Modelos mesoscópicos para DNA mediado por metal e na presença de solventes que simulam o meio molecular

Abstract

Nucleic acids, in special DNA and RNA, have undeniable technological and biological importance. Here, we develop two projects linked to the stability of nucleic acids. In the first, we discuss the effects of metallic ions that bind between the base pairs of DNA (M-DNA), and which has technological potential to be used as biosensors. In the second, we address the effect of polyethylene glycol, that simulates the crowding effect in the denaturation of DNA, an important problem in molecular biology. For the development of these projects we use UV published melting. In addition, we use mesoscopic models, mainly the Peyrard-Bishop (PB), to calculate the interactions present in the system. M-DNA is a DNA molecule where the hydrogen bond between the bases is changed due to the presence of a metallic ion. In natural DNA, only Ag+ and Hg2+ have a stabilizing effect. However, there is no clear understanding if these ions stabilize the DNA via a direct bond, covalent bond base-metal-base, or via another mechanism such as interplanar interactions, stacking between the nearest-neighbouring bases. We know that Ag+ stabilizes the cytosine-cytosine (CC) mismatch, while Hg2+ stabilize the thymine-thymine (TT) mismatch. In other words, the cations stabilize mismatches, who are otherwise quite unstable. Our results show that Hg2+ stabilizes the DNA via the pair base-metal-base which has a strength similar to a CG pair, while for Ag+ the stabilization process is the same, however with a strength somewhat weaker than an AT base pair. The small stacking interactions of CC-CC for Ag+ and TT-TT for Hg 2+ does not support interplanar interactions which were hypothesized by some authors. Therefore, mesoscopic models can conclusively explain the origin of the metal ion stabilizing effect from experimental data available. To understand the intramolecular effects in DNA and RNA, in vitro experiments are com- monly performed in saline solutions, free of other compounds. This is very different from the cell environment that has a myriad of molecules, the cell volume is composed of 20-40% of micro- and macro-molecules. Therefore, it is not know how far the results of the experiments can explain what really happens on the molecular environment. To study these effects it is customary to add polyetylene glycol (PEG), which is available in a variety of molecular weights and therefore offers a simple and controlled way to experimentally simulate the effect of micro-and macro-molecules on the nucleotides. PEGs with molecular weights lower than 1000 (PEG200, for instance) behave like micromolecules and destabilize the DNA, while PEGs with molecular weights greater act as macromolecules and stabilize the DNA. Several authors proposed modifications to the PB model by adding a term in the Morse potential to describe the interaction of nucleotides with the solvent, water in particular, but these models have never been subject to validation with experimental data. Only recently have enough experimental data become available to study whether the models actually describe the effect that H2O molecules have on nucleotides, giving us the opportunity to apply the PB model in this type of system. In this thesis, we compare DNA (low salt concentration, [Na+ ]=100 mM) and RNA (low, [Na+ ]=100 mM, and high, [Na+ ]=1000 mM, salt concentration) aqueous solution in two situations: with PEG200 and without PEG200. Our results confirms that RNA is more hydrated than DNA. The energy difference between AT and CG, DNA, and AU e CG, RNA, increases when we add PEG200, but in RNA the energy difference is much smaller. Large energy differences between the base pairs cause distortions in the conformation of the double helix. In this case, DNA in the presence of PEG200 may be assuming another type of conformation. DNA B-type helix, characteristic in water solutions, changes to an A-type helix, characteristic in solutions with less water available. AT energy values with and without PEG200 are practically the same. This result confirms that homogeneous AT DNA helices assumes only B-type conformation regardless of the hydration change. Among the barrier-modified PB models analysed, only the HMS− was found to adequately predict melting temperatures. In the absence of PEG200 and low salt concentrations the barrier energies were found to be the same. Different to what happens at high salt concentrations, where the CG barrier is a little higher than the AU barrier. RNA concentrates more ions and it is more hydrated than DNA, for this particularly reason we observe a reduction in the barrier energy with PEG200. The HMS− model was found to provide a satisfactorily description of the nucleotide-solvent-nucleotide interaction. Therefore, we found a modified PB model that can explain the intermediate state in the duplex separation process, absent of the standard PB model.