Dual Protein Corona-Mediated Target Recognition System for Visual Detection and Single-Molecule Counting of Nucleic Acids

ACS Nano, Год журнала: 2025, Номер unknown

Опубликована: Фев. 14, 2025

Rapid, highly sensitive, and specific nucleic acid detection plays a crucial role in advancing point-of-care (POC) diagnostics for pathogens viruses, cancer monitoring, optimizing clinical treatments. Herein, leveraging the precise recognition ability of CRISPR/dCas9 powerful localized surface plasmon resonance (LSPR) gold nanoparticles (AuNPs), we report design dual protein corona-mediated platform to simultaneously fulfill rapid POC testing single-molecule counting acids one-pot one-step manner. This system uses guide RNA as molecular bridge anchor dCas9 onto AuNPs, forming artificial coronas. Upon recognizing target, interaction between two coronas on same molecule triggers cross-linked aggregation AuNPs. Then, target low 100 aM can be visually detected within 30 min, making particularly well-suited application screening emerging epidemics. Additionally, superior LSPR properties AuNPs increase light-scattering signal generated during target-induced aggregation, enabling visualization aggregated diffraction-limited spots under confocal microscopy. By these spots, achieves unprecedented sensitivity, identifying 1 aM, which is equivalent just 6 molecules 10 μL system, demonstrating capability. offers exceptional promise large-scale pathogenic viruses early cancer, applications requiring ultrahigh sensitivity at level.

Язык: Английский

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Recent progress in molecular diagnostics: The synergy of rolling circle amplification and CRISPR/Cas systems (2018–2024) – A concise review

TrAC Trends in Analytical Chemistry, Год журнала: 2024, Номер 180, С. 117902 - 117902

Опубликована: Авг. 8, 2024

Язык: Английский

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RNA-triggered CRISPR-Cas12a2 Preferentially and Cooperatively Cleaves Collateral DNA

bioRxiv (Cold Spring Harbor Laboratory), Год журнала: 2025, Номер unknown

Опубликована: Янв. 5, 2025

Abstract CRISPR-Cas systems often rely on collateral cleavage activities against nucleic-acid substrates to combat recognized mobile genetic elements. Of the associated RNA-guided Cas effector nucleases, Cas12a2 stands out as only known example exhibiting RNA-triggered of three distinct substrates: single-stranded (ss)RNA, ssDNA, and double-stranded (ds)DNA. However, little is about underlying mechanisms cleavage, including which these dominates during Cas12a2-mediated immune response. Here, we show that cleaves DNA over RNA substrates, even when are more abundant. This preference relies a positive cooperativity mechanism requires four “aromatic clamp” residues stabilize unwound distorted dsDNA in RuvC nuclease active site. Leveraging for DNA, demonstrate RNA-activated can cleave ssDNA probe presence high concentrations non-target RNA, while RNA-targeting Cas13a cannot. work thus reveals how decides amongst different with immediate implications understanding Cas12a2-based immunity, improving molecular diagnostics, laying mechnastic foundation future technologies.

Язык: Английский

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Tunable control of Cas12 activity promotes universal and fast one-pot nucleic acid detection

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

Опубликована: Янв. 30, 2025

The CRISPR-based detection methods have been widely applied, yet they remain limited by the non-universal nature of one-pot diagnostic approaches. Here, we report a universal fluorescent method for epidemic pathogens, delivering results within 15-20 min. This uses heparin sodium to precisely tunes cis-cleavage capability Cas12 via interference with Cas12a-crRNA binding process, thereby generating significant fluorescence due accumulation isothermal amplification products. Additionally, this assay accommodates both classic and suboptimal PAMs, as well various Cas12a subtypes such LbCas12a, AsCas12a, AapCas12b. Such robust demonstrates sensitivity specificity exceeding 95% in monkeypox pseudovirus, influenza A virus, SARS-CoV-2 from saliva or wastewater samples, when compared qPCR RT-qPCR. Moreover, cost per thousand is $0.01 $0.04 only. Collectively, fast approach based on offers potential possibilities point-of-care testing.

Язык: Английский

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Direct repeat region 3′ end modifications regulate Cas12a activity and expand its applications

Nucleic Acids Research, Год журнала: 2025, Номер 53(3)

Опубликована: Янв. 24, 2025

Abstract CRISPR-Cas12a technology has transformative potential, but as its applications grow, enhancing inherent functionalities is essential to meet diverse demands. Here, we reveal a regulatory mechanism for LbCas12a through direct repeat (DR) region 3′ end modifications and de-modifications, which can regulate LbCas12a’s cis- trans-cleavage activities. We extensively explored the effects of introducing phosphorylation, DNA, photo-cleavable linker, DNA at DR on functionality. find that temporary inhibitory function Cas12a be reactivated by modification corresponding substances, such alkaline phosphatase (ALP), immunoglobulin G (IgG), alpha-fetoprotein (AFP), exonucleases, ultraviolet radiation, glycosylases, greatly expand scope application Cas12a. Clinical demonstrated promising results in ALP, AFP, trace Epstein–Barr virus detection compared gold standard methods. Our research provides valuable insights into regulating activity significantly expands potential clinical targets, paving way future universal clustered regularly interspaced short palindromic repeats (CRISPR) diagnostic strategies.

Язык: Английский

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Using CRISPR for viral nucleic acid detection

Methods in enzymology on CD-ROM/Methods in enzymology, Год журнала: 2025, Номер unknown, С. 245 - 275

Опубликована: Янв. 1, 2025

Язык: Английский

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Engineering MOF microenvironments to enhance Bi-directional identification circuits for reliable electrochemiluminescence α-thalassemia detection strategy

Chemical Engineering Journal, Год журнала: 2025, Номер unknown, С. 160654 - 160654

Опубликована: Фев. 1, 2025

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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Optimization of CRISPR/Cas12f1 guide RNAs using AlphaFold 3 for enhanced nucleic acid detection

Microchemical Journal, Год журнала: 2025, Номер unknown, С. 113194 - 113194

Опубликована: Фев. 1, 2025

Язык: Английский

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High-fidelity telomerase activity assay based on light-triggered nucleic acid separation system for the diagnosis of bladder cancer

Biosensors and Bioelectronics, Год журнала: 2025, Номер 278, С. 117355 - 117355

Опубликована: Март 7, 2025

Язык: Английский

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