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.

FASTmiR: an RNA-based sensor for in vitro quantification and live-cell localization of small RNAs

“…described a Spinach-based Pandan sensor to detect miRNAs in vitro . Pandan is a circular bimolecular sensor with a destabilized G-quadruplex and base triplet required for DFHBI binding ( 21 ). Binding of miRNA re-forms the DFHBI-binding pocket of the sensor which leads to increased fluorescence in vitro ( 21 ).…”

Reappraisal of Spinach-based RNA Visualization in Plants

“…This could be achieved via expressing more tandem repeats of spinach, as demonstrated in this work, or its derivatives in plants. Indeed, since the development of Spinach (Paige et al, 2011), several new aptamers such as Spinach2 (Strack et al, 2013), Baby Spinach (Huang et al, 2014), iSpinach (Autour et al, 2016), Pandan (Aw et al, 2016), Broccoli (Filonov et al, 2014), RNA-Mango (Dolgosheina et al, 2014), Corn-DFHO (Warner et al, 2017) and Pepper (Chen et al, 2019) have been uncovered and used for RNA visualization.…”

Metallic Nanoparticle-Based Optical Cell Chip for Nondestructive Monitoring of Intra/Extracellular Signals

“…These fluorescence images have provided cell-specific information. The advantages of fluorescence-based analytical methods include high sensitivity, selectivity, and reproducibility [46][47][48][49]. Furthermore, fluorescence detection can be easily applied to the inside and outside of cells for noninvasive and multi-analytes detection [50][51][52].…”