Patient sample collection

Patient samples were collected by swabbing the back of the throat (oropharyngeal swab) of patients, as previously described²⁶. The samples were collected from patients with the COVID-19-like clinical picture and were tested with qRT-PCR after nucleic acid extraction. Briefly, after collection, swabs were placed into a labelled sample tube containing a lysis buffer (4 M guanidine thiocyanate, 25 mM Tris–HCl, 0.5% β-mercaptoethanol and MS2 RNA (200 ng µl^–1; Roche)). The tube was gently agitated to ensure the even distribution of lysis buffer. The safety steps have been previously described and were performed in a certified CL2 laboratory²⁶.

Nucleic acid extraction

The total nucleic acid was extracted using spin-column-based systems and as employed by standardized qRT-PCR testing²⁶. The internal amplification control (MS2 (~6 × 10⁴ PFU ml^–1) per 10 ml of lysis buffer) was added in the top-up lysis buffer (25 µl per 10 ml of lysis buffer). The sample was eluted in 100 µl of nuclease-free water (nfH₂O; Invitrogen) and left to stand for 1 min before centrifugation for 1 min at 21,130×g (15,000 rpm) in a benchtop microfuge. The eluted samples were directly subjected to qRT-PCR. The remaining nucleic acid extracts were stored at −80 °C and further used for nanobait–nanopore sensing.

qRT-PCR for SARS-CoV-2

SARS-CoV-2 detection was performed as previously described²⁶. Per reaction, the master mix contained 12.5 µl of 2× Luna Universal Probe One-Step reaction mix, 0.5 µl of 20 µM Wu forward primer (5′-ATGGGTTGGGATTATCCTAAATGTGA-3′), 0.5 µl of 20 µM Wu reverse primer (5′-GCAGTTGTGGCATCTCCTGATGAG-3′), 0.3 µl of 10 µM MGB Probe 3 fluorescein (5′-ATGCTTAGAATTATGGCCTCAC-3′), 0.5 µl of 10 µM of internal control forward primer for MS2 RNA, 0.5 µl of 10 µM internal control reverse primer for MS2 RNA, 0.3 µl of 10 µM internal probe (MS2 ROX), 1 µl of Luna WarmStart RT Enzyme Mix and 3.9 µl of nfH₂O. Then, 20 µl of the master mix was aliquoted into each well of a 96-well plate and then combined with 5 µl of each extract. The MS2 internal extraction and amplification control that underwent the full extraction protocol was included as the negative extraction control in a minimum of two wells on the qRT-PCR plate. To determine potential contamination in the qRT-PCR process, 5 µl nfH₂O was included as the qRT-PCR negative control. Then, 5 µl of spiked SARS-CoV-2 template plasmid was included in a single well as the qRT-PCR positive control. After adding 5 µl of each sample to its designated well, the plate was sealed with an optically clear plastic seal. The plate was centrifuged for 1 min at 2,000×g (1,000 rpm) at 4 °C and then inserted in the qRT-PCR machine (QuantStudio, Thermo Fisher Scientific) and the run was parametrized. Signals for fluorescein (FAM) and carboxyrhodamine (ROX) were acquired. ROX was used to detect the internal MS2 control and fluorescein was used to detect SARS-CoV-2 RNA. The assay was performed for 2 min at 25 °C, 15 min at 50 °C (for the reverse transcriptase), 2 min at 90 °C, before 45 cycles of 95 °C for 3 s followed by 60 °C for 30 s. The results were determined by the confirmation of correct positive controls (amplification of the plasmid), extraction and amplification controls of all the samples (ROX channel), no amplification in the negative controls and consistent mean values of controls. SARS-CoV-2 positivity was confirmed by amplification in the fluorescein channel with an appropriate sigmoidal curve with a CT value of ≤36. The CT values of MS2 and MGB probe 3 were maintained to track the quality and reproducibility of the assay⁴⁴.

Programmable RNase H cutting for nanobait

For nanopore sensing, SARS-CoV-2 RNA controls, nucleic acid extracts (patient samples) or MS2 viral RNA were used further for detection with nanobait. First, we mixed guide oligos with the sample and heated it to 70 °C for 5 min. RNase H (5,000 units per ml; NEB) was added, mixed and heated for 20 min at 37 °C to allow the enzyme to cut RNA in the DNA: RNA hybrid that effectively releases the target RNA. RNase H was thermally inactivated by incubation at 65 °C for 10 min. Guide oligos were validated to not form intramolecular structures, homo- or heterodimers using the NUPACK software⁴⁵. For the measurement with the absent target, the same protocol including guide oligos was used. The control measurements show no displacement, and hence, we can exclude any substantial cross-binding from guide oligos.

Viral target sequence properties for nanobait

The length of target, toehold length and GC content were selected to ensure optimal hybridization²¹. For the DNA nanobait designs, the target sequences were selected to be in the conserved regions of a viral genome and had 40–60% GC content to form a stable 20 bp duplex. The toehold length was selected to be 6 nt long and have 40–60% GC content. We tested all the sequences for potential undesirable highly stable intramolecular interactions or homodimers using the NUPACK software (web application 2020)⁴⁵. Then, we performed a cross-reactivity check between multiple sites employed in each experiment⁴⁵.

Preparation of DNA flower for nanobait

We designed a DNA flower as another label for SARS-CoV-2 RNA detection from the patient samples. Three DNA flowers specific for each SARS-CoV-2 target (seven-way junctions, 7WJa, 7WJb and 7WJc) were separately prepared. Taking 7WJc as an example, 4 μM DNA strand J1, J2, J3 and J4c (Supplementary Table 1) were mixed together in TM buffer (10 mM Tris–HCl, 10 mM MgCl₂, pH 8.0) and heated to 90 °C for 5 min, then cooled down to 65 °C for 15 min, 45 °C for 15 min, 37 °C for 20 min, 25 °C for 20 min and finally to 4 °C for 20 min. Strand J4c was substituted by J4b to prepare 7WJb. For 7WJa, to avoid self-folding at site 43 on the nanobait, J1, J2, J3 J4a and C43 were mixed together before annealing.

Self-assembly of DNA nanobait

The DNA nanobait was assembled by mixing linearized single-stranded M13 DNA (M13mp18, 7,249 nt, Guild Biosciences, 100 nM) with short complementary oligonucleotides¹² (some of which harboured reference structures and capture strands) and by adding partially complementary strands that were 3′-biotinylated for toehold-mediated strand displacement reaction. The linearized M13 DNA (7,228 nt in length) was complemented by oligonucleotides, thereby creating a nicked double-stranded nanobait with two-terminal four deoxythymidine overhangs that prevent multimerization¹². The mix contained 20 nM of linearized M13 DNA, 60 nM of oligonucleotides (three times excess to M13 DNA), 3′-biotinylated strands in the concentration of 180 nM, 10 mM MgCl₂ and 1× TE (10 mM Tris–HCl, 1 mM EDTA, pH 8.0). It was mixed by pipetting and spun down before heating to 70 °C for 30 s and cooled down over 45 min to ambient temperature. Excess oligonucleotides were removed using Amicon Ultra 0.5 ml centrifugal filters with 100 kDa cutoff with a washing buffer (10.0 mM Tris–HCl pH 8.0, 0.5 mM MgCl₂). If DNA flowers were employed as a label, the partially complementary strands that carry it were incubated in 10 mM MgCl₂ for 2 h at ambient temperature, and subsequently, Amicon filtration was performed as described above. The asymmetry of the nanobait design allows for the unambiguous identification of the binding sites. The nanobait was stored until used for further experiments under 4–10 °C in 0.5 mM MgCl₂, 10.0 mM Tris–HCl, pH 8.0. The nanobait design was checked by nanopore readout before each measurement.

Nanopore readout of DNA nanobait

The nanobait was mixed with a sample (nucleic acid extract or purified viral targets at ten times excess) in 10 mM MgCl₂ and 100 mM NaCl. The mixture (5 μl) was incubated at room temperature (~10 min) until prepared for nanopore measurement. The difference in the target sequence composition and its physical characteristics might lead to variability in hybridization and hence the displacement efficiency of sensing sites²¹. We have used htRNA (100 ng μl^–1; Invitrogen) as a background where indicated, to show that there are no non-specific signals induced by human native RNAs. For nanopore measurement, the sample was diluted to <0.5 nM nanobait (for purified viral targets) or 4.7 μl of RNase-H-cut patient sample was mixed with 0.3 μl of monovalent streptavidin (SAe1D3)¹⁸ (1 μM), 5 μl of LiCl (4.0 M) and 5.0 μl of LiCl (8.0 M). We have fabricated 14 ± 3 nm (mean ± standard deviation) nanopores¹² using quartz glass capillaries with 0.5 mm outer diameter and 0.2 mm inner diameter (Sutter Instrument) by laser-assisted puller P-2000 (Sutter Instrument). The mix was pipetted in a nanopore polydimethylsiloxane chip, and all the measurements were performed at a constant voltage of 600 mV. Nanopore measurement details are shown in Supplementary Table 30.

Real-time nanopore data analysis

Nanopore data analysis is explained in detail in Supplementary Section 14. Briefly, nanobait events were filtered out of raw ionic current traces and then the detection region was determined, and information of the spike’s presence at each specific site was extracted. The plotted displacement efficiency was calculated as a displacement efficiency for a measurement subtracted to a no-target control for each site (50 nanobait events for each of the three nanopore recordings), unless stated otherwise:

We verified that the convolutional neural network QuipuNet²⁷ was capable of the real-time analysis of nanopore data following the described procedure. Previously, we demonstrated that with around ten events, we reach 99% confidence in a positive detection of our designed DNA structures⁴⁶.

AFM imaging

AFM (Nanosurf Mobile S) imaging of nanobaits was performed in air in the non-contact mode. The nanobait structures were diluted to 1 ng μl^–1 in 1 mM MgCl₂ and 10 μl was added to freshly cleaved mica, incubated for 1 min, rinsed with filtered Milli-Q water and then blow dried with nitrogen. Before scanning, the mica plate was affixed to the AFM sample stage using double-sided adhesive tape. Image visualization and analysis were performed using Gwyddion (version 2.60).

Statistical analysis

For all the measurements, 99.9% confidence intervals for displacement efficiencies were calculated. Statistical significance between two sites without and with the target was tested using a two-sided Student’s t-test.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

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• Source: https://www.nature.com/articles/s41565-022-01287-x