Nucleic acid folding determined by mesoscale modeling and NMR spectroscopy: solution structure of d(GCGAAAGC).

Determination of DNA solution structure is a difficult task even with the high-sensitivity method used here based on simulated annealing with 35 restraints/residue (Cryoprobe 750 MHz NMR). The conformations of both the phosphodiester linkages and the dinucleotide segment encompassing the sharp turn in single-stranded DNA are often underdetermined. To obtain higher quality structures of a DNA GNRA loop, 5'-d(GCGAAAGC)-3', we have used a mesoscopic molecular modeling approach, called Biopolymer Chain Elasticity (BCE), to provide reference conformations. By construction, these models are the least deformed hairpin loop conformation derived from canonical B-DNA at the nucleotide level. We have further explored this molecular conformation at the torsion angle level with AMBER molecular mechanics using different possible (epsilon,zeta) constraints to interpret the 31P NMR data. This combined approach yields a more accurate molecular conformation, compatible with all the NMR data, than each method taken separately, NMR/DYANA or BCE/AMBER. In agreement with the principle of minimal deformation of the backbone, the hairpin motif is stabilized by maximal base-stacking interactions on both the 5'- and 3'-sides and by a sheared G.A mismatch base pair between the first and last loop nucleotides. The sharp turn is located between the third and fourth loop nucleotides, and only two torsion angles beta6 and gamma6 deviate strongly with respect to canonical B-DNA structure. Two other torsion angle pairs epsilon3,zeta3 and epsilon5,zeta5 exhibit the newly recognized stable conformation BIIzeta+ (-70 degrees, 140 degrees). This combined approach has proven to be useful for the interpretation of an unusual 31P chemical shift in the 5'-d(GCGAAAGC)-3' hairpin.

Dinucleotide TpT and its 2'-O-Me analogue possess different backbone conformations and flexibilities but similar stacked geometries.

UV irradiation at 254 nm of 2'-O,5-dimethyluridylyl(3'-5')-2'-O,5-dimethyluridine (1a) and of natural thymidylyl(3'-5')thymidine (1b) generates the same photoproducts (CPD and (6-4)PP; responsible for cell death and skin cancer). The ratios of quantum yields of photoproducts obtained from 1a (determined herein) to that from 1b are in a proportion close to the approximately threefold increase of stacked dinucleotides for 1a compared with those of 1b (from previous circular dichroism results). 1a and 1b however are endowed with different predominant sugar conformations, C3'-endo (1a) and C2'-endo (1b). The present investigation of the stacked conformation of these molecules, by unrestrained state-of-the-art molecular simulation in explicit solvent and salt, resolves this apparent paradox and suggests the following main conclusions. Stacked dinucleotides 1a and 1b adopt the main characteristic features of a single-stranded A and B form, respectively, where the relative positions of the backbone and the bases are very different. Unexpectedly, the geometry of the stacking of two thymine bases, within each dinucleotide, is very similar and is in excellent agreement with photochemical and circular dichroism results. Analyses of molecular dynamics trajectories with conformational adiabatic mapping show that 1a and 1b explore two different regions of conformational space and possess very different flexibilities. Therefore, even though their base stacking is very similar, these molecules possess different geometrical, mechanical, and dynamical properties that may account for the discrepancy observed between increased stacking and increased photoproduct formations. The computed average stacked conformations of 1a and 1b are well-defined and could serve as starting models to investigate photochemical reactions with quantum dynamics simulations.

Spectroscopic and structural impact of a stem base-pair change in DNA hairpins: GTTC-ACA-GAAC versus GTAC-ACA-GTAC.

Successive investigations over the last decade have revealed and confirmed a stable loop closure in a family of d-[GTAC-5Pur6N7N-GTAC] hairpins, where 5Pur6N7N is a AAA, GAG and AXC loop (X being any nucleotide). The trinucleotide loop is characterized by a well defined 5Pur-7N mispairing mode, and by upfield chemical shifts for three sugar protons of the apical nucleotide 6N. The GTTC-ACA-GAAC DNA hairpin, of interest for its likely involvement in Vibrio cholerae genome mutations, has now been investigated. The GTAC-ACA-GTAC DNA hairpin has also been studied because it is intermediate between the other structures, as it contains the loop of the hairpin under consideration and the stem of the above family. The two hairpins with the ACA loop are stable. They show the same mispairing mode and similar upfield shifts as the previous family, but GTTC-ACA-GAAC seems to be slightly less compact than any other. GTTC-ACA-GAAC is remarkable in that it exhibits a B(II) character for the phosphate-ester conformation at 8Gp9A, together with a swing of the upper hairpin into the major groove that, in particular, brings 6CH1' roughly as close to 7AH2 as to 6CH6. These unexpected structural features are qualitatively deduced from (1)H and (31)P NMR spectra, and confirmed by Raman spectroscopy. This comparative study shows that not only the loop sequence but also the stem sequence may control hairpin structures.

DNA tri- and tetra-loops and RNA tetra-loops hairpins fold as elastic biopolymer chains in agreement with PDB coordinates.

The biopolymer chain elasticity (BCE) approach and the new molecular modelling methodology presented previously are used to predict the tri- dimensional backbones of DNA and RNA hairpin loops. The structures of eight remarkably stable DNA or RNA hairpin molecules closed by a mispair, recently determined in solution by NMR and deposited in the PDB, are shown to verify the predicted trajectories by an analysis automated for large numbers of PDB conformations. They encompass: one DNA tetraloop, -GTTA-; three DNA triloops, -AAA- or -GCA-; and four RNA tetraloops, -UUCG-. Folding generates no distortions and bond lengths and bond angles of main atoms of the sugar-phosphate backbone are well restored upon energy refinement. Three different methods (superpositions, distance of main chain atoms to the elastic line and RMSd) are used to show a very good agreement between the trajectories of sugar-phosphate backbones and between entire molecules of theoretical models and of PDB conformations. The geometry of end conditions imposed by the stem is sufficient to dictate the different characteristic DNA or RNA folding shapes. The reduced angular space, consisting of the new parameter, angle Omega, together with the chi angle offers a simple, coherent and quantitative description of hairpin loops.