SHAPE Selection (SHAPES) enrich for RNA structure signal in SHAPE sequencing-based probing data
| Autor: | Anders Krogh, Lukasz Jan Kielpinski, Jeppe Vinther, Line Dahl Poulsen, Sofie R. Salama |
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| Rok vydání: | 2015 |
| Předmět: |
RNA Caps
Streptavidin Acylation Biotin Biology Signal Ribonuclease P Primer extension Nucleic acid secondary structure chemistry.chemical_compound RNA Ribosomal 16S Methods Escherichia coli Nucleic acid structure Molecular Biology Hydroxyl Radical Sequence Analysis RNA RNA RNA Probes Molecular biology RNA Bacterial chemistry RNA editing Nucleic Acid Conformation RNA extraction Transcription Initiation Site Biological system Bacillus subtilis |
| Zdroj: | Poulsen, L D, Kielpinski, L J, Salama, S R, Krogh, A & Vinther, J 2015, ' SHAPE selection (SHAPES) enrich for RNA structure signal in SHAPE sequencing-based probing data ', R N A, vol. 21, no. 5, pp. 1042-1052 . https://doi.org/10.1261/rna.047068.114 |
| ISSN: | 1469-9001 1355-8382 |
| Popis: | Selective 2′ Hydroxyl Acylation analyzed by Primer Extension (SHAPE) is an accurate method for probing of RNA secondary structure. In existing SHAPE methods, the SHAPE probing signal is normalized to a no-reagent control to correct for the background caused by premature termination of the reverse transcriptase. Here, we introduce a SHAPE Selection (SHAPES) reagent, N-propanone isatoic anhydride (NPIA), which retains the ability of SHAPE reagents to accurately probe RNA structure, but also allows covalent coupling between the SHAPES reagent and a biotin molecule. We demonstrate that SHAPES-based selection of cDNA–RNA hybrids on streptavidin beads effectively removes the large majority of background signal present in SHAPE probing data and that sequencing-based SHAPES data contain the same amount of RNA structure data as regular sequencing-based SHAPE data obtained through normalization to a no-reagent control. Moreover, the selection efficiently enriches for probed RNAs, suggesting that the SHAPES strategy will be useful for applications with high-background and low-probing signal such as in vivo RNA structure probing. |
| Databáze: | OpenAIRE |
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