Gel-based Detection Efficiency Analysis

The detection samples were run in 25-ml 0.8% agarose gels unless otherwise noted, cast from molecular biology grade agarose (Fisher BioReagents) dissolved in 0.5× TBE buffer. Typical running conditions were 75 V for 45 to 70 min at room temperature or cold room. Samples were mixed with a Ficoll-based blue loading dye before loading. Imaging was completed on a Bio-Rad Gel Doc XR+ imager with different exposure times based on the brightness of the detection bands. The detection efficiency was analyzed using included Image Lab software (Fig. 2E). The profiles of detection bands were obtained in ImageJ (57 (link)), and then their integrated intensities were obtained by using the peak analysis function in Origin (OriginLab Corporation), such as the data presented in Figs. 2F, 4C, and 5 (A and B). The detailed analysis procedure can be found in our previous publication (21 (link)). For the E-Gel–related experiments, we used Invitrogen E-Gel agarose system (Thermo Fisher Scientific) and its precast agarose gel (1.0%, SYBR stained). Ten microliters of nanoswitch detection sample was loaded to each lane, and the gel was run at 48 V for 1 hour at room temperature. Because the E-Gel system does not allow user control of the voltage, we used an external power supply connected with the negative and positive electrodes of the precast agarose gel to supply 48 V.