SMART strategy-driven engineering of multifunctional aptamers: Multifaceted applications in biosensing, toxicity...

The development of functional nucleic acids (FNAs) targeting small molecules has been challenging due to the limited number of functional groups available, hindering the creation of effective binding elements and functional regulation. This limitation significantly impacts both the in vivo and in vitro applications of small-molecule aptamers and impedes their pharmaceutical potential. In this study, we presented the Stepwise Molecular Aptamer Refinement and Tailoring (SMART) strategy and applied it to optimize the malachite green (MG)-binding DNA aptamer as an example, enhancing its multifunctional properties, including stability, fluorescence activation, and binding affinity. The refined "light bulb aptamer", CAG-T-ML-S4-TA-AT+1-4L, exhibited a 27-fold improvement in affinity (Kd: 1.173 μmol·L^-1 → 0.044 μmol·L^-1), a substantial enhancement of thermal stability (Tm: 42.77℃→ 61.55℃), and a nearly 7-fold increase in fluorescence intensity compared to the parent sequence, along with a 100-fold improvement relative to free MG molecules. Molecular docking and dynamic simulations revealed critical structural features pivotal for target-aptamer interactions. The "light bulb aptamer", as a powerful molecular tool, was further explored for its versatility, which expanded its multidimensional applications in: (1) Biosensing, where we developed a ratiometric, label-free aptasensor with a broad detection range (5 nmol·L^-1 to 8 µmol·L^-1) and a low detection limit (1.12 nmol·L^-1) with high specificity for environmental hazards; (2) Toxicity Masking, where it improved cell viability by mitigating MG-induced toxicity across various cell models; and (3) Sustained-Release Antibacterial Effects, prolonging bactericidal activity and minimizing antimicrobial resistance. The successful application of the SMART strategy enhances the potential of nucleic acids as molecular tools, offering new opportunities in point-of-care testing, toxicity masking, and antimicrobial resistance management. This work establishes a universal optimization method and powerful molecular tools to drive advancements in biosensing and biomedicine.