Cascaded and Localized Assembly of DNA Nanospheres for Efficient mRNA Imaging.

The pursuit of advanced mRNA detection methods has been driven by the need for sensitive, accurate approaches that are particularly suited for live-cell analysis. Herein, we proposed a cascaded and localized assembly (CLA) system, integrating branched catalytic hairpin assembly (bCHA) with a localized hybridization chain reaction (LHCR) for enhanced mRNA imaging. The CLA system employed a dual-nanosphere (NS) platform, NSABC and NS12, and the interaction between the target and NSABC initiated the bCHA process and activated a split trigger. The newly generated trigger served as the initiator for the LHCR on NS12, leading to amplified fluorescent signals. Notably, this work introduced the first integration of a splitting strategy in a bCHA-HCR cascaded system, reducing false-positive signals and enhancing specific detection. The dual-NS platform further minimized background noise and improved the reaction kinetics through spatial confinement. As a result, the system achieved a detection limit of 1.23 pM. With these advantages, the CLA system demonstrated successful application in both living cells and clinical tissues, underscoring its potential in biomolecular research and clinical diagnostics.

Three-Dimensional CHA-HCR System Using DNA Nanospheres for Sensitive and Rapid Imaging of miRNA in Live Cells and Tissues.

Isothermal, enzyme-free amplification techniques, such as the hybridization chain reaction (HCR) and catalytic hairpin assembly (CHA), have gained increasing attention for miRNA analysis. However, current methodological challenges, including slow kinetics, low amplification efficiency, difficulties in efficient cellular internalization of DNA probes, and concerns regarding the intracellular stability of nucleic acids, need to be addressed. To this end, we propose a novel strategy for sensitive miRNA detection based on a three-dimensional (3D) CHA-HCR system. This system comprises two DNA nanospheres, named DS-13 and DS-24, which are functionalized with CHA and HCR hairpins. Target miR-21 initiates CHA between the two nanospheres, thereby activating downstream HCR and bringing cyanine 3 (Cy3) and cyanine 5 (Cy5) into proximity. The 3D CHA-HCR process leads to the formation of large DNA aggregates and the generation of fluorescence resonance energy transfer signals. In this strategy, the employment of a cascaded reaction and spatial confinement effect improve sensitivity and kinetics, while the use of DNA nanocarriers facilitates cellular delivery and protects nucleic acid probes. The experimental results in vitro, in living cells, and in clinical tissue samples demonstrated the desirable sensing performance. Collectively, this approach holds promise as a valuable tool for cancer diagnosis and biomedical research.

Enhancing RNA detection and breast cancer subtyping with a universal 3D-hybridization chain reaction system.

Breast cancer, a globally prevalent malignancy, is characterized by pronounced heterogeneity. Accurate subtyping requires the simultaneous detection of different biomarkers, which is crucial for personalized treatment strategies. However, existing methodologies are hindered by limited versatility and sensing performance. To overcome these hurdles, this study presents a universal 3D-Hybridization Chain Reaction (3D-HCR) system for RNA detection and subtype-specific diagnosis of breast cancer. The system integrated a universal trigger for HCR, thereby circumventing the need for complex sequence design and enabling the analysis of various RNA targets. Leveraging the spatial-confinement effect offered by DNA nanocarriers, this system exhibited superior amplification efficiency, achieving detection limits of 3.83 pM and 4.96 pM for PD-L1 mRNA and miR-21, respectively. Importantly, the system could differentiate between triple-negative breast cancer and estrogen receptor-positive breast cancer in both living cells and clinical tissues. These findings underscore the potential of the universal 3D-HCR system as a promising tool in clinical diagnostics. With its proven proficiency in breast cancer diagnostics and versatility in RNA analysis, this system holds the promise of broadening the horizons of precision medicine.

Simultaneous detection and imaging of two specific miRNAs using DNA tetrahedron-based catalytic hairpin assembly.

Improving the accuracy, sensitivity and speed of intracellular miRNA imaging is essential for early diagnosis of cancer. To achieve this goal, we herein present a strategy for imaging two distinct miRNAs by DNA tetrahedron-based catalytic hairpin assembly (DCHA). Two nanoprobes, DTH-13 and DTH-24, were prepared by one-pot synthesis. The resultant structures were DNA tetrahedrons functionalized with two sets of CHA hairpins, which respectively responded to miR-21 and miR-155. Using these structured DNA nanoparticles as the carriers, the probes could easily enter living cells. The presence of miR-21 or miR-155 could trigger CHA between DTH-13 and DTH-24, leading to independent fluorescence signals of FAM and Cy3. In this system, the sensitivity and kinetics were significantly enhanced owing to the strategy of DCHA. The sensing performance of our method was thoroughly investigated in buffers, fetal bovine serum (FBS) solutions, living cells, and clinical tissue samples. The results validated the potential of DTH nanoprobes as a diagnostic tool for early stages of cancer.

Recent progress in the development of DNA-based biosensors integrated with hybridization chain reaction or catalytic hairpin assembly.

As isothermal, enzyme-free signal amplification strategies, hybridization chain reaction (HCR) and catalytic hairpin assembly (CHA) possess the advantages such as high amplification efficiency, excellent biocompatibility, mild reactions, and easy operation. Therefore, they have been widely applied in DNA-based biosensors for detecting small molecules, nucleic acids, and proteins. In this review, we summarize the recent progress of DNA-based sensors employing typical and advanced HCR and CHA strategies, including branched HCR or CHA, localized HCR or CHA, and cascaded reactions. In addition, the bottlenecks of implementing HCR and CHA in biosensing applications are discussed, such as high background signals, lower amplification efficiency than enzyme-assisted techniques, slow kinetics, poor stability, and internalization of DNA probes in cellular applications.