Hairpin Form Research Articles

Structural diversity induced by pyrene intercalators in homogeneous oligodeoxyguanylates

Homogeneous oligodeoxyguanylates incorporating small-molecule intercalator pyrene moieties in a 1,5 relationship form various structures of dimer, trimer, tetramer and internal hairpin form that are yellowish in color because of the intermolecular interactions between the pyrene units.

The Discodermolide Hairpin Structure Flows from Conformationally Stable Modular Motifs

(+)-Discodermolide (DDM), a polyketide macrolide from marine sponge, is a potent microtubule assembly promoter. Reported solid-state, solution, and protein-bound DDM conformations reveal the unusual result that a common hairpin conformational motif exists in all three microenvironments. No other flexible microtubule binding agent exhibits such constancy of conformation. In the present study, we combine force-field conformational searches with NMR deconvolution in different solvents to compare DDM conformers with those observed in other environments. While several conformational families are perceived, the hairpin form dominates. The stability of this motif is dictated primarily by steric factors arising from repeated modular segments in DDM composed of the C(Me)-CHX-C(Me) fragment. Furthermore, docking protocols were utilized to probe the DDM binding mode in beta-tubulin. A previously suggested pose is substantiated (Pose-1), while an alternative (Pose-2) has been identified. SAR analysis for DDM analogues differentiates the two poses and suggests that Pose-2 is better able to accommodate the biodata.

Novel Molecular Mechanism for Actinomycin D Activity as an Oncogenic Promoter G-Quadruplex Binder

Actinomycin D (ActD) is a natural antibiotic that inhibits the transcription of genes by interacting with a GC-rich duplex, a single-stranded or hairpin form of DNA, and then interfering with the action of RNA polymerase. In this study, we identified a novel molecular mechanism of anticancer activity of ActD as an oncogenic c-Myc promoter G-quadruplex binder. ActD selectively inhibits the elongation of oligonucleotides containing c-Myc promoter G-quadruplex sequence in PCR-stop assays. UV-vis spectroscopic and circular dichroism studies suggest that ActD interacts with c-Myc promoter G-quadruplex via a surface end stacking interaction, inducing a mixed-type conformation of the G-quadruplex. ActD selectively inhibits the cellular growth and synthesis of c-Myc mRNA in Ramos cells having the NHEIII(1) region in the translocated c-Myc gene. In addition, the results of promoter assays using two kinds of NHEIII(1) region mutants and wild-type constructs strongly support the idea that binding of ActD with G-quadruplex formed in the promoter region results in the reporter gene being turned off. Our study reveals a novel mechanism underlying the anticancer activity of ActD, whereby ActD interacts with oncogenic promoter G-quadruplex DNA to repress gene expression.

Photocleavage of Peptides and Oligodeoxynucleotides Carrying 2‐Nitrobenzyl Groups

Abstractmagnified imagePeptides and oligodeoxynucleotides containing photolabile 2‐nitrobenzyl groups as mid‐sequences were prepared. Photocleavage of aqueous solutions of these compounds neared completion within 30 min to a few hours depending on the photolabile group used. A photolabile group was introduced in the loop of an intramolecular oligodeoxynucleotide hairpin. Melting curves of the hairpin with and without the complementary oligodeoxynucleotide showed a preference for the intramolecular hairpin form, but an intermolecular duplex was observed after photolysis. These results open the possibility of using photolabile DNA hairpins for the fabrication of patterned surfaces.

A disease‐linked DNA repeat array rich in potential guanine quadruplexes

Potential G‐quadruplex sequences (PQS) can form non‐B structures containing four equal‐length runs of Hoogsteen‐paired G residues. They have extremely high thermodynamic stability and form hairpin structures from four clustered G runs, bimolecular‐type structures from two clustered G runs, or tetramolecular‐type structures (e.g., at telomeres) from four G runs on different strands. D4Z4 arrays at 4q35.2, which are linked to facioscapulohumeral muscular dystrophy (FSHD), have a high density of PQS. In D4Z4's 3.3‐kb repeat unit we found seven hairpin (G3‐5N1‐7G3‐5N1‐7G3‐5 N1‐7G3‐5) PQS. Our evidence for G‐quadruplex formation from these consensus sequences and other D4Z4 oligonucleotide sequences that contain runs of only 2 G's or only two runs of 4 or 5 G's came from characteristic circular dichroism (CD), gel electrophoresis, partial resistance to PCR, and highly targeted deletion and mutation products arising spontaneously upon DNA replication in vitro. D4Z4 arrays of 3.3 ‐ 33 kb at 4q are usually pathogenic, and those of >36 kb are almost always phenotypically neutral. Given the overrepresentation of PQS in promoters of myogenic genes, it is attractive to propose that G‐quadruplexes help organize D4Z4 chromatin structure as part of special changes in D4Z4's conformation accounting for the near‐threshold effect of D4Z4 size on FSHD status. (Supported in part by NIH Grant NS048859)

Observation of a dynamic equilibrium between DNA hairpin and duplex forms of covalent adducts of a minor groove binding agent

A dynamic equilibrium between covalent 1:1 hairpin and 2:1 duplex DNA adducts of a pyrrolobenzodiazepine (PBD) minor groove binding agent () has been observed for the first time. The equilibrium, which establishes over 1 hour and must require unfolding of both types of adducts, is surprising given that PBDs normally require DNA minor groove structure for binding and take 24 hours for complete reaction with duplex DNA. The equilibrium is interesting from an energetics perspective due to the well known DNA stabilizing effect of PBDs. This observation could have significance for the in vitro and in vivo biological activity of PBDs, as DNA hairpin and loop structures are known to be important in cellular processes such as transcription and replication.

An Analytical Study on Hairpin Type LPFs Using MSL Resonators Loaded with Dielectric Rods

This paper focuses on realizability of a reduced-size LPF using a substrate loaded width high relative dielectric constant rods by LTCC technologies. We have examined two sections form hairpin type LPF and one section form hairpin. As a result, in comparison with the characteristic of one section, we could confirm that good out-of-band characteristic was provided with the realization of a steep skirt characteristic.

What drives the binding of minor groove-directed ligands to DNA hairpins?

Understanding the molecular basis of ligand–DNA-binding events, and its application to the rational design of novel drugs, requires knowledge of the structural features and forces that drive the corresponding recognition processes. Existing structural evidence on DNA complexation with classical minor groove-directed ligands and the corresponding studies of binding energetics have suggested that this type of binding can be described as a rigid-body association. In contrast, we show here that the binding-coupled conformational changes may be crucial for the interpretation of DNA (hairpin) association with a classical minor groove binder (netropsin). We found that, although the hairpin form is the only accessible state of ligand-free DNA, its association with the ligand may lead to its transition into a duplex conformation. It appears that formation of the fully ligated duplex from the ligand-free hairpin, occurring via two pathways, is enthalpically driven and accompanied by a significant contribution of the hydrophobic effect. Our thermodynamic and structure-based analysis, together with corresponding theoretical studies, shows that none of the predicted binding steps can be considered as a rigid-body association. In this light we anticipate our thermodynamic approach to be the basis of more sophisticated nucleic acid recognition mechanisms, which take into account the dynamic nature of both the nucleic acid and the ligand molecule.

Preferential Integration of Adeno-Associated Virus Type 2 into a Polypyrimidine/Polypurine-Rich Region within AAVS1

Adeno-associated virus type 2 (AAV2) preferentially integrates its genome into the AAVS1 locus on human chromosome 19. Preferential integration requires the AAV2 Rep68 or Rep78 protein (Rep68/78), a Rep68/78 binding site (RBS), and a nicking site within AAVS1 and may also require an RBS within the virus genome. To obtain further information that might help to elucidate the mechanism and preferred substrate configurations of preferential integration, we amplified junctions between AAV2 DNA and AAVS1 from AAV2-infected HeLaJW cells and cells with defective Artemis or xeroderma pigmentosum group A genes. We sequenced 61 distinct junctions. The integration junction sequences show the three classical types of nonhomologous-end-joining joints: microhomology at junctions (57%), insertion of sequences that are not normally contiguous with either the AAV2 or the AAVS1 sequences at the junction (31%), and direct joining (11%). These junctions were spread over 750 bases and were all downstream of the Rep68/78 nicking site within AAVS1. Two-thirds of the junctions map to 350 bases of AAVS1 that are rich in polypyrimidine tracts on the nicked strand. The majority of AAV2 breakpoints were within the inverted terminal repeat (ITR) sequences, which contain RBSs. We never detected a complete ITR at a junction. Residual ITRs at junctions never contained more than one RBS, suggesting that the hairpin form, rather than the linear ITR, is the more frequent integration substrate. Our data are consistent with a model in which a cellular protein other than Artemis cleaves AAV2 hairpins to produce free ends for integration.

Molecular Dynamics and Free Energy Study of the Conformational Equilibria in the UUUU RNA Hairpin.

A series of molecular dynamics (MD) simulations was performed to elucidate the thermodynamic basis for the relative stabilities of hairpin, duplex, and single stranded forms of the 5'-CGC(UUUU)GCG-3' oligonucleotide. According to a recent NMR study this sequence exhibits dynamic conformational equilibrium in aqueous solution in the vicinity of room temperature. Free energy calculations using the molecular mechanics-Poisson Boltzmann-surface area (MM-PB/SA) approach support a shift in the conformational equilibrium from duplex to hairpin as the temperature is increased from 276 to 300 K, in agreement with the NMR results. The effect of added salt on the relative stabilities of RNA conformers is also reproduced by our calculations. The calculated ΔH° for the equilibrium between hairpin and single stranded forms is estimated to be -23.4 kcal/mol, in reasonable agreement with experimental values. Our results reveal that the conformational equilibrium strongly depends on the solute entropy and the electrostatic interactions modulated by added salt. Simulations of hairpin loop conducted at two different temperatures converged to the same lowest energy loop conformation. This conformer is stabilized by favorable van der Waals interactions as a result of U5-U6-U7 base stacking, a hydrogen bond between the U4 base and the phosphate linking U6 and U7, and hydrogen bonds involving the 2'OH groups at U4 and U6. However, the sugar pucker of the four uridines in the lowest energy conformer is different from that reported by a NMR study. While the NMR study found that U5 and U7 adopt the C2'-endo conformation, the simulation results suggests that overall the structure with the U5 and U7 in the C3'-endo conformation is thermodynamically more stable than the structure containing the C2'-endo pucker by approximately 8 kcal/mol. Calculations based on the MM-PB/SA scheme show that although the electrostatic solvation free energy favors the C2'-endo conformation for the U5 and U7 riboses, it is offset by the less favorable intramolecular electrostatic and van der Waals energies. To enhance the conformational sampling, a replica exchange molecular dynamics (REMD) simulation was conducted in a generalized Born (GB) continuum solvent for the hairpin loop. This simulation indicates that the stable loop structure observed in the explicit solvent simulations corresponds to the free energy minimum. It also reveals that while the U4, U5, and U6 sugar rings are predominantly in the C3'-endo conformation, there is considerable variation in the sugar pucker of the U7 ribose ring.

Capillary electrophoresis is a sensitive monitor of the hairpin–random coil transition in DNA oligomers

Capillary electrophoresis (CE) has been used to characterize the hairpin–random coil transition of four octamers in the GCxxxxGC minihairpin family, where xxxx is GAAA, TTTC, TTTT, or AAAA. The transition can be monitored by CE because differences in the frictional coefficients of the hairpin and coil forms of each octamer lead to a difference of approximately 9% in the free solution mobilities of the two conformations. The GAAA octamer is unusually stable, with a melting temperature of 65 °C. The TTTT octamer forms a minihairpin with a melting temperature of 29 °C, the TTTC octamer has a melting temperature of 16 °C, and the AAAA octamer has a melting temperature below 0 °C. The thermal transitions of the TTTT, TTTC, and AAAA octamers are well fitted by a structure prediction algorithm; however, the GAAA minihairpin is considerably more stable than predicted. The melting temperature of the GAAA minihairpin is reduced to 47 °C in aqueous buffers containing 7.2 M urea and to 33 °C in buffers containing 7.2 M urea plus 40% (v/v) formamide. The combined results indicate that CE is a sensitive technique for monitoring conformational transitions in small DNA molecules.

The Impact of Dyskeratosis Congenita Mutations on the Structure and Dynamics of the Human Telomerase RNA Pseudoknot Domain

The pseudoknot domain is a functionally crucial part of telomerase RNA and influences the activity and stability of the ribonucleoprotein complex. Autosomal dominant dyskeratosis congenita (DKC) is an inherited disease that is linked to mutations in telomerase RNA and impairs telomerase function. In this paper, we present a computational prediction of the influence of two base DKC mutations on the structure, dynamics, and stability of the pseudoknot domain. We use molecular dynamics simulations, MM-GBSA free energy calculations, static analysis, and melting simulations analysis. Our results show that the DKC mutations stabilize the hairpin form and destabilize the pseudoknot form of telomerase RNA. Moreover, the P3 region of the predicted DKC-mutated pseudoknot structure is unstable and fails to form as a defined helical stem. We directly compare our predictions with experimental observations by calculating the enthalpy of folding and melting profiles for each structure. The enthalpy values are in very good agreement with values determined by thermal denaturation experiments. The melting simulations and simulations at elevated temperatures show the existence of an intermediate structure, which involves the formation of two UU base pairs observed in the hairpin form of the pseudoknot domain.

Investigation of the complexation of the anti-cancer drug novantrone with the hairpin structure of the deoxyheptanucleotide 5′-d(GpCpGpApApGpC)

In aqueous solution the deoxyheptanucleotide, 5′-d(GpCpGpApApGpC), exists as a very stable hairpin structure in equilibrium with small proportions of the single-stranded and duplex forms. Complexation of the anti-cancer drug novantrone (mitoxantrone) with the DNA heptamer was investigated by one- and two-dimensional 500 MHz 1H NMR spectroscopy (2M-TOCSY, 2M-NOESY) and molecular dynamics simulations. The proton chemical shifts of NOV in mixed solutions with the heptamer were measured as a function of concentration and temperature and the equilibrium association parameters were determined for complexation of NOV with the three forms of the heptamer. The spatial structure of the complex of the antibiotic with the hairpin form of the heptamer was built on the basis of 2D-NOE data. The conformational dynamics of the complex and its interaction with the water environment were investigated by molecular dynamics methods. The results suggest that NOV complexes with the hairpin form of the heptamer in solution by intercalation. Complexation of NOV with the hairpin stem results in a disruption of about one half of the intramolecular water bridges of the hairpin, which is considered to be the main reason for the observed decrease in the thermodynamical stability of the hairpin on binding with the ligand.

Inhibition of DNA replication by a d(CAG) repeat binding ligand

Trinucleotide repeat expansions in genomic DNA are the molecular basis of a number of genetic diseases. The (CAG)n, (CTG)n, and (CGG)n repeats share the common sequence CXG, and inherently produce a hairpin structure involving X-X mismatch base pairs flanked by two G-C base pairs. The chance of a strand slippage leading to the repeat expansion is considered to increase with increasing stability of the hairpin form. Here we show our synthetic ligand naphthyridine-azaquinolone (NA) stabilized a hairpin form of the (CAG)n repeat and inhibited the polymerase-mediated DNA synthesis.

Partial melting of metapelites in the Gagnon terrane below the high-pressure belt in the Manicouagan area (Grenville Province): pressure–temperature (P–T) and U–Pb age constraints and implications

Metapelites of the parautochthonous Gagnon terrane at the footwall of the high-pressure (high-P) belt in the Manicouagan area (central Grenville Province) preserve an impressive textural record of partial melting reactions, mainly in polymineralic inclusions within garnet. The dominant textures were developed within the pressure–temperature (P–T) field of the continuous dehydration melting reaction biotite + kyanite (or sillimanite) + plagioclase + quartz = garnet + K-feldspar + melt, with sillimanite instead of kyanite in the southern part of the footwall. Inferred P–T paths have a hair-pin form in the range of 750–850 °C and 1000–1500 MPa for the kyanite-bearing rocks. Monazite crystallization ages are consistently late Grenvillian, either 995 or 985 Ma, and one sample contains monazite of both ages. Two of these samples also contain inherited monazite with ages of 1738 ± 5 and 1719 ± 30 Ma, indicative of an earlier metamorphic event. Tonalite and diorite from the same area yield Archean zircon crystallization ages and titanite ages of 961 ± 3 and 956 ± 4 Ma, the youngest in the Manicouagan region. The late Grenvillian metamorphism was of comparable intensity but ~50 Ma younger than in the overlying high-P belt in this area and therefore seems to be unrelated to the emplacement of the latter over the Parautochthonous Belt, as previously suggested. Rather, this younger metamorphism suggests a reactivation of the footwall by underthrusting of the Gagnon terrane during the waning stages of convergence, and a link with a major coeval post-tectonic thermal event farther south in the hinterland.

New, extended hairpin form of the TAR-2 RNA domain points to the structural polymorphism at the 5' end of the HIV-2 leader RNA

The HIV-2 TAR RNA domain (TAR-2) plays a key role in the trans-activation of HIV-2 transcription as it is the target for the Tat-2 protein and several cell factors. Here, we show that the TAR-2 domain exists in vitro in two global, alternative forms: a new, extended hairpin form with two conformers and the already proposed branched hairpins form. This points strongly to the structural polymorphism of the 5′ end of the HIV-2 leader RNA. The evidence comes from the non-denaturing PAGE mobility assay, 2D structure prediction, enzymatic and Pb2+- or Mg2+-induced RNA cleavages. Existence of the TAR-2 extended form was further proved by the examination of engineered TAR-2 mutants stabilized either in the branched or extended structure. The TAR-2 extended form predominates with an increasing magnesium concentration. Gel retardation assays reveal that both TAR-2 wt and its mutant, unable to form branched structure, bind Tat-2 protein with comparable, high affinity, while RNA hairpins I and II, derived from TAR-2 branched structure model, show much less protein binding. We propose that an internal loop region of the TAR-2 extended hairpin form is a potential Tat-2 binding site.

A Three-State Mechanism for DNA Hairpin Folding Characterized by Multiparameter Fluorescence Fluctuation Spectroscopy

The folding of a dye-quencher labeled DNA hairpin molecule was investigated using fluorescence autocorrelation and cross-correlation spectroscopy (FCS) and photon counting histogram analysis (PCH). The autocorrelation and cross-correlation measurements revealed the flow and diffusion times of the DNA molecules through two spatially offset detection volumes, the relaxation time of the folding reaction, and the total concentration of DNA molecules participating in the reaction. The PCH measurements revealed the equilibrium distribution of DNA molecules in folded and unfolded conformations and the specific brightnesses of the fluorophore in each conformational state. These measurements were carried out over a range of NaCl concentrations, from those that favored the open form of the DNA hairpin to those that favored the closed form. DNA melting curves obtained from each sample were also analyzed for comparison. It was found that the reactant concentrations were depleted as the reaction progressed and that the equilibrium distributions measured by FCS and PCH deviated from those obtained from the melting curve analyses. These observations suggest a three-state mechanism for the DNA hairpin folding reaction that involves a stable intermediate form of the DNA hairpin. The reaction being probed by FCS and PCH is suggested to be a rapid equilibrium between open and intermediate conformations. Formation of the fully closed DNA hairpin is suggested to occur on a much longer time scale than the FCS and PCH measurement time. The closed form of the hairpin thus serves as a sink into which the reactants are depleted as the reaction progresses.

Mechanism of Hairpin-Duplex Conversion for the HIV-1 Dimerization Initiation Site

We have used the dimerization initiation site of HIV-1 genomic RNA as a model to investigate hairpin-duplex interconversion with a combination of fluorescence, UV melting, gel electrophoresis, and x-ray crystallographic techniques. Fluorescence studies with molecular beacons and crystallization experiments with 23-nucleotide dimerization initiation site fragments showed that the ratio of hairpin to duplex formed after annealing in water essentially depends on RNA concentration and not on cooling kinetics. With natural sequences allowing to form the most stable duplex, and thus also the loop-loop complex (or "kissing complex"), concentrations as low as 3 mum in strands are necessary to obtain a majority of the hairpin form. With a mutated sequence preventing kissing complex formation, a majority of hairpins was even obtained at 80 mum in strands. However, this did not prevent an efficient conversion from hairpin to duplex in the presence of salts. Kinetic considerations are in favor of duplex formation from intermediates involving hairpins engaged in cruciform dimers rather than from free strands. The very first step of formation of such a cruciform intermediate could be trapped in a crystal structure. This mechanism might be significant for the dynamics of small RNAs beyond the strict field of HIV-1.

Targeted activation of transcription in vivo through hairpin-triplex forming oligonucleotide in Saccharomyces cerevisiae

Triplex forming oligonucleotides (TFO) are known to be potential agents for modifying gene function. In most instances they are utilized for repression of transcription. However hybrid molecules containing cis-acting elements in a duplex DNA in a hairpin form contiguously with the TFO can bind transcription factors in vitro. In the present manuscript we demonstrate that hairpin-TFO can be employed in vivo for targeted activation of gene expression of two genes mapping on chromosome XI of Saccharomyces cerevisiae. The cis-acting GAL4 protein-binding site contained in the hairpin-TFO is targeted in vivo to the 5' upstream sequence of STE6 and CBT1 genes that are transcribed in opposite directions and share a poly(pu/py) sequence that can form triple helical structure. The hairpin-TFO is targeted to this site and promotes the activation of both the genes. These results demonstrate four important aspects relating to activation of gene expression: (i) accessibility of duplex DNA packaged into chromatin to triplex forming sequences in vivo, (ii) the potential use of hairpin-TFO in therapeutics by activation of transcription in vivo, (iii) Sharing of transcription factors between two genes transcribed in opposite directions and (iv) specific activation of genes even when their cognate site is not covalently linked to the gene being activated.

Homodimeric MyoD Preferentially Binds Tetraplex Structures of Regulatory Sequences of Muscle-specific Genes

Myogenic transcription is activated by the binding of heterodimers of the basic helix-loop-helix proteins MyoD and E12 or E47 to a consensus E-box sequence, d(CANNTG), in promoter or enhancer regions of muscle-specific genes. Homodimers of MyoD bind E-box less tightly and are less efficient activators of transcription. Recent results from our laboratory (Yafe, A., Etzioni, S., Weisman-Shomer, P., and Fry, M. (2005) Nucleic Acids Res. 33, 2887-2900) indicate that regulatory sequences of several muscle-specific genes contain a disproportionate high content of guanine clusters that readily form hairpin and parallel-stranded unimolecular and bimolecular tetraplex structures. Here we have shown that homodimers of full-length recombinant MyoD formed complexes with bimolecular tetraplex structures of muscle-specific regulatory sequences but not with their double-stranded, hairpin, or unimolecular tetraplex forms. Preferential binding of homodimeric MyoD to bimolecular tetraplex DNA structures over E-box DNA was reflected by the 18.7-39.9-fold lower dissociation constants, Kd, of the MyoD-tetraplex DNA complexes. Conversely, MyoD-E47 heterodimers formed tighter complexes with E-box as indicated by their 6.8-19.0-fold lower Kd relative to complexes with bimolecular tetraplex DNA structures. Similarly, homodimers of the 60-amino acid basic helix-loop-helix domain of MyoD bound E-box more efficiently and tetraplex DNA less efficiently than homodimers of full-length MyoD. It might be that the favored binding of MyoD homodimers to tetraplex DNA structures lowers their ability to activate muscle-specific gene transcription, whereas the formation of MyoD-E47 heterodimers and their preferential binding to E-box DNA enhance transcription.