Stacking heterogeneity: a model for the sequence dependent melting cooperativity of duplex DNA

A microscopic Potts-like one-dimensional model with many particle interactions [referred as the generalized model of polypeptide chains (GMPCs)] is developed to investigate cooperativity of DNA sequence dependent melting. For modeling sequence, regular homogeneous sequences were arranged in heterogeneous blocks of various lengths. Within the framework of the GMPC the authors show that the inclusion of stacking interaction heterogeneity relative to homogeneous hydrogen bond interactions leads to an unexpected and quite remarkable increase in melting cooperativity for small blocks. In some cases this tendency persists for long blocks having sharp sequence heterogeneity.     

Influence of microwave irradiation on DNA hybridization and polymerase reactions

We report herein that exposure of DNA to microwave irradiation at constant temperature leads to faster strand exchange, as compared with same experiments carried out in a water bath at the same temperature. Furthermore, polymerase chain reactions carried out under microwave irradiation were faster than those in a water bath at the same temperature as well. While the causes of these differences are unclear at this time, this research suggests that microwave irradiation can lead to subtle changes in DNA structural dynamics and functions.     

The influence of sequence context and length on the kinetics of DNA duplex formation from complementary hairpins possessing (CNG) repeats

The formation of unusual structures during DNA replication has been invoked for gene expansion in genomes possessing triplet repeat sequences, CNG, where N = A, C, G, or T. In particular, it has been suggested that the daughter strand of the leading strand partially dissociates from the parent strand and forms a hairpin. The equilibrium between the fully duplexed parent:daugter species and the parent:hairpin species is dependent upon their relative stabilities and the rates of reannealing of the daughter strand back to the parent. These stabilities and rates are ultimately influenced by the sequence context of the DNA and its length. Previous work has demonstrated that longer strands are more stable than shorter strands and that the identity of N also influences the thermal stability [Paiva, A. M.; Sheardy, R. D. Biochemistry 2004, 43, 14218-14227]. Here, we show that the rate of duplex formation from complementary hairpins is also sequence context and length dependent. In particular, longer duplexes have higher activation energies than shorter duplexes of the same sequence context. Further, [(CCG):(GGC)] duplexes have lower activation energies than corresponding [(CAG):(GTC)] duplexes of the same length. Hence, hairpins formed from long CNG sequences are more thermodynamically stable and have slower kinetics for reannealing to their complement than shorter analogues. Gene expansion can now be explained in terms of thermodynamics and kinetics.     

Metal-salen-base-pair complexes inside DNA: complexation overrides sequence information

Two isomeric salicylic aldehyde nucleobases have been prepared and incorporated into various DNA duplexes. Reaction with ethylenediamine leads to formation of the well-known salen ligand inside the DNA double helix. Addition of transition-metal ions such as Cu(2+), Mn(2+), Ni(2+), Fe(2+), or VO(2+) results in the formation of metal-salen-base-pair complexes, which were studied by using UV and circular dichroism (CD) spectroscopy. HPLC and ESI mass spectrometric measurements reveal an unusually high stability of the DNA-metal system. These metal-salen complexes act as interstrand cross-links and thereby lead to a strong stabilization of the DNA duplexes, as studied by thermal de- and renaturing experiments. Complex formation is strong enough to override sequence information even when the preorganization of the ligand precursors is unfavorable and the DNA duplex is distorted by the metal complexation. Furthermore, melting-point studies show that the salen complex derived from ligand 2 fits better into the DNA duplex, in accordance with results obtained from the crystal structure of the corresponding copper-salen complex 8.     

Photoactive Ru(II) -polypyridyl complexes that display sequence selectivity and high-affinity binding to duplex DNA through groove binding

The duplex-DNA binding properties of a nonintercalating polypyridyl ruthenium(II) complex that incorporates a linear extended ligand with a catechol moiety has been probed with a variety of photo- and biophysical techniques. These studies reveal that the complex groove binds to DNA sequences biphasically, and displays binding constants equivalent to those of high-affinity metallointercalators. The complex also displays preferential binding to AT-rich sequences. Changes in the structure of the coordinated catechol ligand and the incorporation of intercalating ancillary ligands into the complex were found to modulate both the optical-binding response and binding parameters of the system, which indicates that the catechol moiety plays a crucial role in the observed enhancement to binding affinities.     

A comparison of the binding of metal complexes to duplex and quadruplex DNA

Electrospray ionisation mass spectrometry (ESI-MS) and circular dichroism (CD) spectroscopy were used to compare the binding of mononuclear nickel, ruthenium and platinum complexes to double stranded DNA (dsDNA) and quadruplex DNA (qDNA). CD studies provided evidence for the binding of intact complexes of all three metal ions to qDNA. ESI mass spectra of solutions containing platinum or ruthenium complexes and qDNA showed evidence for the formation of non-covalent complexes consisting of intact metal molecules bound to DNA. However, the corresponding spectra of solutions containing nickel complexes principally contained ions consisting of fragments of the initial nickel molecule bound to qDNA. In contrast ESI mass spectra of solutions containing nickel, ruthenium or platinum complexes and dsDNA only showed the presence of ions attributable to intact metal molecules bound to DNA. The fragmentation observed in mass spectral studies of solutions containing nickel complexes and qDNA is attributable to the lower thermodynamic stability of the former metal complexes relative to those containing platinum or ruthenium, as well as the slightly harsher instrumental conditions required to obtain spectra of qDNA. This conclusion is supported by the results of tandem mass spectral studies, which showed that ions consisting of intact nickel complexes bound to qDNA readily undergo fragmentation by loss of one of the ligands initially bound to the metal. The ESI-MS results also demonstrate that the binding affinity of each of the platinum and ruthenium complexes towards qDNA is significantly less than that towards dsDNA.     

Molecular dynamics studies of ion distributions for DNA duplexes and DNA clusters: salt effects and connection to DNA melting

We present extensive molecular dynamics simulations of the ion distributions for DNA duplexes and DNA clusters using the Amber force field with implicit water. The distribution of ions and the electrostatic energy of ions around an isolated DNA duplex and clusters of DNA duplexes in different salt (NaCl) concentrations over the range 0.2-1.0 mol/L are determined on the basis of the simulation results. Using the electrostatic energy profile, we determine a local net charge fraction phi, which is found to increase with increasing of salt concentration. For DNA clusters containing two DNA duplexes (DNA pair) or four DNA duplexes, phi increases as the distance between the duplexes decreases. Combining this result with experimental results for the dependence of the DNA melting temperature on bulk salt concentration, we conclude that for a pair of DNA duplexes the melting temperature increases by 5-10 K for interaxis separations of 25-40 A. For a cluster of four DNA duplexes, an even larger melting temperature increase should occur. We argue that this melting temperature increase in dense DNA clusters is responsible for the cooperative melting mechanism in DNA-linked nanoparticle aggregates and DNA-linked polymer aggregates.     

Observation of daunomycin and nogalamycin complexes with duplex DNA using electrospray ionisation mass spectrometry

The noncovalent binding of the antitumour drugs daunomycin and nogalamycin to duplex DNA has been studied using electrospray ionisation mass spectrometry (ESI-MS). The conditions for the preparation of drug/duplex DNA complexes and for their detection by ESI-MS have been optimised. Ions corresponding to these complexes were most abundant relative to free DNA when prepared in the pH range 8-9, and using gentle ESI interface conditions. Self-complementary oligonucleotides, 5'-d(GGCTAGCC)-3' or 5'-d(CGGCGCCG)-3', annealed in the presence of a 5-fold molar excess of either nogalamycin or daunomycin gave ESI mass spectra in which the most intense ions corresponded to three molecules of drug bound to duplex DNA, with some evidence for four drug molecules bound. For binding to 5'-d(TGAGCTAGCTCA)(2)-3', complexes containing up to four nogalamycin and six daunomycin molecules were observed. These data are consistent with the neighbour exclusion principle whereby intercalation occurs between every other base pair such that up to four bound drugs would be expected for the 8 mers and up to six for the 12 mer. Competition experiments involving a single drug in an equimolar mixture of two oligonucleotides (5'-d(TGAGCTAGCTCA)(2)-3' with either 5'-d(CGGCGCCG)(2)-3' or 5'-d(GGCTAGCC)(2)-3') showed ions arising from complexes of drug/5'-d(CGGCGCCG)(2)-3' were more intense than complexes of drug/5'-d(GGCTAGCC)(2)-3', relative to those from the 12 mer in each mixture. While this suggests ESI-MS has the potential to detect differences in sequence selectivity, more detailed experiments involving a comparison of the relative ionisation efficiency of different oligonucleotides and a wider range of intercalators are required to establish this definitively. ESI mass spectra from experiments in which both drugs were reacted with the same oligonucleotide were more complex, such that a clear preference for one drug could not be established.     

Dinuclear monointercalating RuII complexes that display high affinity binding to duplex and quadruplex DNA

The DNA duplex binding properties of previously reported dinuclear Ru(II) complexes based on the ditopic ligands tetrapyrido[3,2-a:2',3'-c:3',2'-h:2',3'-j]phenazine (tppz) and tetraazatetrapyrido[3,2-a:2'3'-c:3',2'-l:2',3'-n]pentacene (tatpp) are reported. Photophysical and biophysical studies indicate that, even at high ionic strengths, these complexes bind to duplex DNA, through intercalation, with affinities that are higher than any other monointercalating complex and are only equalled by DNA-threaded bisintercalating complexes. Additional studies at high ionic strengths using the 22-mer d(AG(3)[T(2)AG(3)](3)) [G3] human telomeric sequence reveal that the dinuclear tppz-based systems also bind with high affinity to quadruplex DNA. Furthermore, for these complexes, quadruplex binding is accompanied by a distinctive blue-shifted "light-switch" effect, characterized by higher emission enhancements than those observed in the analogous duplex effect. Calorimetry studies reveal that the thermodynamics of duplex and quadruplex binding is distinctly different, with the former being entirely entropically driven and the latter being both enthalpically and entropically favored.     

Immobilization of oligonucleotides onto silica nanoparticles for DNA hybridization studies

This paper describes the development of oligonucleotide-functionalized nanoparticles. We used disulfide-coupling chemistry for the immobilization of oligonucleotides onto silica nanoparticles and subsequently demonstrated the properties of the resulting DNA nanoparticles. Factors influencing the immobilization and hybridization processes were examined and optimized. The oligonucleotide-modified silica nanoparticles provide an efficient substrate for hybridization and can be used in the development of DNA biosensors and biochips.     

Kinetic studies on the reactions of [Pd(dach)(X-Y)] complexes with some DNA constituents

The kinetics of complex-formation reactions of six Pd(dach) complexes, dach = 1,2-trans-R,R-diaminocyclohexane, viz. [Pd(dach)Cl2], [Pd(dach)(H2O)2]2+, and four complexes with different chelating leaving groups X-Y, viz. [Pd(dach)(O,O-cyclobutane-1,1-dicarboxylate)], [Pd(dach)(N,O-glycine)](+), [Pd(dach)(N,S-methionine)]+ and [Pd(dach)(O,O-oxalate)], were studied. The effect of the leaving group on the lability of the resulting Pd(ii) complexes was studied for the nucleophiles inosine, inosine-5'-monophosphate and guanosine-5'-monophosphate under pseudo-first-order conditions as a function of nucleophile concentration, temperature and pressure using stopped-flow techniques. Two consecutive reaction steps, which both depend on the nucleophile concentration, were observed. The rate constants for all reactions indicate a direct substitution of the X-Y chelate by the selected nucleophiles, thereby showing that the nature of the chelate, viz. O-O (cbdca), (ox), N-O (gly) or S-N (l-met), plays an important role in the kinetic and mechanistic behavior of the Pd(ii) complexes. The mechanism of the substitution reactions is associative in nature as supported by the large and negative values of DeltaS(double dagger) and DeltaV(double dagger).     

Development of a multiplex system to assess DNA persistence in taphonomic studies

In this study, we have developed a PCR multiplex that can be used to assess DNA degradation and at the same time monitor for inhibition: primers have been designed to amplify human, pig, and rabbit DNA, allowing pig and rabbit to be used as experimental models for taphonomic research, but also enabling studies on human DNA persistence in forensic evidence. Internal amplified controls have been added to monitor for inhibition, allowing the effects of degradation and inhibition to be differentiated. Sequence data for single‐copy nuclear recombination activation gene (RAG‐1) from human, pig, and rabbit were aligned to identify conserved regions and primers were designed that targeted amplicons of 70, 194, 305, and 384 bp. Robust amplification in all three species was possible using as little as 0.3 ng of template DNA. These have been combined with primers that will amplify a bacterial DNA template within the PCR. The multiplex has been evaluated in a series of experiments to gain more knowledge of DNA persistence in soft tissues, which can be important when assessing what material to collect following events such as mass disasters or conflict, when muscle or bone material can be used to aid with the identification of human remains. The experiments used pigs as a model species. When whole pig bodies were exposed to the environment in Northwest England, DNA in muscle tissue persisted for over 24 days in the summer and over 77 days in the winter, with full profiles generated from these samples. In addition to time, accumulated degree days (ADD) were also used as a measure that combines both time and temperature—24 days was in summer equivalent to 295 ADD whereas 77 days in winter was equivalent to 494 ADD.     

Active microeletronic chip devices which utilize controlled electrophoretic fields for multiplex DNA hybridization and other genomic applications

Microelectronic DNA chip devices that contain planar arrays of microelectrodes have been developed for multiplex DNA hybridization and a variety of genomic research and DNA diagnostic applications. These devices are able to produce almost any desired electric field configuration on their surface. This ability to produce well-defined electric fields allows charged molecules (DNA, RNA, proteins, enzymes, antibodies, nanobeads, and even micron scale semiconductor devices) to be electrophoretically transported to or from any microlocation on the planar surface of the device. Of key importance to the device function is the permeation layer which overcoats the microelectrodes. The permeation layer is generally a porous hydrogel material that allows water molecules and small ions (Na+, CI-, etc.) to freely contact the microelectrode surface, but impedes the transport of the larger analytes (oligonucleotides, DNA, RNA, proteins, etc.). The permeation layer prevents the destruction of DNA at the active microelectrode surface, ameliorates the adverse effects of electrolysis products on the sensitive hybridization reactions, and serves as a porous support structure for attaching DNA probes and other molecules to the array. In order to maintain rapid transport of DNA molecules, facilitate hybridization, and work within constrained current and voltage ranges, low conductance buffers and various electronic pulsing scenarios have also been developed. These active microelectronic array devices allow electrophoretic fields to be used to carry out accelerated DNA hybridization reactions and to improve selectivity for single nucleotide polymorphism (SNP), short tandem repeat (STR), and point mutation analysis.     

Base sequence effects in radical cation migration in duplex DNA: support for the polaron-like hopping model

A series of anthraquinone-linked (AQ) duplex DNA oligomers were prepared and investigated. Irradiation of the AQ injects a radical cation into the DNA. The radical cation migrates through the DNA and reacts selectively at GG steps, which leads to strand cleavage after treatment with piperidine. The oligomers investigated in this work were selected to assess the effect on long-distance charge transport of placing a T base (or bases) in a strand of repeating purine bases. With notable exceptions, the amount of strand scission decreases with the distance between the AQ and the GG step. The results are consistent only with models for long-distance transport, such as thermally activated polaron-like hopping, that incorporate radical cation delocalization over two or more adjacent bases.     

Influence of initial charge state on fragmentation patterns for noncovalent drug/DNA duplex complexes

The charge state-dependent dissociation of various DNA duplexes and drug/duplex complexes has been investigated using collisionally activated dissociation (CAD) in a quadrupole ion trap mass spectrometer (QIT-MS). Several non-self-complementary 14-residue oligonucleotides were employed, in addition to an array of known DNA-interactive ligands, including the intercalators daunomycin and nogalamycin, as well as the minor groove binding agents distamycin, netropsin, 4',6-diamidino-2-phenylindole, and Hoechst 33342. In general, the dissociation pathways exhibited by both the duplexes and the drug/duplex complexes were found to be markedly sensitive to initial charge state. Time- and activation voltage-independent duplex strand separation predominated for higher charge states, which was interpreted to be a result of internal Coulombic repulsion or partial unzipping in the interface, while time- and activation voltage-dependent covalent cleavage predominated for lower charge states. The identity of the drug and the sequence of the duplex were both found to affect the competition between different dissociation processes. The dissociation pathways for the lower charge state complexes are probably more reflective of specific drug-DNA interactions because Coulombic and/or conformational effects are less marked for these precursors.     

Time-resolved studies of CN radical reactions and the role of complexes in solution

Time-resolved studies using 100 fs laser pulses generate CN radicals photolytically in solution and probe their subsequent reaction with solvent molecules by monitoring both radical loss and product formation. The experiments follow the CN reactants by transient electronic spectroscopy at 400 nm and monitor the HCN products by transient vibrational spectroscopy near 3.07 microm. The observation that CN disappears more slowly than HCN appears shows that the two processes are decoupled kinetically and suggests that the CN radicals rapidly form two different types of complexes that have different reactivities. Electronic structure calculations find two bound complexes between CN and a typical solvent molecule (CH(2)Cl(2)) that are consistent with this picture. The more weakly bound complex is linear with CN bound to an H atom through the N atom, and the more strongly bound complex has a structure in which the CN bridges Cl and H atoms of the solvent. Fitting the transient absorption data with a kinetic model containing two uncoupled complexes reproduces the data for seven different chlorinated alkane solvents and yields rate constants for the reaction of each type of complex. Depending on the solvent, the linear complex reacts between 2.5 and 12 times faster than the bridging complex and is the primary source of the HCN reaction product. Increasing the Cl atom content of the solvents decreases the reaction rate for both complexes.     

ESI-MS and thermal melting studies of nanoscale platinum(II) metallomacrocycles with DNA

The hydrophilic, long-chain diamine PEGda (O,O'-bis(2-aminoethyl)octadeca(ethylene glycol)), when complexed with cis-protected Pt(II) ions afforded water-soluble complexes of the type [Pt(N,N)(PEGda)](NO(3))(2) (N,N = N,N,N',N'-tetramethyl-1,2-diaminoethane (tmeda), 1,2-diaminoethane (en), and 2,2'-bipyridine (2,2'-bipy)) featuring unusual 62-membered chelate rings. Equimolar mixtures containing either the 16-mer duplex DNA D2 or the single-stranded D2a and [Pt(N,N)(PEGda)](2+) were analyzed by negative-ion ESI-MS. Analysis of D2-Pt(II) mixtures showed the formation of 1 : 1 adducts of [Pt(en)(PEGda)](2+), [Pt(tmeda)(PEGda)](2+) and the previously-described metallomacrocycle [Pt(2)(2,2'-bipy)(2){4,4'-bipy(CH(2))(4)4,4'-bipy}(2)](8+) with D2; the dinuclear species bound to D2 most strongly, consistent with its greater charge and aromatic surface area. D2 formed 1 : 2 complexes with the acyclic species [Pt(2,2'-bipy)(Mebipy)(2)](4+) and [Pt(2,2'-bipy)(NH(3))(2)](2+). Analyses of D2a-Pt(II) mixtures gave results similar to those obtained with D2, although fragmentation was more pronounced, indicating that the nucleobases in D2a play more significant roles in mediating the decomposition of complexes than those in D2, in which they are paired in a complementary manner. Investigations were also conducted into the effects of selected platinum(II) complexes on the thermal denaturation of calf thymus DNA (CT-DNA) in buffered solution. Both [Pt(2)(2,2'-bipy)(2){4,4'-bipy(CH(2))(6)4,4'-bipy}(2)](8+) and [Pt(2,2'-bipy)(Mebipy)(2)](4+) stabilized CT-DNA. In contrast, [Pt(tmeda)(PEGda)](2+) and [Pt(en)(PEGda)](2+) (as well as free PEGda) caused negligible changes in melting temperature (ΔT(m)), suggesting that these species interact weakly with CT-DNA.     

Towards sequence selective DNA binding: design, synthesis and DNA binding studies of novel bis-porphyrin peptidic nanostructures

A new series of peptidic nanostructures bearing two intercalating moieties was designed and synthesized to achieve selective recognition of DNA sequences. A cationic porphyrin was attached to a glutamic acid side chain and the latter introduced into a peptidic sequence by standard solid-phase peptide synthesis methodology. Conformation of the hydrosoluble peptidic structures bearing two cationic porphyrins was studied by circular dichroism. Using UV-visible spectroscopy and induced circular dichroism, we demonstrate that the compounds are fully intercalated upon binding to double-stranded DNA and that the compounds exhibit a tremendous preference for GC over AT sequences for intercalation.