Solution structure of a purine rich hexaloop hairpin belonging to PGY/MDR1 mRNA and targeted by antisense oligonucleotides.

A preferential target of antisense oligonucleotides directed against human PGY/MDR1 mRNA is a hairpin containing a stem with a G*U wobble pair, capped by the purine-rich 5'r(GGGAUG)3' hexaloop. This hairpin is studied by multidimensional NMR and restrained molecular dynamics, with special emphasis on the conformation of south sugars and non-standard phosphate linkages evidenced in both the stem and the loop. The hairpin is found to be highly structured. The G*U wobble pair, a strong counterion binding site, displays structural particularities that are characteristic of this type of mismatch. The upper part of the stem undergoes distortions that optimize its interactions with the beginning of the loop. The loop adopts a new fold in which the single-stranded GGGA purine tract is structured in A-like conformation stacked in continuity of the stem and displays an extensive hydrogen bonding surface for recognition. The remarkable hairpin stability results from classical inter- and intra-strand interactions reinforced by numerous hydrogen bonds involving unusual backbone conformations and ribose 2'-hydroxyl groups. Overall, this work emphasizes numerous features that account for the well-ordered structure of the whole hairpin and highlights the loop properties that facilitate interaction with antisense oligonucleotides.

Structure and dynamics of phosphate linkages and sugars in an abasic hexaloop RNA hairpin.

Hairpins containing hexaloops are well represented among the diverse conformations adopted by the RNA molecules. To investigate the intrinsic properties of a backbone submitted to a hexaloop fold, we present here a molecular dynamics study of an abasic hexaloop closed by an A-form 6 basepair stem. The analysis of the 23 ns trajectory made in explicit solvent shows that both the sugars and the torsion angles in the loop undergo numerous conformational transitions. The south sugars, although not in a majority, are the major actors of the loop stretching. The five torsion angles, epsilon, zeta, alpha, beta, and gamma, are unequally variable, and only zeta and alpha exhibit trimodal distributions. The analysis of the phosphate linkages in terms of epsilonzeta'-alpha'-beta'-gamma-combinations allows us to define five conformational families, each one composed of one major substate in equilibrium with several less populated ones. The transitions between the substates within a family follow specific pathways involving the angles epsilon, zeta, and alpha. Thus, this work reveals that the backbone conformational space is both reduced and ordered even in a hexaloop devoid of bases.