Computational study of DNA in non-canonical environment

During my PhD thesis used theoretical techniques, in particular Molecular Dynamics to study the structural properties of nucleie acids in non-canonical environment, especially in the gas phase and apolar conditions. The highly charged nature of the nucleic acids backbone clearly suggests that the environrnent plays a key role in the behavior of these molecules. As the DNA is found in aqueous solution under physiological conditían, the vast majority of published works on nudeic acids naturally investigates its behavior under these conditions. Water is excellent to stabilize DNA, but it is not the ideal solvent for favoring specific recognition, certain reactions or physical processes such as charge transfer. Interest exists then to explore the nature of nucleic acids, particularly DNA, in non-•aqueous solvents, where the universe of nucleic acids applications will expand even more. In the first part of this thesis I used atomistic molecular dynamics simulations to investiga te the structural and thermodynamics changes of a DNA hairpin when transferred from an aqueous solution to a low dielectric media, carbon tetrachloride (CTC), under different DNA charge states. I simulated the pulling of a short DNA hairpin from a water compartment through a CTC slap and estimated the free energy related to the transfer of the DNA from water to CTC through atomistic Umbrella Sampling simulation. The second part of my thesis is centered in the challenge af the mast recent experimental techniques, such as Mass Spectrometry and X-Ray Free Electron Laser (XFEL) which use gas phase ions to provide structural information of macromolecules. They are fast and require low sample consumption but the question is to what extent does gas phase structural information renect the most populated conformation in solution. A series of both experimental and theoretical studies with proteins have demonstrated that gas phase ensembles can be used to accurately madel the solution structure. The question is then, whether or not these findings also stand for a highly flexible and charged non-globular molecule as DNA, depends on the solvent environment. An analysis of the energetics oE DNA suggests that in the absence of solvent screening DNA should unfold, but experimental studies clearly points in the opposite. I used MD simulations to characterize the conformational ensemble of non- canonical structure of DNA, such as triple-stands and hairpins DNA, in the gas phase, validating the results by means of state of the art mass spectroscopy experiments. My results suggest that the ensemble of DNA triplex-strand structures in the gas phase is well defined over the experimental time scale, with the three strands tightly bound. Triplex DNA in the gas phase maintains memory of the solution structure, well-preserved helicity, and a significant number of native contacts. As a breakthrough I considered a very small model of a DNA hairpin containing 2 duplex steps, and combined extended Replica Exchange MD simulations, quantum mechanical Car-Parrinello MD calculations and IMS-MS experiments, to reveal a picture of unprecedented quality on the nature of DNA in the gas phase. I explored the whole ensemble of DNA duplex in the vacuum conditions on millisecond timescale and studied the impact of quantum effects in the structural ensemble of DNA in the gas phase to investigate on proton-transfer process which occurs during vaporization, crucial to understand structural distortions of nudeic acids in vacuum conditions. I conclude that the conformational life of DNA in the gas phase seems richer than previously anticipated. The classical picture of DNA in the gas phase as a frozen structure with rigid topology must be revisited. A mobile proton model, such as now widely accepted to explain peptide fragmentation, also entirely make sense for oligonucleotides.Durante mi tesis doctoral he utilizado técnicas teóricas, en particular dinámica molecular, para estudiar las propiedades estructurales del ADN en medios no canónicos, como medios apolares y el vacío. La naturaleza altamente cargada del esqueleto del ADN sugiere que el solvente juega un papel clave en el comportamiento de esta molécula. El agua, su medio natural, es un excelente estabilizador de su estructura, pero no es el ideal para favorecer ciertas reacciones o procesos físicos, como la transferencia de carga. Existe un fuerte interés en explorar la naturaleza de los ácidos nucleicos en solventes no acuosos, donde el universo de aplicaciones del ADN se expandirá aún más. En la primera parte de mi tesis he usado métodos computacionales, en concreto Dinámica Molecular y Umbrella Sampling, para investigar los cambios estructurales y termodinámicos de una horquilla de ADN al transferirse de una solución acuosa a otra de tetrac1oruro de carbono (TCC) cuya constante dieléctrica es 40 veces menor, estimando el coste en energía libre relativa asociado a dicho proceso. La segunda parte de la tesis se centra en técnicas experimentales recientes como la Espectrometría de Masas y X•Ray Fee Electron Laser que utilizan iones en fase gas para obtener información estructural de las macromoléculas. Tales técnicas son rápidas y requieren una poca cantidad de muestra, pero ¿hasta qué punto la información estructural en fase gas es representativa de la conformación más poblada en solución? Estudios experimentales y teóricos con proteínas han demostrado que la estructura en fase gas puede representar con precisión la estructura en solución. ¿Estos hallazgos valen también para una molécula no globular, flexible y altamente cargada como el ADN, cuya estructura depende mucho más del entorno? Gracias a la combinación de extensas simulaciones de Dinámica Molecular clásica y cuántica (Car-Parrinello MO) y la validación de los resultados por espectroscopia de masas, en esta tesis doy una descripción completa y sin precedentes de la estructura y la naturaleza del ADN en el vacío