Controlling dopamine binding by the new aptamer for a FRET-based biosensor.

Dopamine is one of the most important neurotransmitters. A high-quality DNA aptamer for dopamine was reported in 2018. However, fundamental understanding of its binding and folding is lacking, which is critical for related biosensor design. Herein, we performed careful assays using a label-free technique called isothermal titration calorimetry (ITC) to study its secondary structure. We divided this aptamer into four regions and individually examined each of them. We confirmed two stems, but the third stem is believed to be part of a loop. The aptamer was then truncated. The original aptamer had a Kd of 2.2 ± 0.3 μM at 25 °C. Shortening the structure by one or two base pairs increased the Kd to 6.9 and 44.4 μM, respectively. Dopamine binding was promoted by both increasing the Mg2+ concentration and decreasing the temperature. At 5 °C, a Kd of 0.4 μM was achieved. Based on this understanding, we designed two fluorescence resonance energy transfer (FRET) quenching biosensors that differ only by a base pair. The shorter sensor had 3-fold higher sensitivity and a detection limit of 0.9 μM. In 1% fetal bovine serum, the sensor retained a similar limit of detection of 1.14 μM. A two-fluorophore ratiometric FRET sensor was also demonstrated with a low detection limit of 0.12 μM. This work indicated the feasibility of designing folding-based sensors for dopamine, and this design can be extended to other sensing modalities such as electrochemistry and colorimetry.