Stepwise tuning of a molecular beacon coupled Y probe regulates ternary DNA nanomachine-based microRNA determination

Abstract

The construction of DNA nanodevices is vital for disease diagnosis and biomedical study. Herein, we proposed a ternary DNA nanomachine for microRNA determination using a molecular beacon (MB) coupled Y shape probe (MB-YP). The MB-YP was a tripartite junction scaffold connected with MB-YI, YII, and YIII. The target miRNA firstly recognizes MB-YI, activating the MB-YI-DNA nanomachine to produce endogenous primer I (EPI) responsible for the activation of YII-DNA nanomachine. The YII-DNA nanomachine then allows the production of numerous EPII, which in turn activates the YIII-DNA nanomachine to produce EPIII for re-activating MB-YI-DNA nanomachine. The stepwise operated MB-YI-DNA nanomachine, YII-DNA nanomachine, and YIII-DNA nanomachine constituted the ternary DNA nanomachine and the repeated use of EPI, EPII, and EPIII permits it an all-in amplification. These features lead to the opening of MB-YI for emitting strong fluorescence. As low as 1 fM miRNA can thus be quantitatively detected and the linear response of the miRNA is from 1 fM to 100 pM (six orders of magnitude). Moreover, the nanomachine is specific to distinguish target miRNA from one-base mutant, inserted, and deleted target analogs. Its practicability was demonstrated by confirming miRNA-21 is overexpressed in clinical human serums from lung cancer patients than healthy donors.