Sensitive, specific and rapid methods for detecting microRNAs (miRNAs) play critical roles in disease diagnosis and therapy. Enzyme-free amplification techniques based on DNAzyme assembly have recently been developed for the highly specific miRNA analysis. However, traditional DNAzyme-based assembly (free DNAzyme) amplifiers is mainly dependent on the target-induced split DNAzyme fragments to assemble into activated DNAzyme structures, which have made a compromise between the sensitivity and specificity due to the random diffusion of dissociative probes in a bulk solution with poor kinetics. Herein, based on a rationally designed DNA probe, we developed an intramolecular DNAzyme assembly (intra-DNAzyme) method to overcome these challenges. The miR-373 is used as model analyte for our current proof-of-concept experiments. Compared with the free-DNAzyme method, our method showed significantly improved analytical performance in terms of dynamic range, assay sensitivity and speed. This method can detect miR-373 specifically with a detection limit as low as 4.3 fM, which is about 83.7 times lower than the previous free-DNAzyme method. This intra-DNAzyme strategy would be of great value in both basic research and clinical diagnosis.

用于检测 microRNA (miRNA) 的灵敏、特异和快速的方法在疾病诊断和治疗中发挥着关键作用。最近开发了基于 DNAzyme 组装的无酶扩增技术，用于高度特异性的 miRNA 分析。然而，传统的基于 DNAzyme 的组装（游离 DNAzyme）放大器主要依赖于靶诱导的分裂 DNAzyme 片段组装成活化的 DNAzyme 结构，由于解离探针的随机扩散，在灵敏度和特异性之间做出了折衷。动力学较差的散装溶液。在此，基于合理设计的 DNA 探针，我们开发了一种分子内 DNAzyme 组装（intra-DNAzyme）方法来克服这些挑战。miR-373 用作我们当前概念验证实验的模型分析物。与游离 DNAzyme 方法相比，我们的方法在动态范围、测定灵敏度和速度方面显示出显着提高的分析性能。该方法可特异性检测miR-373，检测限低至4.3 fM，比以往的游离DNAzyme方法降低约83.7倍。这种 DNAzyme 内策略在基础研究和临床诊断中都具有重要价值。