In general, assays were performed by incubating the purified nanoswitches (~100 pM) with the target solution containing miRNAs. Concentration measurements were determined by measuring A260 absorbance with a NanoDrop spectrophotometer (Thermo Fisher Scientific) and applying either molar extinction coefficients (for oligos) or using the standard single-stranded RNA setting (for RNA extracts). Concentrations of final solutions were determined by the volume ratios during dilutions. Typical reactions were carried out in PCR tubes with 10-μl final volumes. Staining was performed by prestaining the samples with GelRed (Biotium Inc.) at 1× concentration and running a 25-ml (0.8%) agarose gel in 0.5× tris-borate EDTA at 75 V at room temperature for 45 min, unless otherwise noted. Specific protocol variations for each experiment are detailed as follows.

For the nanoswitch characterization tests, the target miRNAs were spiked into a 500 nM solution of off-target “blocking” oligos to minimize loss to the tubes. For the mismatch detection (Fig. 2A), the final concentration of target strands was 100 pM and the incubation time was 5 hours at room temperature in a solution containing nominally 1× PBS and 10 mM MgCl2. For sensitivity tests (Fig. 2, B to D), microcentrifuge tubes and pipette tips were additionally preincubated in blocking oligo solution to minimize loss of the target miRNA. The nanoswitch and miRNA solution were incubated either overnight (Fig. 2B) or for various times (Fig. 2, C and D) at room temperature in a solution containing nominally 1× PBS and 10 mM MgCl2. GelRed and a Ficoll-based loading dye were added to each sample to get a 1× concentration of each (for Fig. 2D, GelRed was added before incubation). Either 2.5 μl (Fig. 2, B and C) or 5 μl (Fig. 2D) of each sample was loaded and run in an unstained gel.

For detection of miRNAs from the small RNA samples (Fig. 3D), small RNAs were incubated with the nanoswitches overnight at room temperature in a solution containing nominally 1× PBS, 10 mM MgCl2, and 1× GelRed. Ficoll-based loading dye was added to the samples before loading and running in an unstained gel. Detection from total RNAs (Fig. 3E) proceeded identically except without magnesium and with increased GelRed (3.3×) to prevent understaining with the high-added RNA content and incubated in a decreasing temperature ramp from 40° to 25°C with 5 min for each 0.1°C (total, ~12.5 hours). The limit-of-detection measurements in small and total RNAs (Fig. 3, H and I) proceeded similarly but with the addition of 500 nM blocking oligos to prevent loss to the tubes. In addition, both of those were incubated with magnesium but with GelRed added after incubation. For detection of other miRNAs from different cell lines, total RNA extracts were normalized to a concentration of 500 ng/μl.

For the multiplexed detection (Fig. 4), the five individual nanoswitches were concentration-normalized and mixed in equal volumes. Nanoswitches at ~100 pM each were mixed with small RNAs, 1× PBS, and 10 mM MgCl2 and were incubated using the temperature ramp described above. GelRed and loading dye were added after incubation, and samples were run in an unstained gel. Gels were run at 4°C for 3 hours at 55 V, with the longer time facilitating easier recognition of the six individual bands.

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