A conformation-induced fluorescence method for microRNA detection.

Abstract

MicroRNAs play important roles in a large variety of biological systems and processes through their regulation of target mRNA expression, and show promise as clinical biomarkers. However, their small size presents challenges for tagging or direct detection. Innovation in techniques to sense and quantify microRNAs may aid research into novel aspects of microRNA biology and contribute to the development of diagnostics. By introducing an additional stem loop into the fluorescent RNA Spinach and altering its 3′ and 5′ ends, we have generated a new RNA, Pandan, that functions as the basis for a microRNA sensor. Pandan contains two sequence-variable stem loops that encode complementary sequence for a target microRNA of interest. In its sensor form, it requires the binding of a target microRNA in order to reconstitute the RNA scaffold for fluorophore binding and fluorescence. Binding of the target microRNA resulted in large changes in fluorescence intensity. The median fold change in fluorescence observed for the sensors tested was ∼50-fold. Pandan RNA sensors exhibit good signal-to-noise ratios, and can detect their target microRNAs within complex RNA mixtures.