Abstract
Despite having many key roles in cellular biology, directly imaging biologically important RNAs has been hindered by a lack of fluorescent tools equivalent to the fluorescent proteins available to study cellular proteins. Ideal RNA labelling systems must preserve biological function, have photophysical properties similar to existing fluorescent proteins, and be compatible with established live and fixed cell protein labelling strategies. Here, we report a microfluidics-based selection of three new high-affinity RNA Mango fluorogenic aptamers. Two of these are as bright or brighter than enhanced GFP when bound to TO1-Biotin. Furthermore, we show that the new Mangos can accurately image the subcellular localization of three small non-coding RNAs (5S, U6, and a box C/D scaRNA) in fixed and live mammalian cells. These new aptamers have many potential applications to study RNA function and dynamics both in vitro and in mammalian cells.
Highlights
Despite having many key roles in cellular biology, directly imaging biologically important RNAs has been hindered by a lack of fluorescent tools equivalent to the fluorescent proteins available to study cellular proteins
Fluorogenic RNA aptamers that enhance the fluorescence of an unbound fluorophore have sparked significant interest and hold great potential to enable the visualization of RNA molecules within a cell[1,2,3,4]
Stepwise photobleaching in fixed cell images indicate that as few as 4–17 molecules can be detected in each foci, and photobleaching rates in live cells or in vitro were at least an order of magnitude slower than found for Broccoli. These new aptamers work well with existing fluorescence microscopy techniques and we demonstrate their applicability by imaging the correct localization of 5S, U6 and the box C/D scaRNA in fixed and live mammalian cells
Summary
Despite having many key roles in cellular biology, directly imaging biologically important RNAs has been hindered by a lack of fluorescent tools equivalent to the fluorescent proteins available to study cellular proteins. Despite the inability to simultaneously optimize aptamer-fluorophore brightness and binding affinity, existing fluorogenic systems have achieved some notable successes in bacteria, yeast and mammalian cells[1,2,13,14,15] This suggests that using newly developed screening methodologies to select brighter fluorogenic RNA aptamers either by FACS9 or droplet-based microfluidics platforms[10] can provide powerful and easy to use fluorescent RNA imaging tags to study cellular RNAs. Here, we have used a competitive ligand binding microfluidic selection to isolate three new aptamers (Mango II, III and IV) with markedly improved fluorescent properties, binding affinities, and salt dependencies compared to the original Mango I aptamer[8]. These findings indicate that the new Mango aptamers offer an interesting alternative to existing fluorogenic aptamers[12]
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