RAPID: Enzyme-free detection of SARS-CoV2 using a PAINT-based single-molecule microscopy assay

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The novel coronavirus that causes COVID-19 is composed of a spherical envelope that surrounds a genome made of ribonucleic acids (RNAs). These RNAs control viral replication and have distinct regions for each coronavirus strain. Most rapid detection tests for COVID-19 amplify three unique RNA regions with a protein called an enzyme. The activity of enzymes greatly increases the sensitivity of COVID-19 detection, but have disadvantages of requiring cold storage, short shelf life, and potential for false negative results due to the loss of enzymatic activity. This research develops an enzyme-free COVID-19 test that uses the binding of stabilized deoxyribonucleic acid (DNA) molecules that are locked into place along the novel coronavirus genome. The novel coronavirus can be visualized using a micrscope when another marker DNA molecule constantly docks and undocks from the DNA attached to the coronavirus genetic material. The project will deliver a sensitive and stable test for rapid detection of COVID-19. A major goal of this project is to develop a flexible enzyme-free, shelf-stable viral test that can be readily adapted for the current as well as future viral outbreaks.The goal of this project is to develop an enzyme-free detection method for SARS-CoV-2 with single-molecule sensitivity that can detect single nucleotide polymorphisms of mutated strains in the United States. To achieve this goal, viral capture coverglasses will be engineered by covalently linking oligonucleotide (oligo) sequences that are reverse complementary to the 70-nt leader sequence (LS) and sequences 5’ to key regions of SARS-CoV-2 genome. Extracted viral RNA will be applied to the coverglass to pull down all viral transcripts that bind to the LS capture anchors. The specific regions for SARS-CoV-2 detection suggested by the Center of Disease Control & Prevention (CDC) will be used as target sequences to design probes for small RNA detection via DNA-based points accumulation in nanoscale topography (sRNA-PAINT). Single-molecule viral detection and amplification of target regions will be achieved with sRNA-PAINT by deploying highly specific locked-nucleic acid (LNA) oligonucleotide probes linked to oligo “docking strands”. Amplification is enzyme-free, and instead, the signal for any individual probe is amplified by the predictable binding and unbinding of oligo “imager strands” to the docking strands of the probe. Imager strands have a conjugated fluorophore and their binding is detected in thousands of images taken over 15 minutes on a microscope using a sensitive camera. The detection assay or kit will consist of an oligonucleotide (oligo)-linked capture coverglass, COVID-19 -specific oligo probes, fluorescent oligo imaging probes, buffers, and instructions on how to either deploy the assay on any microscope with single molecule sensitivity or build one.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.