Computational Probing of Watson–Crick/Hoogsteen Breathing in a DNA Duplex Containing N1-Methylated Adenine

Abstract

DNA breathing is a local conformational fluctuation spontaneously occurring in double-stranded DNAs. In particular, the possibility of individual base pairs (bps) in duplex DNA to flip between alternate bp modes, i.e., Watson–Crick (WC)-like and Hoogsteen (HG)-like, at relevant time scales has impacted DNA research fields for many years. In this study, to computationally probe effects of chemical modification on the DNA breathing, we present a free energy landscape of spontaneous thermal transitions between WC and HG bps in a free DNA duplex containing N1-methylated adenine (m1A). For the current free energy computation, a variant of well-tempered metadynamics simulation was extensively performed for a total of 40 μs to produce free energy surfaces. The free energy profile indicated that, upon the chemical modification of adenine, the HG bp (m1A·T) was located in the most favorable conformation (96.7%); however, the canonical WC bp (m1A·T) was distorted into two WC-like bps of WC* (2.8%) and WC** (0.5%). The conformational exchange between these two minor WC-like bps occurs with the first hundred nanoseconds. The transition between WC-like and HG bp features multiple transition pathways displaying various extents of base flipping in combination with glycosidic rotation. Analysis of the simulated ensemble showed that the m1A-induced changes of the backbone and sugar pucker were in a reasonable agreement with previous results inferred from NMR experiments. Also, this study revealed that the formation of the stable HG bp upon the mutation alters the characteristics of dynamic fluctuations of the neighboring WC residues of m1A. We expect this simulation approach to be a robust computational scheme to complement and guide future high-resolution experiments on many outstanding issues of duplex DNA breathing.

Free Energy Landscape and Conformational Kinetics of Hoogsteen Base-Pairing in DNA vs RNA

“…Enhanced sampling simulation: The newly developed meta-eABF method (39) implemented in NAMD 2.12 through the colvars module (55) has been used to obtain the potential of mean force. The glycosidic angle χ and the pseudo-dihedral angle Θ of the A16-T9 base pair were chosen as order parameters following earlier studies (2,23,26,56). The χ angle denotes torsion of the adenine base with respect to the deoxyribose sugar (defined as the dihedral angle between O4 − C1 − N9 − C4), while the Θ angle captures the flip-out motion of adenine, which leads to extra-helical conformations through base pair breaking (26).…”

“…The glycosidic angle χ and the pseudo-dihedral angle Θ of the A16-T9 base pair were chosen as order parameters following earlier studies (2,23,26,56). The χ angle denotes torsion of the adenine base with respect to the deoxyribose sugar (defined as the dihedral angle between O4 − C1 − N9 − C4), while the Θ angle captures the flip-out motion of adenine, which leads to extra-helical conformations through base pair breaking (26). The collective variable space was spanned from -180 • to 180 • with a 5 • bin width for both the dihedral angles.…”

“…In a nutshell, HG base pairing therefore expands the structural and functional repertoire of reaction coordinate is relatively arbitrary as other collective variables might as well contribute to the conformational transition. Apart from the torsion angles of DNA (2,25,26), H-bond donor acceptor distances can also be considered as a viable reaction coordinate. The conformation of the thymine base is also not included in the current torsion angle based description.…”

“…By simultaneously depositing Gaussian bias and applying force against the potential gradient this method has been shown to be capable of sampling configurational space and predicting reasonably accurate free energy surface in orders of magnitude less simulation time (39).We have used meta-eABF method to calculate the potential mean force (PMF) for the WC to HG transition for the duplex of A6-DNA fragment and also for A6-RNA hairpin, a synthetic construct with the same base sequence used by Zhou et al in their experimental studies (24). Unlike 26) there is no report of the free energy profile of the WC to HG base pair transition in A6-RNA in the literature. Moreover, revisiting DNA HG base pairing with the fast converging PMF calculation technique opens up the possibility to apply this technique in future for a large scale comparison of thermodynamics of HG base pairing in various protein and drug bound DNA complexes.…”

“…In the present paper, we perform a computational comparison of the free energy psurface and transition paths of DNA vs RNA WC to HG transitions, seeking to understand the relative stability of the HG state and the factors that modulate the dynamical equilibrium in DNA vs RNA.The first report (2) of the NMR measured WC-HG dynamic equilibrium also included our preliminary work on searching pathways of conformational transition using a biased molecular dynamics simulation. Further computational work from our group and others (12,(23)(24)(25)(26) have since uncovered several other pieces of information, including energetic details, transition pathways and kinetics of the transition between WC and HG base pairs in naked A-T rich DNA helical duplexes. The paths predicted in our initial study were also recently sampled by Vreede et al(27), who have calculated the rate constants of back and forth transitions between WC and HG base pairs using Transition Path Sampling (TPS) (28).Experimental estimates put the WC-HG transition in DNA on a timescale of 50-250 ms (2).…”