-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.

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... in DNA origami, a long single stranded DNA is folded into specific two-or three-dimensional shapes using short complementary strands. [34] Here, we create a simple linear duplex (the "off" state) using a 7249-nt long scaffold strand (commercially available M13 bacteriophage viral genome) tiled with short complementary backbone oligonucleotides ( Figure S1). [35,36] Two of the oligonucleotides contain single stranded extensions (detectors) that are complementary to parts of a target nucleic acid (Figure 1a). ...

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... Here, we create a simple linear duplex (the "off" state) using a 7249-nt long scaffold strand (commercially available M13 bacteriophage viral genome) tiled with short complementary backbone oligonucleotides ( Figure S1). [35,36] Two of the oligonucleotides contain single stranded extensions (detectors) that are complementary to parts of a target nucleic acid (Figure 1a). On binding the target sequence, the nanoswitch is reconfigured from the linear "off" state to a looped "on" state. ...

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... binding the target sequence, the nanoswitch is reconfigured from the linear "off" state to a looped "on" state. [37] This conformational change is easily read out on an agarose gel where the on and off states of the nanoswitch migrate differently due to the topological difference (Figure 1a, inset). Importantly, this approach requires no complex equipment or enzymatic amplification. ...

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... a first step, we designed a nanoswitch with detectors that respond to a target sequence in SARS-CoV-2 that was expanded from one of the original CDC N2 primer targets [42]. Using a synthetic RNA target, we confirmed that our nanoswitches could detect this SARS-CoV-2 fragment (Figure 1b). Next, we moved to address one of the key challenges in detection of SARS-CoV-2 RNA: achieving a short detection time for a low concentration target. ...

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... not yet been achieved for nucleic acids, where secondary structures can slow reaction kinetics. To address this issue, we screened the kinetics of the nanoswitch assay for a variety of conditions using a 30 nt synthetic RNA target that corresponds to a region in the SARS-CoV-2 genome nucleocapsid (N) gene (Figure 1c). First, we performed kinetic experiments at room temperature in phosphate buffered saline (PBS), which took nearly 2 hours to reach completion (Figure 1c). ...

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... address this issue, we screened the kinetics of the nanoswitch assay for a variety of conditions using a 30 nt synthetic RNA target that corresponds to a region in the SARS-CoV-2 genome nucleocapsid (N) gene (Figure 1c). First, we performed kinetic experiments at room temperature in phosphate buffered saline (PBS), which took nearly 2 hours to reach completion (Figure 1c). We then considered the . ...

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... a Tris-HCl buffer, we tested different concentrations of magnesium. We found that achieving the highest detection signal required at least 10 mM MgCl2 and that kinetics were enhanced up to 30 mM MgCl2 (Figure 1d and Figure S2). Choosing 30 mM MgCl2, we then measured kinetics at different temperatures, which showed a continued increase until ~50˚C~50˚C (Figure 1e and Figure S3). ...

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... found that achieving the highest detection signal required at least 10 mM MgCl2 and that kinetics were enhanced up to 30 mM MgCl2 (Figure 1d and Figure S2). Choosing 30 mM MgCl2, we then measured kinetics at different temperatures, which showed a continued increase until ~50˚C~50˚C (Figure 1e and Figure S3). With a 1 nM target RNA concentration, the magnesium and elevated temperature enabled complete reactions in less than 2 minutes, nearly two orders of magnitude faster than our starting condition (Figure 1c). ...

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... 30 mM MgCl2, we then measured kinetics at different temperatures, which showed a continued increase until ~50˚C~50˚C (Figure 1e and Figure S3). With a 1 nM target RNA concentration, the magnesium and elevated temperature enabled complete reactions in less than 2 minutes, nearly two orders of magnitude faster than our starting condition (Figure 1c). ...

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... a typical viral detection assay, we expect the target concentration (~fM level) to generally be lower than the nanoswitch concentration (~200-500 pM level). To assess more realistic conditions, we evaluated the kinetics for reactions with excess nanoswitch (Figure 1f). First, we confirmed that the rate of the signal accumulation relative to maximum signal is independent of the target concentration under these conditions ( Figure S4). ...

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... expected, we found that increasing the nanoswitch concentration increased the reaction rate. At the highest nanoswitch concentration tested (~600 pM), it took less than 2 minutes to reach half signal and less than 10 minutes to reach 90% signal (Figure 1f). ...

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... maximized band separation at a given voltage by minimizing buffer height above the gel (Figure S5), which nearly doubled the separation. We then imaged looped nanoswitches at different voltages and running times, reducing the gel running time from 45 minutes to 5-20 minutes with minimal signal loss (Figure 1g). These optimizations enabled detection of a 50 pM RNA target with an end-to-end assay time of as little as 10 minutes without sacrificing more than 20% of the signal (Figure 1h). ...

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... then imaged looped nanoswitches at different voltages and running times, reducing the gel running time from 45 minutes to 5-20 minutes with minimal signal loss (Figure 1g). These optimizations enabled detection of a 50 pM RNA target with an end-to-end assay time of as little as 10 minutes without sacrificing more than 20% of the signal (Figure 1h). ...

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... design a multi-detector nanoswitch, we incorporated pairwise detectors on the nanoswitch, with each pair offset along the length of the nanoswitch. We designed these pairwise detectors to be a fixed distance apart so that they all form the same loop size on binding their specific target ( Figure S1). Under typical conditions for SARS-CoV-2 detection (when nanoswitch concentration >> target concentration), this results in the ability to detect multiple SARS-CoV-2 fragments with an additive signal (Figure 2b). ...

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... this project, evidence in our lab emerged that longer detector regions enhanced capture of a 401 nt RNA [50]. To see if that result applied to our fragmented viral RNA, we made and tested nanoswitches with detection regions of 15, 20, 25 and 30 nt and found that overall detection signal clearly increased with longer detector lengths ( Figure S10). This result was substantial enough to warrant redesign and reconstruction of our five 24 detector nanoswitches with the 30nt detector pairs. ...

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... result was substantial enough to warrant redesign and reconstruction of our five 24 detector nanoswitches with the 30nt detector pairs. We redesigned our target regions and detectors for the new nanoswitches, which we again validated for each target ( Figure S11). One unintended consequence of this change was false positive detection, likely due to detectors weak cross-reactivity of detectors. ...

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... found that our nanoswitch assay was able to similarly detect all SARS-CoV-2 variants but had no detection of hCoV229E. We believe that intensity variations in some variants was due to differing concentrations in the source material, which was supported by RTqPCR data on the same material that showed nearly a 4-fold variation across samples, with BQ.1 having the lowest concentration ( Figure S13). Our unique approach of wide genome coverage makes our system robust against new variants. ...

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... first target in figure 1 was chosen based on an original CDC primer target [42], which was expanded to 30 nt. For the first set of multiple targets with 30 nt length, we used the previously reported Matlab code [33] based on minimum distance between targets and minimal secondary structure. ...

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... were incubated for 2 hours at 50C unless otherwise noted. For nanoswitches with 15 nt detector regions (Figures 1 and 2), samples were prestained before running the gel with gels imaged immediately after. Samples were brought to 10 uL with water and added 2 uL of 6x loading dye and 1 uL of 10x GelRed (Biotium). ...