Aptamer-Based Sensing with Fluorescent Light-Up DNA

Abstract DNA aptamers are widely used in the construction of fluorescent sensors, typically employing labelled fluorophores as signaling indicators. However, this covalent labeling approach suffers from several limitations, including complex and costly chemical modification, incompatibility with long aptamer sequences, and susceptibility to false-positive signals. To address these limitations, we constructed a novel aptamer-based fluorescent sensor using Lettuce indicator, a light-up DNA aptamer that adopts a precise three-dimensional structure, which enables it to selectively bind and activate the otherwise non-fluorescent small-molecule fluorophores. In our design, we destabilized Lettuce by fusing it with a target-binding aptamer via a transducer sequence. Upon target binding, structural rearrangement is triggered through the transducer, leading to the folding of Lettuce and restoration of its ability to activate the fluorophore, generating a target-dependent fluorescent signal. Through systematic optimization of the transducer and target-binding aptamer sequences, we created sensors for diverse targets, including small molecules, proteins, and metal ions. These sensors exhibit high signal-to-noise ratios, sensitivity and selectivity, and a wide dynamic range. As a proof-of-concept demonstration, a paper-based test strip for cost-effective and rapid detection of small molecule mycotoxins was developed. This versatile design provides a generalizable platform for the development of aptamer-based sensors, opening the way for future detection of diverse analytes.