The design of small complexes that bind and react at specific sequences of DNA becomes important as we begin to delineate, on a molecular level, how genetic information is expressed. A more complete understanding of how to target DNA sites with specificity will lead not only to novel chemotherapeutics but also to a greatly expanded ability for chemists to probe DNA and to develop highly sensitive diagnostic agents.

Recognition and reaction of metallointercalators with DNA.

Here we review studies that provided important information about conformational properties of DNA using circular dichroic (CD) spectroscopy. The conformational properties include the B-family of structures, A-form, Z-form, guanine quadruplexes, cytosine quadruplexes, triplexes and other less characterized structures. CD spectroscopy is extremely sensitive and relatively inexpensive. This fast and simple method can be used at low- as well as high-DNA concentrations and with short- as well as long-DNA molecules. The samples can easily be titrated with various agents to cause conformational isomerizations of DNA. The course of detected CD spectral changes makes possible to distinguish between gradual changes within a single DNA conformation and cooperative isomerizations between discrete structural states. It enables measuring kinetics of the appearance of particular conformers and determination of their thermodynamic parameters. In careful hands, CD spectroscopy is a valuable tool for mapping conformational properties of particular DNA molecules. Due to its numerous advantages, CD spectroscopy significantly participated in all basic conformational findings on DNA.

/pdf/circular-dichroism-and-conformational-polymorphism-of-dna-1k7nf4rrob.pdf

Circular dichroism and conformational polymorphism of DNA

The assembly of synthetic, controllable molecular mechanical systems1,2,3,4,5,6,7 is one of the goals of nanotechnology. Protein-based molecular machines, often driven by an energy source such as ATP, are abundant in biology8,9. It has been shown previously that branched motifs of DNA can provide components for the assembly of nanoscale objects10, links11 and arrays12. Here we show that such structures can also provide the basis for dynamic assemblies: switchable molecular machines. We have constructed a supramolecular device consisting of two rigid DNA ‘double-crossover’ (DX) molecules connected by 4.5 double-helical turns. One domain of each DX molecule is attached to the connecting helix. To effect switchable motion in this assembly, we use the transition between the B and Z13,14 forms of DNA. In conditions that favour B-DNA, the two unconnected domains of the DX molecules lie on the same side of the central helix. In Z-DNA-promoting conditions, however, these domains switch to opposite sides of the helix. This relative repositioning is detected by means of fluorescence resonance energy transfer spectroscopy, which measures the relative proximity of two dye molecules attached to the free ends of the DX molecules. The switching event induces atomic displacements of 20–60 A.

A nanomechanical device based on the B–Z transition of DNA

Transition metal complexes that reversibly bind to DNA have been studied for almost 30 years. In the last few years a variety of new systems have been developed, employing a range of metal ions and ligand architectures. In many cases, high affinity binding and specific selectivities have been observed. These complexes display properties that make them attractive as probes of DNA structure and function, suggesting that they may find a role as prototypical tools for a spectrum of applications, from basic molecular biology to medicine. This review presents an overview of some of the structures and properties of such complexes.

Kinetically inert transition metal complexes that reversibly bind to DNA

In this review, we give an overview of recent literature on the structure and stability of unimolecular G-rich quadruplex structures that are relevant to drug design and for in vivo function The unifying theme in this review is energetics The thermodynamic stability of quadruplexes has not been studied in the same detail as DNA and RNA duplexes, and there are important differences in the balance of forces between these classes of folded oligonucleotides We provide an overview of the principles of stability and where available the experimental data that report on these principles Significant gaps in the literature have been identified, that should be filled by a systematic study of well-defined quadruplexes not only to provide the basic understanding of stability both for design purposes, but also as it relates to in vivo occurrence of quadruplexes Techniques that are commonly applied to the determination of the structure, stability and folding are discussed in terms of information content and limitations Quadruplex structures fold and unfold comparatively slowly, and DNA unwinding events associated with transcription and replication may be operating far from equilibrium The kinetics of formation and resolution of quadruplexes, and methodologies are discussed in the context of stability and their possible biological occurrence

Stability and kinetics of G-quadruplex structures

We have recently reported the cation-induced self-assembly of DNA oligomers of the general sequence C4T4G4T1-4G4 into high-molecular weight multistranded structures [Marotta, S.P., Tamburri, P.A., and Sheardy, R.D. (1996) Biochemistry 35, 10484-10492]. The architecture of the proposed structure consists of a series of four leafed G4 tetrads tethered together via one or two T1-4 strands and thus resembles a long four-sided hollow tube with periodic "pockets". These pockets possess electrostatic, hydrogen bonding, and hydrophobic contact points and should be ideal candidates for the binding of small molecules. To assess the potential of using porphyrins as probes for these structures, we have investigated the interaction of tetrakis(4-N-methylpyridyl)porphine (H2TMPyP) with the simple quadruplex formed by T4G4 and with the duplex formed by CGCGATATCGCG. Visible absorption, circular dichroism, and fluorescent energy transfer studies indicate that H2TMPyP binds to both the duplex and quadruplex via intercalation at low [porphyrin]/[DNA molecule] ratios, i.e., in the presence of excess potential DNA binding sites. Analyses of Scatchard plots show that H2TMpyP binds with high affinity to both DNA secondary structures but binds to the quadruplex with an affinity 2 times greater than that of the duplex.

Porphyrin Binding to Quadruplexed T4G4

Quadruplex structures arise from four coplanar G bases arranged in a Hoogsteen base pairing motif to create a central pore that can coordinate cations. The termini of eukaryotic chromosomes contain structures, known as telomeres, which are capable of forming quadruplex structures. Quadruplexes have been implicated in a variety of disease states, including cancer. The literature seems to agree that the human telomeric repeat containing four stretches of three guanines displays conformational states that are different in the presence of Na+ and K+ and an unknown number of species involved in the quadruplex to single strand transition. Using circular dichroism spectroscopy, differential scanning calorimetry, and singular-value decomposition, the number of species present in the dissociation process is assessed. The results indicate that three species exist in equilibria during the melting process. We present a model for the heat-induced denaturation from the folded to the unfolded state, whereby the hybrid p...

Biophysical Characterization of the Human Telomeric (TTAGGG)4 Repeat in a Potassium Solution

Genetic expansion diseases have been linked to the properties of triplet repeat DNA sequences during replication. The most common triplet repeats associated with such diseases are CAG, CCG, CGG, and CTG. It has been suggested that gene expansion occurs as a result of hairpin formation of long stretches of these sequences on the leading daughter strand synthesized during DNA replication [Gellibolian, R., Bacolla, A., and Wells, R. D. (1997) J. Biol. Chem. 272, 16793-7]. To test the biophysical basis for this model, oligonucleotides of general sequence (CNG)(n), where N = A, C, G, or T and n = 4, 5, 10, 15, or 25, were synthesized and characterized by circular dichroism (CD) spectropolarimetry, optical melting studies, and differential scanning calorimetry (DSC). The goal of these studies was to evaluate the influence of sequence context and oligomer length on their secondary structures and stabilities. The results indicate that all single oligomers, even those as short as 12 nucleotides, form stable hairpin structures at 25 degrees C. Such hairpins are characterized by the presence of N:N mismatched base pairs sandwiched between G:C base pairs in the stems and loops of three to four unpaired bases. Thermodynamic analysis of these structures reveals that their stabilities are influenced by both the sequence of the particular oligomer and its length. Specifically, the stability order of CGG > CTG > CAG > CCG was observed. In addition, longer oligomers were found to be more stable than shorter oligomers of the same sequence. However, a stability plateau above 45 nucleotides suggests that the length dependence reaches a maximum value where the stability of the G:C base pairs can no longer compensate the instability of the N:N mismatches in the stems of the hairpins. The results are discussed in terms of the above model proposed for gene expansion.

Influence of sequence context and length on the structure and stability of triplet repeat DNA oligomers.

A new class of DNA metallobinders showing spectator ligand size selectivity: binding of ligand-bridged bimetallic complexes of ruthenium(II) to calf thymus DNA

Richard D. Sheardy

Papers

Porphyrin Binding to Quadruplexed T4G4

Biophysical Characterization of the Human Telomeric (TTAGGG)4 Repeat in a Potassium Solution

Influence of sequence context and length on the structure and stability of triplet repeat DNA oligomers.

A new class of DNA metallobinders showing spectator ligand size selectivity: binding of ligand-bridged bimetallic complexes of ruthenium(II) to calf thymus DNA

Porphyrin Binding to Quadruplexed T 4 G 4