Direct Analysis of HIV mRNA m6A Methylation by Nanopore Sequencing.
Methods Mol Biol
; 2807: 209-227, 2024.
Article
em En
| MEDLINE
| ID: mdl-38743231
ABSTRACT
The post-transcriptional processing and chemical modification of HIV RNA are understudied aspects of HIV virology, primarily due to the limited ability to accurately map and quantify RNA modifications. Modification-specific antibodies or modification-sensitive endonucleases coupled with short-read RNA sequencing technologies have allowed for low-resolution or limited mapping of important regulatory modifications of HIV RNA such as N6-methyladenosine (m6A). However, a high-resolution map of where these sites occur on HIV transcripts is needed for detailed mechanistic understanding. This has recently become possible with new sequencing technologies. Here, we describe the direct RNA sequencing of HIV transcripts using an Oxford Nanopore Technologies sequencer and the use of this technique to map m6A at near single nucleotide resolution. This technology also provides the ability to identify splice variants with long RNA reads and thus, can provide high-resolution RNA modification maps that distinguish between overlapping splice variants. The protocols outlined here for m6A also provide a powerful paradigm for studying any other RNA modifications that can be detected on the nanopore platform.
Palavras-chave
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
RNA Mensageiro
/
RNA Viral
/
Adenosina
/
Sequenciamento por Nanoporos
Limite:
Humans
Idioma:
En
Revista:
Methods Mol Biol
Assunto da revista:
BIOLOGIA MOLECULAR
Ano de publicação:
2024
Tipo de documento:
Article
País de afiliação:
Estados Unidos