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

Abstract

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.

Figure 1

Detection of DNA target using DNA dumbbell nanoswitch-nanopore sensor. (a) Schematic of the self-assembly of DNA carrier with probes (green) and two groups of DNA dumbbells (blue). (b) Topological change of DNA dumbbell nanoswitch in the presence of a DNA target (strand T1) from “open” state to “close” state and the readout by nanopores with two example events. (c) Dependence of single peak fraction (SPF) on target concentrations. 0.125 nM DNA carrier is used for all measurements. SPF represents the percentage of “close” state in the two states of switch. (d) Detection of the single base substitution (strand T2) and missing (strand T3) in the target strand comparing with the target strand T1 and blank control by the DNA dumbbell nanoswitch. Sequences of the strands are given above the bar graph for comparison. The concentrations of DNA carrier and target strands are 0.125 nM and 2.5 nM, respectively. Error bars in (c) and (d) represent the standard error of the mean obtained from three repeated measurements (Table S5). (e) Atomic force microscopy (AFM) image of DNA carriers with target T1 in “open” state (top) and “close” state (bottom).

Figure 2

Multiplexed sensing of four different DNA target sequences by a carrier with four DNA dumbbell nanoswitches. (a) Sketch of a DNA carrier with four binding sites for target sequences W, X, Y, and Z. (b) A typical unfolded event showing four double peak patterns for the “blank” carrier without target. (c) From top to bottom: addition of strands W and Y, X and Z, and all four with corresponding ionic current signals in (d). Please note that the example signal for all four shows a carrier translocation in the opposite direction. The concentrations of DNA carrier and target strands are 0.125 nM and 2.5 nM, respectively. (e) Bar chart showing the SPF of the experiments from (a) and (c). Error bars represent the standard error of the mean obtained from three repeated measurements (Table S6).

Figure 3

Identification of single base variation by DNA dumbbell nanoswitch-nanopore sensor. (a) Schematic of four barcoded carriers with a single DNA dumbbell nanoswitch as indicated in the sketch. We designed four carriers with barcodes 000, 001, 010, and 011 to test the effect of the four dG, dT, dA, and dC, respectively. The position of the base in the target strand is indicated by the dashed box. The RNA target rG can also be detected by carrier 000. (b) Detection of dG is shown as an example and the example events of the four carriers are given with only the carrier 000 showing a large single downward peak. (c) SPFs of each carrier obtained from different targets. The concentrations of DNA carrier and target strands are 0.125 nM and 2.5 nM, respectively. Error bars represent the standard error of the mean obtained from three repeated measurements (Table S7).

Figure 4

Bacterial identification by detection of 16S rRNA fragments using barcoded carriers with multiple dumbbell nanoswitches. (a) Schematic of the detection of E. coli DH5α 16S rRNA fragments. Two DNA dumbbell nanoswitches are located at two sensing sites on each carrier to detect two target RNA fragments from the same bacterium. Carrier 1, 2, and 3 are designed for E. coli DH5α, Salmonella, and E. coli O104:H4, respectively. (b) Target rRNA fragments highlighted by colored frames from 16S rRNA of the three bacterial species for identification. (c–e) Average SPFs of the two sensing sites on each carrier obtained from different 16S rRNA fragments. Results of E. coli DH5α, Salmonella, and Acinetobacter baumannii (negative control) are given in (c), (d), and (e), respectively. Error bars for the standard error of the mean in (c), (d), and (e) are based on three repeated measurements (Table S8).