Molecular dynamics simulations of RNA bulges, loops and base flipping

RNA molecules have several important functions in a cell. They carry the genetic information from DNA to protein, act as catalysts and regulate gene expression. Both DNA and RNA consist of nucleotides, DNA molecules are mainly double stranded while RNA molecules often have single stranded regions that interrupt the double stranded structure. RNA molecules can take a variety of three dimensional structures and the single stranded regions are commonly used as recognition sites and building blocks. In this thesis the structure and dynamics of different RNA molecules have been studied by molecular dynamics (MD) simulations. The thesis is based on four papers where the RNA structures contain nucleotides that do not form Watson-Crick base pairs. Base flipping is studied by umbrella sampling in two of the papers. In the first paper terminal loop motifs are studied. Some of the loop motifs are known to be very stable in thermal melting experiments and we have studied if the loops have an intrinsic stability. The loop motifs with purine-purine stacking and hydrogen bonds across the loop are shown to be most capable of retaining the loop structure. In the second paper an adenosine to inosine (A-to-I) editing site is studied. The A at the editing site is mismatched with a cytidine (C). The editing site (R/G site) is targeted selectively by an editing enzyme from the adenosine deaminases acting on RNA (ADAR) family. This editing site was studied and compared with another A-C mismatch (46 site), a few base pairs away, that is not selectively targeted. The A at the R/G site is shown to be more flexible than the A at the 46 site in the MD simulations. Base flipping of the A at the R/G site and at the 46 site are investigated and the minor groove pathway is found to be preferred over the major groove. In the third paper the focus is on a part of the spliceosome, the U6 RNA intramolecular stem loop which contains an unpaired uridine (U). In NMR structures this U is in the stack at pH 7.0 and flipped out at pH 5.7. The pathway of this base flipping is studied. We demonstrate that the minor groove pathway is preferred over the major groove pathway, and that protonation on an A adjacent to the flipping U lowers the energy barrier ~3.5 kcal/mol. In the fourth paper, RNA bulges are studied. The RNA molecules consist of an antisense oligonucleotide forming a complex with a target RNA with an internal bulge loop of different sizes. O2 -methylations are performed on the antisense strand and these methylations are found to influence the minor groove hydration