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

Abstract

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.

Intrinsic Turn‐On Response of Thioflavin T in Complexes

“…The utility of ThT is rooted in its “turn‐on” response—a dramatic enhancement (up to thousand‐fold increase) in fluorescence intensity in the presence of amyloids. Complexes with certain macrocycles, oligonucleotides, and other non‐amyloid‐forming proteins also show substantial turn‐on responses. Fluorescence of ThT is also sensitive to other environmental factors including temperature, pressure, solvent viscosity, and pH .…”

“…We also used SILIF to examine complexation of ThT + with other binding partnersi nt he gas phase. We tested two molecules known to inducet urn-on responses in solution (Figure S5, SI): as econd macrocycle-cucurbit [7]uril (CB7), [3] and a biomolecule-the 22AG human telomeric G-quadruplex DNA. [6] [ThT+ +CB7] + ,[ ThT+ +CB7] 2 + and [ThT+ +22AG] 5À ,w hich were the most abundantc omplexes detected under our mass spectrometry conditions, were each individually stored and irradiated at multiple wavelengths.H owever,f luorescencee mission was not detected from any of these complexes ( Figure S6, SI).…”

Symmetric exponential amplification reaction-based DNA nanomachine for the fluorescent detection of nucleic acids

“…Circular dichroism (CD) measurements were performed on a Chirascan CD spectrometer (Applied Photophysics Ltd., UK) using a 1 mm path length quartz cuvette at room temperature under the following parameters: range 200-500 nm, scanning speed 200 nm min À1 , bandwidth 1 nm, response time 0.5 s. 37 The 12% native polyacrylamide gel electrophoresis (nPAGE) was operated at the Bio-Rad electrophoresis analyzer (Bio-Rad, USA) using 80 V constant voltage at room temperature for 1 hour in 1Â TBE buffer (90 mM Tris-boric acid, 2 mM EDTA, pH 8.0). Then, the gel was stained with GoldView I for another 30 minutes with continuous rotation and nally photographed with a Bio-Rad ChemDoc XRS (Bio-Rad, USA).…”

“…36 Fortunately, Zhou et al reported a new G-rich sequence-based DNA nanostructure, G-triplex, which has similar functions to G-quadruplex but only consists of 13 bases. 37 Herein, we propose a label-free, ultra-low background signal and ultra-high signal-to-noise ratio DNA nanomachine, which is based on the palindromic fragment-incorporated multifunctional hairpin probe (P-HP)-assisted symmetric exponential amplication reaction (S-EXPAR), with G-triplex/ThT complex as the signal reporter. In this experiment, the p53 gene was selected as the model analyte because it is mutated in more than 50% of malignant tumors.…”

Facile and Sensitive Fluorescence Assay of DNA Polymerase Activity Using Cu2+ and Ascorbate as Signal Developers

“…Fluorescence method is widely used in the biosensing field because of its simplicity and high‐throughput capability ,. Fluorescence method has been explored to detect DNA polymerase activity .…”