2. Moleclular recognition of DNA:RNA hybrid and multi-stranded structures in bioanalytical and in vitro systems
The biological activity of many drugs is based on the interactions of small organic molecules with DNA and RNA macromolecules. DNA:RNA hybrid and multistranded structures are formed as intermediate structures during many biologically important processes such as DNA replication, transcription, telomere replication and replication of HIV virus. DNA:RNA hybrids can form R-loops that have been detected in various organisms from bacteria to mammals and play crucial roles in regulating gene expression, DNA and histone modifications, immunoglobulin class switch recombination, DNA replication and genome stability. In some cases, hybrid duplexes can be associated with a third single strand, while studied compounds themselves can induce the formation of DNA:RNA hybrid triplexes or the duplex disproportionation. Also, since the telomerase activity can be inhibited either through the binding of ligands to DNA:RNA hybrids or to G-quadruplex structures, it's essential to study the interaction with both targets.
Therefore, DNA:RNA hybrid and multistranded structures represent attractive targets for small molecules, especially since the literature citations of the ligands with selective binding to these targets are scarce.
The main aim of this project is the search for new structural motives/compounds with preferential binding to DNA:RNA hybrids and multistranded structures (triplexes and quadruplexes) in regard to regular (non-hybrid) DNA and RNA duplexes and single-stranded forms and the investigation of mode of binding of selected ligands to DNA and RNA structures first in simple bioanalytical systems (aqueous buffered solutions in „reaction vessel“, e.g. the cuvette, the instrument cell) and then in a more complex, in vitro system (a tumor cell line).
To fulfill the main project goal, other aims have to be fulfilled first: forming a library of compounds, with structural motif necessary for interaction with these targets, introduction and implementation of new DNA and RNA targets (DNA:RNA hybrid and multistranded structures) and high-throughput methods (competition dialysis, thermal denaturation of mixtures, RNaseH assay) in the research study of our project team, which would enable fast screening of compounds for recognition of these targets.
Another aim is to investigate in detail the mode of binding of selected ligands to DNA and RNA structures in bioanalytical systems (aqueous buffered solutions) by spectroscopic, calorimetric and computational methods (UV/Vis- and fluorescence spectroscopy, CD/LD spectrometry, viscometry, DSC, ITC microcalorimetry, NMR, mass spectrometry, molecular modelling calculations by Amber program suit). Further, we will investigate the possible correlation between the strong binding potential of selected compounds and their antiproliferative activity in cell culture (MTT, flow cytometry, confocal microscopy).