A one-pot CRISPR-Cas12a-based toolbox enables determination of terminal deoxynucleotidyl transferase activity for acute leukemia screening.
Anal Chim Acta
; 1254: 341115, 2023 May 08.
Article
em En
| MEDLINE
| ID: mdl-37005025
ABSTRACT
An isothermal, one-pot toolbox (called OPT-Cas) based on CRISPR-Cas12a collateral cleavage capability is proposed for highly sensitive and selective determination of terminal deoxynucleotidyl transferase (TdT) activity. Oligonucleotide primers with 3'-hydroxyl (OH) terminal were randomly introduced for TdT-induced elongation. In the presence of TdT, dTTP nucleotides polymerized at the 3' terminals of the primers to generate abundant polyT-tails, which function as triggers for the synchronous activation of Cas12a proteins. Finally, the activated Cas12a trans-cleaved FAM and BHQ1 dual-labeled single-stranded DNA (ssDNA-FQ) reporters, producing significantly amplified fluorescence signals. This one-pot assay, that is primer, crRNA, Cas12a protein and ssDNA-FQ reporter are all in one tube, allows simple but high-sensitive quantification of TdT activity with a low detection limit of 6.16 × 10-5 U µL-1 in the concentration scope from 1 × 10-4 U µL-1 to 1 × 10-1 U µL-1, and achieves extraordinary selectivity with other interfering proteins. Furthermore, the OPT-Cas was successfully used to detect TdT in complex matrices and accurate determination of TdT activity in acute lymphoblastic leukemia cells, which might be a reliable technique platform for the diagnosis of TdT-related diseases and biomedical research applications.
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Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Técnicas Biossensoriais
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Pesquisa Biomédica
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Leucemia-Linfoma Linfoblástico de Células Precursoras
Tipo de estudo:
Diagnostic_studies
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Screening_studies
Limite:
Humans
Idioma:
En
Revista:
Anal Chim Acta
Ano de publicação:
2023
Tipo de documento:
Article