Characterizing excited conformational states of RNA by NMR spectroscopy

Zhao, Bo; Zhang, Qi

doi:10.1016/j.sbi.2015.02.011

Cited by 45 publications

(44 citation statements)

References 68 publications

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“…Chemical modifications that stabilize the tautomeric and anionic WC-like species also result in increased misincorporation and base substitution probabilities. 10,16–18 Recently, studies based on NMR relaxation dispersion (RD) 19–21 targeting the imino nitrogen of guanine (G-N1) and thymidine / uridine (T-N3 / U-N3) in DNA and RNA duplexes provided evidence for spontaneous transitions from wobble (WB) G•T/U mismatches (Figure 1b) toward short-lived and low-populated WC-like tautomeric (Figure 1c) and anionic (Figure 1d) mismatches. 22 Consistent with the Topal and Fresco hypothesis, these transitions toward WC-like mismatches occur with probabilities (10 −3 –10 −5 ) that are comparable to the probabilities of misincorporation.…”

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Direct NMR Evidence that Transient Tautomeric and Anionic States in dG·dT Form Watson–Crick-like Base Pairs

2017

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The replicative and translational machinery utilizes the unique geometry of canonical G•C and A•T/U Watson-Crick base pairs to discriminate against DNA and RNA mismatches in order to ensure high fidelity replication, transcription, and translation. There is growing evidence that spontaneous errors occur when mismatches adopt a Watson-Crick-like geometry through tautomerization and/or ionization of the bases. Studies employing NMR relaxation dispersion recently showed that wobble dG•dT and rG•rU mismatches in DNA and RNA duplexes transiently form tautomeric and anionic species with probabilities (≈0.01–0.40%) that are in concordance with replicative and translational errors. While computational studies indicate that these exceptionally short-lived and low-abundance species form Watson-Crick like base pairs, their conformation could not be directly deduced from the experimental data, and alternative pairing geometries could not be ruled out. Here, we report direct NMR evidence that the transient tautomeric and anionic species form hydrogenbonded Watson-Crick like base pairs. A guanine-to-inosine substitution, which selectively knocks out a Watson-Crick type (G)N2H2•••O2(T) hydrogen bond, significantly destabilized the transient tautomeric and anionic species, as assessed by lack of any detectable chemical exchange by imino nitrogen rotating frame spin relaxation (R1ρ) experiments. An 15N R1ρ NMR experiments targeting the amino nitrogen of guanine (dG-N2) provides direct evidence for Watson-Crick (G)N2H2•••O2(T) hydrogen bonding in the transient tautomeric state. The strategy presented in this work can be generally applied to examine hydrogen-bonding patterns in nucleic acid transient states including in other tautomeric and anionic species that are believed to play roles in replication and translational errors.

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“…19,22–25 The pH-independent ES1 is characterized by significantly downfield shifted dG-N1 (Δ ω N1 = ω ES1 – ω GS ≈ 36–40 p.p.m.) and dT-N3 (Δ ω N3 ≈ 9–19 p.p.m.)…”

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confidence: 99%

Direct NMR Evidence that Transient Tautomeric and Anionic States in dG·dT Form Watson–Crick-like Base Pairs

2017

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“…Collectively, our results demonstrate that the information content in assigned chemical shift data can be leveraged to conditionally predict the secondary structure of an RNA by combining machine learning tools and existing structure prediction algorithms. With access to the assigned chemical shift fingerprints of individual conformational states of an RNA, the hybrid modeling approach described in this study could be used to generate a hypothetical map of its conformational landscape, which will be particularly powerful when some of these states correspond to difficultto-characterize transient states (16,17).…”

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confidence: 99%

Conditional Prediction of RNA Secondary Structure Using NMR Chemical Shifts

Zhang

Frank

2019

Preprint

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Inspired by methods that utilize chemical-mapping data to guide secondary structure prediction, we sought to develop a framework for using assigned chemical shift data to guide RNA secondary structure prediction. We first used machine learning to develop classifiers which predict the base-pairing status of individual residues in an RNA based on their assigned chemical shifts. Then, we used these base-pairing status predictions as restraints to guide RNA folding algorithms. Our results showed that we could recover the correct secondary folds for nearly all of the 108 RNAs in our dataset with remarkable accuracy. Finally, we assessed whether we could conditionally predict the structure of the model RNA, microRNA-20b (miR-20b), by folding it using folding restraints derived from chemical shifts associated with two distinct conformational states, one a free (apo) state and the other a protein-bound (holo) state. For this test, we found that by using folding restraints derived from chemical shifts, we could recover the two distinct structures of the miR-20b, confirming our ability to conditionally predict its secondary structure. A commandline tool for Chemical Shifts to Base-Pairing Status (CS2BPS) predictions in RNA has been incorporated into our CS2Structure Git repository and can be accessed via: https://github.com/atfrank/CS2Structure.

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“…These transitions between structurally different conformational states add an additional layer of functional adaptability of nucleic acids. NMR spectroscopy has proven to be very well suited to investigate such transitions and to detected and characterize so called excited states 1e, 2. These low populated sub‐states represent high free‐energy conformations, that are speculated to be involved in important processes, such as ligand binding, protein–nucleic acid recognition or catalysis by ribozymes.…”

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Excited States of Nucleic Acids Probed by Proton Relaxation Dispersion NMR Spectroscopy

Juen

Wunderlich²,

Nußbaumer

et al. 2016

Angew Chem Int Ed

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In this work an improved stable isotope labeling protocol for nucleic acids is introduced. The novel building blocks eliminate/minimize homonuclear 13C and 1H scalar couplings thus allowing proton relaxation dispersion (RD) experiments to report accurately on the chemical exchange of nucleic acids. Using site‐specific 2H and 13C labeling, spin topologies are introduced into DNA and RNA that make 1H relaxation dispersion experiments applicable in a straightforward manner. The novel RNA/DNA building blocks were successfully incorporated into two nucleic acids. The A‐site RNA was previously shown to undergo a two site exchange process in the micro‐ to millisecond time regime. Using proton relaxation dispersion experiments the exchange parameters determined earlier could be recapitulated, thus validating the proposed approach. We further investigated the dynamics of the cTAR DNA, a DNA transcript that is involved in the viral replication cycle of HIV‐1. Again, an exchange process could be characterized and quantified. This shows the general applicablility of the novel labeling scheme for 1H RD experiments of nucleic acids.

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Characterizing excited conformational states of RNA by NMR spectroscopy

Cited by 45 publications

References 68 publications

Direct NMR Evidence that Transient Tautomeric and Anionic States in dG·dT Form Watson–Crick-like Base Pairs

Direct NMR Evidence that Transient Tautomeric and Anionic States in dG·dT Form Watson–Crick-like Base Pairs

Conditional Prediction of RNA Secondary Structure Using NMR Chemical Shifts

Excited States of Nucleic Acids Probed by Proton Relaxation Dispersion NMR Spectroscopy

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