-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.

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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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... improve our sensitivity, we developed a new strategy for simultaneous targeting of different fragments of viral RNA. The concept is based on the idea that fragmenting the viral RNA produces many discrete targets from a single viral RNA genome (Figure 2a). A typical assay with a single target sequence can capture one target RNA per viral RNA, but an assay with multiple target sequences can capture multiple target RNAs per viral RNA to increase signal. ...

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... typical assay with a single target sequence can capture one target RNA per viral RNA, but an assay with multiple target sequences can capture multiple target RNAs per viral RNA to increase signal. In a nanoswitch assay, this can be accomplished by targeting several different SARS-CoV-2 sequences with identical loop sizes to essentially add the signal from multiple targets (Figure 2b). To achieve this, we developed a new nanoswitch design where multiple detectors that target distinct regions of the viral genome can be integrated onto a single nanoswitch, improving on previous ideas from combining individual single target nanoswitches. ...

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... 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). To implement this concept, we first identified loops that could be repositioned along the length of the nanoswitch with similar gel migrations. ...

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... targets. We show that for a constant target pool, we achieved a nearly stoichiometric increase in the signal as we increase the targeting capability (Figure 2c). ...

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... designed and built five such 24-target nanoswitches to provide detection of up to 120 fragments that span the SARS-CoV-2 viral genome. We confirmed the positive detection of each of the 120 individual targets using DNA oligonucleotides (Figure 2d) and showed that maximum detection efficiency appears to be a gaussian distribution with a mean 76% (Figure 2e). To measure the signal enhancement, we used an equimolar pool of the 120 targets and measured the detection signal using single nanoswitches with 1, 10, or 24 detector pairs and mixtures of 1, 2, 3, 4, and 5 24-target nanoswitches. ...

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... designed and built five such 24-target nanoswitches to provide detection of up to 120 fragments that span the SARS-CoV-2 viral genome. We confirmed the positive detection of each of the 120 individual targets using DNA oligonucleotides (Figure 2d) and showed that maximum detection efficiency appears to be a gaussian distribution with a mean 76% (Figure 2e). To measure the signal enhancement, we used an equimolar pool of the 120 targets and measured the detection signal using single nanoswitches with 1, 10, or 24 detector pairs and mixtures of 1, 2, 3, 4, and 5 24-target nanoswitches. ...

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... measure the signal enhancement, we used an equimolar pool of the 120 targets and measured the detection signal using single nanoswitches with 1, 10, or 24 detector pairs and mixtures of 1, 2, 3, 4, and 5 24-target nanoswitches. We found that the signal increases nearly linearly with the number of detectors in the mixture (Figure 2f). Using serial dilutions of the 120-target mixture, we tested the sensitivity of the assay with the five multi-detector nanoswitch mixture and a single target nanoswitch. ...

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... serial dilutions of the 120-target mixture, we tested the sensitivity of the assay with the five multi-detector nanoswitch mixture and a single target nanoswitch. Compared to the LoD of 122 fM or 0.76 amol for a single target sequence, our signal multiplication strategy provides a ~65-fold improvement to 1.9 fM or 0.01 amol, corresponding to ~6,000 genome copies (Figure 2g). ...