RNA Tracking & Visualization (RNA Mango)

Overview

The most advanced RNA tracking, visualization and pull down technology.

Technology for studying the diverse cellular roles of RNA has lagged behind the tools for studying DNA and proteins, but innovative researchers are working to change that! One such researcher is Dr. Peter Unrau of Simon Fraser University. He and his team have created RNA Mango, a novel technology with a number of useful applications.

Documents

Product Name	Cat. No.	Size
TO1-3PEG-Biotin Fluorophore	G955	0.5 mg/ml (500 µl)
TO1-3PEG-Desthiobiotin Fluorophore	G956	0.5 mg/ml (500 µl)
TO3-3PEG-Biotin Fluorophore	G959	0.5 mg/ml (500 µl)
YO3-3PEG-Biotin Fluorophore	G957	0.5 mg/ml (500 µl)
* Click here for data on binding affinity to Mango and Peach aptamers

Key Features

RNA Mango technology is based on the specific binding of the RNA Mango Aptamer and a Thizole Orange (TO) bi-functional dye. The main features of this technology is the tight binding between the dye and aptamer (KD ≈ 3nM) , and the strong ~1000X enhancement of the dye’s fluorescence when bound to the Mango aptamer (Fluorescent enhancement FE=1,100).

The Thizole Orange (TO) dye has a number of other desirable properties including:

Small size
Lack of toxicity
Plasma and nuclear membrane permeability
Short intracellular half-life
The accessibility of a broad wavelength range simply via substitutions and alterations to the TO structure
The TO1 and TO3 dyes can be used in a 2-color reporter assay system using both the RNA Mango and RNA Peach aptamer systems (Kong et. al. 2021)

TO1-biotin is the standard variety of TO dye for RNA Mango experiments. View our complete list of RNA Mango dyes.

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The RNA Mango Workflow

The RNA Mango Aptamer
The system has two components: the RNA Mango aptamer, and the TO-1 dye. The dye only fluoresces when bound to the Mango aptamer.
Tagging Your RNA
For mRNA, insert the tag into the 3'UTR. For structured non-coding RNA, replace a stem-loop that is not essential to the RNA function.
Application: RNA Visualization
Express RNA of interest (with Mango Tag) using a vector or CRISPR Knock-In. Soak cells in TO-1 Biotin dye to illuminate the localization of the RNA of interest.
Application: RNA-RNA or RNA-protein complex pulldown
Express RNA of interest (with Mango Tag) using a vector or CRISPR Knock-In. Lyse the cells, and recover RNA of interest with bound RNA or proteins using streptavidin beads

Laboratory Results

Yeast U1 Ribonucleoprotein (RNP) Complex Pulldown Purification
Left: RNAs present in native extract in which U1M migrates as doublet. Right: U1M is a single band RNP present after mango-based purification using TO1 3PEG-Desthiobiotin Fluorophore
RNA Mango in a Test Tube
Mango dye binding to in vitro transcribed RNA Mango-tagged sgRNA. Stable binding and fluorescence even after leaving the tube for 1 month at room temperature.

RNA Mango in Action
Transcription reaction were carried out in 300 µL volumes using T7 RNA polymerase (400 U, 50U/µL, applied biological materials), 0.5 µM TO1-3PEG-Biotin (applied biological materials), in 8 mM GTP, 5 mM CTP and ATP, 2 mM UTP, 40 mM TRIS buffer pH 7.9, 2.5 mM spermidine, 26 mM MgCl2, 20 mM KCl, Pyrophosphatase (0.5 U, 0.1 U/µL, ThermoFisher Scientific), and 0.01% Triton X-100. To each sample, either water (Negative), 0.33 µM DNA template (Mango Transcription), or 500 nM final Mango III A10U RNA (Positive) was added. Samples were visualized in a blue light box, movie is played back at 30X speed.
RNA Mango Binding, Fluorescence, and Resistance Properties
Includes extensive citations list.

TO1 and TO3 dyes can be used in a 2-color reporter assay system using both the RNA Mango and RNA Peach aptamer systems (Kong et. al. 2021)

Top Publications

Ribonucleoprotein purification and characterization using RNA Mango.

Panchapakesan SSS, Ferguson ML, Hayden EJ, Chen X, Hoskins AA, Unrau PJ. et al.
RNA. 2017 Oct;23(10):1592-1599.

DOI: 10.1261/rna.062166.117. PubMed: 28747322. PubMed Central PMCID: PMC5602116.

Structural basis for high-affinity fluorophore binding and activation by RNA Mango.

Trachman RJ 3rd, Demeshkina NA, Lau MWL, Panchapakesan SSS, Jeng SCY, Unrau PJ, Ferré-D'Amaré AR. et al.
Nat Chem Biol. 2017 Jul;13(7):807-813.

DOI: 10.1038/nchembio.2392. Epub 2017 May 29. PubMed : 28553947.PubMed Central PMCID: PMC5550021.

Fluorophore ligand binding and complex stabilization of the RNA Mango and RNA Spinach aptamers.

Jeng SC, Chan HH, Booy EP, McKenna SA, Unrau PJ. et al
RNA. 2016 Dec;22(12):1884-1892. Epub 2016 Oct 24

PubMed : 27777365. PubMed Central PMCID: PMC5113208.