The three‐dimensional structure of a DNA hairpin in solution

Abstract

The hairpin formed by d(ATCCTATTTATAGGAT) was studied by means of two-dimensional NMR spectroscopy and conformational analysis. Almost all 'H resonances of the stem region could be assigned, while the 'H and 31P spectra of the loop region were interpreted completely; this includes the stereospecific assignment of the H5' and H5" resonances. The derivation of the detailed loop structure was carried out in a stepwise fashion including some improved and new methods for structure determination from NMR data. In the first step, the mononucleotide structures were examined. The conformational space available to the mononucleotide was scanned systematically by varying the glycosidic torsion angle and pseudorotational parameters. Each generated conformer was tested against the experimental J coupling constants and NOE parameters. In the following stage, the structures of dinucleotides and longer fragments were derived. Inter-residue distances between protons were calculated by means of a procedure in which the simulated NOEs, obtained via a relaxation-matrix approach, were fitted to the experimental NOEs without the introduction of a molecular model. In addition, the backbone torsion angles fl, y and c were deduced from homocoupling and heterocoupling constants. These data served as constraints in the next step, in which the loop sequence was subjected to a multi-conformer generation procedure. The resulting structures were tested against the mentioned constraints and disregarded if these constraints were violated. This yielded a family of structures for the loop region, confined to a relatively narrow conformational space. A representative conformation was subsequently docked on a B-type stem which fulfilled the structural constraints (derived from the NMR experiments for the stem region) to yield the hairpin structure. Results obtained from subsequent restrained-molecular-mechanics as well as free-molecular-mechanics calculations are in accordance with those obtained by means of the analysis described above.The structure of the hairpin loop is a compactly folded conformation and the first base of the central TTTA region forms a Hoogsteen T-A pair with the fourth base. This Hoogsteen base pair is stacked upon the sixth base pair of the B-type double-helical stem. The second base of the loop is folded into the minor groove, whereas the third base of the loop is partly stacked on the first and fourth bases. The phosphate backbone exhibits a sharp turn between the third and fourth nucleotides of the loop. The peculiar structure of this hairpin loop is discussed in relation to loop folding in DNA and RNA hairpins and in relation to a general model for loop folding.Loop structures in RNA and DNA hairpins have been the subject of numerous studies. We have been interested in these structures because they may serve as models for the folding which occurs in RNA and DNA molecules in the cell, and also because they may provide insight into the mechanism underlying the folding. Our initial studies were concerned with the hairpin fo...

Unusual structures of TTTA repeats in icaC gene of Staphylococcus aureus

“…3D). In addition, a small and broad thymine imino signal at $11 ppm appears at 0°C (Supporting Information, S6b), which is consistent with those observed in TT loops [9,19]. Through variable temperature 1D 1 H NMR experiments, six melting curves were constructed based on the chemical shifts of the wellresolved thymine H7 signals of T1, T2, T3, T5, T6 and T7 (Supporting Information, S7a and S7b).…”

“…In addition to the two hydrogen bonds, the base stacked on top of the closing Hoogsteen base pair also contributes to the stability of the mini-loop [20]. Owing to the shorter interstrand C1 0 -C1 0 distance, the stacking interactions are more effective than those involving a closing Watson-Crick base pair [19,21]. In (TTTA) 3 , the base orientation of A8 also possesses the syn feature as evidenced by the similar NOE intensities of H8-H1 0 and H8-H2 0 /H2 00 (Fig.…”

“…For the TT loop in the DNA hairpin [19], it was closed by a TA Hoogsteen base pair of which the adenine adopts a syn orientation (Fig. 2C).…”

Kinetics of conformational fluctuations in DNA hairpin-loops

“…Most of the previous studies of hairpins deal with the structure of the folded state or with the thermodynamics of the folding-unfolding transitions (5)(6)(7). Knowledge about the kinetics of the hairpin transitions is limited to a few isolated measurements of the characteristic rates (8,9).…”

Similar Conformations of Hairpins with TTT and TTTT Sequences NMR and Molecular Modeling Evidence for T · T base pairs in the TTTT hairpin

“…On the basis of NOESY, DQF-COSY and 'H-"P-RCSC cross-peak patterns and "P shifts, we classified the backbone torsion angles as normal B-DNA or non-B-DNA backbone torsion angles (see Gorenstein, 1984;Nikonowicz and Gorenstein, 1990;Blommers et al, 1991 ;Kim et al, 1992).…”