MicroRNAs (miRNAs) are regulatory small RNAs that have important roles in numerous developmental, metabolic, and disease processes of plants and animals. The individual levels of miRNAs can be useful biomarkers for cellular events or disease diagnosis. Thus, innovative new tools for rapid, specific, and sensitive detection of miRNAs are an important field of research. Using the fluorescence properties of DNA-nanosilver clusters (DNA/AgNC), we have designed a DNA/AgNC probe that can detect the presence of target miRNA. Here, we show that the red fluorescence of the DNA/AgNC probe is diminished upon the presence of target miRNA without pre- or postmodification, addition of extra enhancer molecules, and labeling. The DNA/AgNC probe emission was lowest when the complementary miRNA target was present and was significantly higher for four other control miRNA sequences. Also, when adding whole plant endogenous RNA to the DNA/AgNC probe, the emission was significantly higher for the mutant where miRNA was deficient. On the basis of these findings, we suggest that these DNA/AgNC probes could be developed into a new, simple, inexpensive, and instant technique for miRNAs detection.

SUMMARY The Arabidopsis HYPONASTIC LEAVES1 (HYL1) is a double-stranded RNA-binding protein that forms a complex with DICER-LIKE1 (DCL1) and SERRATE to facilitate processing of primary miRNAs into micro-RNAs (miRNAs). However, the structural mechanisms of miRNA maturation by this complex are poorly understood. Here, we present the crystal structures of double-stranded RNA binding domains (dsRBD1 and dsRBD2) of HYL1 and HYL1 dsRBD1 (HR1)/dsRNA complex as well as human TRBP2 dsRBD2 (TR2)/dsRNA complex for comparison analysis. Structural and functional study demonstrates that both HR1 and TR2 are canonical dsRBDs for dsRNA binding, whereas HR2 of HYL1 is a non-canonical dsRBD harboring a putative dimerization interface. Domain swapping within the context of HYL1 demonstrates that TR2 can supplant the function of HR1 in vitro and in vivo. Further biochemical analyses suggest that HYL1 probably binds to the miRNA/miRNA* region of precursors as a dimer-mediated by HR2.

The influence of the nucleic acid secondary structure on the fast (1 h) formation of bright red emissive silver nanoclusters (AgNCs) in a DNA sequence (DNA-12nt-RED-160), designed for the detection of a microRNA sequence (RNA-miR160), was investigated. The findings show that especially the propensity for mismatch self-dimer formation of the DNA probes can be a good indicator for the creation and stabilization of red emissive AgNCs. Also, the role of the thermal stability of the secondary DNA structures (mismatch self-dimer and hairpin monomers) and the observed AgNC red emission intensity were investigated. These findings can form the basis for a rationale to design new red emissive AgNC-based probes. As an example, a bright red emissive AgNC-based DNA probe was designed for RNA-miR172 detection. The latter opens the possibility to create a variety of AgNC-based DNA probes for the specific detection of plant and animal miRNAs.