A non-enzymatic test for SARS-CoV-2 RNA using DNA nanoswitches

Abstract

The emergence of a highly contagious novel coronavirus in 2019 led to an unprecedented need for large scale diagnostic testing. The associated challenges including reagent shortages, cost, deployment delays, and turnaround time have all highlighted the need for an alternative suite of low-cost tests. Here, we demonstrate a diagnostic test for SARS-CoV-2 RNA that provides direct detection of viral RNA and eliminates the need for costly enzymes. We employ DNA nanoswitches that respond to segments of the viral RNA by a change in shape that is readable by gel electrophoresis. A new multi-targeting approach samples 120 different viral regions to improve the limit of detection and provide robust detection of viral variants. We apply our approach to a cohort of clinical samples, positively identifying a subset of samples with high viral loads. Since our method directly detects multiple regions of viral RNA without amplification, it eliminates the risk of amplicon contamination and renders the method less susceptible to false positives. This new tool can benefit the COVID-19 pandemic and future emerging outbreaks, providing a third option between amplification-based RNA detection and protein antigen detection. Ultimately, we believe this tool can be adapted both for low-resource onsite testing as well as for monitoring viral loads in recovering patients.

Figure 1. DNA nanoswitch detection of SARS-CoV-2 RNA.

A) The DNA nanoswitch is a self-assembled DNA construct designed to form a loop upon interacting with a specific target sequence. The nanoswitch is stained with intercalating dye and imaged on a gel for detection readout. B) Design and validation of a DNA nanoswitch targeting a 30 nt portion of the N-gene. C) Reaction kinetics for a 30 nt RNA target in excess shows nearly two orders of magnitude improvement with optimal magnesium and temperature. D) Effect of various magnesium concentrations on room temperature kinetics. E) Effect of various temperatures on kinetics. F) Reaction kinetics with limited target. G) Gel separation of nanoswitch with different times and voltages. H) Overall assay time for a 50 pM target.

Figure 2. Improving sensitivity with multi-detector nanoswitches.

A) Long viral RNA can have many target regions that separate into discrete strands after fragmentation. B) Concept of single-detector and multi-detector sensing. C) Validation of multi-detector sensing concept by targeting one to five sequences in a five-sequence pool. D) Development and validation of five 24 detector nanoswitches to enable targeting of 120 different regions. Gels demonstrate detection of each individual target. E) Quantified looped % of each nanoswitch target, with a histogram showing the distribution. F) Detection of a mock viral RNA using 120 equimolar targets and corresponding signal increase with increased detectors. G) Overall sensitivity of the 120 detector nanoswitch approach compared with single detector.

Figure 3. Detection of SARS-CoV-2 RNA.

A) Scheme for SARS-CoV-2 fragmentation. B) In vitro transcribed N-gene RNA is fragmented using heat for various times with progressively smaller fragments over time. C) Distribution of apparent fragment sizes as a function of fragmentation time. D) Multi-detector nanoswitch detection of fragmented SARS-CoV-2 full genome RNA controls show optimal performance in the 2–16 minutes range. E) Detection of full genome SARS-CoV-2 with each of five 24 detector nanoswitches and the mixture of all five. F) Analytical sensitivity of the 120 multi-detector assay against full genome SARS-CoV-2 RNA. G) Prominence of different variants during the first 3 years of the pandemic in the United States (data from GISAID). H) Detection of major variants, and no detection of a non-SARS human coronavirus hCoV 229E.

Figure 4. Detection of clinical SARS-CoV-2.

Nanoswitch based detection of RNA extracts from clinical positive samples and clinical negative samples.