https://pubs.rsc.org/en/content/articlehtml/2025/nr/d5nr02731d

Fundamental investigations of ice nucleation, a key process in fields from environmental science to cryobiology, require model systems with chemical and physical structures that are well defined and easily varied. DNA origami is an especially promising model because of the exquisite control that it offers over the physical geometry of the nucleating agent at the nano-scale. Here we compare ice nucleation by solutions of a rectangular DNA origami tile, formed by annealing a 2.6 kbase single-stranded DNA scaffold with ninety shorter ‘staple’ oligonucleotides, to ice nucleation when these components are mixed at the same concentrations but not annealed. Isothermal measurements show that the molecular conformation has a dramatic effect on the ice nucleating efficiency. For an array of droplets containing annealed, well-folded tiles the freezing rate is constant, whereas for unannealed DNA the freezing rate decreases with time. Despite the freezing rate measured at low temperature being higher for the annealed DNA origami samples than for a significant proportion of the unannealed ones, in slow temperature-ramp measurements the latter generally freeze at higher temperatures. We show that this behaviour is consistent with the formation of small numbers of highly efficient nucleating agents in the unannealed samples, likely through molecular aggregation.

Conclusion:

The ice nucleating efficiencies of fixed concentrations of unannealed DNA scaffold and staples are very different from those of the same molecules folded into origami tiles. This is a direct demonstration of the importance of molecular conformation for ice-nucleating biomolecules. Isothermal measurements show time-dependent (decreasing) freezing rates for the unannealed DNA sample and constant freezing rates for annealed DNA origami, indicating that the INAs that dominate ice nucleation in the former are at much lower concentration than those in the latter. Although at low temperatures the freezing rate for annealed DNA origami is higher than for unannealed samples, in temperature ramp measurements, on average, the latter freeze first, if the cooling rate is sufficiently slow. Classical nucleation theory (CNT) explains this phenomenon and provides approximate fits to the data. Our results show the potential of DNA origami as a model ice nucleating agent. DNA origami offers the possibility of studying more complex INA geometries and is amenable to tailored surface functionalization: we believe that it will prove a very valuable tool for future ice nucleation studies.