Engineering thermostable fluorescent DNA aptamer for the isothermal amplification of nucleic acids

Isothermal amplification-based nucleic acid detection technologies have become rapid and efficient tools for molecular diagnostics. Sequence-specific monitoring methods are crucial for isothermal amplification, as they help identify the occurrence of extended primer dimers, which can lead to false positive results. Fluorescent aptamers are promising tools for real-time monitoring of isothermal amplification but are inherently limited by thermostability. Here, we report an engineered fluorescent DNA aptamer variant, named thermostable Lettuce (TS-Lettuce), with a 5 °C higher melting temperature and 20 times greater fluorescence at 60 °C, ideal for real-time monitoring of sequence-specific isothermal amplification. Using molecular dynamics simulations for structural analyses, we introduced mutations to wild-type Lettuce to redesign the non-core sequences of the aptamer structure for tightly stabilizing its folding, thereby enhancing thermostability. The TS-Lettuce offers greater versatility and ease of design for coupling with isothermal amplification for all-in-one nucleic acid detection. We demonstrated three applications of TS-Lettuce in isothermal amplification: fluorescent turn-off, fluorescent turn-on, and fluorescent aptamer switch, facilitating the sequence-specific detection of nucleic acids. In addition, the results generated by TS-Lettuce are visible to the naked eye, enhancing the utility of isothermal amplification reactions in resource-constrained areas. The thermostable fluorescent DNA aptamers can be further utilized in more isothermal amplification methods.