Multiplexed Nanopore-Based Nucleic Acid Sensing and Bacterial Identification Using DNA Dumbbell Nanoswitches

Multiplexed nucleic acid sensing methods with high specificity are vital for clinical diagnostics and infectious disease control, especially in the postpandemic era. Nanopore sensing techniques have developed in the past two decades, offering versatile tools for biosensing while enabling highly sensitive analyte measurements at the single-molecule level. Here, we establish a nanopore sensor based on DNA dumbbell nanoswitches for multiplexed nucleic acid detection and bacterial identification. The DNA nanotechnology-based sensor switches from an “open” into a “closed” state when a target strand hybridizes to two sequence-specific sensing overhangs. The loop in the DNA pulls two groups of dumbbells together. The change in topology results in an easily recognized peak in the current trace. Simultaneous detection of four different sequences was achieved by assembling four DNA dumbbell nanoswitches on one carrier. The high specificity of the dumbbell nanoswitch was verified by distinguishing single base variants in DNA and RNA targets using four barcoded carriers in multiplexed measurements. By combining multiple dumbbell nanoswitches with barcoded DNA carriers, we identified different bacterial species even with high sequence similarity by detecting strain specific 16S ribosomal RNA (rRNA) fragments.


S1.1 Materials
Oligonucleotides were purchased from Integrated DNA Technologies, Inc. (IDT). The sequences of DNA and RNA strands are listed in Table S1-3. M13mp18 ssDNA, RNA fragmentation buffer, BamHI-HF, and EcoRI-HF were purchased from New England Biolabs. MS2 RNA was purchased from Sigma-Aldrich. E. coli DH5α total RNA was purchased from Thermo Fisher Scientific. Synthetic SARS-CoV-2 RNA (880 nt, EURM-019) was purchased from European Commission, Joint Research Centre. Salmonella total RNA was extracted from bacterium cultured in laboratory. Other chemicals were of reagent grade and were used without further purification.

S1.2 Preparation of DNA carriers
190 DNA oligonucleotides (staples) designed in our previous work [1] were used here for M13 linearization. The staples were accurately mixed before preparation of the DNA carriers. Design of the carriers used in this work are shown in Figure S1. The staples at sensing sites are replaced with dumbbell and probe strands as listed in Table S1 and S3. For DNA carriers with barcodes, the staples at coding sites are replaced with dumbbell strands as listed in Table S2.
The carrier synthesis follows our previous work. [1] The 7228 nt DNA scaffold was linearized from M13mp18 ssDNA using the published protocol. [1] After mixing the staples of relevant carriers following the design in Figure S1 and Table S4, the cut M13 scaffold was added into the solution (20 nM M13 scaffold, 60 nM staples and 120 nM dumbbell strands or probes) and heated to 70°C followed by a linear cooling ramp to 25°C over 50 minutes. Finally, the resulting solution was diluted with a washing buffer (10 mM Tris-HCl, 0.5 MgCl 2 , pH 8.0) to 500 μL and centrifuged with an Amicon Ultra 100kDa filter to remove the excess DNA strands at 6000 g for 10 mins (repeated 3 times). About 35 μL solution of DNA carrier was obtained and quantified with NanoDrop 2000 spectrophotometer. S1.3 Fragmentation of 16S ribosome RNA (rRNA) and SARS-CoV-2 RNA 0.5 μL of E. coli DH5 total RNA (1.0 μg/μL), 0.5 μL of Salmonella total RNA (0.57 μg/μL), 0.5 μL of Acinetobacter baumannii total RNA (1.1 μg/μL), or 0.5 μL of MS2 RNA at different concentrations was mixed with 1 μL of 10× RNA fragmentation buffer, and then nuclease-free water was added to 10 μL.
30 μL of Synthetic SARS-CoV-2 RNA (880 nt, Cq value 21) was dried in vacuum concentrator (SpeedVac DNA 130, Thermo Fisher Scientific) and rehydrated with 9 μL nuclease free water before mixed with 1 μL of 10× RNA fragmentation buffer. The mixture was incubated in a preheated thermal cycler for 5 minutes at 94℃ then cooled down to 4℃. Finally, 1 μL of 10× RNA fragmentation stop solution and 0.5 μL of RNAsecure reagent were added into the mixture.

S1.4 Nanopore measurement
The fabrication and measurement of the glass nanopore follows the former study. [1] Glass nanopores with diameters 14±3 nm were generated on a laser-assisted pipette puller (P-2000, Sutter Instrument) by pulling quartz capillaries (outer diameter 0.5 mm and inner diameter 0.2 mm, Sutter Instrument). The resulting nanopores were fixed on a PDMS chip. I-V curve was measured in 4 M LiCl to check the size and current noise of nanopore before the addition of sample.
Target strand was incubated with 1 nM DNA carrier (concentration for each carrier) in TM buffer (10 mM Tris-HCl, 10 mM MgCl 2 , pH 8.0) at room temperature for 10 minutes. For bacterial or MS2 RNA detection, 10 μL of the RNA fragments mixture was mixed with 5 μL of 1 nM DNA carrier (concentration for each carrier) and incubated at room temperature for 10 minutes. Then carrier solution was diluted with Tris-LiCl buffer solution (10 mM Tris-HCl, 4 M LiCl, pH 9.0) to 0.125 nM and then added to the tip side of the glass nanopore. Two electrodes were placed at the two sides of the nanopore. The electrodes were connected to an Axon Axopatch 200B amplifier (Molecular Devices), which applied a voltage of 600 mV to drive the DNA through nanopores and recorded the current signal. For 0.125 nM carrier, the measurement lasted for 2-3 hours, but more time (~12h) was needed for carrier at low 10s of picomolar level in Figure S8. The current signal was filtered with an external Bessel filter (Frequency Devices) at 50 kHz and digitized at a 250 kHz sampling rate with a data card (PCI-6251, National Instruments). S1.5 Atomic force microscopy (AFM) imaging DNA carrier investigated in Figure 1 with target T1 was diluted to 0.1 nM in TM buffer for AFM measurement. 10 μL of 0.1 nM carrier sample was dropped onto a freshly cleaved mica surface for 1 minute, rinsed three times with 100 μL of water, and dried gently by nitrogen flow. The mica plate was affixed to the AFM sample stage using double-sided adhesive tape before the scan. Gwyddion was used for image visualization and analysis. S1.6 Nanopore data analysis Home-made LabVIEW algorithms were used for data collection and analysis. The raw data was analyzed by following the steps described in the previous works [1,2] : (1) filter and search for the translocation events from the raw current trace; (2) remove the folded and uncertain events; (3) barcode and sensing peak determination. Single peak fraction (SPF) was calculated as below to indicate the presence of target sequence in the sample.

= ℎ ℎ
Average SPF was used for bacterial identification. It is the average of the SPFs of the two sensing sites on the same carrier in  Table S4.      (Table S1) predicted by RNAstructure (version 4.6). P2X at sensing site X2 has the most stable self-folding structure, which will affect its binding to M13 scaffold and target. Figure S7. Detection of MS2 viral RNA after fragmentation at different concentrations using DNA dumbbell nanoswitch. 0.125 nM carrier is used in all measurements. More detailed nanopore data can be found in Table S9. Figure S8. Detection of target T1 at 50 pM and synthetic SARS-CoV-2 RNA (880 nt) with Cq value at 21 using single DNA dumbbell nanoswitch on carrier shown in Figure 1. Example events without target and with target are given in (a) and (b), respectively. (c) SPFs of blank, SARS-CoV-2 RNA, and 50 pM T1. 28 translocation events (6 events with single peak) and 58 translocation events (17 events with single peak) were analyzed to calculate the SPFs of SARS-CoV-2 RNA and T1, respectively. 5.0 pM carrier was used for 50 pM T1 and 2.2 pM carrier was used for SARS-CoV-2 RNA. SARS-CoV-2 RNA was concentrated 3.3 times and fragmented in RNA fragmentation buffer before mixed with carrier.    Figure S13. Raw current data collected from DNA carriers in Figure 4 with E. coli 16S rRNA fragments. The translocation event of carrier 2 was cropped and enlarged in the orange box. The translocation of RNA fragments can also be observed in the current trace.     Table S1.       [2] Zhu, J.; Ermann, N.; Chen, K.; Keyser, U. F. Image encoding using multi-level DNA barcodes with nanopore readout. Small 2021, 17, 2100711.