pi-stacking interaction of nucleobases in DNA: damage, conformation, and biological function

• Applicant: Dr. Alessandra Picchiotti
• Subject Area: Physical Chemistry of Molecules, Liquids and Interfaces, Biophysical Chemistry ; Experimental and Theoretical Physics of Polymers ; Preparatory and Physical Chemistry of Polymers ; Statistical Physics, Nonlinear Dynamics, Complex Systems, Soft and Fluid Matter, Biological Physics
• Term: since 2024
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 545555208

Pi-stacking interaction in DNA is pivotal for biological functions. The mechanisms proposed in the literature on its disruption through reactive oxygen species (ROS) remain unsatisfactory, due to technological limitations regarding space and time resolution, and lack of control over DNA secondary structures. To clarify pi-stacking interaction in DNA, its chemical-physical nature, and its live-stream biological disruption, we need to: 1) Trap DNA molecules: Track and trap DNA molecules with high fidelity of localization, and in an environment that mimics the crowdedness in cells. 2) Monitor the time evolution: Track the structural changes of pi-stacking in DNA as the molecule unfolds, at the natural ultrafast time scales (femto- to picosecond). 3) Control the Pi-stacking: Controlled change of DNA secondary structure (pi-stacking) by electromagnetic fields, and molecular tracking of ROS damage. Here we introduce the Seek and Search technique (S&S), a novel technique capable of answering the open questions and filling the research gaps in our understanding of pi-stacking disruption in DNA. S&S merges concepts from nano-tweezers (biotin-streptavidin functionalization of the bead-DNA complex) with microfluidic chips equipped with pillars that create a laminar flow and trap the bead-DNA complex. An external electric field induces the transition between B-DNA and S-DNA form by pulling the DNA. By varying the voltage, the DNA is overstretched to various degrees until it overwinds, pulling the nucleotides apart while simultaneously rotating them and modifying/disrupting the pi-stacking in DNA. S&S allows for an unprecedented level of control over the chemical-physical interactions with ROS. Time-resolved transient absorption spectroscopy (TR) disentangles the complex steady state spectra that monitor pi-stacking in DNA. It captures a "molecular movie" that tracks the structural changes of pi-stacking interaction by observing the variations in the absorption over time. Finally, with S&S we can investigate the disruption of pi-stacking in real-time by TR spectroscopy, as the ROS molecules damage DNA. We initiate the damage by: -direct UV irradiation with a second continuous UV laser source exceeding the intensity limits for DNA damage, -chemical reactions with solvated electrons and superoxide anion radicals, produced by focused laser light in the trap area of the S&S chip, but not on the DNA molecule, and -hydrogen peroxide, through a specifically designed extra microchannel, in the S&S chip.

• DFG Programme: Research Grants