Nmr studies of the chiral and chemical interactions of self-assembled guanosine monophosphate

Among the nucleobases in RNA and DNA, guanine alone exhibits a reversible self-assembly through Hoogsteen hydrogen bonding. The resulting "G-tetrads" are building blocks of higher-order structures formed by guanosine compounds, including guanosine-5'-monophosphate (GMP). These tetrads can associate through pi-pi stacking to form chiral, columnar assemblies that may be further stabilized through centrally-located cations, typically K+. This alternative arrangement of GMP competes with its incorporation into RNA, an interesting factor for the abiotic polymerization of RNA. This dissertation explores the chiral selectivity exhibited by GMP assemblies, and their interactions in nucleotide mixtures. Previous work in the McGown group demonstrated that GMP can separate enantiomers using capillary electrophoresis (CE). However, the quality of chiral selection was dependent on the extended structure of each solute, for example, easily resolving enantiomers of tryptophan, but not amino acids with analogous, but truncated aromatic side chains (phenylalanine and tyrosine). Thus, investigated here are the structural features of GMP assemblies and their interactions with chiral compounds to reveal the basis for enantioselectivity. Solution-state 1H nuclear magnetic resonance (NMR) was used to map site-specific changes in the chemical shifts within both GMP and the chiral solutes. The results presented here are consistent with the CE elution profiles through assembled GMP. Substrates exhibiting longer migration times showed the largest absolute chemical-shift response (Deltadelta). Likewise, those with the best chiral resolution exhibit the greatest enantiomeric contrast by NMR, i.e. Delta(Deltadelta). The site specificity of chemical-shift changes suggests that grooves in stacked GMP assemblies are the templates for chiral selection. Molecular models are presented in order to visualize and evaluate the proposed mode of selection. These models are prepared using templates of self-assembled GMP, including previously reported structures of helical 5'-GMP and stacked G-quadruplex RNA. These models emphasize the logical basis behind and the attributes of the hypothesized modes of chiral discrimination by GMP assemblies, and provide a starting point for computational work. Finally, 1H NMR was used to study the interactions of GMP in mixtures of adenosine 5'-monophosphate (AMP) and cytidine 5'-monophosphate (CMP). Site-specific changes in chemical shift were used to identify the most interactive sites of each species, thereby providing insight into their interaction.