This thesis is based on nine research publications (I – IX) on structure and reactivity of RNA vis-à-vis DNA. The DNA and RNA are made of flexible pentose sugar units, polyelectrolytic phosphodiester backbone, and heterocyclic nucleobases. DNA stores our genetic code, whereas RNA is involved both in protein biosynthesis and catalysis. Various ligand-binding and recognition properties of DNA/RNA are mediated through inter- and intra-molecular H-bonding and stacking interactions, beside hydration, van der Waal and London dispersion forces. In this work the pH dependant chemical shift, pKa values of 2′-OH group as well as those the nucleobases in different sequence context, alkaline hydrolysis of the internucleotidic phosphodiester bonds and analysis of NOESY footprints along with NMR constrained molecular dynamics simulation were used as tools to explore and understand the physico-chemical behavior of various nucleic acid sequences, and the forces involved in their self-assembly process. Papers I – II showed that the ionization of 2′-OH group is nucleobase-dependant. Paper III showed that the chemical characters of internucleotidic phosphate are non-identical in RNA compared to that of DNA…
Contents
1. Physicochemical properties of Nucleic acids
1.1 Structure of Nucleic acids
1.2 Reactive groups in Nucleic acids
1.3 Forces underlying the stacking interactions of nucleobases in DNA and RNA
2. Reactivity of the 2′-hydroxyl group in RNA
2.1 Importance of the 2′-hydroxyl group in RNA
2.1.1 Role of the 2′-hydroxyl group in recognition
2.1.2 Role of the 2′-hydroxyl group in processing and catalytic properties of RNA
2.1.3 Role of 2′-hydroxyl group in stabilization of RNA tertiary structure
2.2 Variability in the experimentally determined pKa values of 2′-OH group of RNA
2.3 Present work (Papers I – II)
2.3.1 Determination of pKa of the 2′-OH group in nucleosides, mono- and dinucleotides and 3’ĺ5′ monophosphates by pH titration studies
2.3.2 Variation in pKa values of 2′-OH group in nucleosides and mononucleotides with varying 3′ substituents and aglycones
2.3.3 The effect of aglycone on the pKa of the internucleotidic 2′-OH group in diribonucleoside (3’ĺ5′) monophosphates
2.4 Implications
3. Reactivity of phosphodiester group in RNA
3.1 Factors affecting nonenzymatic base-promoted degradation of RNA
3.2 Importance of electron density around phosphate in RNA
3.3 Present work (Paper III)
3.3.1 Reflection of ionization of pseudoaromatic 9-guaninyl group on neighboring phosphate groups in ssDNA and ssRNA
3.3.2 Deshielding of phosphorus resonances in the alkaline pH
3.3.3 Non-identical electronic environment around internucleotidic phosphates in ssRNA compared to isosequential ssDNA
3.3.4 Variable 9-guaninyl pKa values from different phosphate markers in heptameric ssRNA
3.3.5 Study of alkaline hydrolysis of heptameric ssRNAs in
comparison with their G N1-methylated counterparts
3.4 Implications
4 S-S interactions between stacked nucleobases in ssRNA
4.1 Different types of aromatic interactions
4.1.1 Predominating forces involved in inter and intramolecular aromatic interactions
4.1.2 Aromatic interactions in nucleic acids
4.2 Stabilization of nucleic acid structure by base stacking
4.3 pKa perturbation in biomolecules
4.4 Present work (Papers IV – VI)
4.4.1 Cross-modulation between nucleobases of dimeric and oligomeric ssRNA
4.4.2 pH titration studies of dimeric and oligomeric ssRNAs
4.4.3 Nearest-neighbor interaction between nucleobases and free energy of offset stacking in ssRNA
4.4.4 An explanatory model for pKa perturbation in single stranded oligonucleotides
4.4.5 Shift in pKa value of the 9-guanylate in a ssRNA sequence
compared to its corresponding monomer unit
4.4.6 Different pKa value in nucleobase due to dissimilar electrostatic
interaction in 3′- versus 5′-phosphate
4.4.7 Variation in the pKa values of 9-guaninyl as obtained from different marker protons of nucleobases across the ssRNA single strand
4.5 Implications
5 Sequence specific recognition in nucleic acids
5.1 Sequence specific interactions of ssDNA and ssRNA with proteins46
5.2 Sequence specific ligand binding of aptamers
5.3 Present work (Paper VII)
5.3.1 pH titration studies on trimeric and heptameric ssRNA as well as ssDNA
5.3.2 Sequence dependant pKa modulation of the central 9-guaninyl (pKa1) in heptameric ssRNA and ssDNA sequence
5.3.3 The electrostatic cross modulation and pKa perturbation of 9-guaninyl (pKa2) among neighboring nucleobases in heptameric ssRNA and ssDNA sequence
5.4 Implications
6 Importance of single-stranded and duplex structures in functioning of nucleic acids
6.1 Role of single and double stranded nucleic acids in biological process
6.2 Characterization of single-stranded nucleic acid structures using NMR spectroscopy and NMR constrained Molecular Dynamics
6.3 Present work (Papers VII-IX)
6.3.1 Observation of right handed helical pattern in ssDNA and ssRNA
6.3.2 NMR constraints and Molecular dynamics protocol for structural analysis of ssDNA and ssRNA
6.3.3 Difference in stacking geometry of ssDNA vs. ssRNA as observed from NMR constrained MD simulation
6.3.4 Extent of stacking versus base pairing forces involved in relative
stability of RNA-RNA compared to DNA-DNA duplex, estimated from the pKa calculation of the model mononucleotides
6.4 Implications
Acknowledgements
Summary in Swedish
References
Author: Acharya, Sandipta
Source: Uppsala University Library
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