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- W4386323474 abstract "Double-stranded (ds) oligonucleotide probes composed of quencher-dye sequence pairs outperform analogous single-stranded (ss) probes due to their superior target sequence specificity without any prerequisite target labeling. Optimizing sequence combinations for dsprobe design requires promoting a fast, accurate response to a specific target sequence while minimizing spontaneous dsprobe dissociation events. Here, flow cytometry is used to rapidly interrogate the stability and selective responsiveness of 20 candidate LNA and DNA dsprobes to a 24 base-long segment of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA and ∼243 degenerate RNA sequences serving as model variants. Importantly, in contrast to quantifying binding events of dye-labeled targets via flow cytometry, the current work employs the Förster resonance energy transfer (FRET)-based detection of unlabeled RNA targets. One DNA dsprobe with a 15-base-long hybridization partner containing a central abasic site emerged as very stable yet responsive only to the SARS-CoV-2 RNA segment. Separate displacement experiments, however, indicated that ∼12% of these quencher-capped hybridization partners remain bound, even in the presence of an excess SARS-CoV-2 RNA target. To examine their quenching range, additional titration studies varied the ratios and spatial placement of nonquencher and quencher-capped hybridization partners in the dsprobes. These titration studies indicate that these residual, bound quencher-capped partners, even at low percentages, act as nodes, enabling both static quenching effects within each residual dsprobe as well as longer-range quenching effects on neighboring FAM moieties. Overall, these studies provide insight into practical implications for rapid dsprobe screening and target detection by combining flow cytometry with FRET-based detection." @default.
- W4386323474 created "2023-09-01" @default.
- W4386323474 creator A5043488897 @default.
- W4386323474 creator A5090358456 @default.
- W4386323474 date "2023-08-31" @default.
- W4386323474 modified "2023-09-30" @default.
- W4386323474 title "Uncovering Molecular Quencher Effects on FRET Phenomena in Microsphere-Immobilized Probe Systems" @default.
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- W4386323474 doi "https://doi.org/10.1021/acs.analchem.3c01064" @default.
- W4386323474 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/37651319" @default.
- W4386323474 hasPublicationYear "2023" @default.
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