Periodic Reporting for period 1 - G4invivo (Probing intracellular folding and dynamics of telomeric DNA structures with single-molecule FRET)
Periodo di rendicontazione: 2016-01-01 al 2017-12-31
Non-canonical DNA secondary structures can participate in several biological processes, such as transcription, replication and telomere lengthening, etc. G-quadruplexes are examples of such non-canonical DNA structures that, in particular, are expected to occur at telomeres and gene promoters regions of the human genome. Several recent observations strongly support the occurrence and functional relevance of G-quadruplexes in vivo, however a detailed description of their behaviour under cellular conditions is currently missing. Their investigation under physiological settings is important in order to understand the mechanisms of their action at the cellular level and furthermore use them as potential drug targets.
Telomeric G-quadruplexes, in particular, are polymorphic and dynamical structures that can form a variety of molecular structures under different experimental conditions. This intrinsic polymorphism and dynamics is difficult to resolve with the majority of classical biophysical techniques that provide both structure and time averaged ensemble results. In order to overcome these complications, we used high-resolution single-molecule fluorescence microscopy techniques. These methods allow probing the behavior of G-quadruplex molecules one by one and thus allow obtaining a direct view of both their structure and dynamics. The objective of the project was to probe the conformation and structural dynamics of G-quadruplex DNA under a range of cell-mimicking milieu to obtain a mechanistic understanding of their behavior.
Telomeric G-quadruplexes, in particular, are polymorphic and dynamical structures that can form a variety of molecular structures under different experimental conditions. This intrinsic polymorphism and dynamics is difficult to resolve with the majority of classical biophysical techniques that provide both structure and time averaged ensemble results. In order to overcome these complications, we used high-resolution single-molecule fluorescence microscopy techniques. These methods allow probing the behavior of G-quadruplex molecules one by one and thus allow obtaining a direct view of both their structure and dynamics. The objective of the project was to probe the conformation and structural dynamics of G-quadruplex DNA under a range of cell-mimicking milieu to obtain a mechanistic understanding of their behavior.
In this project, we have investigated the behavior of G-quadruplexes under different conditions in a bottom-up approach - starting from physiologically relevant buffers, towards more cell-like conditions introduced by the presence of polymer-based crowding agents and human cell lysates. Our investigations allowed obtaining a direct view of the folding and underlying conformational dynamics of G-quadruplexes in physiological salt solutions. We found that G-quadruplex folding is very complicated and proceeds through a complex multi-route pathway, involving several marginally stable conformational states.
We could also directly follow the ligand binding to individual G-quadruplex structures and identified two important mechanisms that are determinant to G-quadruplex-ligand interactions: ligand-induced G-quadruplex folding and ligand trapping of transiently folded structures.
Our experiments in molecularly crowded solutions indicate that crowding significantly affects the folding dynamics of G-quadruplexes. Importantly, we find that under near physiological conditions (crowding levels >25 %) G-quadruplexes are expected to be largely stabilized, which points towards the fact that G-quadruplexes are likely to be folded inside cells.
In addition, we have developed new protocols for preparation of cell extracts suitable for single-molecule experiments. Based on our single-molecule experiments in cell extracts we find that the proteins present in cell extracts affect the G-quadruplex folding.
Thus, our experiments established a global view of the behaviour of human telomeric G-quadruplexes under broad experimental conditions, bridging the dilute aqueous solutions and molecularly crowded cellular environments. Altogether, we find that under cellular level the behaviour of G-quadruplexes will be determined and fine-tuned by the combination of the effect of excluded volume due to high levels of cellular crowding and direct specific protein-DNA interactions.
We could also directly follow the ligand binding to individual G-quadruplex structures and identified two important mechanisms that are determinant to G-quadruplex-ligand interactions: ligand-induced G-quadruplex folding and ligand trapping of transiently folded structures.
Our experiments in molecularly crowded solutions indicate that crowding significantly affects the folding dynamics of G-quadruplexes. Importantly, we find that under near physiological conditions (crowding levels >25 %) G-quadruplexes are expected to be largely stabilized, which points towards the fact that G-quadruplexes are likely to be folded inside cells.
In addition, we have developed new protocols for preparation of cell extracts suitable for single-molecule experiments. Based on our single-molecule experiments in cell extracts we find that the proteins present in cell extracts affect the G-quadruplex folding.
Thus, our experiments established a global view of the behaviour of human telomeric G-quadruplexes under broad experimental conditions, bridging the dilute aqueous solutions and molecularly crowded cellular environments. Altogether, we find that under cellular level the behaviour of G-quadruplexes will be determined and fine-tuned by the combination of the effect of excluded volume due to high levels of cellular crowding and direct specific protein-DNA interactions.
This project provides a direct and integrated view of the behaviour of G-quadruplexes in physiologically relevant milieux that will greatly enhance our understanding of these important biological molecules. The results of this project provide new opportunities to design novel and alternative anti-cancer therapies using G-quadruplex structures as drug targets. They can be used by specialists interested in either the chemistry and/or biology of these systems as well as by medical specialists that can potentially use these structures as targets for pharmacological intervention.