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Investigation of Structural and Chemical Perturbations on Structure-Function Relationships of Biomolecules Using Computational ApproachesAuthor: Tanashree Jaganade Date: 2021-04-23 Report no: IIIT/TH/2021/64 Advisor:Deva U Priyakumar AbstractAccurate prediction of structures and understanding the underlying mechanism behind the dynamics of biopolymers are invaluable to understand various biological applications. The structure and dynamics of molecules are related to its function and have major roles in disease biology, gene regulation, and many other cellular processes. To pave the way towards disease diagnosis, treatment, and faster drug discovery process, it is essential to understand the mechanism behind complex biological processes at the molecular level. Molecular dynamics (MD) simulations and various enhanced sampling simulations are capable of capturing atomistic details of various biological processes such as protein/RNA unfolding, DNA repair mechanism, enzyme kinetics, targeted gene therapy, and membrane transport mechanism. Moreover, molecular simulations provide useful insights to unravel the dynamic nature and thermodynamic stabilities of nucleic acids, proteins, and other complex macromolecules. The findings from this study, provide a detailed understanding of the mechanism behind selected biological phenomena such as urea mediated RNA unfolding, protein unfolding, the role of modified nucleic acids in antisense therapy, and DNA repair mechanism using biomolecular simulation techniques and various theoretical, computational approaches. Techniques like all-atom MD simulations, thermodynamic integration, and umbrella sampling simulations have been employed to elucidate the molecular-level mechanism of these biological processes. The addition of external perturbations to the system of interest is useful to examine the chemical nature, structural details, and dynamic progress of molecules with respect to time. In the current work, we aim to probe the structural, energetic, and thermodynamic aspects of various biomolecules such as nucleobases, amino acids, DNA duplexes, chemically modified nucleic acids, and DNA-protein complexes. The main objective of the current work is to understand the effect of chemical and structural perturbations on the dynamic behaviour and thermodynamic properties of the biomolecules mentioned above to understand their roles in various biological processes. Chemical perturbations like osmolyte (urea) effects, ionic strength effects, and structural perturbations like nucleic acid modifications (backbone or nucleobase modifications) were used to understand their roles in modulating the dynamic behaviour of these micro and macromolecules. The effect of urea on building blocks of RNA and protein i.e. nucleobases and amino acids, respectively, have been investigated to understand the urea mediated RNA and protein unfolding mechanism. Another chemical perturbation such as ionic strength effect on the backbone modified nucleic acid i.e. Peptide Nucleic Acid (PNA) has been studied. In this thesis work, we mainly focus on backbone modified nucleic acids such as PNA-DNA duplexes which are generally known as antisense oligonucleotides. Antisense oligonucleotides are at the forefront of targeted gene delivery for emerging disease therapeutics as it holds promising features like resistance to nuclease degradation, longer halflife compared to naturally occurring nucleic acids, and higher stability in the cellular environment. To study the effect of structural perturbations, the positively charged (Lys-modified) and negatively charged (Asp-modified) side-chains were incorporated on the neutral backbone of the PNA molecules in PNA-DNA duplexes, which has a direct correlation with the binding affinity of these molecules. Apart from the chemically modified nucleic acids, this thesis work also focuses on one of the naturally occurring nucleobase lesions known as thymine glycol (TG). TG lesion is repaired by an enzyme known as DNA Glycosylase through a process called base-flipping. vii viii The DNA-repair mechanism associated with the TG lesion in the absence and presence of enzyme is studied at the molecular level. The first part of the thesis gives detailed understanding about energetics, structural and dynamic properties of building blocks of RNA and proteins i.e. nucleobases and amino acids respectively, in different concentrations of urea solution. With this study, we could elucidate the chemical nature of solute-cosolvent interactions and the driving force behind these interactions. This provides fundamental insights into the comprehensive molecular mechanism behind the urea-assisted RNA and protein unfolding. The next part of the thesis focuses on chemical modifications of nucleic acids. The PNA work showed the binding affinity of PNA strands towards DNA strands is dependent on the chemical nature of the PNA strands. Moreover, our simulation results showed the diverse effect of salt concentrations on the binding affinity of PNA strands towards DNA. This study shows that molecular simulations can help in choosing the correct choice of modification and the optimum salt concentration to get the improved binding affinity of antisense oligonucleotides. This helped us to understand the correlation of binding properties of modified nucleic acids with the type of chemical modification. This also provides, the molecular basis behind this correlation i.e. several factors such as type and position of the modification are crucial to understand the thermodynamic stabilities of these chemically modified nucleic acids. Finally, the study on protein facilitated thymine glycol containing base-flipping dynamics shows the strength of enhanced sampling simulations to get better insights of DNA repair mechanism which involves high-energy barriers. The approach followed in this proposed work to understand various biological processes shows that molecular simulation techniques are effective not only in understating the structure-function relationships at the molecular level but also can be efficiently used to provide a basis for validating experimental observations. Full thesis: pdf Centre for Computational Natural Sciences and Bioinformatics |
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