Native B-DNA Research Articles

Molecular Dynamics and Multi-Spectroscopic of the Interaction Behavior between Bladder Cancer Cells and Calf Thymus DNA with Rebeccamycin: Apoptosis through the Down Regulation of PI3K/AKT Signaling Pathway.

The interaction of Rebeccamycin with calf thymus (ctDNA) in the absence and presence of H1 was investigated by molecular dynamics, multi-spectroscopic, and cellular techniques. According to fluorescence and circular dichroism spectroscopies, Rebeccamycin interacted with ctDNA in the absence of H1 through intercalator or binding modes, while the presence of H1 resulted in revealing theintercalator, as the dominant role, and groove binding modes of ctDNA-Rebeccamycin complex. The binding constants, which were calculated to be 1.22 × 104M-1 and 7.92 × 105M-1 in the absence and presence of H1, respectively, denoted the strong binding of Rebeccamycin with ctDNA. The binding constants of Rebeccamycin with ct DNA in the absence and presence of H1 were calculated at 298, 303 and 308K. Considering the thermodynamic parameters (ΔH0 and ΔS0), both vander waals forces and hydrogen bonds played predominant roles throughout the binding of Rebeccamycin to ctDNA in the absence and presence of H1. The outcomes of circular dichroism suggested the lack of any major conformational changes in ctDNA upon interacting with Rebeccamycin, except some perturbations in native B-DNA at local level. Additionally, the effect of NaCl and KI on ctDNA-Rebeccamycin complex provided further evidence for the reliance of their interaction modes on substituted groups. The observed increase in the relative viscosity of ctDNA caused by the enhancement of Rebeccamycin confirmed their intercalation and groove binding modes in the absence and presence of H1. Moreover, the assessments of molecular docking simulation corroborated these experimental results and also elucidated the effectiveness of Rebeccamycinin inhibiting and proliferating T24 and 5637 cells. Meanwhile, the ability of Rebeccamycin in inhibiting cell proliferation and tumor growth through the induction of apoptosis by down regulating the PI3K/AKT signaling pathway were provided.

A spectroscopic deciphering of the differential interaction behavior of alkaloid drugs with native B-DNA and protonated DNA

The present study demonstrates a detailed spectroscopic investigation on the differential extent and modes of interaction of two alkaloid drugs, Sanguinarine (SG) and Berberine (BR), with biologically significant protonated and B-forms of herring sperm DNA in aqueous buffer medium of pH 3.5 and 7.4 respectively. The steady-state spectroscopic results substantiate the binding interaction of either drug molecule with protonated as well as B-form of DNA. The drug:DNA binding strength obtained employing the Benesi-Hildebrand equation reveals a significantly higher binding affinity of BR with B-DNA at pH 7.4 compared to that with protonated DNA at pH 3.5. However, the binding constant obtained for interaction of iminium form of SG with protonated DNA is found to be an order of magnitude higher compared to that in case of interaction with B-DNA. Moreover, the steady-state emission results validate the role of preferential electrostatic interaction between the cationic forms of the drugs (cationic BR and the iminium form of SG) and the negatively charged phosphate backbone of B-DNA under physiological conditions at pH 7.4 which has been further corroborated by monitoring the effect of a strong electrolyte on drug:DNA interaction under the various experimental conditions. The circular dichroic (CD) spectroscopic studies reveal the probable intercalative binding of the drug molecules within DNA at either pH. The time-resolved fluorescence decay measurements along with the time-resolved fluorescence anisotropy studies further substantiate the binding interaction of the drugs with protonated as well as B-form of herring sperm DNA.

Monitoring the Conformation and Concentration of DNA in Live Cells using Fourier Transform Infrared Spectroscopy

The ability to accurately detect DNA both quantitatively and qualitatively inside cells using Fourier transform infrared (FTIR) spectroscopy has been disputed. Recently, we have demonstrated that the variability of DNA absorptions is due to the dehydrated nature of biological samples prepared for FTIR spectroscopic measurement [1]. We have further demonstrated that in the dehydrated and fixed state DNA in cells assumes an A-DNA conformation instead of the native B-DNA form. Importantly, as well as being detected in eukaryotes which were invariably destroyed during dehydration, this B-A DNA transition has also been observed in desiccation-resistant, dormant bacteria and the native B- conformation has been detected upon rehydration of these cells. This previously undetected reversible transition raises several interesting questions about the role of A-DNA as a defense mechanism and its role in the evolution of nucleic acids.The B-A conformational transition has also been identified as causing changes to the molar extinction coefficients of several DNA bands explaining previous observations of unexpectedly low DNA absorption intensities. The Beer-Lambert nature of these absorptions was demonstrated by infrared spectroscopy of avian erythrocytes and extracted nuclei in conjunction with Partial Least Squares regression analysis to quantify cellular DNA [2]. Furthermore, spectra of hydrated single cells throughout interphase were also used to investigate the quantitative and qualitative biochemical changes involved in the G1, S and G2 phases of the cell cycle [3]. Using Principal Component Analysis cells only two hours apart were successfully clustered based on changes to the concentration and conformation of lipid, protein and DNA.1. D. R. Whelan, et. al., Nucleic Acids Research, 39, 5439-5448 (2011).2. D. R. Whelan, et. al., Journal of Biophotonics, 10, 775-784 (2013).3. D. R. Whelan, et al., Analyst, 138, 3891-3899 (2013).

Spectroscopic studies of the effects of anticancer drug mitoxantrone interaction with calf-thymus DNA

Mitoxantrone (MTX) (1,4-dihydroxy-5,8-bis[[2-[(2-hydroxyethyl)amino]ethyl]amino]-9,10-anthracenedione) is a synthetic antineoplastic drug, widely used as a potent chemotherapeutic agent in the treatment of various types of cancer. It is structurally similar to classical anthracyclines. Widespread interest in the anticancer agent mitoxantrone has arisen because of its apparent lower risk of cardio-toxic effects compared to the naturally occurring anthracyclines. In the present work, we investigated the interaction of mitoxantrone with DNA in the buffer solution at physiological pH using Fourier transform infrared (FTIR), UV–Visible absorption and circular dichroism spectroscopic techniques. FTIR analysis revealed the intercalation of mitoxantrone between the DNA base pairs along with its external binding with phosphate–sugar backbone. The binding constant calculated for mitoxantrone–DNA association was found to be 3.88×105M−1 indicating high affinity of drug with DNA double helix. Circular dichroism spectroscopic results suggest that there are no major conformational changes in DNA upon interaction with drug except some perturbations in native B-DNA at local level. The present work shows the capability of spectroscopic analysis to characterize the nature of drug–biomolecule complex and the effects of such interaction on the structure of biomolecule.

Defects in DNA: Lessons from Molecular Motor Design

The degree of localization of structural damage in DNA is computationally investigated in the framework of molecular motors. Damage is induced on DNA hairpins with two and three guanine-cytosine (GC) base pairs by the photoinduced isomerization of their azobenzene containing cap. Light-induced changes in elasticity of such hairpins can be used to transduce photon energy into mechanical work in a single-molecule pulling setup through optomechanical cycles. The maximum extractable work per cycle is, in fact, a good measure of the degree of disruption of the hairpin structure upon isomerization. The extractable work was quantified by means of free-energy reconstruction techniques and several microseconds of molecular dynamics simulations. The maximum work that can be extracted from the 2GC and 3GC systems starting from their native B-DNA conformation (dO3′–O5′ ≈ 16 Å) is 2.70 kcal/mol in both cases. The fact that the extractable work does not increase when transitioning from the dimer to the trimer implies that the DNA damage induced by azobenzene isomerization is localized to the two base pairs adjacent to the photoswitchable unit. From the perspective of DNA-based molecular motors, these findings indicate that a dense azobenzene arrangement would be required for effective actuation. From a biological perspective, the results highlight the remarkable ability of the DNA design to mitigate the propagation of damage, thus limiting detrimental effects that this may have on healthy cell function.

Encoding Phenotype in Bacteria with an Alternative Genetic Set

An unnatural base-pair architecture with base pairs 2.4 Å larger than the natural DNA-based genetic system (xDNA) is evaluated for its ability to function like DNA, encoding amino acids in the context of living cells. xDNA bases are structurally analogous to natural bases but homologated by the width of a benzene ring, increasing their sizes and resulting in a duplex that is wider than native B-DNA. Plasmids encoding green fluorescent protein were constructed to contain single and multiple xDNA bases (as many as eight) in both strands and were transformed into Escherichia coli. Although they yielded fewer colonies than the natural control plasmid, in all cases in which a modified plasmid (containing one, two, three, or four consecutive size-expanded base pairs) was used, the correct codon bases were substituted, yielding green colonies. All four xDNA bases (xA, xC, xG, and xT) were found to encode the correct partners in the replicated plasmid DNA, both alone and in longer segments of xDNA. Controls with mutant cell lines having repair functions deleted were found to express the gene correctly, ruling out repair of xDNA and confirming polymerase reading of the unnatural bases. Preliminary experiments with polymerase deletion mutants suggested combined roles of replicative and lesion-bypass polymerases in inserting correct bases opposite xDNA bases and in bypassing the xDNA segments. These experiments demonstrate a biologically functioning synthetic genetic set with larger-than-natural architecture.

A post‐SCF quantum chemistry study on local minima of 8‐oxo‐guanine stacked with all four nucleic acid bases in B‐DNA conformations

Abstractmagnified imageThe post SCF MP2/6‐31G*(d=0.25) method was applied to obtain potential energy surface of 8‐oxoguanine stacked with all four canonical DNA bases. The spatial neighbourhood was scanned of stacked complexes found in the native B‐DNA. The presented results suggest that the hydroxyl radical modification of guanine at C8 position has significant impact on structural, energetic, orbital and electrostatic properties of stacked complexes with canonical DNA bases. The pair stabilization energy, including electron correlation terms, suggests that the 5′‐A/GA‐3′ pair is the most stable among all of the studied complexes. The 8‐oxo‐guanine has been found as a source of significant changes in electroaccepting properties compared to stacked pairs formed by canonical guanine since both electron affinities and localization of HOMO orbital were altered. However, electro‐donation abilities are not modified after replacement of guanine with 8‐oxo‐guanine irrespectively on the context of B‐DNA bases.

Biophysical characterization of anionic lipoplexes

Transfection efficiency of liposomal gene delivery vectors depends on an optimal balance in the electro-chemical and structural properties of the transfection-capable complexes. We have recently reported a novel anionic lipoplex DNA delivery system composed of a ternary complex of endogenous occurring non-toxic anionic lipids, physiological Ca 2+ cations, and plasmid DNA encoding a gene of interest with high transfection efficiency and low toxicity. In this work, we investigate the electro-chemical and structural properties anionic lipoplexes and compare them with those of Ca 2+–DNA complexes. Biophysical characterization is used to explain the transfection efficiency of anionic lipoplexes in mammalian CHO-K1 cells. Circular dichroism and fluorescence spectroscopy showed that the plasmid DNA underwent conformational transition from native B-DNA to Z-DNA due to compaction and condensation upon Ca 2+-mediated complexation with anionic liposomes. Zeta potential measurements and gel electrophoresis studies demonstrated that Ca 2+ interaction with plasmid DNA during the formation of lipoplexes also led to increased association of supercoiled plasmid DNA with the lipoplexes, leading to charge neutralization which is expected to facilitate transfection. However, even 10-fold higher concentrations of Ca 2+ alone (in the absence of the anionic liposomes) were unable to induce these changes in plasmid DNA molecules. A model explaining the possible mechanism of anionic lipoplex formation and the correlation of high transfection efficiency to biophysical properties was proposed. These studies confirm the utility of biophysical studies to identify optimal formulation conditions to design efficient liposomal gene delivery vectors.

First evidence on induced topological changes in supercoiled DNA by an aluminium D-aspartate complex.

Aluminium is a debatable and suspected etiological factor in neurodegenerative disorders. Aluminium-amino acid complexes also play an important role in the complex biology of the metal. Recent reports indicate the presence of D-aspartate and D-glutamate in aging brain, human breast tumors, core amyloid plaques and neurofibrillary tangles of Alzheimer's brain. This stereoinversion from the L- to the D-enantiomer is enhanced by Al. Further, the observation that Al is localized in the chromatin region encouraged the present study of the interaction of Al-amino acid complexes with DNA. This study used circular dichroism of supercoiled DNA and showed that Al- D-Asp caused a native B-DNA to C-DNA conformational change, while Al- L-Asp, Al- L-Glu and Al- D-Glu did not alter the native B-DNA conformation. This differential DNA binding property of Al-amino acid complexes is assigned to the stereoisomerism and chirality of the complexes. Interestingly, polyamines like spermine further induced an asymmetric condensation of the "limit C-motif" induced by Al- D-Asp to a Psi-DNA. The results were supported by computer modeling, gel studies and ethidium bromide binding. We also propose a mechanism of Al- D-Asp binding and its ability to modulate DNA topology.

AC Impedance Spectroscopy of Native DNA and M-DNA

Monolayers of thiol-labeled DNA duplexes of 15, 20, and 30 basepairs were assembled on gold electrodes. Electron transfer was investigated by electrochemical impedance spectroscopy with Fe(CN)(6)(3-/4-) as a redox probe. The spectra, in the form of Nyquist plots, were analyzed with a modified Randles circuit which included an additional component in parallel, R(x), for the resistance through the DNA. For native B-DNA R(x) and R(ct), the charge transfer resistance, both increase with increasing length. M-DNA was formed by the addition of Zn(2+) at pH 8.6 and gave rise to characteristic changes in the Nyquist plots which were not observed upon addition of Mg(2+) or at pH 7.0. R(x) and R(ct) also increased with increasing duplex length for M-DNA but both were significantly lower compared to B-DNA. Therefore, electron transfer via the metal DNA film is faster than that of the native DNA film and certain metal ions can modulate the electrochemical properties of DNA monolayers. The results are consistent with an ion-assisted long-range polaron hopping mechanism for electron transfer.

Interligand interactions affecting specific metal bonding to nucleic acid bases. A case of [Rh2(OAc)4], [Rh2(HNOCCF3)4], and [Rh2(OAc)2(HNOCCF3)2] toward purine nucleobases and nucleosides

The reaction of [Rh2(OAc)4] with adenine, 9-methyladenine, 9-ethylguanine, adenosine, guanosine, deoxyguanosine, or inosine in methanolic aqueous solution at room temperature gave specifically and quantitatively pink complexes of adenine or its derivatives (=L), [Rh2(OAc)4(L)] (the dirhodium-core:L ratio of 1:1). The reaction of [Rh2(HNOCCF3)4] or [Rh2(OAc)2(HNOCCF3)2] with these nucleobases or nucleosides (=L) gave non-specifically pink or red complexes with dirhodium-core:L=1:1 for adenine derivatives while 1:2 for guanine derivatives. Recrystallization of a 9-methyladenine adduct from a nitric acid solution yielded [Rh2(OAc)2(HNOCCF3)2(9-methyladeninium)2]·(NO3)2 (1), whose crystal structure was determined by X-ray diffraction, showing that two N(1)-protonated 9-methyladeninium molecules coordinate to both the axial-positions of a [Rh2(OAc)2(HNOCCF3)2] nucleus through N(7) with the formation of an intramolecular interligand N(6)-H⋯O(acetato) hydrogen bond. Crystal structures of guanine derivatives, [Rh2(OAc)2(HNOCCF3)2(9-ethylguanine)2]·2MeOH·2H2O (2), [Rh2(HNOCCF3)4(L)2]·3H2O (L=guanosine (3), deoxyguanosine (4), or inosine (5)), and [Rh2(OAc)2(HNOCCF3)2(guanosine)2]·3H2O (6) were determined, showing that the dirhodium core is occupied at either axial-side by two guanine or hypoxanthine moieties through N(7) with the formation of an intramolecular interligand N(amidato)-H⋯O(6) hydrogen bond. Each of guanosine molecules in 3, 4, and 6 or inosine molecules in 5 adopts the syn, gauche–gauche (or gauche–trans for one molecule in 3), and C(2′)-endo (or C(2′)-endo-C(3′)-exo for one molecule in 4) conformations to form an intramolecular O(5′)-H⋯N(3) hydrogen bond. The formation of interligand N(6)-H⋯O hydrogen-bonding associated with the sterically constrained octahedral coordination geometry about the rhodium atom bonded to N(7), as observed in 1, and/or to N(1) of adenine may give a structural basis for the adenine-specific reactivity of [Rh2(OOCR)4], where the carboxylato ligand could function as hydrogen-bonding acceptor only. Similarly, the formation of interligand N-H⋯O(6) hydrogen-bonding observed in 2–6 suggests that [Rh2(HNHNCR)4] could function as guanine-specific reagents, where the amidinato ligand could function as hydrogen-bonding donor only. The interligand steric constraint due to the octahedral coordination geometry may also explain no reactivity of [Rh2(OOCR)4] toward native B-DNA (right-handed double-stranded structure) but specific bonding to single-stranded and possibly to left-handed double-stranded structures.

The role of water in B-DNAs B I to B II conformer substates interconversion: a combined study by calorimetry, FT-IR spectroscopy and computer simulation

Conformational substates of B-DNA had been observed so far in synthetic oligonucleotides but not in naturally occurring highly polymeric B-DNA. Our low-temperature experiments show that native B-DNA from salmon testes and the d(CGCGAATTCGCG) 2 dodecamer have the same B I and B II substates. Nonequilibrium distribution of conformer population was generated by quenching hydrated nonoriented films or fibers into the glassy state, and isothermal structural relaxation towards equilibrium by interconversion of substates was followed either by differential scanning calorimetry or by Fourier transform infrared spectroscopy. B I converts to B II on isothermal relaxation between 180 and 220 K, whereas on slow cooling from ambient temperature, B II converts to B I. State-of-the-art molecular dynamics simulation of the d(CGCGAATTCGCG) 2 dodecamer revealed that the B I→B II transition involves not only destacking of adjacent base pairs, but is coupled with migration of water from ionic phosphate to the sugar oxygen. These results are consistent with pronounced infrared spectral changes observed upon B I→B II interconversion. The B II substate is stabilized in comparison to B I by enhanced hydrogen-bond interaction with the migrating water. Curve resolution of infrared spectra showed that in hydrated nonoriented films of the d(CGCGAATTCGCG) 2 dodecamer, the B II population is enhanced in comparison to that in single crystals. Thus, the B II substate could be of biological relevance, and the B I to B II substate interconversion could be a major contributor to the protein recognition process.

Unexpected BII Conformer Substate Population in Unoriented Hydrated Films of the d(CGCGAATTCGCG)2 Dodecamer and of Native B-DNA from Salmon Testes

Conformational substates of B-DNA had been observed so far in synthetic oligonucleotides but not in naturally occurring highly polymeric B-DNA. Our low-temperature experiments show that native B-DNA from salmon testes and the d(CGCGAATTCGCG)2 dodecamer have the same BI and BII substates. Nonequilibrium distribution of conformer population was generated by quenching hydrated unoriented films to 200K, and isothermal structural relaxation toward equilibrium by interconversion of substates was followed by Fourier transform infrared spectroscopy. BI interconverts into BII on isothermal relaxation at 200K, whereas on slow cooling from ambient temperature, BII interconverts into BI. Our estimation of the dodecamer's BI-to-BII conformer substate population by curve resolution of the symmetrical stretching vibration of the ionic phosphate is 2.4±0.5 to 1 at 200K, and it is 1.3±0.5 to 1 between 270 and 290K. Pronounced spectral changes upon BI-to-BII interconversion are consistent with base destacking coupled with migration of water from ionic phosphate toward the phosphodiester and sugar moieties. Nonspecific interaction of proteins with the DNA backbone could become specific by induced-fit-type interactions with either BI or BII backbone conformations. This suggests that the BI-to-BII substate interconversion could be a major contributor to the protein recognition process.

Base-pair opening and spermine binding--B-DNA features displayed in the crystal structure of a gal operon fragment: implications for protein-DNA recognition.

A sequence that is represented frequently in functionally important sites involving protein-DNA interactions is GTG/CAC, suggesting that the trimer may play a role in regulatory processes. The 2.5 A resolution structure of d(CGGTGG)/d(CCACCG), a part of the interior operator (OI, nucleotides +44 to +49) of the gal operon, co-crystallized with spermine, is described herein. The crystal packing arrangement in this structure is unprecedented in a crystal of B-DNA, revealing a close packing of columns of stacked DNA resembling a 5-stranded twisted wire cable. The final structure contains one hexamer duplex, 17 water molecules and 1.5 spermine molecules per crystallographic asymmetric unit. The hexamer exhibits base-pair opening and shearing at T.A resulting in a novel non-Watson-Crick hydrogen-bonding scheme between adenine and thymine in the GTG region. The ability of this sequence to adopt unusual conformations in its GTG region may be a critical factor conferring sequence selectivity on the binding of Gal repressor. In addition, this is the first conclusive example of a crystal structure of spermine with native B-DNA, providing insight into the mechanics of polyamine-DNA binding, as well as possible explanations for the biological action of spermine.

Synthesis and characterization of disulfide cross-linked oligonucleotides

Disulfide cross-linked derivatives of the «Dickerson/Drew dodecamer» 5'-d(CGCGAATTCGCG)-3' and the related decamer 5'-d(GCGAATTCGC)-3' have been synthesized, each bearing a dithiobis(ethane) (C 2 ) or dithiobis(propane) (C 3 ) cross-link at the central adenine (A). NMR and CD spectroscopy indicate that these duplexes are essentially undistorted relative to native B-DNA. However, cross-lining does confer a large degree of stabilization upon the duplexes, as reflected in increased melting temperatures of 15-21 o C for the cross-linked oligonucleotides relative to those for the native sequences. In contrast, un-cross-linked tethers at the same adenines cause substantial decreases in the melting temperature