Structural basis for activation of fluorogenic dyes by an RNA aptamer lacking a G-quadruplex motif

Shelke, Sandip A.; Shao, Yaming; Łaski, Artur; Koirala, Deepak; Weissman, Benjamin P.; Fuller, James R.; Tan, Xiaodan; Constantin, T; Waggoner, Alan S.; Bruchez, Marcel P.; Armitage, Bruce A.; Piccirilli, Joseph A.

doi:10.1038/s41467-018-06942-3

Cited by 42 publications

(43 citation statements)

References 64 publications

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“…o-Coral is readily expressed in mammalian cells where it forms a bright complex with Gemini-561 that is otherwise not activated by cell components. Similarly to Malachite Green 48 and DIR2s aptamers 49 , o-Coral does not seem to possess the G-quadruplex organization shared by most of the other structurally characterized light-up aptamers 43 . This is of particular interest when considering a recent report indicating that most of the RNA Gquadruplex domains would be kept globally unfolded in mammalian cells 50 , suggesting that G-quadruplex-based RNA may not be optimal for being used in living cells.…”

Section: Discussionmentioning

confidence: 82%

A dimerization-based fluorogenic dye-aptamer module for RNA imaging in live cells

et al. 2019

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Live-cell imaging of RNA has remained a challenge because of the lack of naturally fluorescent RNAs. Recently developed RNA aptamers that can light-up small fluorogenic dyes could overcome this limitation, but they still suffer from poor brightness and photostability. Here, we propose a concept of cell-permeable fluorogenic dimer of sulforhodamine B dyes (Gemini-561) and corresponding dimerized aptamer (o-Coral) that can drastically enhance performance of the current RNA imaging method. The unprecedented brightness and photostability together with high affinity of this complex allowed, for the first time, direct fluorescence imaging in live mammalian cells of RNA polymerase-III transcription products as well as messenger RNAs labelled with a single copy of the aptamer, i.e. without tag multimerization. The developed fluorogenic module enables fast and sensitive detection of RNA inside live cells, while the proposed design concept opens the route to new generation of ultrabright RNA probes. Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:

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Section: Discussionmentioning

confidence: 82%

A dimerization-based fluorogenic dye-aptamer module for RNA imaging in live cells

et al. 2019

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“…(d) Mango‐I structure with TO1–biotin fluorophore bound in the G‐quadruplex core, flanked by a short helix . (e) DIR2s structure with bound OTB fluorophore between base triple and single unpaired nucleotide in the terminal region of aptamer . Compared to GFP, which has a buried fluorophore, fluorogenic RNA aptamers feature solvent‐exposed binding pockets.…”

Section: Fluorescent Proteinsmentioning

confidence: 99%

“…Comparison of fluorophore binding sites of (a) HBI within GFP, with (b) DFHBI in Spinach, (c) DFHO in Corn, (d) TO1–Biotin in Mango‐I, and (e) OTB in DIR2s . The fluorophore pockets for all molecules are highlighted, and hydrogen bonds are depicted by purple dashed lines.…”

Section: Fluorescent Proteinsmentioning

confidence: 99%

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From fluorescent proteins to fluorogenic RNAs: Tools for imaging cellular macromolecules

Truong

Ferré-D′Amaré

2019

Protein Science

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The explosion in genome-wide sequencing has revealed that noncoding RNAs are ubiquitous and highly conserved in biology. New molecular tools are needed for their study in live cells. Fluorescent RNA-small molecule complexes have emerged as powerful counterparts to fluorescent proteins, which are well established, universal tools in the study of proteins in cell biology. No naturally fluorescent RNAs are known; all current fluorescent RNA tags are in vitro evolved or engineered molecules that bind a conditionally fluorescent small molecule and turn on its fluorescence by up to 5000-fold. Structural analyses of several such fluorescence turn-on aptamers show that these compact (30-100 nucleotides) RNAs have diverse molecular architectures that can restrain their photoexcited fluorophores in their maximally fluorescent states, typically by stacking between planar nucleotide arrangements, such as G-quadruplexes, base triples, or base pairs. The diversity of fluorogenic RNAs as well as fluorophores that are cell permeable and bind weakly to endogenous cellular macromolecules has already produced RNA-fluorophore complexes that span the visual spectrum and are useful for tagging and visualizing RNAs in cells. Because the ligand binding sites of fluorogenic RNAs are not constrained by the need to autocatalytically generate fluorophores as are fluorescent proteins, they may offer more flexibility in molecular engineering to generate photophysical properties that are tailored to experimental needs.

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“…A common structural feature of Spinach and Mango is the presence of a fluorogen-binding G-Quadruplex (GQ) core with mixed parallel and antiparallel connectivity, flanked by A-form duplex stem(s) [15][16][17] . In addition, non-GQ light-up aptamers which can selectively bind to malachite green (MG) 18,19 or cyanine dyes with submicromolar affinity have also been developed 20,21 .…”

mentioning

confidence: 99%

Nanomechanics and co-transcriptional folding of Spinach and Mango

Mitra

2019

Preprint

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Recent advances in fluorogen-binding RNA aptamers known as "light-up" aptamers provide an avenue for protein-free detection of RNA in cells. Crystallographic studies have revealed a G-Quadruplex (GQ) structure at the core of light-up aptamers such as Spinach, Mango and Corn.Detailed biophysical characterization of folding of such aptamers is still lacking despite the potential implications on their in vivo folding and function. We used single-molecule fluorescence-force spectroscopy that combines fluorescence resonance energy transfer with optical tweezers to examine mechanical responses of Spinach2, iMangoIII and MangoIV.Spinach2 unfolded in four discrete steps as force is increased to 7 pN and refolded in reciprocal steps upon force relaxation. Binding of DFHBI-1T fluorogen preserved the step-wise unfolding behavior although at slightly higher forces. In contrast, GQ core unfolding in iMangoIII and MangoIV occurred in one discrete step at forces > 10 pN and refolding occurred at lower forces showing hysteresis. Binding of the cognate fluorogen, TO1, did not significantly alter the mechanical stability of Mangos. In addition to K + , which is needed to stabilize the GQ cores, Mg 2+ was needed to obtain full mechanical stability of the aptamers. Co-transcriptional folding analysis using superhelicases showed that co-transcriptional folding reduces misfolding and allows a folding pathway different from refolding. As the fundamental cellular processes like replication, transcription etc. exert pico-Newton levels of force, these aptamers may unfold in vivo and subsequently misfold.

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Structural basis for activation of fluorogenic dyes by an RNA aptamer lacking a G-quadruplex motif

Cited by 42 publications

References 64 publications

A dimerization-based fluorogenic dye-aptamer module for RNA imaging in live cells

A dimerization-based fluorogenic dye-aptamer module for RNA imaging in live cells

From fluorescent proteins to fluorogenic RNAs: Tools for imaging cellular macromolecules

Nanomechanics and co-transcriptional folding of Spinach and Mango

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