Melting Temperature Of DNA Research Articles

Non-symmetrical furan-amidines as novel leads for the treatment of cancer and malaria

NRH:quinone oxidoreductase 2 enzyme (NQO2) is a potential therapeutic target in cancer and neurodegenerative diseases, with roles in either chemoprevention or chemotherapy. Here we report the design, synthesis and evaluation of non-symmetrical furan-amidines and their analogues as novel selective NQO2 inhibitors with reduced adverse off-target effects, such as binding to DNA. A pathway for the synthesis of the non-symmetrical furan-amidines was established from the corresponding 1,4-diketones. The synthesized non-symmetrical furan-amidines and their analogues showed potent NQO2 inhibition activity with nano-molar IC50 values. The most active compounds were non-symmetrical furan-amidines with meta- and para-nitro substitution on the aromatic ring, with IC50 values of 15 nM. In contrast to the symmetric furan-amidines, which showed potent intercalation in the minor grooves of DNA, the synthesized non-symmetrical furan-amidines showed no affinity towards DNA, as demonstrated by DNA melting temperature experiments. In addition, Plasmodium parasites, which possess their own quinone oxidoreductase PfNDH2, were inhibited by the non-symmetrical furan-amidines, the most active possessing a para-fluoro substituent (IC50 9.6 nM). The high NQO2 inhibition activity and nanomolar antimalarial effect of some of these analogues suggest the lead compounds are worthy of further development and optimization as potential drugs for novel anti-cancer and antimalarial strategies.

The Effect of Common Cations on DNA Degradation

The process of DNA degradation is important to many scientific studies. Heat treatment is the standard procedure used to degrade genetic material by heating DNA-containing media past the DNA melting temperature, but certain chemical alternatives have been explored. More specifically, the presence of cations, such as Mg²⁺, has been linked to increased stability of DNA molecules subjected to high temperatures. However, the possible effects of other ions have not been extensively studied; perhaps cations similar to magnesium may offer more versatile effects. This study examines the effects that NH₄⁺, Ni²⁺, and Li⁺ have on the heat degradation and melting temperature of DNA. Magnesium chloride, magnesium sulfate, ammonium sulfate, nickel chloride, and lithium chloride solutions of different concentrations were mixed with DNA samples; the resulting mixture was heated and subsequently analyzed using gel electrophoresis. Treatment with NH₄⁺ did not yield effects that significantly differed from Mg²⁺, while Li⁺ proved effective at preserving DNA even at high temperatures. Treatment with Ni²⁺ resulted in marked degradation of DNA. These results show that magnesium, ammonium, and especially lithium ions can be used for the preservation of DNA. The specific effects of Li⁺ and Ni²⁺ on DNA are promising subjects for future research.

Photodependent Melting of Unmodified DNA Using a Photosensitive Intercalator: A New and Generic Tool for Photoreversible Assembly of DNA Nanostructures at Constant Temperature.

External control of DNA melting and hybridization, a key step in bio- and DNA nanotechnology, is commonly achieved with temperature. The use of light to direct this process is a challenging alternative, which has been only possible with a DNA modification, such as covalent grafting or mismatch introduction, so far. Here we describe the first photocontrol of DNA melting that relies on the addition of a molecule that noncovalently interacts with unmodified DNA and affects its melting properties in a photoreversible and highly robust manner, without any prerequisite in the length or sequence of the target DNA molecule. We synthesize azobenzene-containing guanidinium derivatives and show that a bivalent molecule with a conformation-dependent binding mode, AzoDiGua, strongly increases the melting temperature (Tm) of DNA under dark conditions because its trans isomer intercalates in the DNA double helix. Upon UV irradiation at 365 nm, the trans-cis isomerization induced the ejection of AzoDiGua from the intercalation binding sites, resulting in a decrease in Tm up to 18 °C. This illumination-dependent Tm shift is observed on many types of DNA, from self-complementary single-stranded or double-stranded oligonucleotides to long genomic duplex DNA molecules. Finally, we show that simply adding AzoDiGua allows us to photoreversibly control the assembly/disassembly of a DNA nanostructure at constant temperature, as demonstrated here with a self-hybridized DNA hairpin. We anticipate that this strategy will be the key ingredient in a new and generic way of placing DNA-based bio- and nanotechnologies under dynamic control by light.

Characterization of the binding of paylean and DNA by fluorescence, UV spectroscopy and molecular docking techniques.

The interaction of paylean (PL) with calf thymus DNA (ctDNA) was investigated using fluorescence spectroscopy, UV absorption, melting studies, ionic strength, viscosity experiments and molecular docking under simulated physiological conditions. Values for the binding constant Ka between PL and DNA were 5.11 × 10(3) , 2.74 × 10(3) and 1.74 × 10(3) L mol(-1) at 19, 29 and 39°C respectively. DNA quenched the intrinsic fluorescence of PL via a static quenching procedure as shown from Stern-Volmer plots. The relative viscosity and the melting temperature of DNA were basically unchanged in the presence of PL. The fluorescence intensity of PL-DNA decreased with increasing ionic strength. The value of Ka for PL with double-stranded DNA (dsDNA) was larger than that for PL with single-stranded DNA (ssDNA). All the results revealed that the binding mode was groove binding, and molecular docking further indicated that PL was preferentially bonded to A-T-rich regions of DNA. The values for ΔH, ΔS and ΔG suggested that van der Waals forces or hydrogen bonding might be the main acting forces between PL and DNA. The binding distance was determined to be 3.37 nm based on the theory of Förster energy transference, which indicated that a non-radiation energy transfer process occurred. Copyright © 2015 John Wiley & Sons, Ltd.

Immobilization-mediated reduction in melting temperatures of DNA–DNA and DNA–RNA hybrids: Immobilized DNA probe hybridization studied by SPR

Abstract The melting temperature of probe-target nucleic acid hybrids plays an important role in sensor performance. This study employed surface plasmon resonance (SPR) techniques to investigate the hybridization of both DNA and RNA targets to their complementary surface-bound DNA oligonucleotide and the corresponding melting temperatures. The target molecule was a 26 nucleotide strand of RNA selected because of its utility in detecting the tobacco mosaic virus, a common plant pathogen with an RNA genome. The melting temperatures of duplexes with immobilized probes were determined by hybridization of the DNA and RNA targets with the bound DNA probe at different temperatures using a custom-built thermostated dual-channel SPR cell. Hybridization conditions and binding efficiencies were compared for both DNA and RNA binding to the gold-coated surface-bound DNA probe. The melting temperature for the DNA–DNA duplex was approximately 15 °C higher than the DNA–RNA in the solid state. The melting temperature in solution was also measured for comparison to the surface values. For both RNA and DNA, the melting transitions were substantially lower (∼20 °C) for solid state binding relative to solution. Particularly for surface immobilized DNA–RNA hybrids, the melting temperature was reduced to room temperature or below. Since most biosensor platforms operate at room temperature, they may consequently exhibit poor sensor performance due to weakened probe-target interactions. This result emphasizes that for optimal sensor performance, the hybrid melting temperature should be considered. The ability to study and optimize the binding of complementary strands of genomic materials to bound DNA probes could facilitate the rapid detection and identification of plant viruses having genomic RNA using various biosensor rapid analytical techniques.

Drosophila muller f elements maintain a distinct set of genomic properties over 40 million years of evolution.

The Muller F element (4.2 Mb, ~80 protein-coding genes) is an unusual autosome of Drosophila melanogaster; it is mostly heterochromatic with a low recombination rate. To investigate how these properties impact the evolution of repeats and genes, we manually improved the sequence and annotated the genes on the D. erecta, D. mojavensis, and D. grimshawi F elements and euchromatic domains from the Muller D element. We find that F elements have greater transposon density (25–50%) than euchromatic reference regions (3–11%). Among the F elements, D. grimshawi has the lowest transposon density (particularly DINE-1: 2% vs. 11–27%). F element genes have larger coding spans, more coding exons, larger introns, and lower codon bias. Comparison of the Effective Number of Codons with the Codon Adaptation Index shows that, in contrast to the other species, codon bias in D. grimshawi F element genes can be attributed primarily to selection instead of mutational biases, suggesting that density and types of transposons affect the degree of local heterochromatin formation. F element genes have lower estimated DNA melting temperatures than D element genes, potentially facilitating transcription through heterochromatin. Most F element genes (~90%) have remained on that element, but the F element has smaller syntenic blocks than genome averages (3.4–3.6 vs. 8.4–8.8 genes per block), indicating greater rates of inversion despite lower rates of recombination. Overall, the F element has maintained characteristics that are distinct from other autosomes in the Drosophila lineage, illuminating the constraints imposed by a heterochromatic milieu.

The interaction of amino acids, peptides, and proteins with DNA

Amino acids that carry charges on their side groups can bind to double stranded DNA (dsDNA) and change the strength of the double helix. Measurement of the DNA melting temperature (Tm) confirmed that acidic amino acids (Glu, Asp) weaken the H-bonds between DNA strands, whereas basic amino acids (Arg, Lys) strengthen the interaction between the strands. A rank correlation exists between the amino acid isoelectric points and the observed changes in Tm. A similar dependence of the hyperchromic effect on the isoelectric point of a protein (pepsin, insulin, cortexin, and protamine) was observed for DNA-protein complexes at room temperature. Short peptides (KE, AEDG, and KEDP) containing a mixture of acidic and basic amino acid residues also affect Tm and the stability of the double helix. A model for binding Glu and Lys to dsDNA was explored by a docking simulation. The model shows that Glu, in an untwisted shape, binds to dsDNA in its major groove and disrupts three H-bonds between the strands, thereby destabilizing the double helix. Lys, in an untwisted shape, binds to the external side of the dsDNA and forms two bonds with O atoms of neighboring phosphodiester groups, thereby strengthening the DNA helix.

DNA melting and genotoxicity induced by silver nanoparticles and graphene.

We have revealed a connection between DNA-nanoparticle (NP) binding and in vitro DNA damage induced by citrate- and branched polyethylenimine-coated silver nanoparticles (c-AgNPs and b-AgNPs) as well as graphene oxide (GO) nanosheets. All three types of nanostructures triggered an early onset of DNA melting, where the extent of the melting point shift depends upon both the type and concentration of the NPs. Specifically, at a DNA/NP weight ratio of 1.1/1, the melting temperature of lambda DNA dropped from 94 °C down to 76 °C, 60 °C, and room temperature for GO, c-AgNPs and b-AgNPs, respectively. Consistently, dynamic light scattering revealed that the largest changes in DNA hydrodynamic size were also associated with the binding of b-AgNPs. Upon introduction to cells, b-AgNPs also exhibited the highest cytotoxicity, at the half-maximal inhibitory (IC50) concentrations of 3.2, 2.9, and 5.2 mg/L for B and T-lymphocyte cell lines and primary lymphocytes, compared to the values of 13.4, 12.2, and 12.5 mg/L for c-AgNPs and 331, 251, and 120 mg/L for GO nanosheets, respectively. At cytotoxic concentrations, all NPs elicited elevated genotoxicities via the increased number of micronuclei in the lymphocyte cells. However, b-AgNPs also induced micronuclei at subtoxic concentrations starting from 0.1 mg/L, likely due to their stronger cellular adhesion and internalization, as well as their subsequent interference with normal DNA synthesis or chromosome segregation during the cell cycle. This study facilitates our understanding of the effects of NP chemical composition, surface charge, and morphology on DNA stability and genotoxicity, with implications ranging from nanotoxicology to nanobiotechnology and nanomedicine.

Identification of acetic acid bacteria in traditionally produced vinegar and mother of vinegar by using different molecular techniques

Culture-dependent and culture-independent methods were combined for the investigation of acetic acid bacteria (AAB) populations in traditionally produced vinegars and mother of vinegar samples obtained from apple and grape. The culture-independent denaturing gradient gel electrophoresis (DGGE) analysis, which targeted the V7–V8 regions of the 16S rRNA gene, showed that Komagataeibacter hansenii and Komagataeibacter europaeus/Komagataeibacter xylinus were the most dominant species in almost all of the samples analyzed directly. The culture-independent GTG5-rep PCR fingerprinting was used in the preliminary characterization of AAB isolates and species-level identification was carried out by sequencing of the 16S rRNA gene, 16S–23S rDNA internally transcribed to the spacer (ITS) region and tuf gene. Acetobacter okinawensis was frequently isolated from samples obtained from apple while K. europaeus was identified as the dominant species, followed by Acetobacter indonesiensis in the samples originating from grape. In addition to common molecular techniques, real-time PCR intercalating dye assays, including DNA melting temperature (Tm) and high resolution melting analysis (HRM), were applied to acetic acid bacterial isolates for the first time. The target sequence of ITS region generated species-specific HRM profiles and Tm values allowed discrimination at species level.

New Porphyrins/Calf Thymus DNA Complexes - Their Thermostability

The paper studies the melting parameters of the complexes of water soluble cationic 3N- and 4N-pyridyl porphyrins with different peripheral substituents (oxyethyl, buthyl, allyl, metallyl) with DNA. The results indicate that the presence of porphyrin changes the shape and parameters of DNA melting curve. The porphyrin concentration increase results in the increase of the melting temperature (Tm) and melting interval (ΔT) of DNA. With the porphyrin-DNA concentration ratio ν = 0.01, changes of melting temperature have not been observed. The melting intervals almost do not change upon the addition of the 4N- porphyrins, while the decrease of ΔT is observed in the presence of 3N-porphyrins. Because of the intercalation binding mechanism occurring in GC-rich regions of DNA, we assume that 3N-porphyrins intercalated in GC-rich regions, reduce the thermal stability of these sites, bringing them closer to the thermal stability of the AT-sites, which is the reason for the decrease in melting interval. While with the relative concentration ν = 0.01 for 4-N porphyrins the external binding mechanism “turns on” and destabilizing effect of porphyrins on GC-pairs compensated by their stabilizing effect on AT-pairs. As a result, no change in the melting parameters of DNA is observed upon complexation with these porphyrins.

Synthesis and characterisation of nickel Schiff base complexes containing the meso-1,2-diphenylethylenediamine moiety: selective interactions with a tetramolecular DNA quadruplex.

As part of a program of preparing metal complexes which exhibit unique affinities towards different DNA structures, we have synthesised the novel Schiff base complex N,N'-bis-4-(hydroxysalicylidine)meso-diphenylethylenediaminenickel(ii) (), via the reaction of meso-1,2-diphenylethylenediamine and 2,4-dihydroxybenzaldehyde. This compound was subsequently reacted with 1-(2-chloroethyl)piperidine or 1-(2-chloropropyl)piperidine, to afford the alkylated complexes N,N'-bis-(4-((1-(2-ethyl)piperidine)oxy)salicylidine)meso-1,2-diphenylethylenediaminenickel(ii) () and N,N'-bis-(4-((1-(3-propyl)piperidine)oxy)-salicylidine)meso-1,2-diphenylethylenediaminenickel(ii) (), respectively. These complexes were characterised by microanalysis and X-ray crystallography in the solid state, and in solution by (1)H and (13)C NMR spectroscopy. Electrospray ionisation mass spectrometry (ESI-MS) was used to confirm the identity of () and (). The affinities of () and () towards a discrete 16 mer duplex DNA molecule, and examples of both tetramolecular and unimolecular DNA quadruplexes, was explored using a variety of techniques. In addition, the affinity of two other complexes () and (), towards the same DNA molecules was examined. Complexes () and () were prepared by methods analogous to those which afforded () and (), however 1,2-phenylenediamine was used instead of meso-1,2-diphenylethylenediamine in the initial step of the synthetic procedure. The results of ESI-MS and DNA melting temperature measurements suggest that () and () exhibit a lower affinity than () and () towards the 16 mer duplex DNA molecule, while circular dichroism (CD) spectroscopy suggested that none of the four complexes had a major effect on the conformation of the nucleic acid. In contrast, ESI-MS and CD spectroscopy suggested that both () and () show significant binding to a tetramolecular DNA quadruplex. The results of ESI-MS and Fluorescence Resonance Energy Transfer (FRET) assays indicated that () and () did not bind as tightly to a unimolecular DNA quadruplex, although both complexes had a major effect on the CD spectrum of the latter. These results highlight that the presence of the meso-1,2-diphenylethylenediamine moiety in metal complexes of this type may provide a general method for instilling selectivity for some DNA quadruplexes over dsDNA.

The relationship of the whole genome sequence identity to DNA hybridization varies between genera of prokaryotes.

In the original proposal of Wayne et al. (Int J Syst Bacteriol 37:463-464, 1987), two measures of genetic relatedness were proposed to set the boundary for prokaryotic species. The first was the change in the melting temperature (ΔTm) of heteroduplex DNA and the second was the extent of DNA-DNA hybridization (DDH). While this approach was justified given the experimental error inherent in these methods, genomic sequencing has the potential to measure both parameters with great precision. The average nucleotide identity (ANIb), a surrogate for the ΔTm, and the calculated DDH (cDDH) were determined from the complete genomes of representatives of 17 genera of prokaryotes. When the ANIb was >75%, the ratio (100-cDDH)/(100-ANIb) was 3.69±0.93 (± SD) and varied from about 2.35 to 4.59 between genera. The differences among genera was highly significant (p<0.001) but not correlated with specific phylogenetic or physiological groups. Moreover, the ANIm was a poor measure of ANIb when ANIb was <75%. Because the ANIb and cDDH provide different measures of relatedness, it is no longer appropiate to consider both when delineating species. For these reasons, measures of relatedness based upon sequence identity should be used for delineating species in the future.

Interaction of doxorubicin with a regulatory element of hmga1 and its in vitro anti-cancer activity associated with decreased HMGA1 expression

High mobility group A1 (HMGA1) non-histone chromatin protein is known as an architectural transcription factor that regulates transcription of various genes. HMGA1 is highly expressed in almost all human cancers and considered as a potent tumor marker. Because of its association with cancers, hmga1 is considered as a critical target for anti-cancer drugs. In the present study, we report interaction of doxorubicin (DOX) with a short deoxyoligonucleotide (−1917 to −1940) within a regulatory element of hmga1 and its subsequent effect on expression of HMGA1 in breast cancer MCF7 cells. Binding of DOX to DNA was found to be strong (Ka, 5.2×105M−1) and thermodynamically favorable by both negative enthalpy (ΔH, −8.1±0.25kcalM−1) and positive entropy changes (TΔS, 21.1±5.2kcalM−1) at 20°C. A significant increase in melting temperature of DNA in presence of DOX by +10°C was accompanied by substantial quenching of fluorescence of DOX (∼85%) at 595nm and hypochromic change (∼40%) at 500nm absorption spectra of DOX along with a bathochromic shift of ∼5nm. Reduced expression of HMGA1 by ∼60% both at mRNA and protein level and associated cell death in presence of DOX was observed in breast cancer cells. Therefore, hmga1 is a promising chemotherapeutic target in treating human malignancies.

Selective stabilization of abasic site-containing DNA by insertion of sterically demanding biaryl ligands.

Biaryl derivatives that consist of one DNA-intercalating unit and a sterically demanding component exhibit a specific behavior towards abasic site-containing DNA (AP-DNA) as determined by thermal DNA denaturation experiments, spectrometric titrations and CD spectroscopic analysis. Specifically, these ligands strongly stabilize AP-DNA towards dissociation, whereas they do not or only marginally affect the melting temperature of regular duplex DNA.

DNA Ligase Mediated Ag+Ion Sensor

Silver and its alloys are widely used as superconductors and dental amalgam as well as in photography, batteries, medicines, lubricants, mirror, and jewelry. Every year, tons of silver and silver compounds are released into the aqueous environment from emissions and industrial waste. Several compounds of silver are toxic to living organisms. Therefore, it is of high importance to develop highly sensitive and selective methods to detect trace amounts of silver ions in an aqueous environment. A number of methods using organic fluorophores, quantum dots, ion selective electrodes, inductively coupled plasma-mass spectroscopy (ICP-MS), atomic absorption spectroscopy, gold nanoparticles, and carbon based materials have been developed for the sensitive, simple, and rapid detection of silver ions in aqueous solution. Although these methods have their own benefits, they still present some limitations such as low water solubility, poor selectivity, and insufficient sensitivity. Recently, Shionoya et al. reported that DNA duplexes possessing metallo-base pairs exchibited higher thermo stability than natural hydrogen-bonded DNA. Furthermore, Ono et al. reported that Ag ions are capable of selectively binding to cytosine (C) bases and forming strong and stable C-Ag-C complexes. This discovery led to the development of oligonucleotidebased sensors to detect Ag ions in an aqueous solution. This approach provides a highly sensitive detection of Ag ions, but still presents a few limitations including a complex process, low sensitivity, and especially, the requirement of fluorophore labeling. Additionally, many metal ions, including Ag ions, can quench fluorescence signals, thus, possibly affecting the sensitivity of fluorescence measurement. To overcome these problems, we designed a novel and highly sensitive strategy involving an enzymatic ligation and quantitative PCR (qPCR) to detect Ag ions. As presented in Scheme 1, the first step was DNA ligation, in which silver ions were detected and the second step was signal amplification of the ligated template DNA. The rate of DNA amplification by qPCR can be directly correlated to the amount of silver ions in the ligation reaction. In this system, three oligonucleotides were used for DNA ligation. One is a template DNA for ligation, and the other two oligomers were a full matched sequence DNA and a mismatched DNA oligomer to the template. The mismatched oligomer had three Cs located near the 3′-terminal site and mismatched to the Cs of the template DNA. The full matched DNA strand has a 5′-phosphate group for ligation. All the three oligomers for ligation reaction could be hybridized at a given temperature. The sequence of each oligomer was designed to adjust the melting temperature (Tm) of the template DNA and matched/mismatched oligomers. The calculated Tm value of the template and matched DNA was 59.7 °C under a salt concentration of 25 mM Na and 5 mM Mg. The calculated Tm of the template and mismatched oligomer was designed to be less than 30 °C. Thus, if the ligation Scheme 1. Ligase-mediated silver ion sensing.

Characterization of single-stranded DNA-binding proteins from the psychrophilic bacteria Desulfotalea psychrophila, Flavobacterium psychrophilum, Psychrobacter arcticus, Psychrobacter cryohalolentis, Psychromonas ingrahamii, Psychroflexus torquis, and Photobacterium profundum.

BackgroundSingle-stranded DNA-binding proteins (SSBs) play essential roles in DNA replication, recombination and repair in Bacteria, Archaea and Eukarya. In recent years, there has been an increasing interest in SSBs, since they find numerous applications in diverse molecular biology and analytical methods.ResultsWe report the characterization of single-stranded DNA-binding proteins from the psychrophilic bacteria Desulfotalea psychrophila (DpsSSB), Flavobacterium psychrophilum (FpsSSB), Psychrobacter arcticus (ParSSB), Psychrobacter cryohalolentis (PcrSSB), Psychromonas ingrahamii (PinSSB), Photobacterium profundum (PprSSB), and Psychroflexus torquis (PtoSSB). The proteins show a high differential within the molecular mass of their monomers and the length of their amino acid sequences. The high level of identity and similarity in respect to the EcoSSB is related to the OB-fold and some of the last amino acid residues. They are functional as homotetramers, with each monomer encoding one single stranded DNA binding domain (OB-fold). The fluorescence titrations indicated that the length of the ssDNA-binding site size is approximately 30 ± 2 nucleotides for the PinSSB, 31 ± 2 nucleotides for the DpsSSB, and 32 ± 2 nucleotides for the ParSSB, PcrSSB, PprSSB and PtoSSB. They also demonstrated that it is salt independent. However, when the ionic strength was changed from low salt to high, binding-mode transition was observed for the FpsSSB, at 31 ± 2 nucleotides and 45 ± 2 nucleotides, respectively. As expected, the SSB proteins under study cause duplex DNA destabilization. The greatest decrease in duplex DNA melting temperature was observed in the presence of the PtoSSB 17°C. The SSBs in question possess relatively high thermostability for proteins derived from cold-adapted bacteria.ConclusionThe results showed that SSB proteins from psychrophilic microorganisms are typical bacterial SSBs and possess relatively high thermostability, offering an attractive alternative to other thermostable SSBs in molecular biology applications.

Quantitative Study of As (V) and As (III) Interaction with Mangrove DNA by Molecular Fluorescence Spectroscopy

This study describes the in vitro study of (1:1) one step nucleophilic displacement ([Formula: see text]) of phosphate by heavier anion arsenate and arsenite in the DNA of arsenic ridden Sundarban mangroves. Mangrove DNA was found to give rise to a broad fluorescence and its integrated fluorescence intensity was enhanced on addition of As (V) and As (III), respectively. Analyses of the fluorescence parameter showed adequacy of 1:1 model to describe substitution of phosphate of mangrove DNA chain exiplex by arsenate and arsenite with equilibrium constant (log Kc) ranging between 4.19 and 4.32 for As (V), and between 3.77 and 3.89 for As (III) at pH 7 and 25°C. In the cases, the melting temperature (Tm) and reassociation rate constant of mangrove DNA was increased on treatment with As (V) and As (III). It is suggested that heavier ion arsenate and arsenite may substitute phosphate in natural DNA.

A Temperature-Controlled Microwell Platform for Electrochemical Melting Analyses

We have designed and fabricated a miniature device for proof-of-concept studies on biomolecular interactions using electrochemical melting analysis. A collection of temperature controlled three-electrode platforms was built on a 100-mm glass wafer, and it includes a total of eight separate probe sites. Each site consists of one 200 nm thick platinum (Pt) heater, one 200 nm gold (Au) electrode and two 200 nm Pt electrodes, as well as a 10 µL polydimethylsiloxane (PDMS) well, all used for probing electrochemical signals as a function of sample temperature. Our Pt heaters have the ability to control sample solution temperatures in the microwell from 4 °C to 120 °C. Heaters were fabricated with different geometries (width and shape) in order to select the best candidate for future device fabrication. Electrochemical melting analysis has been performed by detecting signals derived from changes in methylene blue (MB)-labeled DNA that occur during the heating process. The MB-labeled single strand(ss) DNA was added to hybridize with its matched thiolated ssDNA on Au electrode surface to form duplexes. Electron-transfer(eT) was monitored using square wave voltammetry (SWV) from the beginning of a thermal cycling process. During the heating process the eT was limited significantly when the heater temperature reached the duplex melting temperature. The dehybridized MB-labeled ssDNA left the Au electrode surface causing a current decrease. As a result, a current vs. temperature melting curve was obtained that allowed us to determine the sample DNA melting temperature. The rapid heating capability provided by the Pt heater allows the electrochemical melting measurements to be completed in 20-30 minutes, and that is significantly faster than the typical time for optical melting methods. Figure 1 illustrates the wired microsystem used in the DNA experiment, and includes an enlargement showing the configuration of the miniature electrodes and heater. We plan to build the described probes into a 3x3 array-based microchip for demonstrating the potential for high throughput DNA melting analyses to study biomolecular binding stability.

Recognition of DNA bulges by dinuclear iron(II) metallosupramolecular helicates

Bulged DNA structures are of general biological significance because of their important roles in a number of biochemical processes. Compounds capable of targeting bulged DNA sequences can be used as probes for studying their role in nucleic acid function, or could even have significant therapeutic potential. The interaction of [Fe(2)L(3)](4+) metallosupramolecular helicates (L = C(25)H(20)N(4)) with DNA duplexes containing bulges has been studied by measurement of the DNA melting temperature and gel electrophoresis. This study was aimed at exploring binding affinities of the helicates for DNA bulges of various sizes and nucleotide sequences. The studies reported herein reveal that both enantiomers of [Fe(2)L(3)](4+) bind to DNA bulges containing at least two unpaired nucleotides. In addition, these helicates show considerably enhanced affinity for duplexes containing unpaired pyrimidines in the bulge and/or pyrimidines flanking the bulge on both sides. We suggest that the bulge creates the structural motif, such as the triangular prismatic pocket formed by the unpaired bulge bases, to accommodate the [Fe(2)L(3)](4+) helicate molecule, and is probably responsible for the affinity for duplexes with a varying number of bulge bases. Our results reveal that DNA bulges represent another example of unusual DNA structures recognized by dinuclear iron(II) ([Fe(2)L(3)](4+)) supramolecular helicates.

Calcium phosphate nanoparticles: a study of their synthesis, characterization and mode of interaction with salmon testis DNA

Calcium phosphate nanoparticles (CPNPs) are presently emerging as a second generation vector for efficient delivery and stabilization of nucleic acids inside cells, although the detailed mode of interaction between CPNPs and DNA is still obscure. This study discloses some features of the interaction. For this study, we synthesized CPNPs by a modified co-precipitation method and characterized the particles by different techniques such as dynamic light scattering, X-ray diffraction, electron dispersive spectroscopy, Fourier transform infra-red spectroscopy, differential thermal and thermo-gravimetric analysis, and atomic force, scanning and transmission electron microscopy. The characterization studies showed that the nanoparticles were spherical in shape, about 45 nm in size and were composed of the hydroxyapatite form of calcium phosphate; almost 90% of the starting materials were converted to nanoparticles (NPs). The different aspects of the interaction between CPNPs and salmon testis DNA were investigated using techniques such as UV-Vis spectrophotometry, circular dichroism, Fourier transform infra-red spectrometry, thermal denaturation, microviscometry, agarose gel electrophoresis, cyclic voltammetry and atomic force microscopy. The results revealed that CPNPs interacted with DNA with ~1 : 3.3 stoichiometry with a binding constant of the order of 10(4) M(-1) through groove-interacting mode and a single nanoparticle covered about 6.2 base pairs of the DNA chain. Moreover, the binding interaction was spontaneous, cooperative, exothermic and enthalpy-driven and some electrostatic nature of the binding was also evident; however, the non-polyelectrolyte contribution was dominant. The binding interaction finally caused an increase in the melting temperature of DNA from 70.8 °C to 75 °C and alteration of its secondary structure from the naturally occurring B-form to C-form.