Uncovering Molecular Quencher Effects on FRET Phenomena in Microsphere-Immobilized Probe Systems

• Published in: Analytical chemistry (Washington) 2023-09, Vol.95 (37), p.13796-13803
• Main Authors: Adams, Mary Catherine, Milam, Valeria T.
• Format: Article
• Language: English
• Subjects: Chemistry; Coronaviruses; COVID-19; Deoxyribonucleic acid; Design optimization; DNA; DNA probes; Dyes; Energy transfer; Flow cytometry; Flow stability; Fluorescence resonance energy transfer; Gene sequencing; Hybridization; Oligonucleotides; Probes; Quenching; Ribonucleic acid; RNA; Segments; Severe acute respiratory syndrome coronavirus 2; Target detection; Titration
• ISSN: 0003-2700; 1520-6882
• DOI: 10.1021/acs.analchem.3c01064

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.