High-throughput single-nucleotide structural mapping by capillary automated footprinting analysis

Mitra, Subhasish; Shcherbakova, Inna; Altman, Russ B.; Brenowitz, Michael; Laederach, Alain

doi:10.1093/nar/gkn267

Cited by 93 publications

(96 citation statements)

References 54 publications

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“…This large body of data can be analyzed in minutes instead of in hours using freely available semi-automated software (https://simtk.org/ home/safa) [110]. Still higher-throughput data acquisition is now possible using capillary electrophoresis and a software analysis package called CAFA [111]. Parallel advances have been made with a new and powerful footprinting approach called "SHAPE", which shows protection data for residues in secondary and tertiary structures [112][113][114].…”

Section: Rna: Present and Futurementioning

confidence: 99%

Unwinding RNA's secrets: advances in the biology, physics, and modeling of complex RNAs

Chu

Herschlag

2008

Current Opinion in Structural Biology

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SummaryThe rapid development of our understanding of the diverse biological roles fulfilled by non-coding RNA has motivated interest in the basic macromolecular behavior, structure, and function of RNA. We focus on two areas in the behavior of complex RNAs. First, we present advances in the understanding of how RNA folding is accomplished in vivo by presenting a mechanism for the action of DEAD-box proteins. Members of this family are intimately associated with almost all cellular processes involving RNA, mediating RNA structural rearrangements and chaperoning their folding. Next, we focus on advances in understanding and characterizing the basic biophysical forces that govern the folding of complex RNAs. Ultimately we expect that a confluence and synergy between these approaches will lead to profound understanding of RNA and its biology.The explosion in our knowledge about noncoding RNA (ncRNA) -RNA that is not translated into protein -has expanded interest in RNA beyond the traditional RNA community. Diverse ncRNAs include the recently-discovered riboswitches, whose novel gene-regulation function is induced by the binding of small metabolites [1]; most of the so-called "Human Accelerated Regions" (HAR) of the human genome, whose recent evolution may be linked to the emergence of the human brain [2]; and many ncRNAs of unknown function [3]. These discoveries underscore the need to understand the fundamental macromolecular behavior of RNA, its structure and dynamics, and how these RNAs are handled and exploited in biology.Study of catalytic RNAs, which allow detection of correct folding via activity measurements, has established basic thermodynamic and kinetic properties of RNA folding. Large catalytic RNAs such as the group I intron from Tetrahymena thermophila and the RNase P RNA from Bacillus subtilis fold at vastly different rates under different conditions upon addition of Mg 2+ and have been shown to fold via multiple parallel pathways, in which different molecules follow different routes with different rates and intermediates, to a common final folded structure [4,5]. These observations support the common view that RNAs traverse rugged energy landscapes as they fold [6,7]. However, little is known about the true shape of these

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Section: Rna: Present and Futurementioning

confidence: 99%

Unwinding RNA's secrets: advances in the biology, physics, and modeling of complex RNAs

Chu

Herschlag

2008

Current Opinion in Structural Biology

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“…Extracting quantitative 4 reactivities for each nucleotide requires extensive multistep analytical signal processing. Diverse software tools have been developed to facilitate processing of electropherograms, including CAFA (Mitra et al 2008), ShapeFinder , HiTRACE (Yoon et al 2011), FAST (Pang et al 2011), and SHAPE-CE (Aviran et al 2011b). There is a critical balance to be struck between processing speed, pipeline simplicity, and degree of automation.…”

Section: Introductionmentioning

confidence: 99%

QuShape: Rapid, accurate, and best-practices quantification of nucleic acid probing information, resolved by capillary electrophoresis

Karabiber

McGinnis

Favorov

et al. 2012

RNA

198

248

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Chemical probing of RNA and DNA structure is a widely used and highly informative approach for examining nucleic acid structure and for evaluating interactions with protein and small-molecule ligands. Use of capillary electrophoresis to analyze chemical probing experiments yields hundreds of nucleotides of information per experiment and can be performed on automated instruments. Extraction of the information from capillary electrophoresis electropherograms is a computationally intensive multistep analytical process, and no current software provides rapid, automated, and accurate data analysis. To overcome this bottleneck, we developed a platform-independent, user-friendly software package, QuShape, that yields quantitatively accurate nucleotide reactivity information with minimal user supervision. QuShape incorporates newly developed algorithms for signal decay correction, alignment of time-varying signals within and across capillaries and relative to the RNA nucleotide sequence, and signal scaling across channels or experiments. An analysis-by-reference option enables multiple, related experiments to be fully analyzed in minutes. We illustrate the usefulness and robustness of QuShape by analysis of RNA SHAPE (selective 29-hydroxyl acylation analyzed by primer extension) experiments.

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“…One approach for probing large numbers of RNA molecules involves chemically modifying RNA and reading out these events via electrophoresis or deep sequencing (Mitra et al 2008;Lucks et al 2011;Pang et al 2011;Yoon et al 2011). Numerous reagents, including protein nucleases (Walczak et al 1996;Grover et al 2011;Siegfried et al 2011), alkylating chemicals such as dimethyl sulfate (DMS) (Wells et al 2000;Tijerina et al 2007;Cordero et al 2012a), and hydroxyl radicals (Adilakshmi et al 2006;Das et al 2008;Ding et al 2012), have been leveraged to modify or cleave RNA in a structure-dependent manner.…”

Section: Introductionmentioning

confidence: 99%

“…Numerous reagents, including protein nucleases (Walczak et al 1996;Grover et al 2011;Siegfried et al 2011), alkylating chemicals such as dimethyl sulfate (DMS) (Wells et al 2000;Tijerina et al 2007;Cordero et al 2012a), and hydroxyl radicals (Adilakshmi et al 2006;Das et al 2008;Ding et al 2012), have been leveraged to modify or cleave RNA in a structure-dependent manner. Protection of nucleotides from modification, typically signaling the formation of base pairs, can guide manual or automatic secondary structure inference (Mathews et al 2004;Mitra et al 2008;Vasa et al 2008). Strong cases have been made for using reagents that covalently modify 2 ′ -hydroxyls followed by readout via primer extension (SHAPE) (Merino et al 2005;Mortimer and Weeks 2007;Deigan et al 2009;Watts et al 2009;McGinnis et al 2012) and then applying these data as pseudoenergy bonuses in free-energy minimization algorithms, such as RNAstructure (Mathews et al 2004;Reuter and Mathews 2010;Hajdin et al 2013).…”

Section: Introductionmentioning

confidence: 99%

High-throughput mutate-map-rescue evaluates SHAPE-directed RNA structure and uncovers excited states

Tian

Cordero

Kladwang

et al. 2014

RNA

101

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The three-dimensional conformations of noncoding RNAs underpin their biochemical functions but have largely eluded experimental characterization. Here, we report that integrating a classic mutation/rescue strategy with high-throughput chemical mapping enables rapid RNA structure inference with unusually strong validation. We revisit a 16S rRNA domain for which SHAPE (selective 2 ′ -hydroxyl acylation with primer extension) and limited mutational analysis suggested a conformational change between apo-and holo-ribosome conformations. Computational support estimates, data from alternative chemical probes, and mutate-and-map (M 2 ) experiments highlight issues of prior methodology and instead give a nearcrystallographic secondary structure. Systematic interrogation of single base pairs via a high-throughput mutation/rescue approach then permits incisive validation and refinement of the M 2 -based secondary structure. The data further uncover the functional conformation as an excited state (20 ± 10% population) accessible via a single-nucleotide register shift. These results correct an erroneous SHAPE inference of a ribosomal conformational change, expose critical limitations of conventional structure mapping methods, and illustrate practical steps for more incisively dissecting RNA dynamic structure landscapes.

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High-throughput single-nucleotide structural mapping by capillary automated footprinting analysis

Cited by 93 publications

References 54 publications

Unwinding RNA's secrets: advances in the biology, physics, and modeling of complex RNAs

Unwinding RNA's secrets: advances in the biology, physics, and modeling of complex RNAs

QuShape: Rapid, accurate, and best-practices quantification of nucleic acid probing information, resolved by capillary electrophoresis

High-throughput mutate-map-rescue evaluates SHAPE-directed RNA structure and uncovers excited states

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