Fig. 5: Multiplex detection of let-7 microRNA SNV variants.

a Two-step probe mechanism. miRNA input hybridizes to the first toehold region (T1) on the bottom strand, displacing the barcoded output strand for nanopore readout. Next, a helper strand hybridizes to the second toehold region (T2) on the bottom strand, displacing the quencher strand and enabling spectrofluorometer-based readout. b Multiplex experiment setup. Input strands are synthetic let-7 miRNA SNV variants (sequences shown with SNVs bolded in red) which are added to a mix of probes. A red X denotes the location of bases complementary to SNVs. Hybridization of an input with the correct probe releases its respective barcoded output for nanopore detection. c Seven samples, each containing three let-7 probes, were prepared. A different combination of input strands was added to each sample, as visualized under each cluster of bars. The bar plot shows the capture frequency of each barcoded output in each sample as measured on the nanopore after reaching a steady state. Error bars represent ± standard deviation of three biological replicates.