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.
Using random mutagenesis and high throughput screening by microfluidic-assisted In Vitro Compartmentalization, we report the isolation of an order of magnitude times brighter mutants of the light-up RNA aptamers Spinach that are far less salt-sensitive and with a much higher thermal stability than the parent molecule. Further engineering gave iSpinach, a molecule with folding and fluorescence properties surpassing those of all currently known aptamer based on the fluorogenic co-factor 3,5-difluoro-4-hydroxybenzylidene imidazolinone (DFHBI). We illustrate the potential of iSpinach in a new sensitive and high throughput-compatible fluorogenic assay that measures co-transcriptionally the catalytic constant (kcat) of a model ribozyme.
Several turn-on RNA aptamers that activate small molecule fluorophores have been selected in vitro. Among these, the ~30 nucleotide Mango-III is notable because it binds the thiazole orange derivative TO1-Biotin with high affinity and fluoresces brightly (quantum yield 0.55). Uniquely among related aptamers, Mango-III exhibits biphasic thermal melting, characteristic of molecules with tertiary structure. We report crystal structures of TO1-Biotin complexes of Mango-III, a structure-guided mutant Mango-III(A10U), and a functionally reselected mutant iMango-III. The structures reveal a globular architecture arising from an unprecedented pseudoknot-like connectivity between a G-quadruplex and an embedded non-canonical duplex. The fluorophore is restrained into a planar conformation by the G-quadruplex, a lone, long-range trans-Watson-Crick pair (whose A10U mutation increases quantum yield to 0.66), and a pyrimidine perpendicular to
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