Tunable DNA hairpin springs stretch millions of protein copies under piconewton tension, enabling bulk biochemical discovery of binding partners invisible to single-molecule techniques.
(Nanowerk Spotlight) Some proteins only reveal their full function when they are physically pulled. Under tension, their folded structures open up, exposing surfaces that are otherwise concealed. Other molecules bind to these newly accessible sites, setting off signaling events that influence cell behavior. This mechanism helps cells grip their surroundings, sense tissue stiffness, and coordinate collective movement. When it malfunctions, cells misread mechanical cues, contributing to diseases such as cancer, fibrosis, and atherosclerosis.
Identifying which molecules bind to a force-opened protein, however, has posed a difficult experimental challenge. Instruments such as atomic force microscopes and magnetic tweezers can stretch a single protein with piconewton precision, yet they work one molecule at a time, making them incompatible with the biochemical assays needed to capture and identify new binding partners at scale.
DNA nanotechnology offers a potential way forward. Self-assembling DNA strands can be programmed to form rigid nanoscale structures, and researchers have previously demonstrated DNA nanosprings for measuring protein motor forces. But these devices lacked the ability to directly pull on proteins, limiting their biological applications.
Built from a DNA origami frame fitted with tunable DNA hairpin springs, the nanoscale machine grips a protein at both ends and stretches it under controlled force. Millions of copies can be produced in a single preparation, making the stretched proteins directly accessible to standard laboratory techniques such as pull-down assays, mass spectrometry, and electron microscopy.
Illustration of the DNA nanodevice design. Top: A U-shaped frame (blue) with DNA “handles” (black). Middle: The handles attach to tethers (green) that are attached to a protein of interest (orange). Bottom: When the DNA is triggered to fold (handle on the left), it pulls on the protein, exerting force. (Image: Yale University)
The device consists of a U-shaped DNA origami frame with a cavity roughly 49 nm wide and 17 nm deep. Two DNA handles extend from opposite walls, leaving a gap where a protein can be suspended. One handle functions as a “loaded spring,” initially held extended by a complementary strand.
When a displacement strand is added, it displaces the holding strand through a competitive hybridization reaction, allowing the handle to snap into a hairpin. This contraction stretches whatever protein is tethered between the two attachment points. The hairpin’s sequence and length determine both the force magnitude and the maximum extension.
To test whether the device generates the intended forces, the team mounted a DNA-based tension sensor, a stem-loop labeled with a fluorophore and quencher, in place of a protein. When the spring activated, the sensor unzipped, producing a fluorescence increase consistent with the predicted half-unfolding force of 8.3 pN. The system reached equilibrium within two to three hours.
With force generation confirmed, the researchers turned to a biological test case: the R1-R2 segment of the talin1 rod domain. Talin is a cytoskeletal protein that physically links cell-surface integrins to the internal actin network. Under mechanical load, its helical bundles unfold to expose concealed binding sites for vinculin, a structural protein that reinforces the connection between the cell and its surroundings. The R1-R2 segment contains two such bundles with three vinculin binding sites in total.
To mount R1-R2 with a defined orientation, the team expressed it as a fusion protein flanked by two small protein tags, each chemically linked to a short DNA tether that hybridized to the handles inside the origami frame. Electron microscopy confirmed that about 74% of devices contained a properly suspended protein.
In the relaxed state, the protein had an average end-to-end distance of roughly 9 nm. After spring activation, this distance nearly doubled to about 17 nm, indicating force-induced unfolding. A statistical thermodynamic model placed the applied force at 5 to 9 pN, consistent with physiological tension measured across talin in living cells.
The central question was whether this mechanical extension could activate protein binding in a bulk assay. Using biotinylated devices immobilized on magnetic beads, the researchers performed pull-down experiments with purified vinculin head domain. Before force application, vinculin binding was negligible. After spring activation, binding increased up to six-fold, but only when the initial gap between handles was optimized at 6 or 9 nm. Varying the hairpin strength revealed a force threshold for exposing vinculin binding sites of roughly 6 to 9 pN under sustained load.
An upgraded dual-spring version, with independently activatable hairpins on each handle, extended the protein further and produced correspondingly greater vinculin recruitment. Activating both springs increased the average extension by about 10 nm and yielded more than twice the vinculin pull-down compared to either spring alone, establishing a clear correlation between the degree of protein extension and its capacity to recruit binding partners.
An unbiased proteomic screen produced an unexpected result. The researchers incubated tensioned and relaxed devices with cell lysates and analyzed the pulled-down proteins by mass spectrometry. Vinculin showed the strongest tension-dependent enrichment, as expected. But filamin A and filamin B, actin-crosslinking proteins not previously known to bind talin directly, also appeared among the top hits.
Experiments with purified filamin A confirmed that this interaction is direct and independent of vinculin. A control using an elastic peptide in place of R1-R2 showed no force-dependent filamin binding, supporting the specificity of the result.
This result is notable because previous work had established a functional link between talin and filamin A in cells subjected to mechanical strain, yet no direct binding had been reported. Whether this newly identified interaction mediates known cellular responses to force remains a question for future investigation.
The modular architecture of the device makes it readily adaptable to other mechanosensitive proteins. Wider frames could accommodate larger targets, additional springs in series could drive greater conformational changes, and parallel springs could push forces beyond the roughly 20 pN limit of a single hairpin. By combining programmable mechanical loading with ensemble biochemistry and structural analysis, the platform enables systematic screening for force-regulated protein interactions across a broad range of biological systems.
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