Sequence-dependent collective properties of DNAs and their role in biological systems.

DNA actively interacts with proteins involved in replication, transcription, repair, and regulation processes inside the cell. The base sequence encodes the dynamics of these transformations from the atomic to the nanometre scale length, and over higher spatial scales. In fact, although an important part of the DNA informational content acts locally, it exerts its functions as collective properties of relatively long sequences and manifests as static and dynamic curvature. Physical models that explore different aspects of DNA collective properties associated to such superstructural properties encoded in the sequence will be reviewed. The B-DNA periodicity operates as band-pass-filter; only the local physical-chemical variance associated to the sequence, in phase with the helical periodicity, sums up and reveals at higher scale. In this light, the gel electrophoresis behaviour of DNAs, the nucleosome thermodynamic stability and positioning along genomes were interpreted and discussed. Finally, a part of this review is reserved to describe the ability of some inorganic crystal surfaces to recognize and stabilize certain DNA tracts with peculiar sequences. The collective superstructural properties of DNAs could be involved in the selective interaction between DNA sequence and particular crystal surfaces. It may be conceived that sequences strongly adsorbed on surface could nucleate and expand bits of information in primeval DNA (and/or RNA) chains, early characterized by random sequences, since more protected against the physical-chemical injuries by the environment, and therefore involved in the evolution of their informational content.

Predicting nucleosome positioning in genomes: physical and bioinformatic approaches.

In eukaryotic genomes, nucleosomes are responsible for packaging DNA and controlling gene expression. For this reason, an increasing interest is arising on computational methods capable of predicting the nucleosome positioning along genomes. In this review we describe and compare bioinformatic and physical approaches adopted to predict nucleosome occupancy along genomes. Computational analyses attempt at decoding the experimental nucleosome maps of genomes in terms of certain dinucleotide step periodicity observed along DNA. Such investigations show that highly significant information about the occurrence of a nucleosome along DNA is intrinsic in certain features of the sequence suggesting that DNA of eukaryotic genomes encodes nucleosome organization. Besides the bioinformatic approaches, physical models were proposed based on the sequence dependent conformational features of the DNA chain, which govern the free energy needed to transform recurrent DNA tracts along the genome into the nucleosomal shape.

Self-organization of G-quadruplex structures in the hTERT core promoter stabilized by polyaminic side chain perylene derivatives.

hTERT core promoter regulates telomerase transcription in human cells, thus its structural features are of large interest. We have found that the G-rich hTERT core promoter region, corresponding to the major DNase I hypersensitive site in chromatin organization, contains nine putative G-quadruplex forming sequences (PQS) and is unfavorable for nucleosome formation. Here we show that four PQS are effectively able to form stable parallel intramolecular G-quadruplexes, using PAGE and CD spectroscopy analysis. The PQS-region, as a whole, appears to be organized in three self-interacting G-quadruplexes, probably giving rise to a helicoidal superstructure, as shown by CD and polymerase stop assay. POL-HPDI drugs, that we previously found useful in selectively stabilizing telomeric G-quadruplex, are able to stabilize both the single intramolecular G-quadruplex and the PQS-region superstructure. The features of their induced CD spectra suggest that POL-HPDIs bind to single G-quadruplexes and to whole PQS-region superstructure, mainly by end-stacking interactions.

Prediction of nucleosome positioning in genomes: limits and perspectives of physical and bioinformatic approaches.

Nucleosomes, the fundamental repeating subunits of all eukaryotic chromatin, are responsible for packaging DNA into chromosomes inside the cell nucleus and controlling gene expression. While it has been well established that nucleosomes exhibit higher affinity for select DNA sequences, until recently it was unclear whether such preferences exerted a significant, genome-wide effect on nucleosome positioning in vivo. For this reason, an increasing interest is arising on a wide-ranging series of experimental and computational analyses capable of predicting the nucleosome positioning along genomes. Toward this goal, we propose a theoretical model for predicting nucleosome thermodynamic stability in terms of DNA sequence. Based on a statistical mechanical approach, the model allows the calculation of the sequence-dependent canonical ensemble free energy involved in nucleosome formation. The theoretical free energies were evaluated for 90 single nucleosome DNA tracts and successfully compared with those obtained with nucleosome competitive reconstitution. These results, obtained for single nucleosomes, could in principle allow the calculation of the intrinsic affinity of nucleosomes along DNA sequences virtually opening the possibility of predicting the nucleosome positioning along genomes on physical basis. The theoretical nucleosome distribution was compared and validated with that of yeast and human genome experimentally determined. The results interpret on a physical basis the experimental nucleosome positioning and are comparable with those obtained adopting models based on the identification of some recurrent sequence features obtained from the statistical analysis of a very large pool of nucleosomal DNA sequences provided by the positioning maps of genomes.

Geometrical, conformational and topological restraints in regular nucleosome compaction in chromatin.

The folding of the nucleosome array into a chromatin fiber modulates DNA accessibility and is therefore an important factor for the control of gene expression. The statistical analysis of the nucleosome repeat length in chromatin fibers reveals the presence of a ten-fold periodicity suggesting the existence of orientational constraints of the nucleosome units that provide the geometrical conditions of helical conformations. Recently, the elucidation of the x-ray crystal structure of a nucleosome tetramer array and the interpretation of electron microscopy images of reconstituted nucleosome arrays suggested two different architectures of the chromatin fiber. We approached the problem by integrating the experimental findings with geometrical, conformational and topological restraints, under the hypothesis of the minimum distortion of the nucleosome and linker DNA structures. We show that the excluded volume at linker crossing and the torsional energy limit the possible close packing of the nucleosomes in the chromatin fiber. In particular, the torsional energy of the chromatin fiber appears crucial in determining the kind of nucleosome packing for short nucleosome repeat lengths as in telomeres and yeast chromatin.

A statistical thermodynamic approach for predicting the sequence-dependent nucleosome positioning along genomes.

Nucleosomes are the fundamental repeating unit of chromatin and constitute the structural building blocks of the eukaryotic genome. The distribution of nucleosomes along the genome is a significant aspect of chromatin structure and influences gene regulation through modulation of DNA accessibility. For this reason, an increasing interest is arising in models capable of predicting the nucleosome positioning along genomes. Toward this goal, we propose a theoretical model for predicting nucleosome thermodynamic stability in terms of DNA sequence. The model, based on a statistical mechanical approach allows the calculation of the canonical ensemble free energy involved in nucleosome formation. The theoretical free energies were evaluated for about one hundred nucleosome DNA tracts and successfully compared with those obtained in different laboratories with nucleosome competitive reconstitution (correlation coefficient equal to 0.92). We extended these results to the nucleosome positioning along genomes. To test our model, the theoretical nucleosome distribution was compared with that of yeast genome experimentally determined. The results are comparable with those obtained by different authors adopting models based on identifying some recurrent sequence features obtained from the statistical analysis of a very large pool of nucleosomal DNA sequences provided by the positioning maps of genomes.

Peptides with regular enantiomeric sequences: a wide class of modular self-assembling architectures.

Organic trans-annular assemblies constitute an expanding class of structures with promising applications for the design of nanotechnological devices. Among the strategies developed for the engineering of organic nanotubes, those characterized by regular alternating enantiomeric amino acid sequences have been proven particularly useful. In fact, cyclic peptides with an even number of regularly alternating D- and L-amino acids have the tendency to adopt local beta-conformation that are capable of forming trans-annular self-assembling architectures, hydrogen bond directed. The formation of such structures is the result of the conformational equivalence of the monomer units, a general principle that associates stereo-chemical to chemical equivalence in a polymer chain. For configurationally alternating sequences the conformational equivalence produces cyclic structures, where a monomer unit is related to the adjacent along the chain by a roto-reflection axis, Sn. A slight relaxation of the conformational equivalence can formally transform a cyclic structure into a conformationally quasi-equivalent helical structures characterized by the presence of polar inner channels, which allow the transient binding for an activated flow of specific ions. To prove our early predictions, we synthesized different alternating polypeptide and the corresponding linear and cyclic oligopeptides and investigated their conformations by NMR and CD spectroscopy as well as the formation of self-assembling structures by increasing the concentration in solution. Moreover, their predicted ability to behave as an ion-channel across bilayer membranes are investigated and experimental evidence of single molecule conducting events are reported. Finally, the possibility is suggested to obtain self-assembled trans-annular structures by chemically bridging the amino acid side chains stabilized using different strategies. A complex construct with good perspective for nano-technological applications is proposed in which cyclic DL-lysine side chains are bridged by the formation of salycilaldimmine metal chelates.

A model for triple helix formation on human telomerase reverse transcriptase (hTERT) promoter and stabilization by specific interactions with the water soluble perylene derivative, DAPER.

The promoter of human telomerase reverse transcriptase (hTERT) gene, in the region from -1000 to +1, contains two homopurine-homopyrimidine sequences (-835/-814 and -108/-90), that can be considered as potential targets to triple helix forming oligonucleotides (TFOs) for applying antigene strategy. We have chosen the sequence (-108/-90) on the basis of its unfavorable chromatin organization, evaluated by theoretical nucleosome positioning and nuclease hypersensitive sites mapping. On this sequence, anti-parallel triplex with satisfactory thermodynamic stability is formed by two TFOs, having different lengths. Triplex stability is significantly increased by specific interactions with the perylene derivative N,N'-bis[3,3'-(dimethylamino) propylamine]-3,4,9,10-perylenetetracarboxylic diimide (DAPER). Since DAPER is a symmetric molecule, the induced Circular Dichroism (CD) spectra in the range 400-600 nm allows us to obtain information on drug binding to triplex and duplex DNA. The drug-induced ellipticity is significantly higher in the case of triplex with respect to duplex and, surprisingly, it increases at decreasing of DNA. A model is proposed where self-stacked DAPER binds to triplex or to duplex narrow grooves.

AFM imaging and theoretical modeling studies of sequence-dependent nucleosome positioning.

Telomeric chromatin has different features with respect to bulk chromatin, since nucleosomal repeat along the chain is unusually short. We studied the role of telomeric DNA sequences on nucleosomal spacing in a model system. Nucleosomal arrays, assembled on a 1500-bp-long human telomeric DNA and on a DNA fragment containing 8 copies of the 601 strong nucleosome positioning sequence, have been studied at the single molecule level, by atomic force microscopy imaging. Random nucleosome positioning was found in the case of human telomeric DNA. On the contrary, nucleosome positioning on 601 DNA is characterized by preferential positions of nucleosome dyad axis each 200 bp. The AFM-derived nucleosome organization is in satisfactory agreement with that predicted by theoretical modeling, based on sequence-dependent DNA curvature and flexibility. The reported results show that DNA sequence has a main role, not only in mononucleosome thermodynamic stability, but also in the organization of nucleosomal arrays.

A statistical approach for analyzing structural and regulative information in prokaryotic genomes.

Although DNA is iconized as a straight double helix, it does not exist in this canonical form in biological systems. Instead, it is characterized by sequence dependent structural and dynamic deviations from the monotonous regularity of the canonical B-DNA. Despite the complexity of the system, we showed that DNA structural and dynamics large-scale properties can be predicted starting from the simple knowledge of nucleotide sequence by adopting a statistical approach. The paper reports the statistical analysis of large pools of different prokaryotic genes in terms of the sequence-dependent curvature and flexibility. Conserved features characterize the regions close to the Start Translation Site, which are related to their function in the regulation system. In addition, regular patterns with three-fold periodicity were found in the coding regions. They were reproduced in terms of the nucleotide frequency expected on the basis of the genetic code and the pertinent occurrence of the aminoacid residues.

Theoretical models of possible compact nucleosome structures.

Chromatin structure seems related to the DNA linker length. This paper presents a systematic search of the possible chromatin structure as a function of the linker lengths, starting from three different low-resolution molecular models of the nucleosome. Gay-Berne potential was used to evaluate the relative nucleosome packing energy. Results suggest that linker DNAs, which bridges and orientate nucleosomes, affect both the geometry and the rigidity of the global chromatin structure.

Organization of telomeric nucleosomes: atomic force microscopy imaging and theoretical modeling.

Telomeric chromatin has peculiar features with respect to bulk chromatin, which are not fully clarified to date. Nucleosomal arrays, reconstituted on fragments of human telomeric DNA and on tandemly repeated tetramers of 5S rDNA, have been investigated at single-molecule level by atomic force microscopy and Monte Carlo simulations. A satisfactory correlation emerges between experimental and theoretical internucleosomal distance distributions. However, in the case of telomeric nucleosomal arrays containing two nucleosomes, we found significant differences. Our results show that sequence features of DNA are significant in the basic chromatin organization, but are not the only determinant.

A possible role of DNA superstructures in genome evolution.

The concept of DNA as a simple repository of the gene information has changed in that of a polymorphic macromolecule, which plays a relevant part in the management of the complex biochemical transformations in living matter. As a consequence of the slight stereochemical differences between base pairs, the direction of the DNA double helix axis undergoes deterministic writhing. A useful representation of such sequence-dependent structural distortions is the curvature diagram. Here, it is reported as an evolution simulation obtained by extensive point mutations along a biologically important DNA tract. The curvature changes, consequence of the point mutations. were compared to the related experimental gel electrophoresis mobility. The curvature of most mutants decreases and the mobility increases accordingly, suggesting the curvature of that tract is genetically selected. Moreover, DNA images by scanning force microscopy, show evidence of a sequence-dependent adhesion of curved DNA tracts to inorganic crystal surfaces. In particular, mica shows a large affinity towards the TT-rich dinucleotide sequences. This suggests a possible mechanism of selection of curved DNA regions, characterized by AA.TT dinucleotides in phase with double-helical periodicity, in the very early evolution steps.

Dual role of sequence-dependent DNA curvature in nucleosome stability: the critical test of highly bent Crithidia fasciculata DNA tract.

In spite of the knowledge of the nucleosome molecular structure, the role of DNA intrinsic curvature in determining nucleosome stabilization is still an open question. In this paper, we describe a general model that allows the prediction of the nucleosome stability, tested on 83 different DNA sequences, in surprising good agreement with the experimental data, carried out in ours as well as in many other laboratories. The model is based on the dual role of DNA curvature in nucleosome thermodynamic stabilization. A critical test is the evaluation of the nucleosome free energy relative to a Crithidia fasciculata kinetoplast DNA fragment, which represents the most curved DNA found so far in biological systems and, therefore, is generally believed to form a highly stable nucleosome.

Systematic search for compact structures of telomeric nucleosomes.

Telomeres are structures functionally and structurally distinct from bulk chromatin. They are constituted of highly conserved 5-7 bp tandemly repeated units, organized into nucleosomes with short linkers, whereas the knowledge of the linker histone role in telomeric chromatin is still fragmentary. Experimental evidence suggests the structural organization of telomeric nucleosomes is different from that of the bulk chromatin. This work presents a systematic search of the telomeric nucleosome arrangements. A low-resolution molecular model was used to evaluate the relative nucleosome packing energy. Structures with favorable energy were found, reducing the possible telomeric chromatin conformations to two different three-dimensional folds.

Sequence-dependent DNA dynamics by scanning force microscopy time-resolved imaging.

Scanning force microscopy was used to study in fluid the conformational fluctuations of two double-stranded DNA molecules resulting from differently cut pBR322 circular DNAs. A new approach was conceived to monitor the thermodynamic equilibrium of the chain dynamics on different scale lengths. This method made it possible to demonstrate that both the observed DNA molecules were allowed to equilibrate only on their local small-scale dynamics during the time of the experiment. This capability of monitoring the length scale and the time scale of the equilibration processes in the dynamics of a DNA chain is relevant to give an insight in the thermodynamics of the DNA binding with proteins and synthetic ligands. It was also shown that the small-scale equilibration of the DNA chain during surface-restricted dynamics is enough to allow a valid measurement of the local sequence-dependent curvature.

Sequence-dependent DNA curvature and flexibility from scanning force microscopy images.

This paper reports a study of the sequence-dependent DNA curvature and flexibility based on scanning force microscopy (SFM) images. We used a palindromic dimer of a 1878-bp pBR322 fragment and collected a large pool of SFM images. The curvature of each imaged chain was measured in modulus and direction. It was found that the ensemble curvature modulus does not allow the separation of static and dynamic contributions to the curvature, whereas the curvature, when its direction in the two dimensions is taken into account, permits the direct separation of the intrinsic curvature contributions static and dynamic contributions. The palindromic symmetry also acted as an internal gauge of the validity of the SFM images statistical analysis. DNA static curvature resulted in good agreement with the predicted sequence-dependent intrinsic curvature. Furthermore, DNA sequence-dependent flexibility was found to correlate with the occurrence of A.T-rich dinucleotide steps along the chain and, in general, with the normalized basepair stacking energy distribution.

Recognition of the DNA sequence by an inorganic crystal surface.

The sequence-dependent curvature is generally recognized as an important and biologically relevant property of DNA because it is involved in the formation and stability of association complexes with proteins. When a DNA tract, intrinsically curved for the periodical recurrence on the same strand of A-tracts phased with the B-DNA periodicity, is deposited on a flat surface, it exposes to that surface either a T- or an A-rich face. The surface of a freshly cleaved mica crystal recognizes those two faces and preferentially interacts with the former one. Statistical analysis of scanning force microscopy (SFM) images provides evidence of this recognition between an inorganic crystal surface and nanoscale structures of double-stranded DNA. This finding could open the way toward the use of the sequence-dependent adhesion to specific crystal faces for nanotechnological purposes.