DNA origami cages constrain individual proteins toward preferred orientations on electrodes, dramatically improving electrical measurement precision and enabling detection of subtle structural changes from molecular interactions.
(Nanowerk Spotlight) A single protein molecule, just a few nanometers across, can reveal whether a drug binds its target or whether a disease has begun to alter cellular machinery. The electrical current that flows through such a molecule encodes information about its shape, its chemical state, and the subtle shifts it undergoes when interacting with other molecules. Capturing that current could transform diagnostics and drug discovery.
But scientists must first get proteins to hold still long enough for a reliable measurement. When deposited on an electrode surface, these complex biological structures orient themselves randomly, like scattered dice. Each measurement captures a different angle, a different contact point, producing electrical readings so variable they obscure the very information scientists seek.
Previous efforts to solve this orientation problem achieved only partial success. Some researchers anchored proteins using gold-sulfur bonds formed through cysteine amino acids, but this approach depends on having correctly positioned cysteines, something many proteins lack, and sometimes requires genetic modifications that alter the properties under investigation.
Others employed molecular binding pairs like biotin and streptavidin to position proteins with specific orientations, but this demands substantial structural modifications. Electrostatic methods offer a gentler alternative, yet they yield weak electrical contact and work only for proteins with particular charge patterns.
DNA nanotechnology opened new possibilities. DNA origami, a technique that folds a long viral genome into precisely designed shapes using 203 short helper strands, now allows scientists to construct nanometer-scale scaffolds with nearly arbitrary geometries. Separately, aptamers, short DNA sequences selected through laboratory evolution to bind specific proteins with high affinity, became powerful molecular recognition tools.
Combining these technologies suggested an elegant solution: build a DNA scaffold that uses aptamers to grab and constrain a target protein in a preferred position between two electrodes.
Research published in the journal Advanced Science (“DNA‐Guided Robust Single‐Protein Electronic Readout”) now demonstrates this approach works. Scientists at the University of Science and Technology of China constructed square-shaped DNA origami platforms measuring 80 × 80 × 2 nm³, each featuring a central cavity of 20 × 20 nm². Into this cavity, they extended two DNA arms tipped with different aptamers that recognize the blood-clotting enzyme thrombin at two distinct sites simultaneously. This bivalent (two-point) capture biases the protein toward a preferred orientation. Twenty sulfur-containing chemical groups along the outer edges anchor the entire assembly onto a gold electrode.
Design and characterization of DNA origami nanostructures for site-specific thrombin binding. a) Molecular model of the DNA origami-thrombin complex. The zoomed-in schematic highlights the central cavity of the origami structure, where thrombin binds via two double-stranded DNA stems capped with thrombin-specific aptamers: HD22 (green) and TBA15 (orange). Blue dots indicate the positions of thiol-modified staple strands, which are uniformly distributed along the edges for gold surface anchoring. b) AFM image of DNA origami structures deposited on mica under imaging buffer. c) AFM image of DNA origami incubated with thrombin under the same conditions. Zoomed-in views in b and c show individual nanostructures; corresponding height profiles across their centers confirm the presence of central cavities and thrombin binding. d) AFM image of thrombin-bound DNA origami structures adsorbed on a gold substrate and imaged in air. The overlaid height profiles compare origami with (red) and without (blue) thrombin, confirming successful protein binding and gold surface attachment. (Image: Reproduced from DOI:10.1002/advs.202516711, CC BY) (click on image to enlarge)
The team used conductive atomic force microscopy to measure electrical current flowing through individual trapped thrombin molecules. A conductive tip served as one electrode while the gold substrate served as the other. Sweeping voltage from negative to positive one volt, they recorded current-voltage curves revealing each protein’s conductance, a measure of how readily it carries electrical charge that reflects its internal structure and chemical environment.
Thrombin molecules randomly deposited on gold produced conductance values with substantial variability, showing a standard deviation of 0.42 in logarithmic units. Thrombin captured by two aptamers showed a far narrower distribution, with a standard deviation of just 0.17. This tighter clustering reflects more uniform protein orientation and more consistent electrode contact. Some residual variability remains due to small freedom in tilt and position, intrinsic protein dynamics, and whether the origami lands face up or face down on the surface.
With this improved precision, the researchers detected subtle conductance changes when thrombin interacted with other molecules. Sodium ions bind thrombin at a specific site, inducing a small conformational shift that opens its catalytic pocket. When the team exposed DNA-oriented thrombin to 140 mM sodium chloride, they measured a conductance increase with strong statistical confidence. The p-value of 0.00018 indicated an extremely low probability the result occurred by chance. The same experiment on randomly deposited thrombin showed a similar average shift but with far weaker statistical support.
The researchers attribute the conductance increase to several factors revealed by computational modeling: sodium binding neutralizes a negatively charged region of the protein and stabilizes a more extensive hydrogen bonding network involving 11 water molecules compared to seven in the sodium-free state.
Control experiments with lithium chloride, which binds thrombin weakly and nonspecifically, produced no conductance change. This confirmed the signal reflected genuine sodium-induced structural changes rather than generic ionic effects.
The platform also detected thrombin’s interaction with PPACK, a potent inhibitor that locks the enzyme into a transition-state-like conformation. Oriented thrombin-PPACK complexes showed elevated conductance with narrow distributions. Computational modeling suggested this increase arises from PPACK organizing surrounding amino acid residues into a more compact electronic configuration.
To test generalizability, the researchers redesigned their DNA origami to capture streptavidin, a structurally distinct tetrameric protein. Four identical aptamers extended into the cavity, each targeting one of streptavidin’s binding sites. Streptavidin’s disk-like shape already favors consistent orientation on flat surfaces, so unconfined molecules showed narrower conductance distributions than thrombin. Yet tetravalent (four-point) DNA anchoring further reduced variability, demonstrating the method’s value even for proteins with intrinsically preferred orientations.
The DNA platform also enabled selective protein detection from mixtures. When researchers exposed thrombin-targeting origami to a solution containing equal concentrations of thrombin and streptavidin, atomic force microscopy showed protein occupancy only in origami designed for thrombin.
When both origami types encountered both proteins, conductance measurements revealed two distinct peaks corresponding to the two species. These peaks overlapped and blurred when proteins lacked DNA guidance.
The work carries limitations. Measurements occurred in air at 30% relative humidity rather than in aqueous solution, because current probe insulation technology cannot achieve the sub-picoampere leakage currents required for liquid measurements. Fluorescence studies suggested proteins retained physiologically relevant conformations under these conditions, but advances in tip fabrication will prove necessary for real-time observation of protein dynamics in their native environment. The conductive probe’s relatively large tip radius, about 25 nm, also limits spatial resolution.
This research demonstrates that DNA nanotechnology can overcome a fundamental obstacle in single-molecule bioelectronics. By constraining protein orientation without chemical modification or genetic engineering, the approach preserves native protein structure while dramatically improving measurement reproducibility.
The ability to detect conformational changes induced by ions and inhibitors points toward applications in detecting drug-protein interactions. The capacity to identify proteins from mixtures based on electrical signatures suggests a path toward multiplexed detection in complex biological samples.
Because aptamers exist or can be evolved for virtually any protein target, this label-free platform offers a versatile foundation for single-molecule electronic protein characterization.
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