Stable and Label-Free Fluorescent Probe Based on G-triplex DNA and Thioflavin T.

G-triplexes have recently been identified as a new kind of DNA structures. They perhaps possess specific biological and chemical functions similar as identified G-quadruplex but can be formed by shorter G-rich sequences with only three G-tracts. However, until now, limited G-triplexes sequences have been reported, which might be due to the fact that their stability is one of the biggest concerns during their functional studies and application research. Herein, we found a G-rich sequence (5'-TGGGTAGGGCGGG-3') which can form a stable G-triplex (Tm ∼ 60 °C) at room temperature. The stable G-triplex can combine with thioflavin T and function as an efficient fluorescence light-up probe. Comparing with the traditional G-quadruplex based probe, this triplex based probe was easy to be controlled and excited. Finally, the probe was successfully applied into constructing a label-free molecular beacon for miRNA detection. Taking advantage of these abilities of the G-triplex based fluorescent probe, the challenges faced during designing G-rich sequences based fluorescent biosensors can be efficiently solved. These findings provide important information for the future application of G-triplex.

[Effect and mechanism of miR-124 on osteogenic differentiation of dental pulp mesenchymal stem cells].

PURPOSE: To evaluate the effect of microRNA (miR)-124 on osteogenic differentiation of dental pulp mesenchymal stem cells (DPSCs) and to explore the possible mechanism. METHODS: Logarithmic DPSCs were collected and divided into blank group, no-load group, miR-124 inhibitor group, miR-124 inhibitor combined with N-[N-(3,5-difluorophenacetyl)-1-alanyl]-S-ph (DAPT, Notch signaling pathway inhibitor) group. The blank group was not treated, the empty group was transfected with negative control vector inhibitor-NC, the miR-124 inhibitor group was transfected with miR-124 inhibitor, the miR-124 inhibitor combined with DAPT group was transfected with miR-124 inhibitor, and DAPT was added to make the final concentration of 5 µmol/L. The proliferation ability was tested by CCK-8 method 48 h after transfection. Alkaline phosphatase (ALP) activity was tested by p-nitrophenyl phosphate (P-NPP) method after 2 weeks of induction. The area of calcified nodules was tested by alizarin red staining method. The protein expression of hair-like division-related enhancer 1 (HEY1), hair-like division-related enhancer 2 (HEY2), and cyclin D1 gene (CCND1) were tested by Western blot. The data was analyzed by SPSS 19.0 software package. RESULTS: Compared with the blank group and no-load group, the A450 value at 24, 48, 72 h detected by CCK-8 experiment, A450 value of ALP activity, the area composition ratio of calcified nodules, and expression of HEY1, HEY2, and CCND1 in the miR-124 inhibitor group were increased (P<0.05). Compared with miR-124 inhibitor group, the A450 value at 24, 48, 72 h detected by CCK-8 experiment, A450 value of ALP activity, the area composition ratio of calcified nodules, and the expression of HEY1, HEY2, and CCND1 in the miR-124 inhibitor combined with DAPT group were significantly decreased(P<0.05). CONCLUSIONS: Down-regulation of miR-124 can promote osteogenic differentiation of DPSCs. It is speculated that the mechanism of action is related to the activation of Notch signaling pathway.