Led-Seq: ligation-enhanced double-end sequence-based structure analysis of RNA.
Nucleic Acids Res
; 51(11): e63, 2023 06 23.
Article
em En
| MEDLINE
| ID: mdl-37114986
ABSTRACT
Structural analysis of RNA is an important and versatile tool to investigate the function of this type of molecules in the cell as well as in vitro. Several robust and reliable procedures are available, relying on chemical modification inducing RT stops or nucleotide misincorporations during reverse transcription. Others are based on cleavage reactions and RT stop signals. However, these methods address only one side of the RT stop or misincorporation position. Here, we describe Led-Seq, a new approach based on lead-induced cleavage of unpaired RNA positions, where both resulting cleavage products are investigated. The RNA fragments carrying 2', 3'-cyclic phosphate or 5'-OH ends are selectively ligated to oligonucleotide adapters by specific RNA ligases. In a deep sequencing analysis, the cleavage sites are identified as ligation positions, avoiding possible false positive signals based on premature RT stops. With a benchmark set of transcripts in Escherichia coli, we show that Led-Seq is an improved and reliable approach based on metal ion-induced phosphodiester hydrolysis to investigate RNA structures in vivo.
Texto completo:
1
Base de dados:
MEDLINE
Assunto principal:
RNA
/
Conformação de Ácido Nucleico
Idioma:
En
Ano de publicação:
2023
Tipo de documento:
Article