DNA Hairpin Loops Research Articles

Methylphosphonate Modified DNA Hairpin Loops

We synthesized and analyzed DNA hairpin molecules with methylphosphonate linkages of defined stereochemistry in the loop region. Dinucleotide building blocks ApA and TpT (p indicating methylphosphonate linkage with either Rp or Sp configuration) were synthesized, separated into the diastereomers, and incorporated at three positions of the tetraloops 5′-CGCAAAAGCG-3′ and 5′-CGCTTTTGCG-3′. The oligonucleotides were analyzed for their melting behavior. With a Tm of 67.5°C the molecule 5′-CGCAAApAGCG-3′ with a Sp configurated methylphosphonate is distinctly more stable than the Rp configurated one (Tm = 60.5 °C) and the unmodified oligonucleotide (Tm = 64.5 °C). In contrast to double helical DNA where the substitution of a phosphorodiester by a Sp configurated methylphosphonate results in a lower Tm, in DNA hairpin the introduction of Sp and Rp methylphosphonates at specific positions can lead to a stabilization of the structure.

Asymmetric structure of five and six membered DNA hairpin loops

The tertiary structure of nucleic acid hairpins was elucidated by means of the accessibility of the single-strand-specific nuclease from mung bean. This molecular probe has proven especially useful in determining details of the structural arrangement of the nucleotides within a loop. In this study 3'-labeling is introduced to complement previously used 5'-labeling in order to assess and to exclude possible artifacts of the method. Both labeling procedures result in mutually consistent cleavage patterns. Therefore, methodological artifacts can be excluded and the potential of the nuclease as structural probe is increased. DNA hairpins with five and six membered loops reveal an asymmetric loop structure with a sharp bend of the phosphate-ribose backbone between the second and third nucleotide on the 3'-side of a loop. These hairpin structures differ from smaller loops with 3 or 4 members, which reveal this type of bend between the first and second 3' nucleotide, and resemble with respect to the asymmetry anticodon loops of tRNA.

Stabilities of Nucleotide Loops Bridging the Pyrimidine Strands in DNA Pyrimidine.cntdot.Purine.cntdot.Pyrimidine Triplexes: Special Stability of the CTTTG Loop

Recent studies of DNA hairpin loops have shown considerable dependence of the stability on the sequence of the loop [Senior, M., Jones, R. A., & Breslauer, K. J. (1988a) Proc. Natl. Acad. Sci. U.S.A. 85, 6242-6246; Xodo, L. E., Manzini, G., Quadrifoglio, F., van der Marel, G., & van Boom, J. H. (1989) Biochimie 71, 793-803; Hirao, I., Nishimura, Y., Tagawa, Y., Watanabe, K., & Miura, K. (1992) Nucleic Acids Res. 20, 3891-3896]. Analogous studies have not, until now, been carried out with loops in triple helices. We report the results from experiments in which we examine the relative stabilities of pentanucleotide loops that bridge between the pyrimidine strands in DNA pyr.pur.pyr triple helices. There are two types of loops that are defined by the relative orientation of the purine strand: a 5'-loop and a 3'-loop. The sequences examined in this study are the bimolecular triplexes formed between 5'-dTTCTTTTCL1TTTL5CTTTTCTT (loop nucleotides are underlined, and L1 and L5 represent varied nucleotides) and the two purine strands, 5'-dAAGAAAAG-3' and 5'-dGAAAAGAA-3'. The first and last nucleotides in the loop are varied, since stacking interactions may be strongest at these positions [Senior et al., 1988a; Senior, M., Jones, R. A., & Breslauer, K. J. (1988b) Biochemistry 27, 3879-3885], and we examine 14 sequence combinations for each loop type. Thermal denaturation studies carried out at pH 7.0 indicate considerable variation in the stabilities of these loops.(ABSTRACT TRUNCATED AT 250 WORDS)

Two-base DNA hairpin-loop structures in vivo.

In vitro studies have revealed that DNA hairpin-loops usually contain four unpaired bases. However, a small subset of sequences can form two-base loops. We have previously described an in vivo assay that is sensitive to tight loop formation and have set out to test whether DNA sequences known to form two-base loops in vitro also form tight loops in vivo. It is shown that the sequences 5'dCNNG and 5'dTNNA behave as predicted if they favour two-base loop formation in vivo, a result that is consistent with previously described in vitro studies. The ability of specific DNA sequences to form tight loops in vivo has implications for their potential to form transient structures involved in gene regulation, recombination and mutagenesis.

Polycytosine regions contained in DNA hairpin loops interact via a four-stranded, parallel structure similar to the i-motif.

Thermal denaturation profiles of an oligodeoxynucleotide that forms a hairpin structure with a cytidine-rich loop show an unexpected transition at 60 degrees C at pH 5.0 but not at pH 8.0. Analytical ultracentrifugation shows that this transition reflects dimer formation via the interaction of loops from two molecules to form a novel structure termed the h-dimer. The dependence of this structure on low pH implies the formation of cytosine-protonated cytosine base pairs. NMR spectroscopy, thermal denaturation and ultraviolet absorption spectral analysis suggest a similarity to the i-motif structure recently proposed for the interaction of deoxycytidine oligomers. The use of hairpin loops to form i-motif-like structures may prove useful in searches for cognate proteins and possibly in the production of antibodies.

Theoretical predictions of DNA hairpin loop conformations: correlations with thermodynamic and spectroscopic data.

A computational procedure for generating conformations of DNA hairpin loop structures from a broad range of low-energy starting states is described. The starting point of the modeling is the distribution of oligonucleotide chain conformations obtained from Monte Carlo simulations of feasible dinucleotide steps. Structures which meet the spatial criteria for hairpin loop formation are selected from the distributions and subsequently minimized using all-atom molecular mechanics. Both d(CTnG) and d(CAnG) oligomers, where n = 3, 4, or 5, are modeled. These sequences are chosen because of the large number of published NMR and thermodynamic studies on DNA hairpins containing thymine or adenine residues. The minimized three-dimensional hairpin loop structures are compared with one another as well as analyzed in terms of available experimental data. The computational approach provides the first detailed analysis of DNA hairpin loop structure in terms of a multistate conformational model. Investigation of the minimized conformations reveals several interesting structural features. First, hairpin loops of the same sequence adopt several distinctly different conformations, as opposed to minor variants of the same equilibrium structure, that could potentially interconvert in solution. Second, in contrast to double-helical nucleic acids, the hairpin loop models exhibit hydrophobic and hydrophilic surfaces. The different disposition of hydrophobic groups in loops versus duplexes could modulate both protein-nucleic acid interactions and nucleic acid self-associations. Third, perpendicular aromatic interactions of loop residues are observed in many of the computed hairpins. This sort of interaction might be important in the stabilization of non-hydrogen-bonded nucleic acid secondary and tertiary structures. The predicted structural features in the models help, in addition, to account for the unusual thermodynamic properties of DNA hairpin loops. Comparison of the theoretically-generated NOEs in different structures further reveals that very different molecular structures and interactions can, in principle, produce the same NOEs. The multistate description suggested by this observation differs from the conventional interpretation of DNA solution structure in terms of the fluctuations about a single preferred chain conformation. There is not necessarily only one set of closely related structures consistent with the observed data.

DNA hairpin loops in solution. Correlation between primary structure, thermostability and reactivity with single-strand-specific nuclease from mung bean

Hairpin structures formed by seven DNA inverted repeats have been studied by PAGE, UV(CD)-spectroscopy and nuclease cleavage. The hairpins consisted of (CG)3 stems and loops of 2, 3 and 4 residues. Thermal stabilities (Tm) have been determined in low and high ionic strength buffers, where the hairpins were structured in the B- and Z-DNA form respectively. The thermodynamic parameters of hairpin formation have been obtained by a two-state analysis of the hairpin-coil transitions. It is found that, on increasing the number of bases in the loop from 2 to 3 and 4, the Tms of the B-hairpins decrease, whereas the Tms of the same hairpins in the Z-form increase. This confirms previous evidence (1,2) that in a hairpin molecule the size and structure of the loop are modulated by the conformation of the helical stem. Moreover, B-hairpins with loops comprising 2, 3 and 4 bases have been digested with the single-strand-specific nuclease from mung bean. In our experimental conditions (0 degrees C) the nuclease preferentially cleaves the unbonded nucleotides of the loops. However, the rates of loop hydrolysis, which roughly follow a first-order kinetics, markedly depend on the size of the loop. At a ratio of 3 enzyme units/micrograms DNA, the half-lives of hairpins which are expected to form loops of 4, 3 and 2 residues are 90, 145 and 440 minutes respectively. Thermostability and enzymatic digestion data suggest that two-membered loops can be formed in B-hairpins but not in Z-hairpins.

Conformational Analysis of the 3′-5′-Cyclic Dinucleotide d<pApA> by Means of Molecular Mechanics

The conformational properties of the cyclic dinucleotide d less than pApA greater than were studied by means of molecular mechanics calculations in which a multiconformation analysis was combined with minimum energy calculations. In this approach models of possible conformers are built by varying the torsion angles of the molecule systematically. These models are then subjected to energy minimization; in the present investigation use was made of the AMBER Force field. It followed that the lowest energy conformer has a pseudo-two-fold axis of symmetry. In this conformer the deoxyribose sugars adopt a N-type conformation. The conformation of the sugar-phosphate backbone is determined by the following torsion angles: alpha +, beta t, gamma +, epsilon t and zeta +. The conformation of this ringsystem corresponds to the structure derived earlier by means of NMR spectroscopy and X-ray diffraction. The observation of a preference for N-type sugar conformations in this molecule can be explained by the steric hindrance induced between opposite H3' atoms when one sugar is switched from N- to S-type puckers. The sugars can in principle switch from N- to S-type conformations, but this requires at least the transition of gamma + to gamma -. In this process the molecule obtains an extended shape in which the bases switch from a pseudo-axial to a pseudo-equatorial position. The calculations demonstrate that, apart from the results obtained for the lowest energy conformation, the 180 degrees change in the propagation direction of the phosphate backbone can be achieved by several different combinations of the backbone torsion angles. It appeared that in the low energy conformers five higher order correlations are found. The combination of torsion angles which are involved in changes in the propagation direction of the sugar-phosphate backbone in DNA-hairpin loops and in tRNA, are found in the dataset obtained for cyclic d less than pApA greater than. It turns out, that in the available examples, 180 degrees changes in the backbone direction are localized between two adjacent nucleotides.

Interaction of DNA hairpin loops and a complementary strand by a triplet of base pairs

Hypothesis of non-enzymatic recognition of primordial tRNA and mRNA precursors is experimentally approached. DNA hairpins containing a different number of deoxyguanosine residues in the loop are analyzed for their binding ability to a chemically fixed single-strand of oligo(dC). In presence of small Mg2+ concentration a hairpin with five dG residues in the loop is adsorbed to affinity matrix. Comparison of elution temperatures of hairpin oligonucleotides with those of single-stranded oligoguanylic acids with length of the loop indicates, that smallest loop able to bind forms a triplet of base pairs.

Conformational and model-building studies of the hairpin form of the mismatched DNA octamer d(m5C-G-m5C-G-T-G-m5C-G).

The hairpin form of the mismatched octamer d(m5C-G-m5C-G-T-G-m5C-G) was studied by means of NMR spectroscopy. In a companion study it is shown that the hairpin form of this DNA fragment consists of a structure with a stem of three Watson-Crick-type base pairs and a loop consisting of only two nucleotides. The non-exchangeable proton resonances were assigned by means of two-dimensional correlation spectroscopy and two-dimensional nuclear Overhauser effect spectroscopy. Proton-proton coupling constants were used for the conformational analysis of the deoxyribose ring and for some of the backbone torsion angles. From the two-dimensional NMR spectra and the coupling-constant analysis it is concluded that: (i) the stem of the hairpin exhibits B-DNA characteristics; (ii) the sugar rings are not conformationally pure, but display a certain amount of conformational flexibility; (iii) the stacking interaction in the stem of the hairpin is elongated from the 3'-side in a more or less regular fashion with the two loop nucleotides; (iv) at the 5'-side of the stem a stacking discontinuity occurs between the stem and the loop; (v) at the 5'-side of the stem the loop is closed by means of a sharp backbone turn which involves unusual gamma' and beta+ torsion angles in residue dG(6). The NMR results led to the construction of a hairpin-loop model which was energy-minimized by means of a molecular-mechanics program. The results clearly show that a DNA hairpin-loop structure in which the loop consists of only two nucleotides bridging the minor groove in a straightforward fashion, (i) causes no undue steric strain, and (ii) involves well-known conformational principles throughout the course of the backbone. The hairpin form of the title compound is compared with the hairpin form of d(A-T-C-C-T-A-T4-T-A-G-G-A-T), in which the central -T4- part forms a loop of four nucleotides. Both models display similarities as far as stacking interactions are concerned.