DNA triple helix triggered double amplification for miRNA detection

Triple helix DNA has been exploited in building molecular switches, based on Watson–Crick and Hoogsteen base pairings, and adapted to several biosensing strategies for the detection of nucleic acids and aptasensors development [1-4]. The stability of the triple helix stem can be higher compared to longer duplex, and good sensitivity and selectivity have been generally observed in sensing. Moreover, different recognition elements specific for different targets can be included in the probe on a same single-stranded sequence, allowing in principle a universal assay strategy [5]. Examples in literature in using triple helix DNA probes usually employ only one of the involved oligonucleotides for signal amplification and transduction. We herein propose the use of both the triple helix forming strands in this process as this would give an advantage, such as the chance to combine different transduction approaches leading to a stronger and more reliable detection method. For this reason, we designed triple helix DNA probes specific for different microRNAs, in which both the sequences hidden in the stem would be available for subsequent signal amplification and transduction after target recognition. (Fig.1). To simply test the capacity of our system to induce a double response in the presence of the target, we designed hybridization chain reactions (HCR) [6] triggered by both the oligonucleotides involved in the triple helix. Furthermore, in our peculiar design, the self-assembled nanostructures resulting from a single recognition event can interact with each other forming bigger nanostructures, thus enhancing the recognition signal. After characterizing this strategy in solution (see Fig. 2), we are now in the process of implementing it in a biosensor platform.