SHAMS: Combining chemical modification of RNA with mass spectrometry to examine polypurine tract-containing RNA/DNA hybrids

Turner, Kevin B.; Yi-Brunozzi, Hye Young; Brinson, Robert G.; Marino, John P.; Fabris, Daniele; Grice, Stuart F. J. Le

doi:10.1261/rna.1615409

Cited by 27 publications

(36 citation statements)

References 40 publications

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“…9-12 SHAPE has facilitated development of functional hypotheses in complex biological systems. 13-17 Innovative refinements and applications of SHAPE include time-resolved 7,8,18,19 and temperature-dependent analyses, 5,20 examination of short RNAs 21,22 and interfacing with new readout technologies, 23 detection of long-range through-space interactions, 2,24-27 and identification of optimal sites for attachment of fluorescent reporter probes. 28 …”

Section: Introductionmentioning

confidence: 99%

The Mechanisms of RNA SHAPE Chemistry

et al. 2012

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The biological functions of RNA are ultimately governed by the local environment at each nucleotide. Selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE) chemistry is a powerful approach for measuring nucleotide structure and dynamics in diverse biological environments. SHAPE reagents acylate the 2′-hydroxyl group at flexible nucleotides because unconstrained nucleotides preferentially sample rare conformations that enhance the nucleophilicity of the 2′-hydroxyl. The critical corollary is that some constrained nucleotides must be poised for efficient reaction at the 2′-hydroxyl group. To identify such nucleotides, we performed SHAPE on intact crystals of the E. coli ribosome, monitored the reactivity of 1490 nucleotides in 16S ribosomal RNA, and examined those nucleotides that were hyper-reactive towards SHAPE and had well-defined crystallographic conformations. Analysis of these conformations revealed that 2′-hydroxyl reactivity is broadly facilitated by general base catalysis involving multiple RNA functional groups and by two specific orientations of the bridging 3′-phosphate group. Nucleotide analog studies confirmed the contributions of these mechanisms to SHAPE reactivity. These results provide a strong mechanistic explanation for the relationship between SHAPE reactivity and local RNA dynamics and will facilitate interpretation of SHAPE information in the many technologies that make use of this chemistry.

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Section: Introductionmentioning

confidence: 99%

The Mechanisms of RNA SHAPE Chemistry

et al. 2012

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“…Chemical probing techniques such as hydroxyl radical footprinting (Tullius and Dombroski 1985;Tullius and Greenbaum 2005) or in-line probing (Regulski and Breaker 2008;Wakeman and Winkler 2009a,b) are important approaches for RNA structure determination. In recent years, the development of innovative methods such as SHAPE (selective 29-hydroxyl acylation analyzed by primer extension) (Merino et al 2005;Wilkinson et al 2008), SHAMS (selective 29-hydroxyl acylation analyzed by mass spectrometry) (Turner et al 2009), and antisense interfered aiSHAPE (Legiewicz et al 2010) for probing secondary and tertiary structure of RNA molecules has opened new avenues for RNA research. Certain hydroxylselective electrophiles, such as N-methylisatoic anhydride (NMIA) (Merino et al 2005) and 1-methyl-7-nitroisatoic anhydride (1M7) (Mortimer and Weeks 2007), readily react with 29-OH groups of ribose moieties at nucleotide positions that are single-stranded and unconstrained.…”

Section: Introductionmentioning

confidence: 99%

Correlating SHAPE signatures with three-dimensional RNA structures

Bindewald¹,

Wendeler²,

Łęgiewicz³

et al. 2011

RNA

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Selective 29-hydroxyl acylation analyzed by primer extension (SHAPE) is a facile technique for quantitative analysis of RNA secondary structure. In general, low SHAPE signal values indicate Watson-Crick base-pairing, and high values indicate positions that are single-stranded within the RNA structure. However, the relationship of SHAPE signals to structural properties such as non-Watson-Crick base-pairing or stacking has thus far not been thoroughly investigated. Here, we present results of SHAPE experiments performed on several RNAs with published three-dimensional structures. This strategy allows us to analyze the results in terms of correlations between chemical reactivities and structural properties of the respective nucleotide, such as different types of base-pairing, stacking, and phosphate-backbone interactions. We find that the RNA SHAPE signal is strongly correlated with cis-Watson-Crick/Watson-Crick base-pairing and is to a remarkable degree not dependent on other structural properties with the exception of stacking. We subsequently generated probabilistic models that estimate the likelihood that a residue with a given SHAPE score participates in base-pairing. We show that several models that take SHAPE scores of adjacent residues into account perform better in predicting base-pairing compared with individual SHAPE scores. This underscores the context sensitivity of SHAPE and provides a framework for an improved interpretation of the response of RNA to chemical modification.

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“…Sequencing by CID and IRMPD has been used to locate modified nucleobases,18–29 and modified deoxyribose and phosphate moieties in DNA,30–32 in addition to characterization of the extremely varied modifications of RNA 33–38. Even some 3D structural aspects of DNA and RNA can be obtained by combining the use of chemical probes with tandem mass spectrometry 39–43. Furthermore, the factors governing fragmentation mechanisms of modified oligonucleotides44–48 by CID have been explored.…”

Section: Introductionmentioning

confidence: 99%

Characterization of Oligodeoxynucleotides and Modifications by 193 nm Photodissociation and Electron Photodetachment Dissociation

Smith

Brodbelt

2010

Anal. Chem.

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Ultraviolet photodissociation (UVPD) at 193 nm is compared to collision induced dissociation (CID) for sequencing and determination of modifications of multi-deprotonated 6 -20-mer oligodeoxynucleotides. UVPD at 193 nm causes efficient charge reduction of the deprotonated oligodeoxynucleotides via electron detachment, in addition to extensive backbone cleavages to yield sequence ions of relatively low abundance, including w, x, y, z, a, a-B, b, c, and d ions. Although internal ions populate UVPD spectra, base loss ions from the precursor are absent. Subsequent CID of the charge-reduced oligodeoxynucleotides formed upon electron detachment, in a net process called electron photodetachment dissociation (EPD), results in abundant sequence ions in terms of w, z, a, a-B and d products, with a marked decrease in the abundance of precursor base loss ions and internal fragments. Complete sequencing was possible for virtually all oligodeoxynucleotides studied. EPD of three modified oligodeoxynucleotides, a methylated oligodeoxynucleotide, a phosphorothioate-modified oligodeoxynucleotide, and an ethylated-oligodeoxynucleotide, resulted in specific and extensive backbone cleavages, specifically, w, z, a, a-B and d products, which allowed the modification site(s) to be pinpointed to a more specific location than by conventional CID.

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SHAMS: Combining chemical modification of RNA with mass spectrometry to examine polypurine tract-containing RNA/DNA hybrids

Cited by 27 publications

References 40 publications

The Mechanisms of RNA SHAPE Chemistry

The Mechanisms of RNA SHAPE Chemistry

Correlating SHAPE signatures with three-dimensional RNA structures

Characterization of Oligodeoxynucleotides and Modifications by 193 nm Photodissociation and Electron Photodetachment Dissociation

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