Projects / Programmes
Structural studies of nucleic acids with G-rich repeating sequences
| Code |
Science |
Field |
Subfield |
| 1.04.00 |
Natural sciences and mathematics |
Chemistry |
|
| Code |
Science |
Field |
| B002 |
Biomedical sciences |
Biophysics |
| Code |
Science |
Field |
| 1.04 |
Natural Sciences |
Chemical sciences |
NMR G-quadruplex structure onkogen
Researchers (7)
Organisations (2)
Abstract
Nucleic acids adopt a great number of topologies including multi-stranded helical structures, which enable them to perform different biological functions ranging from transfer of genetic information to catalysis and regulation. G-quadruplex, a four-stranded secondary structure formed by guanine-rich (G-rich) nucleic acids is gaining increasing attention as means for therapeutic intervention. The basic building block of G-quadruplexes is a G-quartet in which four guanine bases are held together by eight Hoogsteen hydrogen bonds in a co-planar arrangement. Within the framework of the current proposal we intend to study structural features of G-rich genomic DNA sequences, which are expected to form G-quadruplex structures. We will primarily focus on promoter regions of oncogenes related to ras proteins that are important in human cancers. G-quadruplex structures formed by promoters have been, in comparison to telomeric regions, addressed in only few studies and their structural features are poorly understood, which raises the need for structural characterization. In many promoter regions of the human genome, including the important oncogenes such as KRAS, HRAS and NRAS, G-rich regions with a potential to form G-quadruplex structures, have been identified. The genes originating from the ras family are expressed in a tissue-specific fashion: HRAS is highly expressed in skin and skeletal muscles, KRAS in colon and thymus and NRAS in male germinal tissue. HRAS, KRAS and NRAS are involved in the pathogenesis of different types of cancer. As such they are considered a primary target for the development of anticancer drugs. Recent studies showed that this role can be attributed to their G-rich promoters, which can potentially fold into a G-quadruplex structure. Numerous findings support the hypothesis that formation and unfolding of specific G-quadruplex structures may be involved in regulation of gene expression. Past studies have shown that G-quadruplexes are highly polymorphic, which has been associated with specifics of oligonucleotide sequences as well as with conditions in solution. Connecting loops, which can span the diagonal or edge of the outer G-quartet or can adopt a double chain reversal orientation along the side of the G-quadruplex core, play a very important role in stabilization of G-quadruplex structures. We will devote special attention to the nature and sequence requirements of loops since their structure is very important in terms of interaction between the DNA molecule and ligands (potential drugs). An attractive therapeutic strategy to inhibit transcription of ras proteins is by the introduction of polycyclic aromatic hydrocarbon insertions such as Twisted Intercalating Nucleic Acids that stabilize G-quadruplexes and repress transcription. Decoy strategy is based on a hypothesis that oligonucleotides mimicking G-quadruplexes compete with the binding of MAZ to the RAS promoter, inhibit its expression and uncontrolled cell growth. To enhance their activity, anti-KRAS decoy oligonucleotides should maintain the 3D structure recognized by the cognate transcription factor and exhibit enhanced resistance to nucleases. The primary research tool will be high-resolution NMR spectroscopy. We plan to complement the results by UV, CD and other (spectroscopic) methods. In addition to the characterization of 3D structures with atomic resolution we will also study dynamics of individual segments of the structure, especially loop regions. Using structure-guided design we expect to optimize the sequence position of intercalating nucleotide residues with pyrene chromophores that supposedly stack on the external G-quartets thus stabilizing the G-quadruplex decoy, and furthermore suggest synthesis of aromatic heterocyclic analogues with potential to regulate transcription of a gene through (de)stabilization of G-quadruplex structures.
Significance for science
The results from our studies will contribute to expansion of knowledge on folding of promoter sequences into G-quadruplex structures and on their dynamic properties. Up recently, very little was known about G-quadruplex structures adopted by G-rich regions found in promoters. Such knowledge is important for understanding the regulation of gene expression and can be of help in design of novel organic molecules that could specifically stabilize certain G-quadruplex structure. This will help against various forms of cancer and many viral diseases. Alternatively, organic molecules with fluorescent properties can be used as in vivo probes. High resolution 3D structures will provide much needed insight into the nature of ligand binding interactions with G-quadruplexes. Currently it is difficult to rationalize ligand affinities due to the absence of appropriate data on geometry of interaction. In order to gain insights into the ligand-quadruplex interaction of individual parts of the structure special attention will be devoted to loop regions. We will also contribute to the development of experimental methods for DNA structure elucidation. Novel ‘in cell’ NMR methods will be developed to facilitate DNA structure elucidation within confines of cell environment.
Significance for the country
Our studies may contribute to development of new areas of research and science in Slovenia, in particular to the studies of G-quadruplex structures that are expected to form in the promoter regions of genes. We expect that our work will allow identification of new structural elements within G-quadruplex structures that may be used as targets to develop new drugs. New small molecule drugs may in turn generate interest within pharmaceutical industry and may ultimately lead to the creation of new jobs in Republic of Slovenia. Novel NMR methodologies that are being developed at the Slovenian NMR centre, which serves a role of regional infrastructural facility, are used in several studies that range from preservation of literature at the national library to identification of objects in museums. NMR centre has been Centre of Excellence since it was awarded this title under FP5 project and has contributed importantly to promotion of Slovenian research and its integration into international exchange and projects. A very important activity envisaged in the proposal is rising awareness of potentials of NMR spectroscopy and in education of younger colleagues at both under- and graduate level.
Most important scientific results
Annual report
2014,
2015,
final report
Most important socioeconomically and culturally relevant results
Annual report
2014,
2015,
final report
