Guo Haijing, Chen Jun, Feng Yaqiang, Dai Zong
College of Chemistry and Materials Science, Northwest University, Xi'an 710127, PR China.
School of Pharmaceutical Sciences, Southern Medical University, Guangzhou 510515, PR China.
Anal Chem. 2024 Feb 13;96(6):2643-2650. doi: 10.1021/acs.analchem.3c05323. Epub 2024 Jan 31.
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 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.
微小RNA的特异性和灵敏检测仍然面临重大挑战,尤其是在为即时检测环境开发快速高效的等温扩增策略方面。指数扩增反应(EXPAR)因其简单性以及能在短时间内快速扩增信号而备受关注。然而,扩增效率大幅损失、难以区分密切相关的同源序列以及使设计的模板适用于其他靶标,严重阻碍了EXPAR的实际应用。在这项工作中,通过在发夹的两条臂上添加相同的触发序列和酶切位点,构建了一种为EXPAR系统量身定制的发夹模板(exp-Hairpin),实现了理论上超过2倍的扩增效率以及EXPAR的最小背景扩增。通过增加茎长度来调节exp-Hairpin模板的稳定性,可以显著提高在高度同源序列中检测靶标微小RNA的特异性。以微小RNA let-7a作为靶标模型,用于EXPAR扩增曲线的茎长度为8 bp的exp-Hairpin能够有效区分靶标let-7a和非靶标let-7b/7c/7f/7g/7i同源序列。该策略能够对稀释的人血清中的let-7a进行灵敏且准确的分析,回收率令人满意。通过简单替换exp-Hairpin的环识别序列,还实现了对miR-200b的特异性检测,证明了该策略的通用性。exp-Hairpin EXPAR加速了针对短核酸的简单快速分子诊断应用。
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