Classical Triplex Molecular Beacons for MicroRNA-21 and Vascular Endothelial Growth Factor Detection

Triplex molecular beacons (tMBs) possess great potential in biological sensing because of the pH responsiveness and controllability of binding strength. Here, we systematically investigate and rationally design a classical tMB for convenient detection of microRNA-21, a well-known biomarker of cardio-cerebrovascular diseases. In the tMB, we employ the complementary sequence of miR-21 as the loop and the sequences of protonated cytosine–guanine–cytosine (C-G•C+) and thymine–adenine–thymine (T-A•T) as the triplex stem, in which both the Watson–Crick and Hoogsteen base-pairing control the binding strength in cooperation. It is demonstrated for the first time that the presence of miR-21 would only break the Hoogsteen base-pairing in the stem and hybridize with the tMB to form the rigid heterozygous hybrid duplex structure. These would hinder the fluorescence resonance energy transfer (FRET) between the fluorophore (FAM) and quencher (BHQ1) labeled at the ends of the oligonucleotide, and the fluorescence recovery degree of FAM can be used as the standard to quantitate the miR-21. More significantly, the excellent adjustability and sensitivity of our tMBs have been confirmed by constructing the corresponding duplex molecular beacon (dMB) for comparison. The fluorophore FAM in the tMB could be replaced by the fluorescent DNA/silver nanoclusters, which exhibits the universal applicability of energy donor and receptor selection for tMB. Furthermore, our proposed tMB could also be developed as an aptasensor for the detection of vascular endothelial growth factor (VEGF) by only introducing the complementary sequence of its aptamer into the tMB. This work is of great significance for the systematic study of tMBs for the detection of biomarkers such as nucleic acids and proteins.