Tandem Spinach Array for mRNA Imaging in Living Bacterial Cells

Abstract

Live cell RNA imaging using genetically encoded fluorescent labels is an important tool for monitoring RNA activities. A recently reported RNA aptamer-fluorogen system, the Spinach, in which an RNA aptamer binds and induces the fluorescence of a GFP-like 3,5-difluoro-4-hydroxybenzylidene imidazolinone (DFHBI) ligand, can be readily tagged to the RNA of interest. Although the aptamer–fluorogen system is sufficient for imaging highly abundant non-coding RNAs (tRNAs, rRNAs, etc.), it performs poorly for mRNA imaging due to low brightness. In addition, whether the aptamer-fluorogen system may perturb the native RNA characteristics has not been systematically characterized at the levels of RNA transcription, translation and degradation. To increase the brightness of these aptamer-fluorogen systems, we constructed and tested tandem arrays containing multiple Spinach aptamers (8–64 aptamer repeats). Such arrays enhanced the brightness of the tagged mRNA molecules by up to ~17 fold in living cells. Strong laser excitation with pulsed illumination further increased the imaging sensitivity of Spinach array-tagged RNAs. Moreover, transcriptional fusion to the Spinach array did not affect mRNA transcription, translation or degradation, indicating that aptamer arrays might be a generalizable labeling method for high-performance and low-perturbation live cell RNA imaging.

RNA Localization in Bacteria

“…Since then, new aptamer systems have been developed, including Spinach 2 (53), Mango (54), Broccoli (55, 56), and Corn (57). Repetitive Spinach aptamers have been shown to increase the brightness by a maximum of 17-fold (with 64 repeats) compared to a single Spinach monomer at a cost of a significant lengthening of the aptamer sequence (58). Alternatively, the fluorogenic small molecules have been designed into a covalently linked fluorophore-quencher pair, and the aptamers are selected to either bind the quencher or the fluorophore.…”

“…(C) The Spinach aptamer and its application to image mRNAs in live E.coli cells. The image is adapted from (58) (licensed under a Creative Commons Attribution 4.0 International License: http://creativecommons.org/licenses/by/4.0/), in which a tandem array of Spinach aptamers is fused to the RNA of interest to enhance the signal, and fluorescent detection upon addition of the organic ligand DFHBI.…”

Genetically Encoded Catalytic Hairpin Assembly for Sensitive RNA Imaging in Live Cells

“…It has been demonstrated that fluorescent RNA molecules, named Spinach and Broccoli, can be employed as genetically encoded reporters for RNAimaging. 20–22 Spinach and Broccoli are RNA aptamers that can bind and activate the fluorescence of small-molecule dyes, such as 3,5-difluoro-4-hydroxybenzylidene-1-trifluoroethyl-imidazolinone (DFHBI-1T). 23–25 Recently, the Ellington group has successfully demonstrated that Spinach can function as a reporter in an RNA-based CHA reaction in vitro.…”

Recent advances in high-performance fluorescent and bioluminescent RNA imaging probes

“…Therefore, DFHBI can bind to Spinach aptamer-fused 5S RNAs and image their live-cell dynamics 47 . Furthermore, a tandem Spinach array shows up to about 17-fold higher fluorescence by accommodating 8–64 repeats of Spinach aptamers in target RNAs 48 . However, the Spinach aptamer-DFHBI system suffers from limited folding efficiency and thermal instability.…”