NMR characterization of RNA small molecule interactions

Thompson, Rhese D.; Baisden, Jared T.; Zhang, Qi

doi:10.1016/j.ymeth.2019.05.015

Cited by 17 publications

(11 citation statements)

References 151 publications

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“…Through the years, a toolbox of biophysical methods by which such insights can be obtained has been assembled. These methods include X-ray crystallography [5,6], cryo-electron microscopy (cryoEM) [7,8], nuclear magnetic resonance spectroscopy (NMR) [9,10], electron paramagnetic resonance (EPR) [11,12], Förster resonance energy transfer (FRET) [13,14], small-angle X-ray scattering (SAXS) [15], and molecular dynamics simulation (MD) [16]. Out of this toolbox, EPR is a method that provides such information but requires the presence of unpaired electrons.…”

Section: Introductionmentioning

confidence: 99%

Site-Directed Spin Labeling of RNA with a Gem-Diethylisoindoline Spin Label: PELDOR, Relaxation, and Reduction Stability

et al. 2019

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Ribonucleic acid function is governed by its structure, dynamics, and interaction with other biomolecules and influenced by the local environment. Thus, methods are needed that enable one to study RNA under conditions as natural as possible, possibly within cells. Site-directed spin-labeling of RNA with nitroxides in combination with, for example, pulsed electron–electron double resonance (PELDOR or DEER) spectroscopy has been shown to provide such information. However, for in-cell measurements, the usually used gem-dimethyl nitroxides are less suited, because they are quickly reduced under in-cell conditions. In contrast, gem-diethyl nitroxides turned out to be more stable, but labeling protocols for binding these to RNA have been sparsely reported. Therefore, we describe here the bioconjugation of an azide functionalized gem-diethyl isoindoline nitroxide to RNA using a copper (I)-catalyzed azide–alkyne cycloaddition (“click”-chemistry). The labeling protocol provides high yields and site selectivity. The analysis of the orientation selective PELDOR data show that the gem-diethyl and gem-dimethyl labels adopt similar conformations. Interestingly, in deuterated buffer, both labels attached to RNA yield TM relaxation times that are considerably longer than observed for the same type of label attached to proteins, enabling PELDOR time windows of up to 20 microseconds. Together with the increased stability in reducing environments, this label is very promising for in-cell Electron Paramagnetic Resonance (EPR) studies.

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Section: Introductionmentioning

confidence: 99%

Site-Directed Spin Labeling of RNA with a Gem-Diethylisoindoline Spin Label: PELDOR, Relaxation, and Reduction Stability

et al. 2019

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“…Despite having lower throughput relative to HTS, NMR spectroscopy is also a powerful tool for identifying and validating small molecules that interact with biomolecules and has played a significant role in protein-targeted drug discovery. Excellent reviews have been published in recent years ( Thompson et al, 2019 ), which provide thorough discussions of various NMR experiments for identifying protein-binding small molecules as well as evaluating strengths and liabilities of individual methods. Since many of these methods are based on observing ligand NMR signals, the nature of a target, whether it is a protein or an RNA, has minor influence on experimental setups of these methods, enabling their direct applications in RNA-binding small molecules identification.…”

Section: Selectivity and Specificity Of Binding For Rnas Moleculesmentioning

confidence: 99%

An overview of structural approaches to study therapeutic RNAs

Mollica

Cupaioli²,

Rossetti³

et al. 2022

Front. Mol. Biosci.

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RNAs provide considerable opportunities as therapeutic agent to expand the plethora of classical therapeutic targets, from extracellular and surface proteins to intracellular nucleic acids and its regulators, in a wide range of diseases. RNA versatility can be exploited to recognize cell types, perform cell therapy, and develop new vaccine classes. Therapeutic RNAs (aptamers, antisense nucleotides, siRNA, miRNA, mRNA and CRISPR-Cas9) can modulate or induce protein expression, inhibit molecular interactions, achieve genome editing as well as exon-skipping. A common RNA thread, which makes it very promising for therapeutic applications, is its structure, flexibility, and binding specificity. Moreover, RNA displays peculiar structural plasticity compared to proteins as well as to DNA. Here we summarize the recent advances and applications of therapeutic RNAs, and the experimental and computational methods to analyze their structure, by biophysical techniques (liquid-state NMR, scattering, reactivity, and computational simulations), with a focus on dynamic and flexibility aspects and to binding analysis. This will provide insights on the currently available RNA therapeutic applications and on the best techniques to evaluate its dynamics and reactivity.

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“…Nuclear magnetic resonance (NMR) spectroscopy accounts for ∼35% of the RNA structures deposited in the PDB and ∼7% of the protein structures, making it competitive with other biophysical tools such as X-ray crystallography and more recently cryo-electron microscopy (cryo-EM) (Figure C) . Moreover, NMR spectroscopy provides high-resolution structural dynamic information in solution, rendering it an ideal tool to study RNA and its interactions with macromolecules or small drug-like compounds or both. − However, unlike proteins, which are made up of 20 unique amino acid building blocks, RNAs are composed of only four aromatic nucleotides [i.e., adenosine (Ade or A), guanosine (Gua or G), cytidine (Cyt or C), and uridine (Uri or U)] that resonate over a very narrow chemical shift region. This poor chemical shift dispersion is further exacerbated with increasing RNA size.…”

Section: Introductionmentioning

confidence: 99%

Isotope Labels Combined with Solution NMR Spectroscopy Make Visible the Invisible Conformations of Small-to-Large RNAs

2022

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RNA is central to the proper function of cellular processes important for life on earth and implicated in various medical dysfunctions. Yet, RNA structural biology lags significantly behind that of proteins, limiting mechanistic understanding of RNA chemical biology. Fortunately, solution NMR spectroscopy can probe the structural dynamics of RNA in solution at atomic resolution, opening the door to their functional understanding. However, NMR analysis of RNA, with only four unique ribonucleotide building blocks, suffers from spectral crowding and broad linewidths, especially as RNAs grow in size. One effective strategy to overcome these challenges is to introduce NMR-active stable isotopes into RNA. However, traditional uniform labeling methods introduce scalar and dipolar couplings that complicate the implementation and analysis of NMR measurements. This challenge can be circumvented with selective isotope labeling. In this review, we outline the development of labeling technologies and their application to study biologically relevant RNAs and their complexes ranging in size from 5 to 300 kDa by NMR spectroscopy.

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NMR characterization of RNA small molecule interactions

Cited by 17 publications

References 151 publications

Site-Directed Spin Labeling of RNA with a Gem-Diethylisoindoline Spin Label: PELDOR, Relaxation, and Reduction Stability

Site-Directed Spin Labeling of RNA with a Gem-Diethylisoindoline Spin Label: PELDOR, Relaxation, and Reduction Stability

An overview of structural approaches to study therapeutic RNAs

Isotope Labels Combined with Solution NMR Spectroscopy Make Visible the Invisible Conformations of Small-to-Large RNAs

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