Molecular electrotatic potential of a triple stranded helix: Poly(dT)·poly(dA)·poly(dT)

The molecular electrostatic potential of the triple helix poly(dT)·tpoly(dA)·poly(dT) is calculated, and the results are examined in relation to those obtained for its component double and single helical parts. For the double helix presenting the standard Watson–Crick hydrogen bonds, the deepest potentials are formed on the side of the major groove, a situation similar to that observed in the A-DNA duplex. For the double helix presenting Hoogsteen-type hydrogen bonds the deepest potentials lie in the major groove, on the side of the pyrimidine strand. In the triple helix the deepest potentials are located in the major groove in a narrow zone over the thymine bases of the Watson–Crick pair.     

Hypochromism of polynucleotides in the nearest neighbour approximation

A method of calculating the hypochromism of polynucleotides in the nearest neighbour approximation is given by perturbation theory. The calculated hypochromism values of the first UV-absorption band of polynucleotides poly(dA)·poly(dT) and poly(dG)·poly(dC) are in good agreement with experiment. In this approximation the origin of the hypochromic effect is studied for the double-stranded polynucleotides; the dependence of the hypochromism upon the length of the polynucleotide is given.     

Calorimetric and thermal analysis studies on the binding of phenothiazinium dye thionine with DNA polynucleotides

Binding of the phenothaizinium dye thionine with four sequence specific deoxyribopolynucleotides, poly(dG-dC).poly(dG-dC), poly(dG).poly(dC), poly(dA-dT).poly(dA-dT), and poly(dA).poly(dT) has been investigated by means of thermal helix melting, isothermal titration calorimetry, and differential scanning calorimetry experiments. The binding affinity values evaluated from isothermal titration calorimetry suggests that thionine exhibits the highest binding affinity to poly(dG-dC).poly(dG-dC). The binding to poly(dG-dC).poly(dG-dC), poly(dA-dT).poly(dA-dT), and poly(dG).poly(dC) is exothermic and favoured by negative enthalpy changes while binding to poly(dA).poly(dT) is endothermic and anomalous. The values of heat capacity changes of the interaction are negative and in the range (?0.4 to ?0.5) kJ · K^?1 · mol^?1. The binding is characterized by strong stabilization of the polynucleotides against thermal strand separation. The binding affinity values derived from thermal melting data are in excellent agreement with those obtained from isothermal titration calorimetry data. Insights into the energetic aspects and guanine–cytosine selectivity of the DNA interaction of thionine have been obtained from these studies.     

Rapid Location of the Preferred Interaction Sites between Small Polar Molecules and Macromolecules. II. Binding of Water to a Model Segment of B-DNA

The procedure developed in Part I of this series is applied to the homopolymeric sequences poly(dA) · poly(dT) and poly(dG) · poly(dC) on the double helical structure of B-DNA. Some aspects of the base sequence influence on the polymer's attraction for water molecule are described. The results are used to discuss the general hydration features of those systems in relation to recent experimental studies of DNA single crystals.     

Naphthalene Imide Conjugates: Formation of Supramolecular Assemblies,and the Encapsulation and Release of Dyes through DNA‐Mediated Disassembly

We report the synthesis of two new amphiphilic conjugates 1 and 2 based on naphthalene di‐ and monoimide chromophores and the investigation of their photophysical, self‐assembly and DNA‐binding properties. These conjugates showed aqueous good solubility and exhibited strong interactions with DNA and polynucleotides such as poly(dG?dC)–poly(dG?dC) and poly(dA?dT)–poly(dA?dT). The interaction of these conjugates with DNA was evaluated by photo‐ and biophysical techniques. These studies revealed that the conjugates interact with DNA through intercalation with association constants in the order of 5–8×10⁴ M ^?1. Of these two conjugates, bolaamphiphile 1 exhibited a supramolecular assembly that formed vesicles with an approximate diameter of 220 nm in the aqueous medium at a critical aggregation concentration of 0.4 mM , which was confirmed by SEM and TEM. These vesicular structures showed a strong affinity for hydrophobic molecules such as Nile red through encapsulation. Uniquely, when exposed to DNA the vesicles disassembled, and therefore this transformation could be utilised for the encapsulation and release of hydrophobic molecules by employing DNA as a stimulus.     

Theoretical study of the interaction of tetramethylammonium with double-stranded oligonucleotides

Computations are performed on the interaction specificities of tetramethylammonium (TMA) for double-stranded oligonucleotides held in the B conformation. The effects of base sequence and chain length are investigated. In the short oligomers (helices formed from dinucleoside monophosphates and trinucleoside diphosphates), the interaction energies of TMA are larger in the major groove of (dG)_n · (dC)_n than in the minor groove of either (dA)_n · (dT)_n or (dA—dT)_n. Upon lengthening the oligomers, and owing to the gradual shaping of the grooves of the helix and cumulative effect of the phosphates, TMA is shown to increasingly favor the minor groove of (dA)_n · (dT)_n with respect to the major groove of (dG)_n · (dC)_n, with a sizeable energy difference computed at the pentanucleoside hexaphosphate level. The binding of TMA in the minor groove of (dA)_n · (dT)_n involves stabilizing contacts with several sites, on the bases and on the deoxyriboses. Configurations locating the cation closer to the thymine strand are slightly preferred over configurations locating it closer to the adenine strand.     

Simultaneous binding of meso-tetrakis(N-methylpyridinium-4-yl)porphyrin and 4',6-diamidino-2-phenylindole at the minor grooves of poly(dA).poly(dT) and poly[d(A-T)(2)]: fluorescence resonance energy transfer between DNA bound drugs

The spectral properties of meso-tetrakis(N-methylpyridinium-4-yl)porphyrin (TMPyP) bound to poly(dA).poly(dT) and poly[d(A-T)(2)] in the presence and in the absence of 4',6-diamidino-2-phenylindole (DAPI) have been studied. DAPI fits deeply into the minor groove of both poly(dA).poly(dT) and poly[d(A-T)(2)], and TMPyP is also situated at the minor groove. The nature of the absorption, circular dichroism (CD), and flow linear dichroism (LD) spectra of the TMPyP-poly(dA).poly(dT) and -poly[d(A-T)(2)] complexes in the Soret band is essentially unaffected whether the minor groove is blocked by DAPI or not, although small variations been noticed in the presence of DAPI. Furthermore, a close analysis of the reduced LD spectrum in the Soret band results in angles of approximately 80 degrees and 55 degrees between transition moments of the TMPyP and DNA helix axes in the absence of DAPI. All these observations indicate that the side of TMPyP whose structure resembles that of classical minor groove binding drugs does not fit deeply into the minor groove. This suggests that TMPyP binds across the minor groove: two positively charged pyridiniumyl rings interact electrostatically with negatively charged phosphate groups of DNA. When DAPI and TMPyP are simultaneously bound to poly(dA).poly(dT) or poly[d(A-T)(2)], the fluorescence intensity of DAPI decreases as TMPyP concentration increases, indicating that the excited energy of DAPI is transferred to TMPyP.     

HPLC ANALYSIS OF 4'',5''-MONOADDUCT FORMATION IN CALF THYMUS DNA and SYNTHETIC POLYNUCLEOTIDES TREATED WITH UVA and 8-METHOXYPSORALEN

Abstract— 8-methoxypsoralen monoadduct formation in calf thymus DNA irradiated with subbands of ultraviolet A light has been quantitated by HPLC analysis of the enzymatic hydrolysates of the DNA. Normalization of the yield of monoadducts for the variation in source output and the absorptivity of 8-MOP at each of the irradiating wavelengths showed that the 4',5'-furan monoadduct was the principal photoproduct and the efficiency of its formation was independent of irradiating wavelength. Synthetic polynucleotides irradiated with ultraviolet A light demonstrated a base composition and sequence dependence for 8-MOP photoreactivity: (poly(dAdT.dAdT) > poly(dA.dT) > poly(dGdC.dGdC) in both the B and Z forms > pofy(dT).     

Fluorescence of the DNA double helix (dA)20 x (dT)20 studied by femtosecond spectroscopy--effect of the duplex size on the properties of the excited states

The fluorescence of the DNA double-stranded oligomer (dA)20 x (dT)20 is studied at room temperature by fluorescence up-conversion at times shorter than 10 ps. The profile of the up-conversion spectra is similar to that of the steady-state fluorescence spectrum, showing that the majority of the photons are emitted within the probed time scale. At all the probed wavelengths, the fluorescence decays are slower than those of the monomeric chromophores dAMP and TMP. The fluorescence anisotropy decays show strong wavelength dependence. These data allow us to conclude that energy transfer takes place in this double helix and that this process involves exciton states. The spectral and dynamical properties of the oligomer are compared to those of the polymer poly(dA) x poly(dT), composed of about 2000 base pairs, reported previously. The oligomer absorption spectrum is characterized by a smaller hypsochromic shift and weaker hypochromism compared to the polymer. Moreover, the fluorescence decays of (dA)20 x (dT)20 are twice as fast as those of poly(dA) x poly(dT), and its fluorescence anisotropy decays more slowly. These differences are the fingerprints of a larger delocalization of the excited states induced by an increase in the size of the duplex.     

Aminoglycoside-nucleic acid interactions: remarkable stabilization of DNA and RNA triple helices by neomycin

The stabilization of poly(dA).2poly(dT) triplex, a 22-base DNA triplex, and poly(rA).2poly(rU) triple helix by neomycin is reported. The melting temperatures, the association and dissociation kinetic parameters, and activation energies (E(on) and E(off)) for the poly(dA).2poly(dT) triplex in the presence of aminoglycosides and other triplex binding ligands were determined by UV thermal analysis. Our results indicate that: (i) neomycin stabilizes DNA triple helices, and the double helical structures composed of poly(dA).poly(dT) are virtually unaffected. (ii) Neomycin is the most active and triplex-selective stabilization agent among all aminoglycosides, previously studied minor groove binders, and polycations. Its selectivity (DeltaT(m3-->2) vs DeltaT(m2)(-->)(1)) exceeds most intercalating drugs that bind to triple helices. (iii) Neomycin selectively stabilizes DeltaT(m3)(-->)(2) for a mixed 22-base DNA triplex containing C and T bases in the pyrimidine strand. (iv) The rate constants of formation of triplex (k(on)) are significantly enhanced upon increasing molar ratios of neomycin, making triplex association rates closer to duplex association rates. (v) E(on) values become more negative upon increasing concentration of aminoglycosides (paromomycin and neomycin). E(off) values do not show any change for most aminoglycosides except neomycin. (vi) Aminoglycosides can effectively stabilize RNA [poly(rA).2poly(rU)] triplex, with neomycin[being one of the most active ligands discovered to date (second only to ellipticine). (vii) The stabilization effect of aminoglycosides on triple helices is parallel to their toxic behavior, suggesting a possible role of intramolecular triple helix (H-DNA) stabilization by the aminoglycosides.     

Neomycin-neomycin dimer: an all-carbohydrate scaffold with high affinity for AT-rich DNA duplexes

A dimeric neomycin-neomycin conjugate 3 with a flexible linker, 2,2'-(ethylenedioxy)bis(ethylamine), has been synthesized and characterized. Dimer 3 can selectively bind to AT-rich DNA duplexes with high affinity. Biophysical studies have been performed between 3 and different nucleic acids with varying base composition and conformation by using ITC (isothermal calorimetry), CD (circular dichroism), FID (fluorescent intercalator displacement), and UV (ultraviolet) thermal denaturation experiments. A few conclusions can be drawn from this study: (1) FID assay with 3 and polynucleotides demonstrates the preference of 3 toward AT-rich sequences over GC-rich sequences. (2) FID assay and UV thermal denaturation experiments show that 3 has a higher affinity for the poly(dA)·poly(dT) DNA duplex than for the poly(dA)·2poly(dT) DNA triplex. Contrary to neomycin, 3 destabilizes poly(dA)·2poly(dT) triplex but stabilizes poly(dA)·poly(dT) duplex, suggesting the major groove as the binding site. (3) UV thermal denaturation studies and ITC experiments show that 3 stabilizes continuous AT-tract DNA better than DNA duplexes with alternating AT bases. (4) CD and FID titration studies show a DNA binding site size of 10-12 base pairs/drug, depending upon the structure/sequence of the duplex for AT-rich DNA duplexes. (5) FID and ITC titration between 3 and an intramolecular DNA duplex [d(5'-A(12)-x-T(12)-3'), x = hexaethylene glycol linker] results in a binding stoichiometry of 1:1 with a binding constant ～10(8) M(-1) at 100 mM KCl. (6) FID assay using 3 and 512 hairpin DNA sequences that vary in their AT base content and placement also show a higher binding selectivity of 3 toward continuous AT-rich than toward DNA duplexes with alternate AT base pairs. (7) Salt-dependent studies indicate the formation of three ion pairs during binding of the DNA duplex d[5'-A(12)-x-T(12)-3'] and 3. (8) ITC-derived binding constants between 3 and DNA duplexes have the following order: AT continuous, d[5'-G(3)A(5)T(5)C(3)-3'] > AT alternate, d[5'-G(3)(AT)(5)C(3)-3'] > GC-rich d[5'-A(3)G(5)C(5)T(3)-3']. (9) 3 binds to the AT-tract-containing DNA duplex (B* DNA, d[5'-G(3)A(5)T(5)C(3)-3']) with 1 order of magnitude higher affinity than to a DNA duplex with alternating AT base pairs (B DNA, d[5'-G(3)(AT)(5)C(3)-3']) and with almost 3 orders of magnitude higher affinity than a GC-rich DNA (A-form, d[5'-A(3)G(5)C(5)T(3)-3']).     

Thermodynamic profiles of the DNA binding of benzophenanthridines sanguinarine and ethidium: A comparative study with sequence specific polynucleotides

Energetics of the binding of two known classical DNA intercalating molecules, ethidium and sanguinarine with four sequence specific polynucleotides, poly(dG-dC).poly(dG-dC), poly(dG).poly(dC), poly(dA-dT).poly(dA-dT), and poly(dA).poly(dT) have been compared under identical conditions. The binding of both the molecules was characterized by strong stabilization of the polynucleotides against thermal strand separation in optical melting as well as differential scanning calorimetry studies. Isothermal titration calorimetry results revealed that the binding of both sanguinarine and ethidium to poly(dG-dC).poly(dG-dC), poly(dA-dT).poly(dA-dT), and poly(dG).poly(dC) was exothermic and favoured by negative enthalpy changes. On the other hand, the binding of both molecules to poly(dA).poly(dT) was endothermic and entropy driven. The binding affinity values obtained from isothermal titration calorimetry data was in close proximity to that derived from thermal melting data. The heat capacity changes obtained from temperature dependence of the enthalpy change gave negative values in the range (?0.4 to 1.25) kJ · mol^?1 · K^?1 for the binding of ethidium and sanguinarine to these polynucleotides. The variations in the values indicate important differences in the formation of the complexes. New insights into the energetics and specificity aspects of interaction of these molecules to DNA have emerged from these studies.     

新的杯[4]芳烃衍生物通过缔合乙腈进行自插入的X-射线衍射和密度泛函理论研究

合成和表征了一个新的杯[4]芳烃衍生物，11,23-二羟亚胺甲基-25,27-二羟基-26,28-二丙氧基杯[4]芳烃 (B)及其与乙腈生成的组成为B·2CH₃CN的化合物。¹H NMR显示，在B·2CH₃CN中B采取锥型构象，X-射线衍射分析确证在溶液中所发现的构象。在晶格网络中存在着B·2CH₃CN以二聚体形式的自插入现象。在B3LYP/6-311G(d)水平上计算了该自插入二聚体中的非共价相互作用能，并对基集叠加误差进行了校正。在二聚体中的B·2CH₃CN，一个CH₃CN通过与羟亚胺基形成氢键使之稳定，结合能为–5.02 kJ·mol^-1，另一个CH₃CN则通过与另一个羟亚胺基形成氢键以及与另一B·2CH₃CN中B苯环空腔间的C–H···π相互作用使之稳定，结合能分别为–14.23 kJ·mol^-1和–3.77 kJ·mol^-1。自插入的驱动能为–7.54 kJ·mol^-1。     

Exploring concerted effects of base pairing and stacking on the excited‐state nature of DNA oligonucleotides by DFT and TD‐DFT studies

We have taken (dA)₅, (dT)₅, and (dA)₅·(dT)₅ as model systems to study concerted effects of base pairing and stacking on excited‐state nature of DNA oligonucleotides using density functional theory (DFT) and time dependent DFT methods. The spectroscopic states are determined to be of a partial A → A charge‐transfer nature in the A·T oligonucleotides. The T → T charge‐transfer transitions produce dark states, which are hidden in the energy region of the steady‐state absorption spectra. This is different from the previous assignment that the T → T charge‐transfer transition is responsible for a shoulder at the red side of the first strong absorption band. The A → T charge‐transfer states were predicted to have relatively high energies in the A·T oligonucleotides. The present calculations predict that the T → A charge‐transfer states are not involved in the spectra and excited‐state dynamics of the A·T oligonucleotides. In addition, the influence of base pairing and stacking on the nature of the ¹nπ* and ¹ππ* states are discussed in detail. © 2010 Wiley Periodicals, Inc. Int J Quantum Chem, 2011     

A cooperative beads-on-a-string approach to exceptionally stable DNA triplexes

A poly(dT) oligomer can serve as the string on which synthetic hairpins possessing poly(dA) and poly(dT) arms connected by a hydrophobic perylene diimide linker are assembled like beads on a string. The synthetic hairpins form head-to-head dimers and trimers, respectively, in the absence of the poly(dT) string and in the presence of a string with inverted polarity at mid-strand. However, they assemble in cooperative head-to-tail fashion on normal poly(dT) oligomers.     

Extent of formation of a dimeric adenine photoproduct in polynucleotides and DNA

Quantum yields are reported for the formation of a dimeric adenine photoproduct, A = A, in adenine homopolymers and DNA irradiated at 254 nm. The A = A content of irradiated samples was assayed by using reversed-phase HPLC to isolate the 4,6-diamino-5-guanidinopyrimidine (DGPY) which is produced from A = A on acid hydrolysis. Acid hydrolysates derived from DNA radiolabelled with [14C] 2'-deoxyadenosine were spiked with unlabelled DGPY before fractionation on HPLC and the recovered material was further purified by chromatography on Sephadex G-10 followed by co-crystallization with DGPY sulphate. Although A = A is formed with a relatively high quantum yield of 1.6 X 10(-3) mol einstein-1 in single-stranded poly(dA) the photoaddition reaction is strongly quenched in base-paired poly(dA).poly(dT) and undetectable in poly(rA).poly(dT). Respective quantum yields of 6 X 10(-5) and 9 X 10(-6) were estimated for the formation of A = A in single- and double-stranded E. coli DNA implying that the photoproduct has very limited biological significance. From studies with d(ApG), d(GpA), ApG, GpA, d(A)20 and d(A4G)4 it is concluded that adjacent guanine and adenine bases do not form a photoadduct analogous to A = A and also that guanine residues have no local or long-range quenching effect on photodimerization within A-A doublets.     

High-performance affinity chromatography of messenger RNA.

A 50-mer of thymidylic acid, (dT)50, was coupled to silica inside prepacked columns using the N-hydroxysuccinimide chemistry. The resulting (dT)50-silica columns were used to resolve oligomers of adenylic acid, (dA)19-24, and to separate poly(A) mRNA (messenger RNA) from Saccharomyces. Oligomers which differed in length by a single nucleotide base were readily resolved. Using either (dT)50- or (dT)18-silica, poly(A) mRNA could be purified in as little as 8 min. The poly(A) mRNA isolated appeared to be full length and could be used directly for T4 RNA ligase and RNAse A and T1 enzymatic reactions. The (dT)50-silica column was used to fractionate total poly(A) mRNA by tail length. While the separation was primarily due to poly(A) tail length, most fractions appeared to contain multiple tail lengths. Whether this represents an intrinsic feature of the RNA or a limitation of the method is discussed. These studies show that polynucleotides in the kilobase size range can be separated rapidly and with good resolution on DNA-silica.     

Site‐Directed Spin‐Labeling of DNA by the Azide–Alkyne ‘Click’ Reaction: Nanometer Distance Measurements on 7‐Deaza‐2′‐deoxyadenosine and 2′‐Deoxyuridine Nitroxide Conjugates Spatially Separated or Linked to a ‘dA‐dT’ Base Pair

Nucleobase‐directed spin‐labeling by the azide‐alkyne ‘click’ (CuAAC) reaction has been performed for the first time with oligonucleotides. 7‐Deaza‐7‐ethynyl‐2′‐deoxyadenosine ( 1 ) and 5‐ethynyl‐2′‐deoxyuridine ( 2 ) were chosen to incorporate terminal triple bonds into DNA. Oligonucleotides containing 1 or 2 were synthesized on a solid phase and spin labeling with 4‐azido‐2,2,6,6‐tetramethylpiperidine 1‐oxyl (4‐azido‐TEMPO, 3 ) was performed by post‐modification in solution. Two spin labels ( 3 ) were incorporated with high efficiency into the DNA duplex at spatially separated positions or into a ‘dA‐dT’ base pair. Modification at the 5‐position of the pyrimidine base or at the 7‐position of the 7‐deazapurine residue gave steric freedom to the spin label in the major groove of duplex DNA. By applying cw and pulse EPR spectroscopy, very accurate distances between spin labels, within the range of 1–2 nm, were measured. The spin–spin distance was 1.8±0.2 nm for DNA duplex 17 ( dA*^7,10 ) ?11 containing two spin labels that are separated by two nucleotides within one individual strand. A distance of 1.4±0.2 nm was found for the spin‐labeled ‘dA‐dT’ base pair 15 ( dA*⁷ ) ?16 ( dT*⁶ ). The ‘click’ approach has the potential to be applied to all four constituents of DNA, which indicates the universal applicability of the method. New insights into the structural changes of canonical or modified DNA are expected to provide additional information on novel DNA structures, protein interaction, DNA architecture, and synthetic biology.     

Exploration of the (ethanol)4–water heteropentamers potential energy surface by simulated annealing and Ab initio molecular dynamics

A DFT electronic structure study of the (ethanol)₄–water heteropentamers at the B3LYP/6‐31+G(d) model chemistry was carried out. To get determine possible configurations, the potential energy surface (PES) was explored with two methods: simulated annealing and ab initio molecular dynamics. The results suggest that the PES is very flat. A total of 81 stable structures were determined. These structures were classified into 16 different geometric patterns according to geometric criteria like the number of hydrogen bonds and their spatial arrangement: cyclic, bicyclic, or lineal patterns. Thermodynamic stability was used for defining the order of such classification. Hydrogen bonds are mutually disturbed due to the existence of cooperative effects. Cooperativity affects the nature of the hydrogen bonds and the overall stability of the ethanol–water system given that the strongest interactions are markedly covalent and the most stable geometric pattern corresponds to the pentagonal arrangement. These observations were supported by the analysis of the loss of atomic charge of the hydrogen atoms involved in hydrogen bonds. These hydrogen bonds were classified as primary and secondary hydrogen bonds: O? H ··· O and C? H ··· O, respectively. For comparative purposes, some (ethanol)₅, (methanol)₅, and (methanol)₄–water clusters were characterized in this study. © 2010 Wiley Periodicals, Inc. Int J Quantum Chem, 2011