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Enhanced SNP-sensing using DNA-templated reactions through confined hybridization of minimal substrates (CHOMS).

ABSTRACT: DNA or RNA templated reactions are attractive for nucleic acid sensing and imaging. As for any hybridization-based sensing, there is a tradeoff between sensitivity (detection threshold) and resolution (single nucleotide discrimination). Longer probes afford better sensitivity but compromise single nucleotide resolution due to the small thermodynamic penalty of a single mismatch. Herein we report a design that overcomes this tradeoff. The reaction is leveraged on the hybridization of a minimal substrate (covering 4 nucleotides) which is confined by two guide DNAs functionalized respectively with a ruthenium photocatalyst. The use of a catalytic reaction is essential to bypass the exchange of guide DNAs while achieving signal amplification through substrate turnover. The guide DNAs restrain the reaction to a unique site and enhance the hybridization of short substrates by providing two π-stacking interactions. The reaction was shown to enable the detection of SNPs and SNVs down to 50 pM with a discrimination factor ranging from 24 to 309 (median 82, 27 examples from 3 oncogenes). The clinical diagnostic potential of the technology was demonstrated with the analysis of RAS amplicons obtained directly from cell culture.

SUBMITTER: Kim KT

PROVIDER: S-EPMC8152519 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Tauopathies are a subset of neurodegenerative diseases characterized by abnormal tau inclusions. Specifically, three-repeat tau and four-repeat tau in Alzheimer's disease, three-repeat tau in Pick's disease (PiD) and four-repeat tau in progressive supranuclear palsy (PSP) and corticobasal degeneration (CBD) form amyloid-like fibrous structures that accumulate in neurons and/or glial cells. Amplification and cell-to-cell transmission of abnormal tau based on the prion hypothesis are believed to explain the onset and progression of tauopathies. Recent studies support not only the self-propagation of abnormal tau, but also the presence of conformationally distinct tau aggregates, namely tau strains. Cryogenic electron microscopy analyses of patient-derived tau filaments have revealed disease-specific ordered tau structures. However, it remains unclear whether the ultrastructural and biochemical properties of tau strains are inherited during the amplification of abnormal tau in the brain. In this study, we investigated template-dependent amplification of tau aggregates using a cellular model of seeded aggregation. Tau strains extracted from human tauopathies caused strain-dependent accumulation of insoluble filamentous tau in SH-SY5Y cells. The seeding activity towards full-length four-repeat tau substrate was highest in CBD-tau seeds, followed by PSP-tau and Alzheimer's disease (AD)-tau seeds, while AD-tau seeds showed higher seeding activity than PiD-tau seeds towards three-repeat tau substrate. Abnormal tau amplified in cells inherited the ultrastructural and biochemical properties of the original seeds. These results strongly suggest that the structural differences of patient-derived tau strains underlie the diversity of tauopathies, and that seeded aggregation and filament formation mimicking the pathogenesis of sporadic tauopathy can be reproduced in cultured cells. Our results indicate that the disease-specific conformation of tau aggregates determines the tau isoform substrate that is recruited for templated amplification, and also influences the prion-like seeding activity.

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Enhanced SNP-sensing using DNA-templated reactions through confined hybridization of minimal substrates (CHOMS).

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