Activatable Self-Dissociation of Watson-Crick Structures with Fluorescent Nucleotides for Sensing Multiple Human Glycosylases at Single-Cell Level.
Anal Chem
; 94(50): 17700-17708, 2022 12 20.
Article
em En
| MEDLINE
| ID: mdl-36475642
ABSTRACT
Nucleobase oxidation and alkylation can destroy Watson-Crick base-pairing to challenge the genomic integrity. Human 8-oxoguanine glycosylase 1 (hOGG1) and alkyladenine glycosylase (hAAG) are evolved to counter these two cytotoxic lesions through base-excision repair, and their deregulations are implicated with multifactorial diseases and cancers. Herein, we demonstrate activatable self-dissociation of Watson-Crick structures with fluorescent nucleotides for sensing multiple human glycosylases at single-cell level. The presence of hOGG1 and hAAG catalyzes 8-oxoG and deoxyinosine removal in functional probe 1 to release two trigger probes (1 and 2). Then, trigger probes hybridize with functional probe 2 to activate the autocatalytic degradation of functional probes 2 (Cycle I) and 3 (Cycle II), replicating abundant trigger probes (1-4) and releasing two fluorophores (2-aminopurine (2-AP) and pyrrolo-dC (P-dC)). New trigger probes (1, 2) and (3, 4), in turn, hybridize with free functional probes 2 and 3, repeating Cycles I and II turnovers. Through multicycle self-dissociation of Watson-Crick structures, 2-AP and P-dC are exponentially accumulated for the simultaneous quantification of hOGG1 and hAAG. This nanodevice exhibits high sensitivity with a detection limit of 2.9 × 10-3 U/mL for hOOG1 and 1.5 × 10-3 U/mL for hAAG, and it can measure enzymatic kinetics, identify potential inhibitors, discriminate glycosylases between cancer and normal cell lines, and even quantify glycosylase activities in a single HeLa cell. Moreover, this assay may be rapidly and isothermally performed in one tube with only one tool enzyme in a quencher-free manner, promising a simple and powerful platform for multiple human glycosylase detection.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Reparo do DNA
/
Nucleotídeos
Limite:
Humans
Idioma:
En
Revista:
Anal Chem
Ano de publicação:
2022
Tipo de documento:
Article
País de afiliação:
China