Structure-based investigation of fluorogenic Pepper aptamer

Huang, Kaiyi; Chen, Xianjun; Li, Chunyan; Song, Qianqian; Li, Huiwen; Zhu, Linyong; Yang, Yi; Ren, Aiming

doi:10.1038/s41589-021-00884-6

Cited by 44 publications

(52 citation statements)

References 52 publications

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“…3 B,C). Inside the junction regions, we observed negligible difference in SHAPE reactivity between the three ligands tested, confirming that the mode of binding is conserved as indicated by the previously determined crystal structures 34 .…”

Section: Resultssupporting

confidence: 88%

“…3 B,C). From the crystal structure 34 and our EM data we would expect this to be one of the more dynamic residues as it has no hydrogen bonding partner and is only supported by base stacking from one adjacent nucleotide. The three next nucleotides (position 5-7) show significant flexibility in the apo-structure that is attenuated in the ligand bound state.…”

Section: Resultsmentioning

confidence: 99%

“…Since we obtained the cryo-EM map before the publication of the Pepper crystal structures 34 , we attempted structure determination using the DRRAFTER pipeline 37,38 . The models generated reached a mean pairwise RMSD (convergence) of 3.5 Angstrom, and out of the top 10 models, 9 had the correct strand path for J3/2.…”

Section: Discussionmentioning

confidence: 99%

“…The C35 and C44 bands appear more intensely in the Benzoyl Cyanide treated samples, suggesting an increase in termination due to the chemical modification. For C44, it is observed that HBC485 and HBC487 have similar intensity as with no ligand, but that HBC620 has a higher signal, which can be explained by its stronger binding affinity (HBC485 KD=8.0 nM, HBC497 KD=6.7 nM, HBC620 KD=6.1 nM) 34 . For C35, we observe that HBC485 terminates at a similar level to no ligand, while HBC487 and HBC620 have a higher signal.…”

Section: Shape Probing Of Pepper Reveals Cooperative Binding Of Hbcmentioning

confidence: 97%

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RNA origami scaffolds as a cryo-EM tool for investigating aptamer-ligand binding of a Broccoli-Pepper FRET pair

Vallina

McRae

Hansen

et al. 2022

Preprint

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RNA nanotechnology uses motifs from nature as well as aptamers from in vitro selection to construct nanostructures and devices for applications in RNA medicine and synthetic biology. The RNA origami method allows cotranscriptional folding of large RNA scaffolds that can position functional motifs in a precise manner, which has been verified by Förster Resonance Energy Transfer (FRET) between fluorescent aptamers. Cryogenic electron microscopy (cryo-EM) is a promising method for characterizing the structure of larger RNA nanostructures. However, the structure of individual aptamers is difficult to solve by cryo-EM due to their low molecular weight. Here, we place aptamers on the RNA origami scaffolds to increase the contrast for cryo-EM and solve the structure of a new Broccoli-Pepper FRET pair. We identify different modes of ligand binding of the two aptamers and verify by selective probing. 3D variability analysis of the cryo-EM data show that the relative position between the two bound fluorophores on the origami fluctuate by only 3.5 Angstrom. Our results demonstrate the use of RNA origami scaffolds for characterizing small RNA motifs by cryo-EM and for positioning functional RNA motifs with high spatial precision. The Broccoli-Pepper apta-FRET pair has potential use for developing advanced sensors that are sensitive to small conformational changes.

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Section: Resultssupporting

confidence: 88%

Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Shape Probing Of Pepper Reveals Cooperative Binding Of Hbcmentioning

confidence: 97%

See 2 more Smart Citations

RNA origami scaffolds as a cryo-EM tool for investigating aptamer-ligand binding of a Broccoli-Pepper FRET pair

Vallina

McRae

Hansen

et al. 2022

Preprint

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“…2 A-B ). Secondly, to select the light-up RNA aptamers, we compare the fluorescence of Peppers, which have superior properties, with that of Broccoli, which is one of the widely used light-up RNA aptamers [28] , [34] , [35] . Pepper exhibits a higher signal-to-noise ratio (S/N) (205.2-fold, Fig.…”

Section: Resultsmentioning

confidence: 99%

Allosteric aptasensor-initiated target cycling and transcription amplification of light-up RNA aptamer for sensitive detection of protein

Song¹,

Yuan²,

Tan³

et al. 2022

Sensors and Actuators B: Chemical

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RNA Control via Redox‐Responsive Acylation

Guo,

Chen,

Onishi

et al. 2024

Angewandte Chemie

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Incorporating stimuli‐responsive components into RNA constructs provides precise spatiotemporal control over RNA structures and functions. Despite considerable advancements, the utilization of redox‐responsive stimuli for the activation of caged RNAs remains scarce. In this context, we present a novel strategy that leverages post‐synthetic acylation coupled with redox‐responsive chemistry to exert reversible control over RNA. To achieve this, we design and synthesize a series of acylating reagents specifically tailored for introducing disulfide‐containing acyl adducts into the 2’‐OH groups of RNA (“cloaking”). Our data reveal that these acyl moieties can be readily appended, effectively blocking RNA catalytic activity and folding. We also demonstrate the traceless release and reactivation of caged RNAs (“uncloaking”) through reducing stimuli. By employing this strategy, RNA exhibits rapid cellular uptake, effective distribution and activation in the cytosol without lysosomal entrapment. We anticipate that our methodology will be accessible to laboratories engaged in RNA biology and holds promise as a versatile platform for RNA‐based applications.

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Structure-based investigation of fluorogenic Pepper aptamer

Cited by 44 publications

References 52 publications

RNA origami scaffolds as a cryo-EM tool for investigating aptamer-ligand binding of a Broccoli-Pepper FRET pair

RNA origami scaffolds as a cryo-EM tool for investigating aptamer-ligand binding of a Broccoli-Pepper FRET pair

Allosteric aptasensor-initiated target cycling and transcription amplification of light-up RNA aptamer for sensitive detection of protein

RNA Control via Redox‐Responsive Acylation

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