Universal Aptamer-Based Real-Time Monitoring of Enzymatic RNA Synthesis

Abstract

In vitro transcription is an essential laboratory technique for enzymatic RNA synthesis. Unfortunately, no methods exist for analyzing quality and quantity of the synthesized RNA while the transcription proceeds. Here we describe a simple, robust, and universal system for monitoring and quantifying the synthesis of any RNA in real time without interference from abortive transcription byproducts. The distinguishing feature is a universal fluorescence module (UFM), consisting of the eGFP-like Spinach aptamer and a highly active hammerhead ribozyme, which is appended to the RNA of interest (ROI). In the transcription mixture, the primary transcript is cleaved rapidly behind the ROI, thereby releasing always the same UFM, independent of the ROI sequence, polymerase, or promoter used. The UFM binds to the target of the Spinach aptamer, the fluorogenic dye DFHBI, and thereby induces a strong fluorescence signal. This design allows real-time quantification, standardization, parallelization, and high-throughput screening.

Live Cell Imaging of Endogenous mRNA Using RNA-Based Fluorescence “Turn-On” Probe

“…Fluorescence will be observed only upon complexation of the fluorophore to the RNA aptamer, hence reducing the background from the unbound fluorophores in the cytoplasm during live cell imaging. 31–33 Nevertheless, adding the RNA aptamer sequences to the target RNA may affect its expression level and functional activities. 14 …”

Illuminating Messengers: An Update and Outlook on RNA Visualization in Bacteria

“…Spinach and its derivatives have found many applications due to the low background signals and the cell-permeating ability of DFHBI. Spinach has also been adapted to sense metabolites such as cyclic d-AMP and to follow transcriptional events (Höfer et al, 2013; Kellenberger et al, 2013; Song et al, 2013; Pothoulakis et al, 2014; Wang X.C. et al, 2016).…”

Detection of human immunodeficiency virus RNAs in living cells using Spinach RNA aptamers

“…Recently, fluorophore binding RNA aptamers have gained attention for their potential to meet these challenges. One of these aptamers, known as Spinach (Paige et al, 2011) or in its more recently improved form, Spinach2 (Strack et al, 2013), has been used for a number of in vitro and synthetic biology applications (Chizzolini et al, 2014; Hofer et al, 2013; Nakayama et al, 2012; Pothoulakis et al, 2014; Shu et al, 2014; van Nies et al, 2013), as well as for detecting triplet repeat RNAs (Strack et al, 2013), protein expression (Song et al, 2013), and metabolites (Paige et al, 2012) in living cells. Both aptamers bind a conditional fluorophore known as DFHBI (3,5-difluoro-4-hydroxybenzylidene imidazolinone), which is reported to have limited fluorescence on its own, but fluoresces similarly to enhanced GFP (EGFP) when bound (Paige et al, 2011).…”