Delayed full opening of bumped switchable molecular probe enables repeated generation of target analogues for mix-to-signaling determination of microRNAs

Herein, we have designed a unique oligonucleotide probe named as bumped switchable molecular probe (BS-MB) and employ it to ultrasensitive detection of miRNA-21. The uniqueness is that the bumped portion renders the BS-MB great design flexibility and divides its long stem into two short stems so that it can play the roles of recognition unit, reporting unit, and polymerization primer and template simultaneously. The opening of BS-MB requires a delayed reaction, allowing the system a better-suppressed background without miRNA-21. In contrast, the introduction of miRNA-21 delayed the full opening of BS-MB, inducing a concurrent three-stage amplification for consuming lots of BS-MBs in one amplification event. During the amplification, lots of target analogues of extended miRNA-21 (E-miRNA-21) and cleavaged miRNA-21 (C-miRNA-21) that can be reused as miRNA-21 to cause further fluorescence enhancement are repeatedly generated. The system retains the simplicity (one oligonucleotide probe) and simplifies the detection procedure (one-step detection). Its mix-to-signaling and one-to-many amplification behaviors are superior to conventional MB based amplification, exhibiting a wide linear response and a low detection limit of 8.1 fM. High specificity against even one-base mutation, desirable recovery rates for testing miRNA-spiked serum samples, and well-defined accuracy for classifying clinically related samples are also demonstrated.