Studies of G-quadruplex DNA structures at the single molecule level

Research output: Book/anthology/dissertation/reportPh.D. thesis

  • Sofie Louise Kragh, Denmark
Folding of G-quaduplex structures adopted by the human telomeric repeat is
here studied by single molecule FRET microscopy. This method allows for the
investigation of G-quadruplex structures and their conformational dynamic.
Telomeres are located at the ends of our chromosomes and end in a single
stranded overhang containing multiple copies of TTAGGG in mammals. This
sequence can fold into G-quadruplex structures that adopt different topologies
depending on experimental conditions such as the presence of cations and
molecular environment. Previous studies have also shown that DNA sequences
with human telomeric repeat adopt several different G-quadruplex conformations in the presence of K+ ions.
G-quadruplexes inhibit telomerase activity and are therefore potential targets
for anti-cancer drugs, which can be small molecule ligands capable of stabilizing
G-quadruplex structures. Understanding folding pathways and dynamics
of G-quadruplex structures may provide clues for the function of G-quadruplex
structures in vivo, which can be used in the improvement of anti-cancer drugs.
Fluorescence Resonance Energy Transfer (FRET) is a very sensitive technique
to measure distance changes in the 2–10nm range. FRET spectroscopy
can be performed on an ensemble of molecules, or on the single molecule level.
In single molecule FRET experiments it is possible to follow the behaviour in
time for each molecule independently, allowing insight into both dynamically
and statistically heterogeneous molecular populations and thus providing more
information than traditional ensemble experiments.
Using single molecule FRET microscopy different aspects of G-quadruplex
folding were investigated.
We have obtained direct insight into G-quadruplex structural polymorphism
both in K+ and Na+ solutions. Polymorphism have previously only been investigated
in K+. Here, we observe significant polymorphism also in Na+.
By investigating the dynamics of these conformational changes and comparing
these findings with other experiments for G-quadruplexes with known topology
we are able to identify different conformations and folding intermediates.
The interaction between the human telomeric repeat and a small molecule
ligand (Phen-DC 3) is investigated with both ensemble and single molecule
techniques. Previous studies have shown that a G-quadruplex with the human
telomeric repeat folds into different conformations depending on the cation
present in the solution. We have found that after the interaction with Phen-
DC 3 the FRET histograms obtained in K+ and Na+ are similar. This indicates
that the G-quadruplex conformations are the same after interaction with Phenii
DC 3 the structures present are independent of the G-quadruplex conformation
before ligand addition.
Previous studies have shown that Topoisomerase I (TOPO I) binds to telomeres
and induces the formation of inter molecular G-quadruplex structures. We
have investigated whether TOPO I stabilize the intra molecular G-quadruplexes
with ensemble FRET measurement. We have also investigated the possibility
of multiple binding sites for DNA on TOPO I.
FRET measurements can be used for determining structural changes of all
kind of nucleic acid nanostructures and we have used it to measure on the
opening and closing of a 3D nanocage. The nanocage was designed for encapsulation and release of specific cargo. Opening and closing of the nanocage
was controlled by temperature. The structural change was controlled by DNA
hairpins unfolding with increasing temperature, resulting in an opened cage
structure. We have investigated the opening of the nanocage with fluorescence
spectroscopy. Our measurements indicate that the cage structures attain a
closed conformation at low temperatures.
Translated title of the contributionEnkelt molekyleniveau studier af G-quadruplexstrukturer
Original languageEnglish
Number of pages209
Publication statusPublished - 24 Feb 2015

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