The promise of cryo-EM to explore RNA structural dynamics

Bonilla, Steve; Kieft, Jeffrey S.

doi:10.1016/j.jmb.2022.167802

Cited by 32 publications

(20 citation statements)

References 59 publications

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“…60,72 The analysis of RNA conformations is typically achieved by either chemical probing with selective 2′ hydroxyl acylation analyzed by primer extension (SHAPE) method 72,75 or biophysical technique like single-molecule Forster resonance energy transfer (smFRET) 76 or cryogenic electron microscopy. 77 These methods are very informative, but sample preparations can be complicated and the measurements and analysis are often time-consuming. Solid-state nanopore based experiments are easy to prepare and quick to perform, and data analysis is less time-consuming; they have been used previously to detect RNA conformations.…”

Section: Resultsmentioning

confidence: 99%

Probing RNA Conformations Using a Polymer–Electrolyte Solid-State Nanopore

et al. 2022

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Nanopore systems have emerged as a leading platform for the analysis of biomolecular complexes with singlemolecule resolution. The conformation of biomolecules, such as RNA, is highly dependent on the electrolyte composition, but solid-state nanopore systems often require high salt concentration to operate, precluding analysis of macromolecular conformations under physiologically relevant conditions. Here, we report the implementation of a polymer−electrolyte solid-state nanopore system based on alkali metal halide salts dissolved in 50% w/v poly(ethylene) glycol (PEG) to augment the performance of our system. We show that polymer− electrolyte bath governs the translocation dynamics of the analyte which correlates with the physical properties of the salt used in the bath. This allowed us to identify CsBr as the optimal salt to complement PEG to generate the largest signal enhancement. Harnessing the effects of the polymer−electrolyte, we probed the conformations of the Chikungunya virus (CHIKV) RNA genome fragments under physiologically relevant conditions. Our system was able to fingerprint CHIKV RNA fragments ranging from ∼300 to ∼2000 nt length and subsequently distinguish conformations between the cotranscriptionally folded and the natively refolded ∼2000 nt CHIKV RNA. We envision that the polymer−electrolyte solidstate nanopore system will further enable structural and conformational analyses of individual biomolecules under physiologically relevant conditions.

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Section: Resultsmentioning

confidence: 99%

Probing RNA Conformations Using a Polymer–Electrolyte Solid-State Nanopore

et al. 2022

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“…Determining long-range connectivity and its variations is challenging. Direct structural techniques, such as X-ray crystallography 8 and cryo-electron microscopy 9 , are not well suited to long and heterogeneous RNA molecules. And indirect techniques, such as chemical probing by SHAPE 10 and DMS 11 , measure the conformational flexibility of each nucleotide in the sequence, which is related to the probability that each nucleotide is connected to another in the sequence but does not directly reveal the endpoint of the connection or if competing connections are present 12 .…”

Section: Main Textmentioning

confidence: 99%

Measuring intramolecular connectivity in long RNA molecules using two-dimensional DNA patch-probe arrays

Chiang

Kimchi

Dhaliwal

et al. 2023

Preprint

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We describe a simple method to infer intramolecular connections in a population of long RNA molecules in vitro. First we add DNA oligonucleotide 'patches' that perturb the RNA connections, then we use a microarray containing a complete set of DNA oligonucleotide 'probes' to record where perturbations occur. The pattern of perturbations reveals couplings between different regions of the RNA sequence, from which we infer connections as well as their prevalences in the population. We validate this patch-probe method using the 1,058-nucleotide RNA genome of satellite tobacco mosaic virus (STMV), which has previously been shown to have multiple long-range connections. Our results not only indicate long duplexes that agree with previous structures but also reveal the prevalence of competing connections. Together, these results suggest that globally-folded and locally-folded structures coexist in solution. We show that the prevalence of connections changes when pseudouridine, an important component of natural and synthetic RNA molecules, is substituted for uridine in STMV RNA.

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“…Therefore, more structural studies of ribosomes [76], spliceosomes [75,77], transfer-RNA synthetases [78], and RNA polymerase complexes [79] will provide valuable insights into understanding the molecular interactions between RNAs and RBPs. The development of structural biology makes it possible to elucidate the structural basis of these structures [80].…”

Section: Domains Of Rbps For Rna Bindingmentioning

confidence: 99%

Targeting RNA‐binding proteins with small molecules: Perspectives, pitfalls and bifunctional molecules

Kang

2023

FEBS Letters

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RNA‐binding proteins (RBPs) play vital roles in organisms through binding with RNAs to regulate their functions. Small molecules affecting the function of RBPs have been developed, providing new avenues for drug discovery. Herein, we describe the perspectives on developing small molecule regulators of RBPs. The following types of small molecule modulators are of great interest in drug discovery: small molecules binding to RBPs to affect interactions with RNA molecules, bifunctional molecules binding to RNA or RBP to influence their interactions, and other types of molecules that affect the stability of RNA or RBPs. Moreover, we emphasize that the bifunctional molecules may play important roles in small molecule development to overcome the challenges encountered in the process of drug discovery.

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The promise of cryo-EM to explore RNA structural dynamics

Cited by 32 publications

References 59 publications

Probing RNA Conformations Using a Polymer–Electrolyte Solid-State Nanopore

Probing RNA Conformations Using a Polymer–Electrolyte Solid-State Nanopore

Measuring intramolecular connectivity in long RNA molecules using two-dimensional DNA patch-probe arrays

Targeting RNA‐binding proteins with small molecules: Perspectives, pitfalls and bifunctional molecules

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