Determining Force Thresholds in ssDNA-ssDNA Interactions for Mechanically Triggered Molecular Devices

Introduction: : Pulmonary fibrosis is a progressive, often fatal disease marked by aberrant extracellular matrix (ECM) remodeling, excessive collagen deposition, and increased tissue stiffness. DNA-based nanotechnology, especially DNA origami and force-responsive oligonucleotide structures, offers a promising platform for targeted therapeutic release and mechanobiological interrogation of those diseases. The mechanical stability of single-stranded DNA (ssDNA) under applied force is a critical design parameter for molecular systems such as force-responsive DNA nanodevices and biosensors. DNA-based mechanical “locks” can be tailored to rupture at defined force thresholds, enabling controlled release of cargo in systems subjected to mechanical stress, such as the extracellular matrix (ECM) during fibrosis or tissue remodeling. However, a detailed understanding of how sequence composition and force-loading geometry affect the rupture behavior of ssDNA is essential for tuning these molecular components. In this study, we systematically investigate the rupture forces of ssDNA constructs with GC content either low (40%) or high (73%) under two geometries: unzipping, where force is applied to separate strands at the base-pair interface, and shearing, where force is applied longitudinally along the strand. These two modes simulate the range of mechanical loads DNA might experience in complex nanostructures. By measuring the most probable rupture forces and calculating the force-dependent dissociation rate constants (k_off), we aim to map the mechanical response of DNA to inform the development of force-threshold DNA switches. These quantitative parameters provide the mechanical foundation for engineering tunable rupture bonds in DNA-based systems for biosensing and therapeutic applications.

Materials and

Methods: : To perform AFM-based ssDNA force spectroscopy, both the AFM probe and glass coverslip were chemically functionalized to enable specific biotin–streptavidin-mediated attachment. For AFM probe functionalization, a 400 µL thiol-PEG solution was prepared by mixing SH-PEG (100 µL), SH-PEG-SC (20 µL), and DMSO (180 µL). The gold-coated cantilever (NPG 10, spring constant 0.06 N/m, Bruker) was immersed in this solution for 1 hour at room temperature. The SH-PEG provides a flexible, non-fouling linker, while the SH-PEG-SC introduces succinimidyl carbonate groups that enable covalent coupling to primary amines. Following functionalization, the probe was incubated overnight at 4 °C in 400 µL amine-functionalized ssDNA, under light-protected conditions, to ensure stable DNA attachment. For glass coverslip functionalization, coverslip was sequentially sonicated in DI water (3X), 0.1 M EDTA (1X), and ethanol (2X), followed by Piranha treatment (H₂SO₄:H₂O₂, 3:1) for 1 hour to introduce hydroxyl groups. Silanization was performed using 0.5 mL APTES and 0.5 mL acetic acid in 49 mL DI water for 2 hours, followed by drying. The coverslip was then activated with 2.7 mM DSS in 0.5% TEA/chloroform and treated sequentially with BSA-Biotin (3:1), streptavidin, and biotinylated ssDNA, each for 15 minutes, and rinsed with PBS. Finally, AFM force measurements were performed in contact mode using the BioScope Resolve (Bruker) at loading rates of 800, 1400, 2800, and 5000 pN/s across four experiments: High GC Unzipping, Low GC Unzipping, High GC Shearing, and Low GC Shearing. A total of 400 force-distance curves were analyzed for rupture force, binding probability, and kinetic parameters.

Results, Conclusions, and Discussions::

Results:
Mechanical unfolding of ssDNA constructs revealed distinct force thresholds dependent on both loading geometry and GC content (Fig.1 (a,b)). Low-GC constructs demonstrated lower average rupture forces (41.7±3.9 pN) under unzipping forces than high-GC variants (55.3±2.2 pN), which is consistent with reduced base-pair stability. Rupture forces were considerably higher in shearing geometries, where base stacking is more important. The average rupture forces were approximately 62.3 ± 2.2 pN for low-GC shearing and 66.2 ± 3.0 pN for high-GC sequences. The most probable rupture force was identified by Gaussian fitting force histograms for each condition to probabilistic rupture models (Fig.1 (b)). Binding probabilities also varied across conditions (Fig.1 (d)), with Low GC Unzipping showing the lowest value (11.9±1.6%), indicating reduced bond formation efficiency due to weaker base pairing and the unzipping geometry. Similarly, dynamic force spectroscopy also made it possible to estimate the dissociation rate constant (k_off), which showed that k_off values varied across different GC contents and loading geometries (Fig.1 (c)). These variations reflect how molecular stability and bond dissociation dynamics are influenced by both sequence composition and the direction of force application. Overall, these findings show that DNA bonds can be tuned mechanically and kinetically for integration into force-sensitive nanodevices. probable rupture force. (d) Binding probabilities under different GC contents, based on n = 400 force–distance curves.

Conclusion:
The study demonstrates the design of DNA-based molecular machines that respond to biomechanical forces. By varying GC content and loading geometry, it enables the creation of force-sensitive DNA origami systems that can localize to ECM fibrils and release cargo in response to mechanical deformation. This versatility is particularly useful in pulmonary fibrosis, where ECM stiffening and remodeling are dynamic. Measuring the binding probability, the most probable rupture forces, dissociation constant of these ssDNA-ssDNA interactions (both low and high GC content, unzipping and shearing mode) are extremely insightful in promoting optimal design of the DNA origami boxes for targeted drug delivery approaches.

Acknowledgements and/or References (Optional):: 1. Koep, A., Masud, N., Van’t Hul, J., Stanley, C., Nilsen-Hamilton, M., Sarkar, A., & Schneider, I. C. (2024). Design and Assembly of a Cargo-agnostic Hollow Two-lidded DNA Origami Box for Drug Delivery. bioRxiv, 2024-03.
2. Koep, A., Masud, N., Sarkar, A., Nilsen-Hamilton, M., & Schneider, I. (2023, October). Collagen localized nanoscale drug delivery system. Poster presented at the Biomedical Engineering Society (BMES) Annual Meeting, Seattle, WA.