DNA Origami as a Platform for Hybrid Nanostructures

By 2022, DNA origami had matured into a versatile three-dimensional construction platform with sub-nanometre positional precision. At Leipzig, Seidel and co-workers had pioneered DNA origami molds for shape-controlled metallic nanoparticle synthesis — casting gold nanorods inside tubular cavities (Helmi et al., 2014) and developing a modular construction kit for complex metal nanostructures. The platform had been extended to palladium nanostructures (Ye et al., 2021). DNA origami nanopores had been demonstrated in solid-state platforms and lipid bilayers. However, the integration of thermal control with these nanostructures — to reversibly gate molecular access or dynamically reconfigure sensing elements — remained unrealised.

DNA Hybridization Kinetics

Equilibrium thermodynamics of DNA duplex formation were well described by the nearest-neighbour model (SantaLucia, 1998), but kinetic parameters — association and dissociation rates — were poorly characterised. Single-molecule techniques (smFRET, DNA-PAINT) provided dwell-time information for specific systems, but systematic temperature-dependent measurements across diverse sequences were absent. Whether association rates increased, decreased, or remained constant with temperature was actively debated. No high-throughput platform existed to measure sequence-specific kon and koff across temperatures simultaneously.

Strand Displacement Dynamics

Toehold-mediated strand displacement (Zhang & Winfree, 2009) had become the standard mechanism for dynamic DNA nanotechnology. While displacement kinetics had been characterised in bulk at room temperature (Srinivas et al., 2013; Irmisch et al., 2020), the temperature dependence of displacement rate constants and the activation barriers governing strand invasion and branch migration were not systematically characterised. This gap was critical for designing thermally switchable DNA devices that must operate across a range of temperatures.

Plasmonic Sensing and SERS

Surface-enhanced Raman scattering (SERS) in plasmonic nanostructures had achieved single-molecule sensitivity. DNA origami provided scaffolds for assembling nanoparticle dimers with defined gap sizes (~2–10 nm), creating electromagnetic hotspots with enhancement factors exceeding 108. However, systematic methods for reproducible analyte positioning and orientation control within hotspots were lacking. The DNA origami mould technology offered a promising route for casting metal nanostructures with programmable gap geometries, but quantitative integration with thermally switchable DNA elements had not been demonstrated.

Temperature Measurement at the Nanoscale

Fluorescence thermometry using Rhodamine B and other temperature-sensitive dyes had been demonstrated in microfluidic contexts, but integration with TIRF microscopy for surface-temperature calibration during single-molecule experiments was not established. Sub-diffraction temperature mapping — essential for characterising thermal gradients around plasmonic heaters — remained an unmet challenge.

Selected Output

• N. Kumar, F. Ricci, R. Seidel, P. Irmisch. "Engineering the temperature response of DNA strand displacement." Nature Communications [Under Review]
• N. Kumar, A. Overchenko, A. Sivaraman, C. Bastiaanssen, P. Irmisch, F. Cichos, R. Seidel, C. Joo. "Temperature- and sequence-dependent DNA hybridization kinetics from high-throughput single-molecule measurements." [In Preparation — Nature Chemistry]
• N. Kumar et al. "Probing sequence-specific DNA hybridization dynamics using ThermoSPARXS." Biophysical Journal, 125(4), 287a (2026).
• S. Banerjee, C. Hadlich, M. Scherf, N. Kumar, R. Seidel, J. Kneipp. "SERS detects site-specific labeling of DNA nanocages via hexynyl modifications." [In Preparation]
• N. Kumar, A. Sivaraman, P. Irmisch, D. Renger, C. Joo, R. Seidel. "DNA Origami-Enabled Single-Molecule Ratiometric Thermometry with Nanoscale Spatial Resolution." [In Preparation]

Advances in DNA Origami–Templated Nanostructures

The DNA origami mould approach has expanded to palladium nanowires with metallic conductivity (Kemper et al., 2023; Rodriguez-Barea et al., 2026) and gold nanoparticle multimers for ultrasensitive SERS. DNA origami nanopore technology has evolved through three structural phases: hybrid designs with solid-state platforms, vertically inserted nanopores, and horizontally arranged nanopores with functional modifications (aptamers, enzymes) for selective detection. The integration of DNA origami with whispering gallery mode (WGM) sensors has enabled label-free single-molecule DNA hybridization detection (Ghamari et al., 2025).

Sequence-Resolved Single-Molecule Kinetics (ThermoSPARXS)

The development of ThermoSPARXS within this project has enabled the first simultaneous measurement of kon and koff for 79 DNA probe sequences across ten temperatures (26–58°C). The key finding — that koff is strongly temperature- and sequence-dependent while kon is nearly invariant — provides quantitative design rules for engineering thermal switching thresholds. Complementary advances include DNA calorimetric force spectroscopy (Ritort group, 2024–2025) and temperature-jump infrared spectroscopy of dinucleotide hybridisation kinetics (Szostak group, 2023).

Temperature-Dependent Strand Displacement

Within this project, the temperature dependence of toehold-mediated strand displacement has been systematically characterised, revealing a striking non-monotonic behaviour: rates initially increase with temperature but decrease at elevated temperatures due to the interplay between reversible toehold hybridisation and irreversible strand replacement. A Markov chain model successfully describes this behaviour across different toehold lengths (5–10 nt). Invader-target mismatches were shown to enhance temperature sensitivity, providing design handles for thermally responsive DNA devices.

Hybrid Plasmonic DNA Nanopores for SERS

Gold nanoparticles have been synthesised inside DNA origami nanopores through templated seeded growth, creating hybrid plasmonic nanocages. Four cage variants with alkyne reporters at different positions (inner cavity, outer lid, outer mold) were characterised by SERS at 633 nm excitation. The terminal alkyne reporter was detected in all functionalised variants, with spectral response strongly sensitive to reporter position — the inner cage showed intimate metal–DNA coupling while the outer lid cage produced the highest signal intensity. These results establish the DNA origami nanocage as a programmable SERS platform. Systematic studies of molecular orientation effects on SERS using DNA origami antennas (Kanehira et al., 2026) have further advanced the field.

Single-Molecule Thermometry

A ratiometric fluorescence thermometry approach using Alexa 647 / Atto 647N dye pairs has been developed and validated at both bulk and single-molecule levels. A DNA origami-based super-resolution thermometry platform — using DNA-PAINT to resolve individual reporter sites with ~30 nm precision — has been demonstrated, enabling sub-diffraction temperature mapping. These tools provide the thermal characterisation infrastructure needed for all temperature-dependent sensing modalities within the DYNAMO framework.

Convergence and Outlook

The current state of the art is characterised by the convergence of previously separate fields. DNA origami–templated plasmonic nanostructures can now be fabricated with increasing complexity. A comprehensive kinetic framework for DNA hybridization and strand displacement exists to rationally design thermal switching behaviour. Programmable SERS detection within DNA origami nanocages has been demonstrated. And validated thermometry tools enable precise nanoscale temperature characterisation. The remaining challenges include scaling to integrated hybrid devices, translating design rules from surface-tethered to nanopore-confined geometries, and combining optical (SERS/fluorescence) with electrical (ionic current) readout in a single platform.

Selected Output

• N. Kumar, F. Ricci, R. Seidel, P. Irmisch. "Engineering the temperature response of DNA strand displacement." Nature Communications [Under Review]
• N. Kumar, A. Overchenko, A. Sivaraman, C. Bastiaanssen, P. Irmisch, F. Cichos, R. Seidel, C. Joo. "Temperature- and sequence-dependent DNA hybridization kinetics from high-throughput single-molecule measurements." [In Preparation — Nature Chemistry]
• N. Kumar et al. "Probing sequence-specific DNA hybridization dynamics using ThermoSPARXS." Biophysical Journal, 125(4), 287a (2026).
• S. Banerjee, C. Hadlich, M. Scherf, N. Kumar, R. Seidel, J. Kneipp. "SERS detects site-specific labeling of DNA nanocages via hexynyl modifications." [In Preparation]
• N. Kumar, A. Sivaraman, P. Irmisch, D. Renger, C. Joo, R. Seidel. "DNA Origami-Enabled Single-Molecule Ratiometric Thermometry with Nanoscale Spatial Resolution." [In Preparation]