Dual amplification strategy triggered by triple helix probe for the detection of microRNAs

none2noThe triple helix is an alternative structure adopted by nucleic acids based on Watson–Crick and Hoogsteen base pairings involving three strands in the correct orientation. Since the stability of the triple helix stem can be higher even compared to longer duplexes, triple stranded DNA has been used in biosensing to build aptasensors or sensors for nucleic acids detection [1-4] and good sensitivity and selectivity have been generally observed. Moreover, different recognition elements specific for different targets can be included in the nucleic acid probe on a same single-stranded sequence, allowing in principle a universal assay strategy [5]. In the literature, the use of only one of the involved oligonucleotides for signal amplification and transduction is generally reported. 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 feasibility of our system to induce a double response triggered by the target, we designed hybridization chain reactions (HCR) [6] started 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.openMiti Andrea; Zuccheri GiampaoloMiti Andrea; Zuccheri Giampaol