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

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-Y I , Y II , and Y III . The target miRNA firstly recognizes MB-Y I , activating the MB-Y I -DNA nanomachine to produce endogenous primer I (EP I ) responsible for the activation of Y II -DNA nanomachine. The Y II -DNA nanomachine then allows the production of numerous EP II , which in turn activates the Y III -DNA nanomachine to produce EP III for re-activating MB-Y I -DNA nanomachine. The stepwise operated MB-Y I -DNA nanomachine, Y II -DNA nanomachine, and Y III -DNA nanomachine constituted the ternary DNA nanomachine and the repeated use of EP I , EP II , and EP III permits it an all-in amplification. These features lead to the opening of MB-Y I 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.