Fluorescent DNA tetrahedral probe with catalytic hairpin self-assembly reaction for imaging of miR-21 and miR-155 in living cells.

A CHA-based fluorescent DNA tetrahedral probe (FDTp) has been designed to detect the microRNAs miR-21 and miR-155 sensitively and specifically in living cells. The design consisted of functional elements (H1, H2, and Protector) connected to a DNA tetrahedron modified with two pairs of fluorophores and quenching groups. In the presence of miR-21, the chain displacement effect was triggered and Cy3 fluorescence was emitted. In the presence of miR-155, the signal of the catalytic hairpin assembly (CHA) between H1 and H2 on FDTp was amplified, making the fluorescence of FAM sensitive to miR-155. Using this method, the detection limit for miR-155 was 5 pM. The FDTp successfully imaged miR-21 and miR-155 in living cells and distinguished a variety of cell lines based on their expression levels of miR-21 and miR-155. The detection and imaging of dual targets in this design ensured the accuracy of tumor diagnosis and provided a new method for early tumor diagnosis.

Label-Free Ratiometric Homogeneous Electrochemical Strategy Based on Exonuclease III-Aided Signal Amplification for Facile and Rapid Detection of miR-378.

MiR-378 is abnormally expressed in various cancers, such as hepatocellular carcinoma, renal cell carcinoma, and nonsmall cell lung cancer. Here, we developed a label- and immobilization-free ratiometric homogeneous electrochemical strategy based on exonuclease III (Exo III) for the facile and rapid determination of miR-378. Two 3'-protruding hairpin DNA probes (HPs) are designed in this strategy. Doxorubicin (DOX) and potassium ferrocyanide (Fe2+) were used as label-free probes to produce a response signal (IDOX) and a reference signal (IFe2+) in the solution phase. When no target was present in the solution, the HP was stable, most of the DOX was intercalated in the stem of the HP, and the diffusion rate of DOX was significantly reduced, resulting in reduced electrochemical signal response. When miR-378 was present, double-cycle signal amplification triggered by Exo III cleavage was initiated, ultimately disrupting the hairpin structures of HP1 and HP2 and releasing a large amount of DOX into the solution, yielding a stronger electrochemical signal, which was low to 50 pM. This detection possesses excellent selectivity, demonstrating high application potential in biological systems, and offers simple and low-cost electrochemical detection for miR-378.

Real-time in situ fluorescence imaging of telomerase and miR378 in living cells using a two-color DNA tetrahedron nanoprobe combined with molecular beacons.

A biosensor that can detect biomarkers accurately, quickly, and conveniently is important for the diagnosis of various diseases. However, most of the existing detection methods require sample extraction, which makes it difficult to detect and image intracellular molecules or to detect two different types of biomarkers simultaneously. In this study, we constructed a DNA tetrahedral nanoprobe (DTP) capable of detecting both miR378 and telomerase, both of which are tumor markers. In the presence of miR378, FAM on the molecular beacon of DTP fluoresced via Förster resonance energy transfer (FRET), and the limit of detection was 476 pM with excellent specificity. When present, telomerase binds to telomerase substrate (TS) primers, extending the repeat sequence (TTAGGG)n to trigger Cy3 fluorescence. A strong linear relationship existed between the fluorescence intensity of Cy3 and the number of HeLa cells. The limit of detection was 800 HeLa cells. In addition, DTP was less cytotoxic to and biocompatible with HeLa cells and fluoresced only in cancer cells, which can help to sensitively distinguish between normal and cancer cells. In conclusion, DTP can simultaneously detect the content of miR378 and activity of telomerase and realize intracellular imaging, which has broad application prospects in early cancer diagnosis and treatment.