A mesophilic Argonaute from Clostridium formicaceticum with efficient DNA cleavage activity guided by small DNA

Structure, Год журнала: 2025, Номер unknown

Опубликована: Март 1, 2025

Язык: Английский

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NAPTUNE: nucleic acids and protein biomarkers testing via ultra-sensitive nucleases escalation

Nature Communications, Год журнала: 2025, Номер 16(1)

Опубликована: Фев. 4, 2025

Язык: Английский

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Pyrococcus furiosus Argonaute-Based Fluorometric Biosensor for One-Tube Detection of Cancer-Associated Single Nucleotide Polymorphisms in MicroRNAs

Analytical Chemistry, Год журнала: 2025, Номер unknown

Опубликована: Фев. 21, 2025

MicroRNA-related single nucleotide polymorphisms (miR-SNPs) are promising biomarkers for cancer diagnostics, yet accurate detection methods remain limited. Here, we introduce a ligation-triggered Pyrococcus furiosus Argonaute (PfAgo) cleavage (LTAC) strategy the sensitive of miR-SNPs, demonstrated using rs11614913 SNP in miR-196a2, which is associated with nonsmall cell lung (NSCLC). The mutant miR-196a2T serves as scaffold formation guide DNA (gDNA) catalyzed by SplintR ligase, leading to PfAgo activation and enhanced fluorescence. In contrast, wild-type miR-196a2C cannot facilitate gDNA thus fails activate PfAgo. This method exhibits linear relationship logarithm concentration over range 0.2 pM 100 nM, achieving low limit 0.15 pM. Analysis NSCLC patient samples LTAC reveals elevated levels miR-196a2 compared healthy controls, underscoring diagnostic potential LTAC.

Язык: Английский

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Structural basis of ssDNA-guided NADase activation of prokaryotic SPARTA system

Nucleic Acids Research, Год журнала: 2025, Номер 53(4)

Опубликована: Фев. 8, 2025

Abstract Short prokaryotic Argonaute and the associated TIR-APAZ (SPARTA) proteins constitute a immune system, mediating RNA- or DNA-guided target single-stranded DNA (ssDNA) to activate NADase activity induce cell death by degrading NAD+ in response invading plasmids. Although guide RNA-mediated targeting mechanism of SPARTA has been established, functional role mechanisms DNA-mediated remain poorly understood. Here, we report two crystal structures Crenotalea thermophila complexes with 5′-phosphorylated 21-nt complementary ssDNA lengths 15 20 nt. The demonstrate specific recognition 5′-OH 3′-OH groups SPARTA, while not recognizing 5′-P group DNA. This suggests distinct models for RNA, indicating different activation mechanisms. Furthermore, these reveal disparate DNA, providing insights into length requirement activation.

Язык: Английский

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Development of RT-RPA-PfAgo-mediated assay for rapid and sensitive detection of influenza B virus

Microchemical Journal, Год журнала: 2025, Номер unknown, С. 113138 - 113138

Опубликована: Фев. 1, 2025

Язык: Английский

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Target DNA-induced filament formation and nuclease activation of SPARDA complex

Cell Research, Год журнала: 2025, Номер unknown

Опубликована: Март 24, 2025

Abstract The short Argonaute-based bacterial defense system, SPARDA ( S hort P rokaryotic Ar gonaute and D Nase/RNase- A PAZ), utilizes guide RNA to target invading complementary DNA exhibits collateral nuclease activity, leading cell death or dormancy. However, its detailed mechanisms remain poorly understood. In this study, we investigated the system from Novosphingopyxis baekryungensis Nba SPARDA) discovered an unexpected filament configuration upon binding, which strongly correlated with activity. Filament formation activation require a guide–target heteroduplex of sufficient length perfect complementarity at central region. series cryo-EM structures complexes, loaded RNA, varying lengths, substrate ssDNA, were determined ~3.0 Å resolution. Structural analyses indicated that binding induces dimerization complex, while engagement disrupts dimerization. Further propagation triggers through checkpoint mechanism. consists backbone formed by interlocking Argonaute proteins, inner layer composed DREN domains. leads tetramerization monomeric domain, activating activity against environmental nucleic acids — feature leveraged for molecular diagnostics. For bacteria heterologously expressing bacteriophages plasmids relies on formation. Collectively, these findings illustrate working mechanism complex highlight importance in host defense.

Язык: Английский

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Trends in the use of Argonaute Proteins in Molecular Diagnosis

TrAC Trends in Analytical Chemistry, Год журнала: 2024, Номер unknown, С. 118081 - 118081

Опубликована: Ноя. 1, 2024

Язык: Английский

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Advancements of Prokaryotic Argonautes in Molecular Diagnostics and Further Perspectives

TrAC Trends in Analytical Chemistry, Год журнала: 2024, Номер unknown, С. 118122 - 118122

Опубликована: Дек. 1, 2024

Язык: Английский

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The Application of RPA-PfAgo Technology Combined with Multidimensional Data Analysis in the Rapid Detection of the MTHFR A1298C Polymorphism

Research Square (Research Square), Год журнала: 2024, Номер unknown

Опубликована: Сен. 5, 2024

This study presents a novel approach that integrates recombinase polymerase amplification (RPA) with PfAgo protein technology for the rapid and precise detection of MTHFR A1298C polymorphism. Although traditional genotyping methods are effective, they often limited by complexity, high cost, need specialized equipment. The RPA-PfAgo technique harnesses swift isothermal RPA specificity sensitivity PfAgo-mediated DNA cleavage, completing entire process from sample collection to within 90 minutes. utility this method has been substantiated through battery optimization experiments, parameter analysis, assessments sensitivity, specificity, repeatability, along clinical validation using oral mucosal samples. findings indicate new not only substantially reduces time cost but also offers an effective tool personalized medicine disease prevention accuracy reliability.

Язык: Английский

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Duplex in-one-tube detection of two important shrimp farming pathogens with color indication using a method combining RPA and PfAgo

Aquaculture, Год журнала: 2024, Номер unknown, С. 741949 - 741949

Опубликована: Ноя. 1, 2024

Язык: Английский

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