Absolute and Direct MicroRNA Quantification Using DNA–Gold Nanoparticle Probes

作者：Federica Degliangeli, Prakash Kshirsagar, Virgilio Brunetti, Pier Paolo Pompa, Roberto Fiammengo · 发表于：Journal of the American Chemical Society · 年份：2014 · DOI：10.1021/ja412152x · 被引用次数：389 · 研究领域：Advanced biosensing and bioanalysis techniques、MicroRNA in disease regulation、RNA Interference and Gene Delivery

DNA-gold nanoparticle probes are implemented in a simple strategy for direct microRNA (miRNA) quantification. Fluorescently labeled DNA-probe strands are immobilized on PEGylated gold nanoparticles (AuNPs). In the presence of target miRNA, DNA-RNA heteroduplexes are formed and become substrate for the endonuclease DSN (duplex-specific nuclease). Enzymatic hydrolysis of the DNA strands yields a fluorescence signal due to diffusion of the fluorophores away from the gold surface. We show that the molecular design of our DNA-AuNP probes, with the DNA strands immobilized on top of the PEG-based passivation layer, results in nearly unaltered enzymatic activity toward immobilized heteroduplexes compared to substrates free in solution. The assay, developed in a real-time format, allows absolute quantification of as little as 0.2 fmol of miR-203. We also show the application of the assay for direct quantification of cancer-related miR-203 and miR-21 in samples of extracted total RNA from cell cultures. The possibility of direct and absolute quantification may significantly advance the use of microRNAs as biomarkers in the clinical praxis.