Safer one-pot synthesis of the ‘SHAPE’ reagent 1-methyl-7-nitroisatoic anhydride (1m7)

SHAPE chemistries exploit small electrophilic reagents that react with the 2′-hydroxyl group to interrogate RNA structure at single-nucleotide resolution. Mutational profiling (MaP) identifies modified residues based on the ability of reverse transcriptase to misread a SHAPE-modified nucleotide and then counting the resulting mutations by massively parallel sequencing. The SHAPE-MaP approach measures the structure of large and transcriptome-wide systems as accurately as for simple model RNAs. This protocol describes the experimental steps, implemented over three days, required to perform SHAPE probing and construct multiplexed SHAPE-MaP libraries suitable for deep sequencing. These steps include RNA folding and SHAPE structure probing, mutational profiling by reverse transcription, library construction, and sequencing. Automated processing of MaP sequencing data is accomplished using two software packages. ShapeMapper converts raw sequencing files into mutational profiles, creates SHAPE reactivity plots, and provides useful troubleshooting information, often within an hour. SuperFold uses these data to model RNA secondary structures, identify regions with well-defined structures, and visualize probable and alternative helices, often in under a day. We illustrate these algorithms with the E. coli thiamine pyrophosphate riboswitch, E. coli 16S rRNA, and HIV-1 genomic RNAs. SHAPE-MaP can be used to make nucleotide-resolution biophysical measurements of individual RNA motifs, rare components of complex RNA ensembles, and entire transcriptomes. The straightforward MaP strategy greatly expands the number, length, and complexity of analyzable RNA structures.

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“…18,56 ) CRITICAL: 1M7 should be stored in a desiccator at 4 °C. When properly stored, 1M7 is stable for at least a year.…”

Section: Methodsmentioning

confidence: 99%

Selective 2′-hydroxyl acylation analyzed by primer extension and mutational profiling (SHAPE-MaP) for direct, versatile and accurate RNA structure analysis

et al. 2015

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“…The following steps outline RNA folding and modification with 1-methyl-7-nitroisatoic anhydride (1M7) [49], which can be synthesized in a one-step reaction [50]. Alternatively, N-methylisatoic anhydride (NMIA) can be commercially purchased and used in these steps with the noted modifications.…”

Section: Methodsmentioning

confidence: 99%

“…For in-cell SHAPE-Seq in E. coli we recommend 1M7 because its half-life is on a shorter time scale than cell division and RNA degradation. All three SHAPE reagents usable in vivo can be synthetized in one (1M7 [50], NAI, and FAI [32]) or a few steps (NAI-N 3 [27]) from commercially available reagents. To use NAI instead of 1M7 for in vivo probing, replace the 13.3 μL of 250 mM 1M7 with 51.2 μL of NAI (or NAI-N 3 ) 1–2 M stock solution and incubate for 15 min in place of 2–3 min before two-phase extraction to quench the reaction.…”

Section: Experimental Considerationsmentioning

confidence: 99%

Characterizing RNA structures in vitro and in vivo with selective 2′-hydroxyl acylation analyzed by primer extension sequencing (SHAPE-Seq)

Watters

Strobel

et al. 2016

Methods

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RNA molecules adopt a wide variety of structures that perform many cellular functions, including, among others, catalysis, small molecule sensing, and cellular defense. Our ability to characterize, predict, and design RNA structures are key factors for understanding and controlling the biological roles of RNAs. Fortunately, there has been rapid progress in this area, especially with respect to experimental methods that can characterize RNA structures in a high throughput fashion using chemical probing and next-generation sequencing. Here, we describe one such method, selective 2′-hydroxyl acylation analyzed by primer extension sequencing (SHAPE-Seq), which measures nucleotide resolution flexibility information for RNAs in vitro and in vivo. We outline the process of designing and performing a SHAPE-Seq experiment and describe methods for using experimental SHAPE-Seq data to restrain computational folding algorithms to generate more accurate predictions of RNA secondary structure. We also provide a number of examples of SHAPE-Seq reactivity spectra obtained in vitro and in vivo and discuss important considerations for performing SHAPE-Seq experiments, both in terms of collecting and analyzing data. Finally, we discuss improvements and extensions of these experimental and computational techniques that promise to deepen our knowledge of RNA folding and function.

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“…1-methyl-7-nitroisatoic anhydride (1M7) was synthesized according to the method described by Turner and co-authors 57 . Empty puromycin treated salt-washed ribosomes were prepared as described previously 58 .…”

Section: Methodsmentioning

confidence: 99%

The Functional Role of eL19 and eB12 Intersubunit Bridge in the Eukaryotic Ribosome

Kisly

Gulay

Mäeorg

et al. 2016

Journal of Molecular Biology

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During translation, the two eukaryotic ribosomal subunits remain associated through 17 intersubunit bridges, five of which are eukaryote-specific. These are mainly localized to the peripheral regions and are believed to stabilise the structure of the ribosome. The functional importance of these bridges remains largely unknown. Here the essentiality of the eukaryote-specific bridge eB12 has been investigated. The main component of this bridge is ribosomal protein eL19 which is composed of an N-terminal globular domain, a middle region and a long C-terminal α-helix. Analysis of deletion mutants demonstrated that the globular domain and middle region of eL19 are essential for cell viability, most likely functioning in ribosome assembly. The eB12 bridge, formed by contacts between the C-terminal α-helix of eL19 and 18S rRNA in concert with additional stabilising interactions involving either eS7 or uS17, is dispensable for viability. Nevertheless, eL19 mutants impaired in eB12 bridge formation displayed slow growth phenotypes, altered sensitivity/resistance to translational inhibitors and enhanced hyperosmotic stress tolerance. Biochemical analyses determined that the eB12 bridge contributes to the stability of ribosome subunit interactions in vitro. 60S subunits containing eL19 variants defective in eB12 bridge formation failed to form 80S ribosomes regardless of Mg2+ concentration. The reassociation of 40S and mutant 60S subunits was markedly improved in the presence of deacetylated tRNA, emphasising the importance of tRNAs during the subunit association. We propose that the eB12 bridge plays an important role in subunit joining and in optimizing ribosome functionality.

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Safer one-pot synthesis of the ‘SHAPE’ reagent 1-methyl-7-nitroisatoic anhydride (1m7)

Cited by 31 publications

References 30 publications

Selective 2′-hydroxyl acylation analyzed by primer extension and mutational profiling (SHAPE-MaP) for direct, versatile and accurate RNA structure analysis

Selective 2′-hydroxyl acylation analyzed by primer extension and mutational profiling (SHAPE-MaP) for direct, versatile and accurate RNA structure analysis

Characterizing RNA structures in vitro and in vivo with selective 2′-hydroxyl acylation analyzed by primer extension sequencing (SHAPE-Seq)

The Functional Role of eL19 and eB12 Intersubunit Bridge in the Eukaryotic Ribosome

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