SHAPE selection (SHAPES) enrich for RNA structure signal in SHAPE sequencing-based probing data

TY - JOUR

T1 - SHAPE selection (SHAPES) enrich for RNA structure signal in SHAPE sequencing-based probing data

AU - Poulsen, Line Dahl

AU - Kielpinski, Lukasz Jan

AU - Salama, Sofie R

AU - Krogh, Anders

AU - Vinther, Jeppe

N1 - © 2015 Poulsen et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

PY - 2015/5/1

Y1 - 2015/5/1

N2 - 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.

AB - 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.

U2 - 10.1261/rna.047068.114

DO - 10.1261/rna.047068.114

M3 - Journal article

C2 - 25805860

SN - 1355-8382

VL - 21

SP - 1042

EP - 1052

JO - R N A

JF - R N A

IS - 5

ER -