tRNA-Fused Strategy for Enhancing RNA Aptamer Sensor Stability

RNA aptamer sensors are promising for environmental and clinical detection, but their poor stability limits practical application. Here, we developed a tRNA-fused strategy to enhance the stability of unmodified RNA aptamer sensors. The tRNA scaffold was fused to the 3′ end of Spinach aptamer to construct tRNA-Spinach. In vitro stability assays showed that tRNA-Spinach retained 50% of its initial fluorescence for 84 days at 25 °C (60% humidity), a 7-fold improvement compared with native Spinach (12 days). The tRNA-fused strategy also doubled the in vivo half-life of Spinach from 20 min to 40 min in KM mice. Based on this strategy, a tobramycin sensor was constructed, which exhibited a LOD of 30 nM, a linear range of 30–100 nM (R2 = 0.9905). The biosensor could be detected with a handheld UV lamp within 10 min. This tRNA-fused strategy enables room-temperature storage of RNA aptamer sensors without chemical modification, providing a scalable and cost-effective platform for point-of-care diagnostics in resource-limited settings.