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RNA is a biopolymer with a negatively charged phosphate backbone. RNA sequentially folds into compact
tertiary structure and performs several biological functions, hence required counterion to mitigate electrostatic
phosphate backbone repulsion. The different mode by which a counterion interacts with RNA microscopically
can be well characterized by using atomistic molecular dynamics simulations, free-energy calculations,
enhanced sampling methods such as well-tempered metadynamics. This thesis abstract contains five
interconnected chapters from application of RNA to the effect of ion-chelation on RNA structure-function. In
the first chapter, we have described the motivation of the thesis, applications, scheme of RNA folding and
preferential ion binding sites in RNA based on charge density of counter ions. In the second chapter we have
discussed some of the bullet points about issues in simulating RNA and force field, so as to minimize issues we
have taken simple dimethyl phosphate molecules (DMP) with direct contact of a magnesium mimicking RNA
phosphate backbone. Activation barrier height calculations for this model system using
amber99_parmbsc0_chioL3_po force fields reflects experimental agreement. The third chapter characterizes
different modes of counter-ion interaction present in the real complex RNA from simple pseudoknot (PK)
containing Beet Western Yellow Virus to multiple PK containing Flaviviral. The radial distribution function
(RDF) of the crystal structure reveals three peaks at distinct positions which are (i) direct Mg 2+ coordination (ii)
outer-sphere (iii) multiple solvent separated Mg 2+ -coordination. We incorporated a little complex system that is
two DMP molecules in direct coordination with magnesium. Since, increase in complexity demands an
enhanced sampling technique like well-tempered metadynamics. It reveals four distinct states (i) Chelated (ii)
Pre-Chelate 1(PC1) (iii) Pre-Chelate 2 (PC2) (iv) outer sphere. Pre-Chelate states (PC1 and PC2) are stabilize by
the “meta-sphere coordination” 1 . Local phosphate oxygen shows dangling behavior and allow inclusion of water
molecules. In fourth chapter we would like to validate the existence of the Pre-Chelate complex in real complex
RNA system like SAM 1 aptamer RNA, for that we have calculated free energy of magnesium chelation, which
reveals five distinct state (i) Chelated (ii) Pre-Chelate 1 (PC1) (iii) Pre-Chelate 2 (PC2) (iv) Pre-Chelate 3 (PC3)
(v) outer sphere magnesium coordination. We observed that local phosphate oxygen showing dangling behavior
as observed in the model system, this validates the existence of the Pre-Chelate complexes in SAM 1 aptamer
RNA. Chapter fifth deals with effect of chelation on the SAM1 aptamer RNA, regulates transcription process.
XRD crystal structure of SAM 1 aptamer RNA revealed two site specific magnesium (i) core chelated (ii)
exposed chelated 2 . But, in a cellular environment there exist outer and inner sphere magnesium coordination.
Our phosphorothioate interference mapping experiment validates magnesium binding sites for simulated
structure as well as XRD crystal structure site specific magnesium chelation. RMSF calculations confirm that
present of both core chelated and exposed magnesium is necessary to maintain tertiary structure of the SAM 1
aptamer riboswitch. Absence of any core chelated or exposed magnesium leads to disordered structure. |