Institute of Bioorganic Chemistry
Polish Academy of Sciences
Structural studies of RNA quadruplexes
composed of AGG and UGG trinucleotide repeats
Magdalena Małgowska
Doctoral thesis written under supervision of prof. dr hab. Zofia Gdaniec
in the Laboratory of Biomolecular NMR
Poznań, 2015
ABSTRACT
The dissertation is devoted to the RNA quadruplexes composed of two and
four AGG and UGG trinucleotide repeats. Quadruplex is a structural motif
characteristic for DNA and RNA sequences rich in guanosine residues. In general,
the NMR structural studies are focused mainly on DNA quadruplexes. Currently,
the RCSB PDB data base contains 100 DNA quadruplex structures and only 5 RNA
quadruplex structures obtained using methods of NMR spectroscopy. This small
number of known RNA quadruplex structures reflects the current level of knowledge
as well as a big potential for this type of research.
The main aim of this work was the determination of the fold of RNA molecules
composed of AGG and UGG repeats. All of the studied molecules exist as
a quadruplexes in solution, regardless of the RNA concentration or the type
of monovalent cation. Nevertheless, only (AGG)2A and p(UGG)2U molecules adopt
single forms in a solution containing potassium cations. For these molecules
the structural models were proposed.
It was also shown, using NMR, UV-Vis, CD spectroscopies and mass
spectrometry, that the quadruplex folds in the solution containing potassium
or ammonium cations are usually similar, but the sodium cations promote
the formation of different quadruplex structures. Additionally, the 1H NMR spectra
were collected for very low RNA concentration, 0.01 mM, typically used
for the UV-Vis, CD or ESI-IMS-MS experiments. For the molecules composed of two
and four AGG or UGG trinucleotide repeats the 1H NMR spectra were also recorded
in the HeLa cells extract in order to test the influence of the cellular environment
on the quadruplex folding. Furthermore, it was demonstrated that the molecules
containing four AGG or UGG repeats are transported through the cell membrane
and are localized in both the cytoplasm and the nucleus.