A Simple and Robust Exponential Amplification Reaction (EXPAR)-Based Hairpin Template (exp-Hairpin) for Highly Specific, Sensitive, and Universal MicroRNA Detection

Specific and sensitive detection of microRNAs continues to encounter significant challenges, especially in the development of rapid and efficient isothermal amplification strategies for point-of-care settings. The exponential amplification reaction (EXPAR) has garnered significant attention owing to its simplicity and rapid amplification of signals within a short period. However, a substantial loss of amplification efficiency, difficulty in distinguishing closely related homologous sequences, and adapting the designed templates to other targets seriously hamper the practical application of the EXPAR. In this work, a hairpin template tailored for the EXPAR system (exp-Hairpin) was constructed by adding identical trigger sequences and enzyme cleavage sites on two arms of the hairpin, achieving theoretically more than 2 n amplification efficiency and minimal background amplification of EXPAR. Modulating the stability of the exp-Hairpin template by increasing the stem length, the specificity of detecting target miRNA in highly homologous sequences could be significantly improved. Using miRNA let-7a as a target model, the exp-Hairpin with 8 bp stem length for EXPAR amplification curves could effectively distinguish target let-7a and nontarget let-7b/7c/7f/7g/7i homologous sequences. This strategy enabled the sensitive and accurate analysis of let-7a in diluted human serum with satisfactory recoveries. By simply replacing the loop recognition sequence of exp-Hairpin, the specific detection of miR-200b was also achieved, demonstrating the universality of this strategy. The exp-Hairpin EXPAR accelerates simple and rapid molecular diagnostic applications for short nucleic acids.