SHAPE Selection (SHAPES) enrich for RNA structure signal in SHAPE sequencing-based probing data.
RNA
; 21(5): 1042-52, 2015 May.
Article
en En
| MEDLINE
| ID: mdl-25805860
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.
Palabras clave
Texto completo:
1
Colección:
01-internacional
Banco de datos:
MEDLINE
Asunto principal:
ARN
/
Sondas ARN
/
Análisis de Secuencia de ARN
/
Radical Hidroxilo
/
Conformación de Ácido Nucleico
Idioma:
En
Revista:
RNA
Asunto de la revista:
BIOLOGIA MOLECULAR
Año:
2015
Tipo del documento:
Article
País de afiliación:
Dinamarca