Apical loops of at least six nucleotide residues are often distinguished by a cross-loop base pair leaving three nucleotides at the cap of the loop forming a well conserved motif called T-loop or lonepair triloop. Our previous studies have indicated the occurrence of such a loop pattern in the HIV-1 TAR RNA. The present project concerns molecular structure and dynamics of sequence-dependent modes of large loop stabilisation. We will focus on apical loop of the dimer initiation site (DIS) of the leader sequence of HIV-2 RNA, composed of 11-nucleotide residues and including a palindromic sequence. We suppose that prior to its involvement into loop-loop interaction via kissing hairpin mode, the HIV-2 DIS RNA hairpin undergoes intra-loop stabilisation.
Advanced molecular dynamics simulation techniques will be used in order to get an insight into the structure and dynamics of this model. In addition, possible contribution of magnesium cations to the loop stabilisation will be investigated by combined Brownian Dynamics/MD methods. The Molecular Dynamics Thermocycler protocol, recently proposed by us, will be simultaneously developed and applied for the project. At the level of conformational analysis, novel methods for description of the conformational space of the loops will be proposed, based on the cluster analysis approach. In order to confront our simulations results with physical data, the RNA models will also be chemically synthesised and their melting thermodynamics will be studied based on magnesium dependence. The picture of RNA/magnesium binding sites emerging from simulations will be confronted with results of Mg2+ ion-promoted RNA cleavages studied by electrophoresis.
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