DNA repair-retarded isothermal CRISPR amplification for rapid, sensitive base excision repair enzyme assay.

Uracil-DNA Glycosidase / metabolism Humans Nucleic Acid Amplification Techniques / methods CRISPR-Cas Systems / genetics DNA Repair Enzyme Assays / methods Excision Repair
Base excision repair Biosensors CRISPR/Cas12a Enzyme assay Isothermal amplification UDG

Journal

Analytica chimica acta
ISSN: 1873-4324
Titre abrégé: Anal Chim Acta
Pays: Netherlands
ID NLM: 0370534

Informations de publication

Date de publication:
25 Jul 2024
Historique:
received: 08 03 2024
revised: 05 05 2024
accepted: 29 05 2024
medline: 15 6 2024
pubmed: 15 6 2024
entrez: 14 6 2024
Statut: ppublish

Résumé

As a core enzyme in the base excision repair system, uracil DNA glycosylase (UDG) is indispensable in maintaining genomic integrity and normal cell cycles. Its abnormal activity intervenes in cancers and neurodegerative diseases. Previous UDG assays based on isothermal amplification and Clustered Regularly Interspaced Short Palindromic Repeats/Cas (CRISPR/Cas) system were fine in sensitivity, but exposed to complications in assay flow, time, and probe design. After isothermal amplification, a CRISPR/Cas reagent should be separately added with extra manual steps and its guide RNA (gRNA) should be designed, considering the presence of protospacer adjacent motif (PAM) site. We herein describe a UDG-REtarded CRISPR Amplification assay, termed 'URECA'. In URECA, isothermal nucleic acid (NA) amplification and CRISPR/Cas12a system were tightly combined to constitute a one-pot, isothermal CRISPR amplification system. Isothermal NA amplification for a UDG substrate (US) with uracil (U) bases was designed to activate and boost CRISPR/Cas12a reaction. Such scheme enabled us to envision that UDG would halt the isothermal CRISPR amplification reaction by excising U bases and messing up the US. Based on this principle, the assay detected the UDG activity down to 9.17 x 10 URECA offers further merits: i) the assay is seamless. Following target recognition, the reactions proceed in one-step without any intervening steps, ii) probe design is simple. Unlike the conventional CRISPR/Cas12a-based assays, URECA does not consider the PAM site in probe design as Cas12a activation relies on instantaneous gRNA binding to single-stranded DNA strands. By rationally designing an enzyme substrate probe to be specific to other enzymes, while keeping a role as a template for isothermal CRISPR amplification, the detection principle of URECA will be expanded to devise biosensors for various enzymes of biological, clinical significance.

Sections du résumé

BACKGROUND BACKGROUND
As a core enzyme in the base excision repair system, uracil DNA glycosylase (UDG) is indispensable in maintaining genomic integrity and normal cell cycles. Its abnormal activity intervenes in cancers and neurodegerative diseases. Previous UDG assays based on isothermal amplification and Clustered Regularly Interspaced Short Palindromic Repeats/Cas (CRISPR/Cas) system were fine in sensitivity, but exposed to complications in assay flow, time, and probe design. After isothermal amplification, a CRISPR/Cas reagent should be separately added with extra manual steps and its guide RNA (gRNA) should be designed, considering the presence of protospacer adjacent motif (PAM) site.
RESULTS RESULTS
We herein describe a UDG-REtarded CRISPR Amplification assay, termed 'URECA'. In URECA, isothermal nucleic acid (NA) amplification and CRISPR/Cas12a system were tightly combined to constitute a one-pot, isothermal CRISPR amplification system. Isothermal NA amplification for a UDG substrate (US) with uracil (U) bases was designed to activate and boost CRISPR/Cas12a reaction. Such scheme enabled us to envision that UDG would halt the isothermal CRISPR amplification reaction by excising U bases and messing up the US. Based on this principle, the assay detected the UDG activity down to 9.17 x 10
SIGNIFICANCE CONCLUSIONS
URECA offers further merits: i) the assay is seamless. Following target recognition, the reactions proceed in one-step without any intervening steps, ii) probe design is simple. Unlike the conventional CRISPR/Cas12a-based assays, URECA does not consider the PAM site in probe design as Cas12a activation relies on instantaneous gRNA binding to single-stranded DNA strands. By rationally designing an enzyme substrate probe to be specific to other enzymes, while keeping a role as a template for isothermal CRISPR amplification, the detection principle of URECA will be expanded to devise biosensors for various enzymes of biological, clinical significance.

Identifiants

DOI: 10.1016/j.aca.2024.342799 PMID: 38876521
pubmed: 38876521
pii: S0003-2670(24)00600-7
doi: 10.1016/j.aca.2024.342799
pii:
doi:

Substances chimiques

Uracil-DNA Glycosidase EC 3.2.2.-

Types de publication

Journal Article

Langues

eng

Sous-ensembles de citation

IM

Pagination

342799

Informations de copyright

Copyright © 2024 Elsevier B.V. All rights reserved.

Déclaration de conflit d'intérêts

Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Auteurs

Hyogu Han (H)

User Convenience Technology R&D Department, Korea Institute of Industrial Technology (KITECH), Ansan, 15588, South Korea; Department of Chemistry, Gangneung-Wonju National University, Gangneung, 25457, South Korea.

Se Hee Jang (SH)

User Convenience Technology R&D Department, Korea Institute of Industrial Technology (KITECH), Ansan, 15588, South Korea; Department of Medical Device Engineering and Management, College of Medicine, Yonsei University, Seoul 03722, South Korea.

Jun Ki Ahn (JK)

User Convenience Technology R&D Department, Korea Institute of Industrial Technology (KITECH), Ansan, 15588, South Korea. Electronic address: jkahn@kitech.re.kr.

Chang Yeol Lee (CY)

Bionanotechnology Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), 125 Gwahak-ro, Yuseong-gu, Daejeon, 34141, South Korea. Electronic address: lcyeol8457a@kribb.re.kr.

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Classifications MeSH

questionsmedicales.fr › Enzymes et coenzymes › Enzymes › Enzymes de réparation de l'ADN › Uracil-DNA glycosidase › DNA repair-retarded isothermal CRISPR...
questionsmedicales.fr › Eucaryotes › Animaux › Chordés › Vertébrés › Mammifères › Eutheria › Primates › Haplorhini › Catarrhini › Hominidae › Humains › DNA repair-retarded isothermal CRISPR...
questionsmedicales.fr › Techniques d'investigation › Techniques génétiques › Techniques d'amplification d'acides nucléiques › DNA repair-retarded isothermal CRISPR...
questionsmedicales.fr › Phénomènes génétiques › Régulation de l'expression des gènes › Épigenèse génétique › Extinction de l'expression des gènes › Systèmes CRISPR-Cas › DNA repair-retarded isothermal CRISPR...
questionsmedicales.fr › Phénomènes génétiques › Réparation de l'ADN › DNA repair-retarded isothermal CRISPR...
questionsmedicales.fr › Techniques d'investigation › Techniques de chimie analytique › Dosages enzymatiques › DNA repair-retarded isothermal CRISPR...