The influence of Ni2+ and other ions on the trigonal structure of DNA.

We present a new structure of a DNA dodecamer obtained in the presence of Ni2+ ions. The DNA forms Ni-guanine cross-links between neighboring molecules. Our results show that an adequate dosage of Ni2+ may help to form well-defined DNA nanostructures. We also compare our structure with other dodecamers which present unique features and also crystallize in trigonal unit cells, strongly influenced by the counterions associated with DNA. In all cases, the DNA duplexes form parallel pseudo-helical columns in the crystal, similar to DNA-protamine and native DNA fibers.

Functional and structural analysis of AT-specific minor groove binders that disrupt DNA-protein interactions and cause disintegration of the Trypanosoma brucei kinetoplast.

Trypanosoma brucei, the causative agent of sleeping sickness (Human African Trypanosomiasis, HAT), contains a kinetoplast with the mitochondrial DNA (kDNA), comprising of >70% AT base pairs. This has prompted studies of drugs interacting with AT-rich DNA, such as the N-phenylbenzamide bis(2-aminoimidazoline) derivatives 1 [4-((4,5-dihydro-1H-imidazol-2-yl)amino)-N-(4-((4,5-dihydro-1H-imidazol-2-yl)amino)phenyl)benzamide dihydrochloride] and 2 [N-(3-chloro-4-((4,5-dihydro-1H-imidazol-2-yl)amino)phenyl)-4-((4,5-dihydro-1H-imidazol-2-yl)amino)benzamide] as potential drugs for HAT. Both compounds show in vitro effects against T. brucei and in vivo curative activity in a mouse model of HAT. The main objective was to identify their cellular target inside the parasite. We were able to demonstrate that the compounds have a clear effect on the S-phase of T. brucei cell cycle by inflicting specific damage on the kinetoplast. Surface plasmon resonance (SPR)-biosensor experiments show that the drug can displace HMG box-containing proteins essential for kDNA function from their kDNA binding sites. The crystal structure of the complex of the oligonucleotide d[AAATTT]2 with compound 1 solved at 1.25 Å (PDB-ID: 5LIT) shows that the drug covers the minor groove of DNA, displaces bound water and interacts with neighbouring DNA molecules as a cross-linking agent. We conclude that 1 and 2 are powerful trypanocides that act directly on the kinetoplast, a structure unique to the order Kinetoplastida.

Effect of Solvent Choice on the Self-Assembly Properties of a Diphenylalanine Amphiphile Stabilized by an Ion Pair.

A diphenylalanine (FF) amphiphile blocked at the C terminus with a benzyl ester (OBzl) and stabilized at the N terminus with a trifluoroacetate (TFA) anion was synthetized and characterized. Aggregation of peptide molecules was studied by considering a peptide solution in an organic solvent and adding pure water, a KCl solution, or another organic solvent as co-solvent. The choice of the organic solvent and co-solvent and the solvent/co-solvent ratio allowed the mixture to be tuned by modulating the polarity, the ionic strength, and the peptide concentration. Differences in the properties of the media used to dissolve the peptides resulted in the formation of different self-assembled microstructures (e.g. fibers, branched-like structures, plates, and spherulites). Furthermore, crystals of TFA⋅FF-OBzl were obtained from the aqueous peptide solutions for X-ray diffraction analysis. The results revealed a hydrophilic core constituted by carboxylate (from TFA), ester, and amide groups, and the core was found to be surrounded by a hydrophobic crown with ten aromatic rings. This segregated organization explains the assemblies observed in the different solvent mixtures as a function of the environmental polarity, ionic strength, and peptide concentration.

Effect of Hydrodynamic Forces on meso-(4-Sulfonatophenyl)-Substituted Porphyrin J-Aggregate Nanoparticles: Elasticity, Plasticity and Breaking.

The J aggregates of 4-sulfonatophenyl meso-substituted porphyrins are non-covalent polymers obtained by self-assembly that form nanoparticles of different morphologies. In the case of high aspect-ratio nanoparticles (bilayered ribbons and monolayered nanotubes), shear hydrodynamic forces may modify their shape and size, as observed by peak force microscopy, transmission electron microscopy of frozen solutions, small-angle X-ray scattering measurements in a disk-plate rotational cell, and cone-plate rotational viscometry. These nanoparticles either show elastic or plastic behaviour: there is plasticity in the ribbons obtained upon nanotube collapse on solid/air interfaces and in viscous concentrated nanotube solutions, whereas elasticity occurs in the case of dilute nanotube solutions. Sonication and strong shear hydrodynamic forces lead to the breaking of the monolayered nanotubes into small particles, which then associate into large colloidal particles.

Structure of the DNA duplex d(ATTAAT)2 with Hoogsteen hydrogen bonds.

The traditional Watson-Crick base pairs in DNA may occasionally adopt a Hoogsteen conformation, with a different organization of hydrogen bonds. Previous crystal structures have shown that the Hoogsteen conformation is favored in alternating AT sequences of DNA. Here we present new data for a different sequence, d(ATTAAT)2, which is also found in the Hoogsteen conformation. Thus we demonstrate that other all-AT sequences of DNA with a different sequence may be found in the Hoogsteen conformation. We conclude that any all-AT sequence might acquire this conformation under appropriate conditions. We also compare the detailed features of DNA in either the Hoogsteen or Watson-Crick conformations.

Polymorphic crystal structures of an all-AT DNA dodecamer.

In this work, we explore the influence of different solvents and ions on the crystallization behavior of an all-AT dodecamer d(AATAAATTTATT)2 In all cases, the oligonucleotides are found as continuous columns of stacked duplexes. The spatial organization of such columns is variable; consequently we have obtained seven different crystal forms. The duplexes can be made to crystallize in either parallel or crossed columns. Such versatility in the formation of a variety of crystal forms is characteristic for this sequence. It had not been previously reported for any other sequence. In all cases, the oligonucleotide duplexes have been found to crystallize in the B form. The crystallization conditions determine the organization of the crystal, although no clear local interactions have been detected. Mg(2+) and Ni(2+) can be used in order to obtain compact crossed structures. DNA-DNA interactions in the crystals of our all-AT duplexes present crossovers which are different from those previously reported for mixed sequence oligonucleotides. Our results demonstrate that changes in the ionic atmosphere and the crystallization solvent have a strong influence on the DNA-DNA interactions. Similar ionic changes will certainly influence the biological activity of DNA. Modulation of the crystal structure by ions should also be explored in DNA crystal engineering. Liquid crystals with a peculiar macroscopic shape have also been observed.

In and out of the minor groove: interaction of an AT-rich DNA with the drug CD27.

The DNA of several pathogens is very rich in AT base pairs. Typical examples include the malaria parasite Plasmodium falciparum and the causative agents of trichomoniasis and trypanosomiases. This fact has prompted studies of drugs which interact with the minor groove of DNA, some of which are used in medical practice. Previous studies have been performed almost exclusively with the AATT sequence. New features should be uncovered through the study of different DNA sequences. In this paper, the crystal structure of the complex of the DNA duplex d(AAAATTTT)2 with the dicationic drug 4,4'-bis(imidazolinylamino)diphenylamine (CD27) is presented. The drug binds to the minor groove of DNA as expected, but it shows two new features that have not previously been described: (i) the drugs protrude from the DNA and interact with neighbouring molecules, so that they may act as cross-linking agents, and (ii) the drugs completely cover the whole minor groove of DNA and displace bound water. Thus, they may prevent the access to DNA of proteins such as AT-hook proteins. These features are also expected for other minor-groove binding drugs when associated with all-AT DNA. These findings allow a better understanding of this family of compounds and will help in the development of new, more effective drugs. New data on the biological interaction of CD27 with the causative agent of trichomoniasis, Trichomonas vaginalis, are also reported.

Crystal structure of a complex of DNA with one AT-hook of HMGA1.

We present here for the first time the crystal structure of an AT-hook domain. We show the structure of an AT-hook of the ubiquitous nuclear protein HMGA1, combined with the oligonucleotide d(CGAATTAATTCG)(2), which has two potential AATT interacting groups. Interaction with only one of them is found. The structure presents analogies and significant differences with previous NMR studies: the AT-hook forms hydrogen bonds between main-chain NH groups and thymines in the minor groove, DNA is bent and the minor groove is widened.

Coiled-coil conformation of a pentamidine-DNA complex.

The coiled-coil structure formed by the complex of the DNA duplex d(ATATATATAT)(2) with pentamidine is presented. The duplex was found to have a mixed structure containing Watson-Crick and Hoogsteen base pairs. The drug stabilizes the coiled coil through the formation of cross-links between neighbouring duplexes. The central part of the drug is found in the minor groove as expected, whereas the charged terminal amidine groups protrude and interact with phosphates from neighbouring molecules. The formation of cross-links may be related to the biological effects of pentamidine, which is used as an antiprotozoal agent in trypanosomiasis, leishmaniasis and pneumonias associated with AIDS. The DNA sequence that was used is highly abundant in most eukaryotic genomes. However, very few data are available on DNA sequences which only contain A.T base pairs.

The interaction of manganese ions with DNA.

We present the structure of the duplex formed by a fragment of auto-complementary DNA with the sequence d(CGTTAATTAACG). The structure was determined by X-ray crystallography. Up to date it is the first structure presenting the interaction between a DNA oligonucleotide and manganese ions. The presence of Mn2+ creates bonds among the N7 atom of guanines and phosphates. These bonds stabilize and determine the crystallographic network in a P3(2) space group, unusual in oligonucleotide crystals. The crystal structure observed is compared with those found in the presence of Mg2+, Ca2+ and Ni2+, which show different kinds of interactions. The double helices show end-to-end interactions, in a manner that the terminal guanines interact with the minor groove of the neighboring duplex, while the terminal cytosines are disordered. We have chosen this sequence since it contains a TTAA repeat. Such repeats are very rare in all genomes. We suggest that this sequence may be very susceptible to the formation of closely spaced thymine dimers.

Stabilization by extra-helical thymines of a DNA duplex with Hoogsteen base pairs.

We present the crystal structure of the DNA duplex formed by d(ATATATCT). The crystals contain seven stacked antiparallel duplexes in the asymmetric unit with A.T Hoogsteen base pairs. The terminal CT sequences bend over so that the thymines enter the minor groove and form a hydrogen bond with thymine 2 of the complementary strand in the Hoogsteen duplex. Cytosines occupy extra-helical positions; they contribute to the crystal lattice through various kinds of interactions, including a unique CAA triplet. The presence of thymine in the minor groove apparently contributes to the stability of the DNA duplex in the Hoogsteen conformation. These observations open the way toward finding under what conditions the Hoogsteen duplex may be stabilized in vivo. The present crystal structure also confirms the tendency of A.T-rich oligonucleotides to crystallize as long helical stacks of duplexes.

Macroscopic DNA helices.

Presently there is great interest in the construction of nanostructures from DNA fragments. Here we report the preparation of much larger helical textures with different shapes from pure DNA fragments. We have observed them while preparing crystals of the dodecamer d(AAAAAATTTTTT), which only contains adenine and thymine. Noncoding regions of the genome are always rich in these two bases. We have found a strong influence of ions either monovalent or divalent, with which different crystalline structures are found. All of them contain long helical stacks of duplexes in the unit cell. The most remarkable structures are macroscopic helices with diameters and pitch in the range of 20-40 microm. Thus, pure DNA oligonucleotides may form a hierarchy of helical structures, going from the B-form double helix (pitch, p = 33 A) to helical stacks of duplexes (p approximately 900 A), and to macroscopic helices (p approximately 300,000 A). These different levels of organization are reminiscent of the different levels of organization of DNA in eukaryotic chromosomes.

A unique vertebrate histone H1-related protamine-like protein results in an unusual sperm chromatin organization.

Protamine-like proteins constitute a group of sperm nuclear basic proteins that have been shown to be related to somatic linker histones (histone H1 family). Like protamines, they usually replace the chromatin somatic histone complement during spermiogenesis; hence their name. Several of these proteins have been characterized to date in invertebrate organisms, but information about their occurrence and characterization in vertebrates is still lacking. In this sense, the genus Mullus is unique, as it is the only known vertebrate that has its sperm chromatin organized by virtually only protamine-like proteins. We show that the sperm chromatin of this organism is organized by two type I protamine-like proteins (PL-I), and we characterize the major protamine-like component of the fish Mullus surmuletus (striped red mullet). The native chromatin structure resulting from the association of these proteins with DNA was studied by micrococcal nuclease digestion as well as electron microscopy and X-ray diffraction. It is shown that the PL-I proteins organize chromatin in parallel DNA bundles of different thickness in a quite distinct arrangement that is reminiscent of the chromatin organization of those organisms that contain protamines (but not histones) in their sperm.