Nucleic acid tests are valuable tools in the diagnosis of viral infection. In this work, we present a rapid, high throughput approach to detection of specific nucleic acid sequences using double-stranded probes immobilized to microspheres. The overall goal was to screen multiple candidate double-stranded probes to identify one or more probes with low fluorescence background in its initially quenched state, but high fluorescence signal in response to perfectly-matched, label-free RNA target. We screened multiple DNA and LNA hybridization partners with varying base lengths and base fidelity to optimize the detection system and found that double-stranded probes possessing a fifteen base-long hybridization partner with a central abasic site exhibited the desired, selective fluorescence response to complementary 24 base-long RNA. Separate experiments with scrambled and mismatched RNA sequences confirmed that the fluorescence activity of this select probe system is due to RNA targets displacing shorter, quencher-capped hybridization partners. In contrast, several other candidate probes exhibited either undesirable high fluorescence background (in the absence of RNA target) or remained unresponsive to complementary RNA.

Exploration of flow cytometry-based detection of label-free RNA

ACS Spring 2022

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