DNA纳米机器的发展为肿瘤标记物的检测提供了一种新策略。在这项工作中，设计了一种智能的三维DNA步行机，其带有多核苷酸激酶（PNK）激活剂，与由DNA纳米线串级扩增反应形成的独特纳米机器相结合，用于通用荧光检测T4 PNK活性和信使RNA（mRNA）。当PNK存在时，通过外切酶的水解裂解形成自由DNA步行器，然后在循环裂解反应中剪切Au纳米粒子（NPs）上的荧光标记报告探针，从而恢复荧光信号以检测PNK。此外，通过滚环扩增产生DNA纳米线，然后目标mRNA通过DNA纳米线顺序启动发夹带探针的间隙杂交，从而实现高“开”信号的DNA纳米机器的DNA级联反应（DCR）进行mRNA检测。这种新型荧光纳米机器开发了一种具有广泛靶标应用前景的新型测定方法，并显示出分子靶向治疗和临床诊断的巨大潜力。版权所有©2023 Elsevier B.V.。保留所有权利。

The development of DNA nanomachines provides a new strategy for the detection of tumor markers. In this work, an intelligent three-dimensional (3D) DNA walking machine with polynucleotide kinase (PNK) activator was designed, which was coupled with unique nanomachine formed by DNA nanowire cascade amplification reaction for versatile fluorescence detection of T4 PNK activity and messenger RNA (mRNA). When PNK exists, the free DNA walker was formed by hydrolysis cleavage of exonuclease, then the fluorophore-labeled report probe on the Au nanoparticles (NPs) was sheared during cycling cleavage reaction, thus the fluorescence signal was recovered for detection of PNK. Moreover, the DNA nanowires were produced by rolling ring amplification, then target mRNA sequentially initiated interval hybridization of hairpin probes through DNA nanowire, thus realizing DNA cascade reaction (DCR) with high "on" signal of DNA nanomachine for mRNA assay. This developed novel fluorescence nanomachine reported a new assay method with promising application for versatile targets and showed great potential for molecular-target therapies, and clinic diagnostics.Copyright © 2023 Elsevier B.V. All rights reserved.