Four-stranded DNA on the move

It is becoming clear that macromolecules need to move in order to perform function or fold into an active form. In recent years, more thorough picture of macromolecular machines in action or folding process is emerging, where macromolecules form dynamic conformational ensembles and low populated, high energy structure represent functional states that may differ in only small changes in structure on a level of a base-pairs, or in complete structural rearrangements. Without detailed knowledge of dynamics, understanding of biological role of molecules, their interactions and regulation is incomplete or even worse, erroneous. NMR methods for detection of dynamics have been shown to be excellent in filling in gaps in understanding of dynamic processes. Their strength lies in possibility to detect dynamic exchange in different timescales from nanoseconds to hours and to give information about structure of alternative conformers in form of chemical shifts. Especially NMR methods for measuring relaxation parameters can detect contribution of extremely low populated and short lived states that were often shown to be triggers of functional structural changes or refolding. We are proposing NMR studies of dynamics for four-stranded DNA structures – G-quadruplexes, which form in G-rich regions of genome, influencing replication, transcription, gene expression and extension of telomeres. Intensive exploration of G-quadruplexes in recent years has led to realization that they are extremely polymorphic and that their dynamic properties are most likely vital part of their function. With NMR methods for detection of dynamics we plan to lead the way in linking quick motions of structural elements of G-quadruplexes with large structural rearrangements during folding, polimerization and binding. Determination of effects that alternative conformers of G-quadrupleks structural ensemble have on folding process, binding accessibility and rates and extent of dimerization will be determined with protein binding assays and in vitro transcription for determination of enzymatic activity on G-rich DNA template. We plan to develop methods for different 15N/13C isotopic labelling schemes for DNA and to implement NMR methods for determination of dynamics especially in the micro-millisecond time scale range for G-kvadruplexes.