UdgX-Mediated Uracil Sequencing at Single-Nucleotide Resolution.
J Am Chem Soc
; 144(3): 1323-1331, 2022 01 26.
Article
em En
| MEDLINE
| ID: mdl-35037455
ABSTRACT
As an aberrant base in DNA, uracil is generated by either deoxyuridine (dU) misincorporation or cytosine deamination, and involved in multiple physiological and pathological processes. Genome-wide profiles of uracil are important for study of these processes. Current methods for whole-genome mapping of uracil all rely on uracil-DNA N-glycosylase (UNG) and are limited in resolution, specificity, and/or sensitivity. Here, we developed a UdgX cross-linking and polymerase stalling sequencing ("Ucaps-seq") method to detect dU at single-nucleotide resolution. First, the specificity of Ucaps-seq was confirmed on synthetic DNA. Then the effectiveness of the approach was verified on two genomes from different sources. Ucaps-seq not only identified the enrichment of dU at dT sites in pemetrexed-treated cancer cells with globally elevated uracil but also detected dU at dC sites within the "WRC" motif in activated B cells which have increased dU in specific regions. Finally, Ucaps-seq was utilized to detect dU introduced by the cytosine base editor (nCas9-APOBEC) and identified a novel off-target site in cellular context. In conclusion, Ucaps-seq is a powerful tool with many potential applications, especially in evaluation of base editing fidelity.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Nucleotídeos
Idioma:
En
Revista:
J Am Chem Soc
Ano de publicação:
2022
Tipo de documento:
Article
País de afiliação:
China