Introduction

From the beginning of the understanding of the biological sciences, there has been an intriguing question about how organisms can inherit the characteristics of their progenitors. This question was answered by Watson and Crick in their seminal article in 1953 when they proposed

information¹. DNA consists of two helices of nucleotides composed of a sugar-phosphate backbone and four nitrogenous bases, where the helices are bonded together through the formation of hydrogen bonds between adenine-thymine (AT) and guanine-cytosine (GC) nitrogenous base pairs (see Figures 2-1 and 2-2). Although DNA is known to be a very stable molecule, the original genetic information can be modified through mutations, which are responsible for new variations of a trait². The mutations in DNA can happen in several ways, including exposure to radiation (fields, ionizing radiation), free radicals (Hydroxyl radicals), metallic centers (Mg), or mutagenic compounds (benzene) and spontaneous mispairing of base pairs^3-7. A base mispairs of the double-strand DNA in one, or several positions can produce malformation of proteins, which can lead to an adaptive improvement, or a total malfunction of the cell causing cellular death or metastasis

8-10.

In 1963, Löwdin suggested that base pairs in DNA could be responsible for the spontaneous mutation of DNA, where this mutation may occur as a consequence of the DPT reaction between the Watson and Crick base pairs forming rare tautomers such as AT2 GC2^11-13 (see Figures 2-1 and 2-2). These tautomers can subsequently cause base mispairing during the replication process that can lead to a mutation in the DNA producing the loss or modification of the genetic information. Löwdin also suggested that the DPT does not follow a classical reaction path because the protons behave more like a quantum particle. Consequently, these particles can quantum tunnel (QT). Even though Löwdin’s mechanism of QT seems reasonable, the DPT mechanism in DNA has not been observed yet under physiological conditions. Nevertheless, an experimental study performed by Limbach and co-workers suggests that these rare tautomers can exist at low concentrations, thus under physiological conditions, DNA is indeed a very stable molecule¹⁴.

Although Löwdin presents an exciting possibility for point mutations in DNA, more theoretical work needs to be done to understand how this process occurs entirely. Because these macromolecules are not isolated under physiological conditions, there is a great need to understand the effects of the environment of the DNA on the DPT reaction of its base pairs.

It is well known that 30% of water by weight is essential for DNA to maintain its native configuration stable^15,16. It is also known that under biological conditions, nucleic acids are considered charged electrolytes because of the presence of deprotonated phosphate groups in the DNA backbone on the lateral chains. The neutrality of these macromolecules, as well as the cellular environment, is achieved by the presence of monovalent and divalent cations such as Na⁺, K⁺, Ca^2+, and Mg²⁺, which can stabilize or destabilize the double helix structure of DNA^17,18. Among these ions, magnesium is considered the most important because it plays a role in most of the nucleic acid activation processes such as RNA three-dimensional folding, DNA replication, and protein codification^19-22.

The simplest model to understand the mechanism of spontaneous mutation in DNA due to DPT between base pairs consists of studying the pairs in “isolation,” i.e., without the DNA backbone. In this model, the Van Der Walls interactions (stacking) between base pairs can increase or decrease the spontaneous mutations in DNA⁶.However, the activity of spontaneous mutations outweighs the contribution of stacking effects. To get a better picture of these and other mechanisms of spontaneous mutation in DNA, in the first approximation, the physiological conditions (determinant for the stability of DNA) are simulated through the inclusion of water

of the two methods described next: 1) A gas-phase micro-hydrated model where water molecules are added around the base pairs. This approach requires the direct comparison of bonds lengths with the experimental values to accurately describe the final geometry, which depends on the number of water molecules, added to the system where the base pair hydrogen bond interaction happens. 2) A simulated solvation medium – separated multiple solvent spheres – of non-interacting molecules surrounding the substrate of interest. This model is called the polarizable continuum model (PCM) and has been successfully used to simulate solvated environments in chemical and biochemical systems^25,26.

Figure 2-1. Double proton transfer (DPT) reaction in the AT base pairs. This reaction could take place via a concerted (CDPT) or through a stepwise mechanism with two different proton transfer steps (SP1 and SP2) achieving the DPT tautomers

A^* and T^* (AT2 complex).

In the 1960s, Löwdin suggested that the DPT mechanism was a concerted mechanism where both protons in the GC or AT complex are transferred from one molecule to another through tunneling¹². This approach is only sound in the gas phase, where the environment does not weaken the hydrogen bonds between the base pairs. However, in solution, the surrounding water molecules can interact with atoms that present lone pairs, thus causing the weakening of the inter-base hydrogen bonds. While in the GC complex the oxygen atoms O1(C) and O2(G) can interact with the water molecules in its surroundings via hydrogen bonds, in the AT complex the interaction occurs at the O4(T) oxygen atom. The interaction between the solvent molecules and the base pairs dramatically affects how the proton transference occurs in both systems. The transfer can happen via i) a concerted mechanism where, in a single step, two protons are exchanged, forming the AT2 and GC2 complexes (see Figures 2-1 and 2-2) or ii) a stepwise mechanism where only one proton is transferred at a time forming AT1 and GC1.

Figure 2-2. Double proton transfer (DPT) reaction in the GC base pairs. This reaction could take place via a concerted (CDPT) or through a stepwise mechanism with two different proton transfer steps (SP1 and SP2) achieving the DPT tautomers

G^* and C^* (GC2 complex).

It is already known that the presence of water molecules in the neighborhood of the base pairs weakens the hydrogen bonds^5,6,27,28. Consequently, the interaction between the base pairs becomes less critical. This effect results in a decrease in the probability of spontaneous mutations in DNA, which depends on the proximity and interaction between the neighboring water molecules and the base pairs. There are two well-known mechanisms based on the interaction between the water molecules and the base pairs that describe the proton transference reaction^{27, 28}. The first one comprises the direct proton transference between the base pairs without any assistance from the water molecules near the base pairs. This scenario is highly probable when the water molecules are not in direct contact with the base pairs due to spatial interference of the DNA backbone and

stacking interactions. This mechanism is typically and satisfactorily modeled using PCM²². The second one considers the assistance of the water molecules in the transfer of exposed protons in the base pairs in a concerted fashion. This type of reaction tends to require more energy compared to the first mechanism because it needs the breaking and formation of bonds in the partaking water molecules.

Using DFT in the Gaussian 03 (G03) suite, Cerón et al. .^{24, 25} proposed a third mechanism of DPT in DNA in AT and GC simulating a micro-hydrated environment without considering stacking interactions (not in the DNA double-strand). Based on the relative free energy of the tautomers with respect to the transition state at 298K and without considering tunneling corrections in the rate constants, Cerón and co-workers’ results suggest that the only base pair vulnerable to spontaneous mutation in DNA is the GC base pair. In their work, the authors were able to achieve a local minimum for the GC2 structure in a micro-hydrated medium, which exhibits a concerted mechanism. They rationalized that during the reaction, the proton H1(G) is transferred in a first step to the N2(C) of the cytosine, and the water-assisted, simultaneously, the transfer of the second proton from N3(C) to O2(G). On the other hand, the DPT on the AT base pair happens in a stepwise mechanism where H6(T) is transferred from Thymine to Adenine in a first step, and then H2(A) is water-assisted transferred from adenine to thymine in a second step.

To pursue a deeper understanding of the actual mechanisms of spontaneous mutation in DNA due to DPT between base pairs in solution, we decided to study the effect of solvent polarity on this process. In this dissertation, we report on the study of DPT of AT and GC base pairs using

PCM and density functional theory at different levels of theory. The study was carried out in water, methanol, ethanol, 1-propanol, 1-butanol, 1-pentanol, 1-hexanol, and 1-heptanol.