Nucleic Acid Structure Characterization by Small Angle X‐Ray Scattering (SAXS)

Burke, Jordan E.; Butcher, Samuel E.

doi:10.1002/0471142700.nc0718s51

Cited by 32 publications

(29 citation statements)

References 62 publications

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“…Large RNA molecules can be difficult to crystallize, and their characterization by NMR is challenging due to spectral overlap and fast relaxation. The SAXS methodology is particularly well suited to study these systems, as the electron-rich phosphate backbone increases the overall contrast between nucleic acid and buffer, and this facilitates outlining the shape of the RNA under study (Lipfert et al 2008;Ali et al 2010;Burke and Butcher 2012;Fang et al 2015). To tackle the study of the 3 ′ X domain and verify whether it formed two or three helical stems, we separately studied by SAXS two smaller subdomains in addition to the full-length sequence: a 46-nt sequence containing the extended SL1 subdomain present in the three-stem domain conformation, and a 55nt construct adopting the extended SL2 ′ stem-loop structure contained in the two-stem domain fold (Fig.…”

Section: Strategy For Saxs Analysesmentioning

confidence: 99%

Three-dimensional structure of the 3′X-tail of hepatitis C virus RNA in monomeric and dimeric states

Cantero-Camacho

Wang

et al. 2017

RNA

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The 3'X domain is a 98-nt region located at the 3' end of hepatitis C virus genomic RNA that plays essential functions in the viral life cycle. It contains an absolutely conserved, 16-base palindromic sequence that promotes viral RNA dimerization, overlapped with a 7-nt tract implicated in a distal contact with a nearby functional sequence. Using small angle X-ray scattering measurements combined with model building guided by NMR spectroscopy, we have studied the stoichiometry, structure, and flexibility of domain 3'X and two smaller subdomain sequences as a function of ionic strength, and obtained a three-dimensional view of the full-length domain in its monomeric and dimeric states. In the monomeric form, the 3'X domain adopted an elongated conformation containing two SL1' and SL2' double-helical stems stabilized by coaxial stacking. This structure was significantly less flexible than that of isolated subdomain SL2' monomers. At higher ionic strength, the 3'X scattering envelope nearly doubled its size, reflecting the formation of extended homodimers containing an antiparallel SL2' duplex flanked by coaxially stacked SL1' helices. Formation of these dimers could initialize and/or regulate the packaging of viral RNA genomes into virions.

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Section: Strategy For Saxs Analysesmentioning

confidence: 99%

Three-dimensional structure of the 3′X-tail of hepatitis C virus RNA in monomeric and dimeric states

Cantero-Camacho

Wang

et al. 2017

RNA

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“…Here, we discuss protocols for acquiring SAXS data, including some methods for determining the homogeneity of the sample. Additional, detailed information about protocols for preparing RNA samples for SAXS and for acquiring high‐quality SAXS data can be found in two recent reviews …”

Section: Protocols For Rna Saxs Experimentsmentioning

confidence: 99%

SAXS studies of RNA: structures, dynamics, and interactions with partners

Chen

Pollack

2016

WIREs RNA

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Small angle x-ray scattering, SAXS, is a powerful and easily employed experimental technique that provides solution structures of macromolecules. The size and shape parameters derived from SAXS provide global structural information about these molecules in solution and essentially complement data acquired by other biophysical methods. As applied to protein systems, SAXS is a relatively mature technology: sophisticated tools exist to acquire and analyze data, and to create structural models that include dynamically flexible ensembles. Given the expanding appreciation of RNA’s biological roles, there is a need to develop comparable tools to characterize solution structures of RNA, including its interactions with important biological partners. We review the progress towards achieving this goal, focusing on experimental and computational innovations. The use of multiphase modeling, absolute calibration and contrast variation methods, among others, provides new and often unique ways of visualizing this important biological molecule and its essential partners: ions, other RNAs or proteins.

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“…This approach has been shown to provide accurate low-resolution RNA structures (42)(43)(44)(45). VAI has a central bulged region flanked by a short arm and a longer, kinked arm (Fig.…”

Section: Structural Model Of Vaimentioning

confidence: 99%

Structural Analysis of Adenovirus VAI RNA Defines the Mechanism of Inhibition of PKR

Launer-Felty

Wong

Cole

2015

Biophysical Journal

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Protein kinase R (PKR) is activated by dsRNA produced during virus replication and plays a major role in the innate immunity response to virus infection. In response, viruses have evolved multiple strategies to evade PKR. Adenovirus virus-associated RNA-I (VAI) is a short, noncoding transcript that functions as an RNA decoy to sequester PKR in an inactive state. VAI consists of an apical stem-loop, a highly structured central domain, and a terminal stem. Chemical probing and mutagenesis demonstrate that the central domain is stabilized by a pseudoknot. A structural model of VAI was obtained from constraints derived from chemical probing and small angle x-ray scattering (SAXS) measurements. VAI adopts a flat, extended conformation with the apical and terminal stems emanating from a protuberance in the center. This model reveals how the apical stem and central domain assemble to produce an extended duplex that is precisely tuned to bind a single PKR monomer with high affinity, thereby inhibiting activation of PKR by viral dsRNA.

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Nucleic Acid Structure Characterization by Small Angle X‐Ray Scattering (SAXS)

Cited by 32 publications

References 62 publications

Three-dimensional structure of the 3′X-tail of hepatitis C virus RNA in monomeric and dimeric states

Three-dimensional structure of the 3′X-tail of hepatitis C virus RNA in monomeric and dimeric states

SAXS studies of RNA: structures, dynamics, and interactions with partners

Structural Analysis of Adenovirus VAI RNA Defines the Mechanism of Inhibition of PKR

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