Real‐Time Atomistic Description of DNA Unfolding

Abstract

Despite recent efforts, the folding/unfolding of DNA (understood as a dramatic conformational change from the native conformation in a significantly large portion of the duplex) is still poorly described. A range of classical physical studies led to the assumption that the unfolding of short DNA fragments is reversible and follows a two-state mechanism, starting at d(A·T) pairs. [1][2][3] Nevertheless, this traditional view has been recently challenged [4][5][6][7][8][9] by ultrafast techniques, which suggested a more complex scenario where compact intermediates are detected during the microsecond-long unfolding process. [4] Unfortunately, these experiments were not able to provide atomistic-detailed information on the process, thus making necessary the use of simulation techniques (mainly molecular dynamics, MD) as complementary tools. For computational reasons MD simulations of unfolding have typically followed indirect approaches, such as the use of multiple short trajectories, [10,11] replica exchange, [12,13] or moderately large (100 ns) simulations under nonphysical denaturing conditions (T = 400 K). [14,15] These simulations provided clear evidence of the complexity of DNA unfolding but were unable to define a mechanistic view, which would require multiple very large unbiased trajectories. Herein, we present a full atomistic description [16] of the unfolding of a full turn of DNA under realistic denaturing conditions. The study, a real "tour de force" for MD, provides for the first time a detailed atomistic picture of DNA unfolding in the microsecond timescale.All simulations were performed using Dickersons dodecamer [17] (DDD; Protein Data Bank (PDB) code 1BNA), a well-studied model of a short nonhairpin duplex. To guarantee unfolding on the microsecond timescale, [10,11] we simulated strong (but realistic) denaturing conditions by adding a high concentration of a chemical denaturant (pyridine; Pyr) and increasing the temperature to nearly the boiling point of water (these simulations are coded as PHT). Control simulations were performed that considered: 1) water at low temperature (WLT), where we expected no signal of unfolding on the microsecond timescale; 2) a mixture of water and Pyr at low temperature (PLT), where we expected the beginning of unfolding; and 3) simulations in water at high temperature (WHT), where we expected partial unfolding. WLT and PLT microsecond simulations were performed at T = 300 K considering pure water (5000 water molecules) and a 1:1 (by mass) Pyr/water mixture (3923 water and 760 Pyr molecules), respectively. Simulations in hot solvent (WHT and PHT) were performed at T = 368 K using the same simulation boxes as before (for simulation details, see the Supporting Information). To gain better statistics, ten replicas were performed under PHT conditions for at least 1 ms. Simulations were performed using state-of-the-art simulation conditions (see the Supporting Information), the parmbsc0/parm99 [18] force field for DNA, TIP3P [16] for water, and a new force field for Pyr der...

Structure and Properties of DNA in Apolar Solvents

“…24 Experiments and theory have shown that this oligonucleotide folds on a (multi)microsecond time scale 25−27 in water, and should unfold on a similar time scale in a denaturant solvent such as pyridine. 22 The small size of the system accelerates calculations, favors sampling, and reduces memory effects, which might be very important for longer oligonucleotides. Previous studies have demonstrated the ability of current force field and MD protocols to simulate this structure properly in a variety of environments.…”

Theoretical studies of nucleic acids folding

“…Because of the long timescales involved in the structure formation process of nucleic acids the reverse process of unfolding stable structures has been studied using MD simulations at elevated temperatures . The increased simulation temperature can significantly accelerate conformational transitions and shifts relative free energies of conformational states and can lower free energy barriers.…”

“…Similar to unfolding of proteins, the study of the unfolding process of nucleic acids can also give insights into putative intermediate states of the folding process. The method has been used extensively to study the coupled dissociation and unfolding of a DNA duplex dodecamer and the unfolding of nucleic acid hairpin structures . Alternatively, unfolding can be induced by applying an external force on the molecule to disrupt a key interaction that stabilizes the 3D conformation.…”

Recent successes in coarse‐grained modeling of DNA

“…In the view of the complexity of problems related to the DNA physics, computer modeling has always played an important role in bridging the gap between the bulk experiments and molecular‐level understanding. The currently available all atom (AA) force fields have significantly evolved since their inception in the 1980s21,22 and are now able to reproduce a diverse set of subtle structural fluctuations that take place from pico‐ to microsecond timescales 23–27…”