Detection and quantification of single mRNA dynamics with the Riboglow fluorescent RNA tag

Abstract

Labeling and tracking biomolecules with fluorescent probes on the single molecule level enables quantitative insights into their dynamics in living cells. We previously developed Riboglow, a platform to label RNAs in live mammalian cells, consisting of a short RNA tag and a small organic probe that increases fluorescence upon binding RNA. Here, we demonstrate that Riboglow is capable of detecting and tracking single RNA molecules. We benchmark RNA tracking by comparing results with the established MS2 RNA tagging system. To demonstrate versatility of Riboglow, we assay translation on the single molecule level, where the translated mRNA is tagged with Riboglow and the nascent polypeptide is labeled with a fluorescent antibody. The growing effort to investigate RNA biology on the single molecule level requires sophisticated and diverse fluorescent probes for multiplexed, multi-color labeling of biomolecules of interest, and we present Riboglow as a new member in this toolbox.

Single mRNA Imaging with Fluorogenic RNA Aptamers and Small‐molecule Fluorophores

“…Single mRNA imaging with RNA aptamers: SFs systems was first reported in 2019 [11, 19] . Since then, RNA aptamers: SFs systems have been inspiring the field of single mRNA imaging.…”

“…Next, the authors engineered Riboglow for tracking single mRNAs in living mammalian cells (Figure 2c and 4c). They first tagged β‐actin mRNA with 12 copies of the Riboglow RNA tag [19] . After transfecting with plasmids in U2OS cells, they detected different punctate dots in the red channel (561 nm exciter) using highly tilted and laminated optical thin section (HILO) microscopy (Figure 2c).…”

“…In 2019, Palmer and co‐workers developed a Riboglow platform as a tool for single mRNA imaging (Figure 2c and 4c). [19] …”

“… [21] Compared to the MS2: GFP system, there is no significant difference in the puncta size of the Riboglow‐tagged β‐actin. In addition, the authors applied Riboglow for the quantification of single mRNA dynamics in mammalian cells [19] …”

Genetically encoded RNA nanodevices for cellular imaging and regulation

“…102 Several Spinach derivatives, such as Spinach2, Baby Spinach, and Broccoli, have been further developed with reduced size, improved brightness and folding capability. 103,109,110 More recently, imaging of single cellular RNAs has been achieved by using a tandem array of exceptionally bright fluorogenic RNA aptamer/chromophore pairs including Mango II/TO1-B, 111 Pepper/HBC, 106 Riboglow/ATTO590, 112 and Broccoli/BI. 113 Meanwhile, a wide spectral range (λ ex , ∼380-650 nm; λ em , ∼420-660 nm) of fluorogenic RNA tags is now available for various imaging purposes (Table 1 and Fig.…”