Light-Up RNA Aptamers and Their Cognate Fluorogens: From Their Development to Their Applications

Bouhedda, Farah; Autour, Alexis; Ryckelynck, Michaël

doi:10.3390/ijms19010044

Cited by 91 publications

(78 citation statements)

References 135 publications

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“…Up until now, the development of a fluorogenic platform has been guided by specificity and affinity of the fluorogen to the RNA aptamer and the resulting fluorescent enhancement and photostability of the module 55 . As the fundamental cellular processes generate and are regulated by pico Newton levels of tension, mechanical stability of these aptamers is potentially another important consideration.…”

Section: Discussionmentioning

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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Section: Discussionmentioning

confidence: 99%

Nanomechanics and co-transcriptional folding of Spinach and Mango

Mitra

2019

Preprint

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“…Specifically, we adapted an in cellulo Pol III transcriptional reporter system based on fluorescent RNA aptamers that are active in human cell lines [24]. RNA aptamers are structural motifs capable of binding to target ligands with high specificity [25]. Here we utilized the Tornado system which contains the Corn aptamer embedded within RNA transcripts for direct reporting of Pol III transcriptional activity ( Fig.…”

Section: Introductionmentioning

confidence: 99%

RNA sequence and structure determinants of Pol III transcriptional termination in human cells

Verosloff¹,

Corcoran²,

Dolberg³

et al. 2020

Preprint

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The precise mechanism of transcription termination of the eukaryotic RNA polymerase III (Pol III) has been a subject of considerable debate. Although previous studies have clearly shown that at the end of RNA transcripts, tracts comprised of multiple uracils are required for Pol III termination, whether upstream RNA secondary structure in the nascent transcript is necessary for robust transcriptional termination is still subject to debate. We sought to address this directly through the development of an in cellulo Pol III transcription termination assay using a synthetic biology approach. Specifically, we utilized the recently developed Tornado expression system and a stabilized Corn RNA aptamer to create a Pol III-transcribed RNA that produces a detectable fluorescent signal when transcribed in human cells. To study the effects of RNA sequence and structure on Pol III termination, we systematically varied the sequence context upstream of the aptamer and identified sequence characteristics that enhance or diminish termination. We found that in the absence of predicted secondary structure, only poly-U tracts longer than then the average length found in the human genome (4–5 nucleotides), efficiently terminate Pol III transcription. We found that shorter poly-U tracts could induce termination when placed in proximity to secondary structural elements, while secondary structure by itself was not sufficient to induce termination. These findings demonstrate a key role for sequence and structural elements within Pol III-transcribed nascent RNA for efficient transcription termination, and demonstrate a generalizable assay for characterizing Pol III transcription in human cells.

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“…Instead, RNA mimics of FPs are molecules that bind to cognate small molecules, that is, aptamers . These aptamers recognize compounds that are minimally fluorescent on their own and increase their fluorescence by as much as 5000 times upon binding . All such described fluorescence turn‐on aptamers are the result of in vitro evolution and design experiments.…”

Section: Introductionmentioning

confidence: 99%

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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Light-Up RNA Aptamers and Their Cognate Fluorogens: From Their Development to Their Applications

Cited by 91 publications

References 135 publications

Nanomechanics and co-transcriptional folding of Spinach and Mango

Nanomechanics and co-transcriptional folding of Spinach and Mango

RNA sequence and structure determinants of Pol III transcriptional termination in human cells

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

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