title: SHAPE Selection (SHAPES) enrich for RNA structure signal in SHAPE sequencing-based probing data.
abstract: 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.
authors: Department of Biology, University of Copenhagen, DK-2200 Copenhagen N, Denmark.
authors: Line Dahl
authors: LD
authors: Poulsen
authors: Department of Biology, University of Copenhagen, DK-2200 Copenhagen N, Denmark.
authors: Lukasz Jan
authors: LJ
authors: Kielpinski
authors: Center for Biomolecular Science and Engineering, and Howard Hughes Medical Institute, University of California Santa Cruz, Santa Cruz, California 95064, USA.
authors: Sofie R
authors: SR
authors: Salama
authors: Department of Biology, University of Copenhagen, DK-2200 Copenhagen N, Denmark.
authors: Anders
authors: A
authors: Krogh
authors: Department of Biology, University of Copenhagen, DK-2200 Copenhagen N, Denmark.
authors: Jeppe
authors: J
authors: Vinther
mesh_terms: D000215:Acylation; D001412:Bacillus subtilis / Q000235:genetics; D001710:Biotin / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics; D017665:Hydroxyl Radical / Q000737:chemistry* / Q000378:metabolism; D009690:Nucleic Acid Conformation; D012313:RNA / Q000032:analysis / Q000737:chemistry*; D012315:RNA Caps / Q000737:chemistry; D015347:RNA Probes / Q000737:chemistry*; D012329:RNA, Bacterial / Q000737:chemistry; D012336:RNA, Ribosomal, 16S / Q000737:chemistry; D043262:Ribonuclease P / Q000235:genetics; D017423:Sequence Analysis, RNA / Q000379:methods*; D024363:Transcription Initiation Site
chemical_list: D012315:RNA Caps; D015347:RNA Probes; D012329:RNA, Bacterial; D012336:RNA, Ribosomal, 16S; D017665:Hydroxyl Radical; D012313:RNA; D001710:Biotin; D043262:Ribonuclease P
keywords: NPIA; RNA structure; SHAPE; Selection; sequencing