one‐pot system allows the target to compete continuously for allosteric sites, and the activated transcription switches continue to amplify MGA forward, achieving accurate Aflatoxin 1 quantification at the picomolar level in 1 h. Due to the programmability of this RNA sensor, the design method of target‐competitive aptamers is standardized, making it universally applicable.