Conformation Of Polynucleotides Research Articles

Antioxidant activity and polyphenol content of endemic plant species Centaurea ragusina L.

The genus of Centaurea (Asteraceae) represents an attractive source of bioactive substances [1]. The goal of this work is to determine polyphenol content and antioxidant activity in endemic Croatian plant species Centaurea ragusina L. cultivated in vitro (½MS 2.9 μM GA3+0.5 μM BA and ½MS 2.5 μM IBA) [2] and collected at natural habitats (Katalinic brig - K and Sustipan - S). To clarify biological activity of C. ragusina, interactions of extracts with double stranded polynucleotides (poly A – poly U and ctDNA) were studied. The highest level of total phenols, flavonoids and flavonols was determined in calli ethanol/aqueous extracts cultivated in vitro (2.9 μM GA3 + 0.5 μM BA) while the highest value of hydroxycinnamic acids and proanthocyanidins was detected in ethanol/aqueous extracts of leaves after acclimatization from culture media (½MS 2.5 μM IBA). Significant antioxidant activity measured by DPPH and ABTS methods was observed in almost all extracts with respect to gallic acid as a standard. Extracts of leaves after acclimatization showed the highest stabilization on poly A - poly U (18.36) and ctDNA (5.75) evaluated by changing of melting temperature of the polynucleotide (ΔTm). Results of circular dichroism (CD spectroscopy) indicate significant impact of all tested extracts on the conformation of both polynucleotides. The results obtained suggest that the plant species C. ragusina grown in vitro can be efficiently used as a potential source of polyphenols and antioxidants in food, pharmacological and cosmetics industry as it is equally rich with phytochemicals as its wild grown counterparts.

Structural waters in the minor and major grooves of DNA--a major factor governing structural adjustments of the A-T mini-helix.

The role of microhydration in structural adjustments of the AT-tract in B-DNA was studied at the B97-D/def2-SV(P) level. The (dA:dT)5 complexes with 10 water molecules in minor and 15 water molecules in major grooves were studied. The obtained network of hydrogen bonds revealed the dependence between the groove width and the types of water patterns. In the minor groove, the following patterns were observed: interstrand one-water bridges similar to that of the Dickerson "water spine" and interstrand two-water bridges. The network of structural waters in the major groove is more diverse than that in the minor groove, which agrees with crystallographic data. As the major groove is wider, it is enriched by water molecules forming two- and three-water bridges. Results suggest the nucleobase-water interactions in both grooves prevent AT-tract twisting and its "collapse" along the minor groove. Whereby, a helix structure with narrow minor and wide major grooves is formed. The structural waters affect the polynucleotide conformation so that it becomes similar to poly(dA)·poly(dT) in fibers and acquires features of the A-tracts in DNA in solution. We suggest that formation of specific water patterns in both grooves is the factor responsible for stabilization of A-tracts with a narrowed minor groove, leading in turn to their strong intrinsic bending in DNA.

Natural or synthetic nucleic acids encapsulated in a closed cavity of amphiphiles

In this review some aspects of the interactions of organized structures of amphiphiles with natural or synthetic DNAs are briefly considered. In particular DNAs encapsulated in closed cavities of amphiphiles, specifically giant vesicles and water-in-oil droplets and reverse micelles, are dealt with. Two main applications of giant vesicles are reviewed in detail, namely their use as microreactors where reactions can be followed by optical microscopy on a single vesicle and in synthetic biology as protocell models or as potential semi-synthetic “living” cells. Water-in-oil droplets uses for rapid and relatively low-cost DNA amplification by PCR reaction are described as well as for in vitro transcription and translation. A large variety of non-Watson–Crick conformations of polynucleotides observed in the aqueous inner core of reverse micelles is illustrated and compared with those observed in matched water solutions.

Influence of preparation method on polynucleotide conformation and pharmacological activity of lipoplex

Conformations of polyinosinic acid [poly(I)] and polycytidylic acid [poly(C)] in liposomes (lipoplex) were investigated by both circular dichroism (CD) spectroscopy and fluorescence resonance energy transfer (FRET) measurements, and compared with those in aqueous solution. The results indicate that poly(I) and poly(C) take double-stranded structure in aqueous solution at pH 6.5–7.5 in the presence of NaCl at higher concentration than 50 mM. Although lipoplex was prepared without NaCl to avoid aggregation of lipoplex particles, poly(I) and poly(C) were double-stranded in pre-mixed poly(I)/poly(C) lipoplex (pre-mixed LIC), prepared by adding a mixed solution of poly(I) and poly(C) to the cationic liposomes. However, poly(I) and poly(C) did not take double-stranded structure in separately mixed LIC, prepared by separate addition of poly(I) solution and poly(C) solution to the cationic liposomes. The physicochemical properties (particle diameter and zeta potential) of pre-mixed LIC and separately mixed LIC were not different, but the anti-proliferative effect of pre-mixed LIC on human epidermoid carcinoma A431 cells was about eight times greater than that of separately mixed LIC. Our results indicate that polynucleotide conformation in lipoplex is markedly influenced by the preparation method, and the polynucleotide conformation in lipoplex has a substantial effect on pharmacological activity.

Evolution of conformational principles in nucleic acids

The analysis of conformations of nucleic acids and polynucleotides have been greatly simplified by the important observations that the nucleotide building blocks exhibit two classes of preferred conformations and that the flexibility in the polynucleotide chain resides mainly in the internucleotide PO linkages.

Mg 2+ ion effect on conformational equilibrium of poly A · 2 poly U and poly A poly U in aqueous solutions

Differential UV spectroscopy and thermal denaturation were used to study the Mg 2+ ion effect on the conformational equilibrium in poly A · 2 poly U (A2U) and poly A · poly U (AU) solutions at low (0.01 M Na +) and high (0.1 M Na +) ionic strengths. Four complete phase diagrams were obtained for Mg 2+–polynucleotide complexes in ranges of temperatures 20–96 °C and concentrations (10 −5–10 −2) M Mg 2+. Three of them have a ‘critical’ point at which the type of the conformational transition changes. The value of the ‘critical’ concentration ([Mg t 2+] cr=(4.5±1.0)×10 −5 M) is nearly independent of the initial conformation of polynucleotides (AU, A2U) and of Na + contents in the solution. Such a value is observed for Ni 2+ ions too. The phase diagram of the (A2U+Mg 2+) complex with 0.01 M Na + has no ‘critical’ point: temperatures of (3→2) and (2→1) transitions increase in the whole Mg 2+ range. In (AU+Mg 2+) phase diagram at 0.01 M Na + the temperature interval in which triple helices are formed and destroyed is several times larger than at 0.1 M Na +. Using the ligand theory, a qualitative thermodynamic analysis of the phase diagrams was performed.

Atomic force microscopy examination of conformations of polynucleotides in response to platinum isomers: significance of GC content at broken ends.

Atomic force microscopy is a technique that enables visualization of macromolecular conformations of polynucleotides at nanometer resolution. We investigated the results of interactions of cisplatin, a DNA binding anticancer drug, and its inactive counterpart, transplatin isomer, on the molecular conformation of polynucleotides: poly d(G-C). poly d(G-C) (polyGC) and poly d(A-T). poly d(A-T) (polyAT). We observed that polyAT exhibited an increased number of enlarged ends of molecules, which we attribute to unwound and/or collapsed regions of polyAT. PolyGC molecules did not show such ends unless cisplatin was added to the PolyGC polymers. Transplatin had the apparent effect of causing overlapping or stacking of the polymer molecules. Addition of exonuclease-III to these polymers removed the visible enlarged ends. The effects of cisplatin as compared to transplatin on the polyGC duplex polymers provide support for the presence of intrastrand covalent linkages, consistent with known N7 guanine interaction of the cis isomer on molecular conformation. Furthermore, our results indicate that the mechanism of interactions of DNA with cisplatin may be dependent on the GC content of the molecules. Int. J. Cancer (Radiat. Oncol. Invest.) 90, 68-72, 2000.

99/01567 Thermodynamics energy-efficiency and economic criteria for evaluation of alternative refrigerants for heat pumps : Kazachki, G. and Gage, C. Heat Pump Syst., Energy Effic. Global Warming, Proc. Conf., 1997, 71–77

Two types of multilayer films of DNA complexes with amphiphilic molecules of dioctadecyldimethylammonium (DODA) and polycations: polyallylamine, polyethylenimine and poly-l-lysine were obtained using Langmuir-Blodgett (LB) technique and successive alternating adsorption method (SA films) correspondingly. Structure and affinity properties of the films were studied by IR, UV-VIS and CD spectroscopy. IR spectral criteria of polynucleotide conformation [M. Ghomi, R. Lettelier, J. Liquier, E. Taillandier, Int. J. Biochem., 22 (1990) 691–699] show DNA conserves its double helical structure in all the films. Low frequency shift of IR absorption bands of DNA in the films indicates electrostatic interaction between PO2- groups of DNA and positively charged groups of polycations or polar heads of DODA molecules. Water molecules were found to penetrate into all types of the films and to bind with DNA hydration centers (phosphatic groups). In contrast to usual DNA films, the hydration in the LB and SA films does not initiate B-to-A conformational transition of double helix. The binding of DNA-contained films with different DNA-intercalated molecules, including antitumor drug—daunomycin, has been also studied.

Spectroscopic study of thin multilayer films of the complexes of nucleic acids with cationic amphiphiles and polycations: their possible use as sensor elements

Two types of multilayer films of DNA complexes with amphiphilic molecules of dioctadecyldimethylammonium (DODA) and polycations: polyallylamine, polyethylenimine and poly- l-lysine were obtained using Langmuir-Blodgett (LB) technique and successive alternating adsorption method (SA films) correspondingly. Structure and affinity properties of the films were studied by IR, UV-VIS and CD spectroscopy. IR spectral criteria of polynucleotide conformation [M. Ghomi, R. Lettelier, J. Liquier, E. Taillandier, Int. J. Biochem., 22 (1990) 691–699] show DNA conserves its double helical structure in all the films. Low frequency shift of IR absorption bands of DNA in the films indicates electrostatic interaction between PO 2 - groups of DNA and positively charged groups of polycations or polar heads of DODA molecules. Water molecules were found to penetrate into all types of the films and to bind with DNA hydration centers (phosphatic groups). In contrast to usual DNA films, the hydration in the LB and SA films does not initiate B-to-A conformational transition of double helix. The binding of DNA-contained films with different DNA-intercalated molecules, including antitumor drug—daunomycin, has been also studied.

UV Light-Induced Crosslinking of the Strands of Poly(dA-dT) and Related Alternating Purine-Pyrimidine DNAs

Alkaline agarose gel electrophoresis was used to detect UV-induced crosslinking of the strands of poly(dA-dT) and related alternating purine-pyrimidine DNAs in solutions stabilizing various polynucleotide conformations. Strands of the B-form and A-form of poly(dA-dT) were not crosslinked but a UV dose-dependent retarded species appeared in the denaturing gels in parallel with the polynucleotide isomerization into the unusual X-form. Most other polynucleotides adopting the X-form were crosslinked as well. The exceptions include the X-forms of poly(dA-butyl5dU) and poly(dA-pentyl5dU) whose strands do not crosslink because the long exocyclic substituents attached to uracil make the photodimerization impossible. Strands of poly(amino2dA-dT) and poly(dA, amino2dA-dT), the latter polynucleotide containing roughly equal amounts of amino2adenine and adenine, also do not crosslink upon UV irradiation because they isomerize into an A-like conformation which is different from the X-form of poly (dA-dT). In contrast, strands of the mixed copolymers of poly(dA, amino2dA-dT) containing low amino2adenine contents are crosslinked upon UV irradiation, in accordance with the observation that they isomerize into the X-form.

Berenil recognizes and changes the characteristics of adenine and thymine polynucleotide structures

Using circular dichroism, differential scanning calorimetry and susceptibility to DNAse I cleavage assays, we show that the interaction of berenil, a minor-groove binding drug, with poly(dA−dT) ṡ poly(dA−dT) and poly(dA) ṡ poly(dt) involves important changes in the polynucleotide conformation. The effect of berenil on poly(dA−dT) ṡ poly(dA−dT) comprises a clear alteration in CD spectra even at drug/DNA ratios smaller than the stoichiometric value. Berenil recognizes and binds to the alternating-B conformation of DNA changing it to a new conformation which appears to show some of the peculiarities of poly(dA) ṡ poly(dT), possibly through a modification in the helical parameters at the TpA and ApT steps. Such alteration is accompanied by a small calorimetric enthalpy change. Moreover, the calorimetric enthalpy does not change significantly whatever the input ratio of drug to poly(dA−dT) ṡ poly(dA−dT), indicating that berenil binding does not substantially alters the enthalpy of transition. In addition to increasing the melting temperature of the polynucleotide, berenil reduces the cooperativity of the poly(dA−dT) ṡ poly(dA−dT) transition slightly more than either distamycin or netropsin.

LOW‐TEMPERATURE LUMINESCENCE SPECTRA AND FLUORESCENCE LIFETIMES OF POLYCYTIDYLIC ACID IN POLYALCOHOLIC GLASSES

Abstract— Corrected emission spectra and fluorescence lifetimes of polycytidylic acid in ethylene glycol: water glass at low temperatures are reported. Luminescence properties observed exhibit a strong dependence on pH and temperature. At neutral pH a vibronic structure of a blue part of the fluorescence spectrum is revealed when temperature is changed from 77 to 10 K, confirming that a monomer component of fluorescence is present. There is also a strong difference in decay of a red‐shifted excimer fluorescence at 10 K at pH 7 and pH 3.9, reflecting a different protonation of cytosine residues and different conformations of polynucleotides in such conditions.

Study of polynucleotide conformation by resolution-enhanced ultraviolet spectroscopy poly(rC) and poly(dC).

Self-deconvolution and the fourth derivative of ultraviolet absorption spectra have been used to study stacked single-stranded and double-helix structures of different cytosine-containing polynucleotides for the first time. These compounds were studied under different solution conditions (pH and organic solvents) and at low temperatures. The red shift of the lower band (B2u band plus possibly some n-->pi* transition) of the absorption spectra in the cytosine-containing polynucleotides and the appearance of new peaks in the deconvoluted and derivative spectra in the 280-310 nm region are attributed mainly to cytosine-cytosine stacking interactions. In particular, the fourth-derivative peaks at wavelengths higher than 290 nm can be associated to coupling of electronic transitions of cytosine bases. The nature of the electronic transitions producing the absorption bands which are resolved in the aforementioned fourth-derivative peaks is discussed. It is concluded that the resolution-enhancement techniques used in this work, i.e. self-deconvolution and fourth derivative, complement each other and are useful methods to study structural changes of single-stranded and double-stranded polynucleotides allowing, at the same time, more information to be obtained about specific stacking interactions than classical absorption spectrophotometry.

Block‐units method for conformational calculations of large nucleic acid chains. II. The two‐hierarchical approach and its application to conformational arrangement of the unusual TψC loop of rabbit tRNAval

The two-level hierarchical methodology is suggested for conformational calculations of large fragments of nucleic acids. The method of the first level is intended for performing a fast screening of the conformational phase space. The high-level method may be used to refine structurally important conformations. The method of the first level is the block-units method, which has been developed specially for these purposes (see part I). It has been shown that the block-units method allows the satisfactory calculation of the structure parameters of the optimal conformations of polynucleotides. The results of the conformational rearrangement calculations of the T psi C loop of the tRNA(Phe) after modification of its sequence are represented.

Quantitative analysis of DNA secondary structure from solvent-accessible surfaces: the B- to Z-DNA transition as a model.

Solvent structure and its interactions have been suggested to play a critical role in defining the conformation of polynucleotides and other macromolecules. In this work, we attempt to quantitate solvent effects on the well-studied conformational transition between right-handed B- and left-handed Z-DNA. The solvent-accessible surfaces of the hexamer sequences d(m5CG)3, d(CG)3, d(CA)3, and d(TA)3 were calculated in their B- and Z-DNA conformations. The difference in hydration free energies between the Z and the B conformations (delta delta GH(Z-B] was determined from these surfaces to be -0.494 kcal/mol for C-5 methylated d(CG), 0.228 kcal/mol for unmethylated d(CG), 0.756 kcal/mol for d(CA)-d(TG), and 0.896 kcal/mol for d(TA) dinucleotides. These delta delta GH(Z-B) values were compared to the experimental B- to Z-DNA transition energies of -0.56 kcal/mol that we measured for C-5 methylated d(CG), 0.69-1.30 kcal/mol reported for unmethylated d(CG), 1.32-1.48 kcal/mol reported for d(CA)-d(TG), and 2.3-2.4 kcal/mol for d(TA) dinucleotides. From this comparison, we found that the calculated delta delta GH(Z-B) of these dinucleotides could account for the previous observation that the dinucleotides were ordered as d(m5CG) greater than d(CG) greater than d(CA)-d(TG) greater than d(TA) in stability as Z-DNA. Furthermore, we predicted that one of the primary reasons for the inability of d(TA) sequences to form Z-DNA results from a decrease in exposed hydrophilic surfaces of adjacent base pairs due to the C-5 methyl group of thymine; thus, d(UA) dinucleotides should be more stable as Z-DNA than the analogous d(TA) dinucleotides.(ABSTRACT TRUNCATED AT 250 WORDS)

Binding of RecA Protein to Z-form DNA Studied with Circular and Linear Dichroism Spectroscopy

Binding of RecA to poly(dG-m5dC) and poly(dG-dC) under B- and Z-form conditions was studied using circular dichroism (CD) and linear dichroism (LD). LD revealed a quantitative binding of RecA to Mg2+-induced Z-form poly(dG-m5dC) with a stoichiometry of 3.1 base pairs/RecA monomer, which is slightly larger than the 2.7 base pairs observed for the B-form. The LD spectra indicate a preferentially perpendicular orientation of DNA bases and a rather parallel orientation of the tryptophan residues relative to the fiber axis in both complexes. The association rate of RecA to Z-form DNA was found to be slower than to B-form. CD measurements showed that the polynucleotide conformation is retained upon RecA binding, and CD and LD confirm that RecA binds to both forms of DNA. The Mg2+-induced Z-form is shown to be retransformed into B-form, both in free and in RecA-complexed polynucleotides by addition of NaCl, whereas the B----Z transition cannot be induced by addition of Mg2+ when the polynucleotide is complexed with RecA. From this it is inferred that RecA does not stabilize the Z-conformation of the polynucleotide but that it can kinetically "freeze" the polynucleotide in its B-conformation. On all essential points, the same conclusions were also reached in a corresponding study of unmethylated poly(dG-dC) with the Z-form induced by Mn2+.

Conformational Characteristics of Mixed Sugar Puckered Deoxytetranucleoside Triphosphate d-GpCpGpC from Energy Minimization Studies

As a continuation of our theoretical studies on nucleic acid subunit systems, in this article we consider the case of the tetranucleoside d-GpCpGpC, the minimally ideal representative unit for analyzing the relative stabilities of different forms of homo- and mixed helical conformation of polynucleotides. The four sugar rings are kept so as to generate B-genus, B+A genus and Z-genus conformations. Twenty five helical conformational states which resulted from judicious mixing of A-, B-, C-, W-, and Z-, states locally are subjected to energy minimization permitting the 19 dihedral angles to vary simultaneously. Conformational states corresponding to regular helical forms and mixed helical forms, when analyzed provide valuable information as to the local conformational flexibility and transitions available to polynucleotides.

Conformational peculiarities of polynucleotides with a nonrandom base sequence according to the 1H----3H exchange rate in C8H groups of purinic residues.

We have determined the 1H----3H exchange rate constants between water and C8H groups of purinic residues of alternating polynucleotides poly(dA-dT).poly(dA-dT), poly(dG-dC).poly(dG-dC) and poly(dA-dC).poly(dG-dT) as well as homopolynucleotides poly(dA).poly(dT) and poly(dG).poly(dC) in aqueous solutions with high-salt concentrations (3 M NaCl and 4-6 M CsF), in water-ethanol (60%) solution and in 0.15 M NaCl at 25 degrees C. The rate constants for adenine (kA) and guanine (kG) of polynucleotides were compared with corresponding constants for E. coli DNA. dGMP nd dAMP at the same conditions. The relation between exchange rates and conformations of polynucleotides permits the study of their conformational peculiarities in solution. Of three alternating polynucleotides examined in 0.15 M NaCl the exchange retardation was observed only for poly(dA-dT).poly(dA-dT) as compared with that in B-DNA, which is in good agreement with the B-alternating "wrinkled" DNA model. The conformations of poly(dG-dC).poly(dG-dC) and poly(dA-dC).poly(dG-dT), according to the exchange data obtained are within the B form. For homopolynucleotides in 0.15 M NaCl, the KA value for poly(dA).poly(dT) is nearly the same as kA for B-DNA, which indicates the similarity of their conformations, whereas the kG value for poly(dG).poly(dC) is 1.7-fold lower in comparison with the kG value in B-DNA. This seems to be connected with the existence of B = A conformation equilibrium for poly(dG).poly(dC) in solution. The increase of NaCl concentration to 3 M results in a B----Z transition in the case of poly(dG-dC).poly(dG-dC) and in the shift of B = A equilibrium towards the A-form in the case of poly(dG).poly(dC) as is evidenced by alterations of their KG values. Poly(dA-dT).poly(dA-dT) in 6 M CsF and poly(dA-dC).poly(dG-dT) in 4.3 M CsF maintain their inherent conformations in 0.15 M NaCl in spite of the fact that they are characterised by the "X-type" CD-spectrum at these conditions. According to the exchange data the conformation of poly(dA).poly(dT) in 6 M CsF corresponds to the "heteronomous" DNA model or some other structure with lower accessibility of C8H groups of adenylic residues.

The photoreactivity of pyrimidine-purine sequences in some deoxydinucleoside monophosphates and alternating DNA copolymers.

Abstract— A reversed‐phase HPLC system has been developed which separates the common nucleo‐bases from the 6‐methylimidazo[4,5‐b]pyridin‐5‐one (6‐MIP) produced on acid hydrolysis of a thymine‐adenine photoadduct (TA*) that is formed between adjacent thymine and adenine bases in UV‐irradiated polydeoxyribonucleotides. By measuring the relative amounts of adenine and 6‐MIP in acid hydrolysates, this system has been used to investigate how polynucleotide conformation affects the yield of TA* in poly(dA‐dT) irradiated at 254 nm. The photoreactivity of other pyrimidine‐purine sequences has been examined with the deoxydinucleoside monophosphates d(TpI) and d(m5CpA) and with the alternating DNA copolymers poly(dA‐dU), poly(dI‐dC), poly(dG‐dC) and poly(dA‐dC).poly(dG‐dT). Samples were irradiated at 254 nm in aqueous solution and in ice, and at wavelengths >290 nm with acetone as photosensitizer. A photoproduct resembling TA*, and giving 6‐MIP on acid hydrolysis, was isolated from d(TpI) irradiated at 254 nm in solution or in ice; d(m5CpA) was comparatively unreactive. Acid hydrolysates of the irradiated DNA copolymers were screened by HPLC and by TLC and paper electrophoresis, for the presence of imidazo[4,5‐b]pyridin‐5‐one, 6‐MIP, or other species attributable to specific photoproduct formation. By this criterion, however, none of the copolymers showed evidence of significant photoreactivity in either their single‐ or double‐stranded conformational states. The formation of mixed pyrimidine‐purine photoadducts in DNA is therefore probably restricted to T‐A doublets.

Interpretation of DNA vibration modes. II--The adenosine and thymidine residues involved in oligonucleotides and polynucleotides.

Normal coordinate analysis of the adenosine and thymidine residues involved in the right- and left-handed conformations of oligonucleotides and polynucleotides has been performed. The valence force field, employed in this work, allowed recently to reproduce the vibrational spectra of 2'-deoxythymidine and 2'-deoxyadenosine. The calculated wavenumbers based on a non-redundant set of internal coordinates have been compared to the Raman and infrared peak positions arising from A, B, C, D and Z conformations, in the 1550-1250 cm-1 and 800-600 cm-1 spectral regions: i.e. characteristic of adenosine and thymidine residues. Moreover, a systematic study has been performed on the evolution of the vibrational wavenumbers as a function of the glycosidic angle (chi) and the sugar pucker conformation.