ISBN 9783843926126

978-3-8439-2612-6, Reihe Organische Chemie

Claudia Gerlach
NMR-Studien zu einem RNA:DNA-Duplex mit molekularer Kappe und zu DNA-Hybriden

196 Seiten, Dissertation Universität Stuttgart (2016), Softcover, A5

Zusammenfassung / Abstract

The three-dimensional structure of a DNA:RNA hairpin, whose terminus was bridged by the so-called ogOA cap, was elucidated by two-dimensional NMR and restrained torsion angle molecular dynamics. ogOA stands for an oxolinic acid residue, linked via a glycine L-prolinol phosphate linker to the 5'-terminus of the DNA strand of a DNA:RNA hybrid duplex. In previous studies, a duplex-stabilizing and sequence selectivity increasing effect of this cap had been demonstrated for DNA probes binding to RNA targets. The NMR spectra of the studied hairpin showed that two conformations with different binding modes for the ogOA cap exist in solution. The elucidated structures differ by the conformation of the prolinol glycine unit of the cap. A cis/ trans isomerization in the linker is the molecular basis of the two conformations that both form without considerable loss of stacking of the cap. In both cases, the oxolinic acid residue stacks on the terminal base pair and has molecular contacts with the ribose moiety of the opposite strand. A shape complementarity to Watson-Crick paired terminus of DNA:RNA duplex explains the stabilizing and selectivity-increasing effect of the ogOA cap, and makes it attractive for applications in hybridization probes binding RNA target strands, such as microRNAs.

The assembly properties of branched oligonucleotide hybrids with self-complementary CG or non-self-complementary TC dimer DNA arms were studied by NMR spectroscopy. It was known that hybrids with CG dimers as arms can assemble in aqueous solution into macroscopic assemblies. The aim of the study was to gain insights into whether the assembly is driven by base pairing, or by aggregation between the lipophilic branching elements or 'cores' of the DNA hybrids. The hybrids with self-complementary CG arms showed line broadening in the NMR spectrum. The more CG arms were present in the molecule and the larger and the more lipophilic the core was, the more pronounced the line broadening was. With increasing temperature, the signals sharpened in the NMR spectrum. The addition of acetonitrile to the NMR samples of the DNA hybrids had a small effect on the line width. When the solution was made basic, a decrease in the line widths was observed, indicating the dissociation of aggregates. The NMR spectra of DNA hybrids with non-self-complementary TC dimer arms show that just eight-armed hybrids also contribute interactions between the cores beside the base pairing to the assembly process.