biostudies-literatureJu ZNational Natural Science Foundation of Chinae16711https://www.ebi.ac.uk/biostudies/studies/S-EPMC12948236biostudies-literatureUnknown13(12)Electronic transport properties of single proteins offer powerful insights into their structure and dynamics. However, the orientation variability of electrode-attached protein molecules poses a big difficulty in achieving precise electric measurements and reliable data interpretation. Here, a DNA origami-based method that promotes preferred orientations of target proteins is reported, enabling more reproducible single-molecule electrical characterization. With two orthogonal DNA aptamers installed inside a DNA origami nanocavity, a thrombin protein can be captured with a prescribed orientation between a gold substrate and a conductive AFM tip. Matrix-patterned I-V measurements reveal that bivalently anchored thrombin molecules exhibit significantly reduced conductance variability comparedAdvanced science (Weinheim, Baden-Wurttemberg, Germany)DNA-Guided Robust Single-Protein Electronic Readout.PMC1294823622434005Li YLi JJu ZDeng ZfalseDNA-Guided Robust Single-Protein Electronic Readout.Electronic transport properties of single proteins offer powerful insights into their structure and dynamics. However, the orientation variability of electrode-attached protein molecules poses a big difficulty in achieving precise electric measurements and reliable data interpretation. Here, a DNA origami-based method that promotes preferred orientations of target proteins is reported, enabling more reproducible single-molecule electrical characterization. With two orthogonal DNA aptamers installed inside a DNA origami nanocavity, a thrombin protein can be captured with a prescribed orientation between a gold substrate and a conductive AFM tip. Matrix-patterned I-V measurements reveal that bivalently anchored thrombin molecules exhibit significantly reduced conductance variability compared2026-01-01T00:00:00Z2026 Feb2026-07-16T22:20:47.34Z2026-07-11T03:12:14.446ZS-EPMC129482364140538610.1002/advs.202516711