Progress Along the Three Research Axes
1 • Thermophoresis
We established a robust strategy to reproduce thermophoretic settings using atomistic molecular dynamics simulations, which we now routinely apply to various small, neutral solutes in aqueous solutions. Our data collection covered many systems, allowing us to assess how the thermophoretic behavior of a molecule depends on factors such as size, water affinity, mass, and concentration. These observations suggested that existing models and theories may not accurately describe molecular solutes in water under thermal gradients. While we attempted to develop new models to predict molecular behavior in these conditions, this work remains ongoing and inconclusive thus far.
2 • Phosphoester Bond Formation
We addressed the complex issue of phosphoester bond reactivity—a reaction with unfavorable energetics and slow kinetics. Initially, we employed a semi-empirical strategy for atomic interaction modeling. After significant effort in designing reaction coordinates and sampling methods, we applied this setup to a simple model reaction involving a small alcohol and phosphate. We identified two distinct reaction pathways and mechanisms but also uncovered key limitations in our approach.
To overcome these limitations, we shifted to machine-learned interatomic potentials, which are increasingly popular in molecular simulations. However, challenges remained in selecting chemical structures for the training dataset and identifying relevant collective variables for sampling. We successfully addressed both issues and studied the mechanism of uncatalyzed phosphoester bond formation in unprecedented detail. We are currently investigating how pH and activating leaving groups affect the reaction.
Additionally, we developed ArcaNN, a freely available active learning protocol and software, which is now widely transposable to other chemical reactions. Lastly, we initiated a related research axis on phosphoester bond formation in autocatalytic RNA networks involving small ribozymes.
3 • RNA Duplex Stability
We focused on the chemical alterations of RNA sugar backbones, beginning with one of RNA’s most crucial properties under abiotic conditions—duplex stability during the final stages of RNA replication. Without enzymes, strand separation is challenging and typically requires high temperatures. To explore how chemical modifications could later influence strand separation, we first developed a robust strategy to assess RNA duplex separation as temperature increases.
Building on a method we had previously applied to proteins, we observed exciting results on small model strands of varying sequences. Our findings showed that the widely-accepted two-state, all-or-nothing model (in which strands are either fully associated or fully separated) needs refinement. Instead, the separation mechanism is more gradual, with significant base fraying at duplex ends well before full unfolding. We characterized the sequence- and size-dependence of this fraying effect.
Finally, we developed forcefield parameters to study chemical modifications of RNA duplex backbones. Although investigations into how these modifications impact separation mechanisms and melting temperatures are ongoing, early results are promising.
