Split activator of CRISPR/Cas12a for direct and sensitive detection of microRNA.

CRISPR/Cas12a-based nucleic acid assays have been increasingly used for molecular diagnostics. However, most current CRISPR/Cas12a-based RNA assays require the conversion of RNA into DNA by preamplification strategies, which increases the complexity of detection. Here, we found certain chimeric DNA-RNA hybrid single strands could activate the trans-cleavage activity of Cas12a, and then discovered the activating effect of split ssDNA and RNA when they are present simultaneously. As proof of concept, split nucleic acid-activated Cas12a (SNA-Cas12a) strategy was developed for direct detection of miR-155. By adding a short ssDNA to the proximal end of the crRNA spacer sequence, we realized the direct detection of RNA targets using Cas12a. With the assistance of ssDNA, we extended the limitation that CRISPR/Cas12a cannot be activated by RNA targets. In addition, by taking advantage of the programmability of crRNA, the length of its binding to DNA and RNA was optimized to achieve the optimal efficiency in activating Cas12a. The SNA-Cas12a method enabled sensitive miR-155 detection at pM level. This method was simple, rapid, and specific. Thus, we proposed a new Cas12a-based RNA detection strategy that expanded the application of CRISPR/Cas12a.

Zipper-Confined DNA Nanoframe for High-Efficient and High-Contrast Imaging of Heterogeneous Tumor Cell.

Current study in the heterogeneity and physiological behavior of tumor cells is limited by the fluorescence in situ hybridization technology in terms of probe assembly efficiency, background suppression capability, and target compatibility. In a typically well-designed assay, hybridization probes are constructed in a confined nanostructure to achieve a rapid assembly for efficient signal response, while the excessively high local concentration between different probes inevitably leads to nonspecific background leakage. Inspired by the fabric zipper, we propose a novel confinement reaction pattern in a zipper-confined DNA nanoframe (ZCDN), where two kinds of hairpin probes are independently anchored respective tracks. The metastable states of the dual tracks can well avoid signal leakage caused by the nonspecific probe configuration change. Biomarker-mediated proximity ligation reduces the local distance of dual tracks, kinetically triggering an efficient allosteric chain reaction between the hairpin probes. This method circumvents nonspecific background leakage while maintaining a high efficiency in responding to targets. ZCDN is employed to track different cancer biomarkers located in both the cytoplasm and cytomembrane, of which the expression level and oligomerization behavior can provide crucial information regarding intratumoral heterogeneity. ZCDN exhibits high target response efficiency and strong background suppression capabilities and is compatible with various types of biological targets, thus providing a desirable tool for advanced molecular diagnostics.