Porphyrin intercalation in G4-DNA quadruplexes by molecular dynamics simulations.

We investigated the intercalation of a porphyrin derivative (TMPyP) between guanine tetrads (G4-tetrads, G4t's) in 4-stranded G4-DNA oligomers by classical molecular dynamics simulations. Contrary to experimental evidence on very short oligomers that contain stabilizing cations, we find that TMPyP can stack with the G4-tetrads in the absence of interplane cations. A high TMPyP/G4t stoichiometric ratio of 1/2 induces strong deformations of the G4-quadruplexes. A lower ratio of 1/8 is better compatible with the helical conformation. When a TMPyP is accommodated between two tetrads, the stacking distance between the intercalated molecule and a G4-tetrad is approximately 4.3-4.7 A. We find that the possibility of regular TMPyP intercalation depends on the length of the quadruplex, on the stoichiometric ratio and on the edge termination motif.

Ab initio optical absorption spectra of size-expanded xDNA base assemblies.

We present the results of time-dependent density functional theory calculations of the optical absorption spectra of synthetic nucleobases and of their hydrogen-bonded and stacked base pairs. We focus on size-expanded analogues of the natural nucleobases obtained through the insertion of a benzene ring bonded to the planar heterocycles (x-bases), according to the protocol designed and realized by the group of Eric Kool (e.g., see: Gao, J.; Liu, H.; Kool, E.T. Angew. Chem., Int. Ed. 2005, 44, 3118, and references therein). We find that the modifications of the frontier electron orbitals with respect to natural bases, which are induced by the presence of the aromatic ring, also affect the optical response. In particular, the absorption onset is pinned by the benzene component of the HOMO of each x-base (xA, xG, xT, xC). In addition, the main trait of the H-bonding interbase coupling is a conspicuous red shift of spectral peaks in the low-energy range. Finally, the hypochromicity, a well-known fingerprint of stacking, is more pronounced in stacked xG-C and xA-T pairs than that in stacked G-C and A-T pairs, an index of enhanced stacking.

Stability and migration of metal ions in G4-wires by molecular dynamics simulations.

We present a molecular dynamics investigation of guanine quadruple helices based on classical force fields. We analyze the dependence of the helical conformation on various compositional factors, such as the length of the G4-wire, as well as the incorporation into the helix channel of alkali ions of different species and in different amounts. In compliance with previous indications, our results suggest that monovalent alkali cations assist the stability of the quadruplex arrangement against disruption on the few nanoseconds time scale in the order of increasing van der Waals radius. Whereas very short G4-wire fragments immediately unfold in the absence of coordinating metal ions or in the presence of tiny ions (e.g., Li+) in agreement with the experimental evidence that empty short guanine quadruplexes are not formed in any synthetic conditions, our simulations show that longer empty helices do not discompose. This finding supports the possibility of producing long G4-wires with different guanine-cation stoichiometries than those routinely known. The classical trajectories allow us to identify different stationary axial sites for the different metal species, which are confirmed by complementary quantum calculations.

Molecular basis of the targeting of topoisomerase II-mediated DNA cleavage by VP16 derivatives conjugated to triplex-forming oligonucleotides.

Human topoisomerase II (topo II) is the cellular target for a number of widely used antitumor agents, such as etoposide (VP16). These agents 'poison' the enzyme and induce it to generate DNA breaks that are lethal to the cell. Topo II-targeted drugs show a limited sequence preference, triggering double-stranded breaks throughout the genome. Circumstantial evidence strongly suggests that some of these breaks induce chromosomal translocations that lead to specific types of leukaemia (called treatment-related or secondary leukaemia). Therefore, efforts are ongoing to decrease these secondary effects. An interesting option is to increase the sequence-specificity of topo II-targeted drugs by attaching them to triplex-forming oligonucleotides (TFO) that bind to DNA in a highly sequence-specific manner. Here five derivatives of VP16 were attached to TFOs. The active topo II poisons, once linked, induced cleavage 13-14 bp from the triplex end where the drug was attached. The use of triple-helical DNA structures offers an efficient strategy for targeting topo II-mediated cleavage to DNA specific sequences. Finally, drug-TFO conjugates are useful tools to investigate the mechanistic details of topo II poisoning.

Improved synthesis of daunomycin conjugates with triplex-forming oligonucleotides. The polypurine tract of HIV-1 as a target.

Triple helix-forming oligonucleotides (TFOs) are promising agents for the control of gene expression, as they can selectively bind to a chosen oligopyrimidine.oligopurine region of a gene of interest thus interfering with its expression. The stability of the triplex formed by the TFO and the duplex is often too poor for successful applications of TFOs in vivo and the conjugation of a DNA intercalating moiety to the TFO is a common way to enhance the TFO affinity for its target. In a previous work, we investigated the properties of daunomycin conjugated TFO (dauno-TFO) and found that this class of compounds showed a higher degree of affinity than native oligonucleotides for an oligopyrimidine.oligopurine duplex target and that the presence of the amino sugar increases such stability. Here, we report a significantly improved synthetic procedure for the preparation of the conjugates, based on the protection of the daunosamine moiety by N-trifluoroacetylation. This protecting group is removed as a final step from the conjugation product by mild basic hydrolysis to give the desired dauno-TFO. Compared to the previous synthetic procedure, the improvement is important. The synthesis is now more reproducible and no side products are formed. Moreover, the thus protected daunomycin derivative is very stable, up to at least one year. Two dauno-TFOs, differing by the length of the oligonucleotide moiety, were prepared to target the polypurine tract (PPT) of HIV-1. Triplex formation by these compounds with model duplexes was studied by UV spectroscopy, thermal gradient gel electrophoresis (TGGE) and gel electrophoretic mobility shift. The experimental results demonstrate that dauno-TFOs bind to the PPT of HIV-1 more strongly than the unconjugated TFOs.

Strain-dependence of the electronic properties in periodic quadruple helical G4-wires.

The electronic structure of periodic quadruple helix guanine wires, which mimic G4-DNA molecules, was studied as a function of the stacking distance between consecutive planes, by means of first principles density functional theory calculations. We show that, whereas for the native DNA interplane separation of 3.4 A the HOMO- and LUMO-derived bands are poorly dispersive, the bandwidths can be significantly increased when compressive strain is applied along the helical axis. Our findings indicate that efficient band conduction for both holes and electrons can be supported by such wires for stacking distances below 2.6 A, which imply a huge axial deformation with respect to double and quadruple helices in solutions and in crystals.

Relative DNA binding affinity of helix 3 homeodomain analogues, major groove binders, can be rapidly screened by displacement of prebound ethidium bromide. A comparative study.

The binding affinity for a 12-bp dsDNA of Antennapedia helix 3 analogues, major groove binders, has been measured by displacement of prebound ethidium bromide, a fluorescent displacement assay proposed for minor groove binders by Boger et al.(J. Am. Chem. Soc., 2000, 122, 6382-6394). Relative binding affinities determined by this method were compared to those obtained by gel mobility shift and footprinting assays for the 12-bp dsDNA and a 178-bp DNA fragment. The present work demonstrates that the fluorescence displacement assay is suitable for rapid screening of major groove binders, even though about 60 to 70% of the prebound ethidium bromide is displaced by these peptides. Total (100%) displacement of ethidium bromide was serendipitously achieved by addition in the peptide sequence, at the N-terminus, of a S-3-nitro-2-pyridinesulfenyl-N-acetyl-cysteine residue. S-3-nitro-2-pyridinesulfenylcysteine was shown to (i) bind to dsDNA with a micromolar affinity and (ii) direct within DNA grooves a peptide with no affinity for dsDNA.

Biomolecular electronic devices based on self-organized deoxyguanosine nanocrystals.

We report on a new class of hybrid electronic devices based on a DNA nucleoside (deoxyguanosine lipophilic derivative) whose assembled polymeric ribbons interconnect a submicron metallic gate. The device exhibits large conductivity at room temperature, rectifying behavior and strong current-voltage hysteresis. The transport mechanism through the molecules is investigated by comparing films with different self-assembling morphology. We found that the main transport mechanism is connected to pi-pi interactions between guanosine molecules and to the formation of a strong dipole along ribbons, consistently with the results of our first-principles calculations.