Comprehensive analysis of RNA-protein interactions by high-throughput sequencing-RNA affinity profiling.
Nat Methods
; 11(6): 683-8, 2014 Jun.
Article
em En
| MEDLINE
| ID: mdl-24809628
ABSTRACT
RNA-protein interactions play critical roles in gene regulation, but methods to quantitatively analyze these interactions at a large scale are lacking. We have developed a high-throughput sequencing-RNA affinity profiling (HiTS-RAP) assay by adapting a high-throughput DNA sequencer to quantify the binding of fluorescently labeled protein to millions of RNAs anchored to sequenced cDNA templates. Using HiTS-RAP, we measured the affinity of mutagenized libraries of GFP-binding and NELF-E-binding aptamers to their respective targets and identified critical regions of interaction. Mutations additively affected the affinity of the NELF-E-binding aptamer, whose interaction depended mainly on a single-stranded RNA motif, but not that of the GFP aptamer, whose interaction depended primarily on secondary structure.
Texto completo:
1
Base de dados:
MEDLINE
Assunto principal:
RNA
/
Proteínas
/
Sequenciamento de Nucleotídeos em Larga Escala
Idioma:
En
Ano de publicação:
2014
Tipo de documento:
Article