Structure of Poly (dT)·Poly (dA)·Poly (dT)

Abstract

The molecular structure of poly (dT)·poly (dA)·poly (dT) has been determined and refined using the continuous x-ray intensity data on layer lines in the diffraction pattern obtained from an oriented fiber of the DNA. The final R-value for the preferred structure is 0.29 significantly lower than that for plausible alternatives. The molecule forms a 12-fold right- handed triple-helix of pitch 38.4 Å and each base triplet is stabilized by a set of four Crick-Watson-Hoogsteen hydrogen bonds. The deoxyribose rings in all the three strands have C2′-endo conformations. The grooveless cylindrical shape of the triple-helix is consistent with the lack of lateral organization in the fiber.     

Charge Transport in Poly(dG)–Poly(dC) and Poly(dA)–Poly(dT) DNA Polymers

We investigate the charge transport in synthetic DNA polymers built up from single type of base pairs. In the context of a polaronlike model, for which an electronic tight-binding system and bond vibrations of the double helix are coupled, we present estimates for the electron-vibration coupling strengths utilizing a quantum-chemical procedure. Subsequent studies concerning the mobility of polaron solutions, representing the state of a localized charge in unison with its associated helix deformation, show that the system for poly(dG)–poly(dC) and poly(dA)–poly(dT) DNA polymers, respectively possess quantitatively distinct transport properties. While the former supports unidirectionally moving electron breathers attributed to highly efficient long-range conductivity, the breather mobility in the latter case is comparatively restrained, inhibiting charge transport. Our results are in agreement with recent experimental results demonstrating that poly(dG)–poly(dC) DNA molecules acts as a semiconducting nanowire and exhibit better conductance than poly(dA)–poly(dT) ones.     

The Structure of Triple Helical Poly(U)·Poly(A)·Poly(U) Studied by Raman Spectroscopy

Abstract

Using Raman spectroscopy, we examined the ribose-phosphate backbone conformation, the hydrogen bonding interactions, and the stacking of the bases of the poly(U)·poly(A) ·poly(U) triple helix. We compared the Raman spectra of poly(U)·poly(A)·poly(U) in H₂O and D₂O with those obtained for single-stranded poly(A) and poly(U) and for double-stranded poly(A)·poly(U). The presence of a Raman band at 863 cm^?1 indicated that the backbone conformations of the two poly(U) chains are different in the triple helix. The sugar conformation of the poly(U) chain held to the poly(A) by Watson-Crick base pairing is C3′ endo; that of the second poly(U) chain may be C2′ endo. Raman hypochromism of the bands associated with base vibrations demonstrated that uracil residues stack to the same extent in double helical poly(A)·poly(U) and in the triple-stranded structure. An increase in the Raman hypochromism of the bands associated with adenine bases indicated that the stacking of adenine residues is greater in the triple helix than in the double helical form. Our data further suggest that the environment of the carbonyls of the uracil residues is different for the different strands.     

Complex-Formation of Ethidium Bromide with Poly[d(A-T)]·Poly[d(A-T)]

Abstract

The interaction of Ethidium Bromide (EtBr) with double-stranded (ds-) and single-stranded (ss-) poly[d(A-T)] was studied in different ionic strengths solutions. Optical spectroscopy and Scatchard analysis results indicate that the ligand interacts to both helix and coiled structures of the polynucleotide by “strong” and “weak” binding modes. The association parameters (binding constant—K—and the number of nucleotides corresponding to a binding site—n) of the strong type of interaction were found to be independent of Na+ concentration. Weak interaction occurs at low ionic strength and/or high EtBr concentration. Estimated binding parameters of EtBr with ss- and ds-polynucleotide are in good agreement with those for EtBr-B-DNA complexes. Data obtained provided an evidence for a stacking interaction of EtBr with single stranded poly[d(A-T)].     

Structure of Poly(dA)·Poly(dT) is not Identical to the AT Rich Regions of the Single Crystal Structure of CGCGAATTBrCGCG. The Consequence of this to Netropsin Binding to Poly(dA)·Poly(dT)

Abstract

The basic assumption of Dickerson and Kopka (J. Biomole. Str. Dyns. 2, 423, 1985) that the conformation of poly(dA)·poly(dT) in solution is identical to the AT rich region of the single crystal structure of the Dickerson dodecamer is not supported by any experimental data. In poly(dA)·poly(dT), NOE and Raman studies indicate that the dA and dT units are conformationally equivalent and display the (anti-S-type sugar)-conformation; incorporation of this nucleotide geometry into a double helix leads to a conventional regular B-helix in which the width of the minor groove is 8A. The derived structure is consistent with all available experimental data on poly(dA)·poly(dT) obtained under solution conditions. In the crystal structure of the dodecamer, the dA and dT units have distinctly different conformations—dA residues adopt (anti, S-type sugar pucker), while dT residues belong to (low anti, N-type sugar pucker). These different conformations of the dA and dT units along with the large propeller twist can be accommodated in a double helix in which the minor groove is shrunk from 8A to less than 4A. In the conventional right handed B-form of poly(dA)·poly(dT) with the 8A wide minor groove, netropsin has to bind asymmetrically along the dA strand to account for the NOE and chemical shift data and to generate a stereochemically sound structure (Sarma et al, J. Biomole. Str. Dyns. 2, 1085, 1985).     

The Z-Conformation of Poly(dA-dT) · Poly(dA-dT) in Solution as Studied by Ultraviolet Resonance Raman Spectroscopy

Abstract

Poly(dA-dT) poly(dA-dT) structures in aqueous solutions with high NaCl concentrations and in the presence of Ni²⁺ ions have been studied with resonance Raman spectroscopy (RRS). In low water activity the effects of added 95 mM NiCl₂ in solution stabilize the syn geometry of the purines and reorganize the water distribution via local interactions of Ni-water charged complexes with the adenine N7 position. It is shown that RRS provides good marker bands for a left-handed helix: i) a purine ring breathing mode around 630 cm″^?1coupled to the deoxyribose vibration in the syn geometry, ii) a 1300-1340 cm^?1 region characterizing local chemical interactions of the Ni²⁺ ions with the adenien N7 position, iii) lines at about 1483-and 1582 cm^?1 correlated to the anti/syn reorientation of the adenine residues on B-Z structure transition, iv) marker bands of the thymidine carbonyl group couplings at 1680-and 1733 cm^?1 due to the disposition of the thymidine residues in the Z helix specific geometry. Hence poly(dA-dT) poly(dA-dT) can adopt a Z form in solution. The Z form observed in alternate purine-pyrimidine sequences does not require G-C base pairs.     

Poly(3-mercaptopropionate): a nonbiodegradable biopolymer?

Polythioesters (PTEs) represent a novel class of biopolymers, which basically can be synthesized with polyhydroxyalkanoate (PHA) biosynthesis systems. Albeit technical applications of PTEs have not been elucidated yet, biodegradability might be an important property of this new thermoplastic material. In this study, extensive approaches were employed to isolate microorganisms capable of degrading poly(3-mercaptopropionate), poly(3MP), as a model compound of PTEs. Screening of 74 different environmental samples using various enrichment techniques were applied, but neither bacteria nor fungi could be isolated hydrolyzing poly(3MP). Furthermore, microcosms such as soil, compost, or activated sludge were applied to search for poly(3MP) degrading microorganisms, considering microbial communities and/or nonculturable bacteria, and the poly(3MP) material was exposed for more than half a year. However, no poly(3MP) degrading organisms were found, indicating an unexpected persistence of this biologically produced polymer.     

Eine Polyäthylen-Glas-Taucherwaage

Zusammenfassung Robuste Taucherwaagen von geringem Reduzierten Gewicht lassen sich leicht herstellen, wenn als Material für den Plastikkopf statt Polystyrol (Dichte 1,05) das spezifisch leichtere Polyäthylen (Dichte 0,92) verwendet wird.Sowohl das Reduzierte Gewicht als auch das Volumen begrenzen die Empfindlichkeit der Taucherwaage.

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Summary The use of polyethylene (density 0.92) instead of polystyrene (density 1.05) as cap material permits the easy construction of relatively strong diver balances of small Reduced Weight.Both Reduced Weight and volume limit the sensitivity of the diver balance.

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Mit 1 Textabbildung

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Mit Unterstützung der Deutschen Forschungsgemeinschaft und des Rask-Ørsted-Fonds.     

Conformational Variability of Poly(dA-dT)·Poly(dA-dT) and Some Other Deoxyribonucleic Acids Includes a Novel Type of Double Helix

Abstract

The article reviews data indicating that poly(dA-dT)?poly (dA-dT) is able of adopting three distinct double helical structures in solution, of which only the A form conforms to classical notions. The other two structures have dinucleotides as double helical repeats. At low salt concentrations poly(dA-dT)?poly(dA-dT) adopts a B-type alternating conformation which is exceptionally variable. Its architecture can gradually move in the limits demarcated by the CD spectra with inverted long wavelength CD bands and the ³¹P NMR spectra with a very low and a 0.6 ppm separation of two resonances. Contrary to Z-DNA, the ³¹P NMR spectrum of the limiting alternating B conformation of poly(dA-dT)?poly(dA-dT) is characterized by an upfield shift of one resonance. We attribute the exceptional conformational flexibility of the alternating B conformation to the unequal tendency of bases in the dA-dT and dT-dA steps to stack.

However, by assuming the limiting alternating B conformation, the variability of the synthetic DNA is not exhausted. Specific agents make it isomerize into another conformation by a fast, two-state mechanism, which is reflected by a further deepening of the negative long wavelength CD band and a downfield shift of the ³¹P NMR resonance of poly (dA-dT)?poly(dA-dT) that was constant in the course of the gradual alterations of the alternating B conformation. These changes are, however, qualitatively different from the way poly(dG-dC)?poly(dG-dC) behaves in the course of the B-Z isomerization. Poly(dG-dC) ?poly(dG-dC) displays purine-pyrimidine (dGpdC) resonance in the characteristic downfield position, while the downfield resonance of poly(dA-dT)?poly(dA-dT) belongs to the pyrimidine-purine (dTpdA) phosphodiester linkages. Consequently, phosphodiester linkages in the purine-pyrimidine steps play a similar role in the appearance of the Z form to the pyrimidine-purine phosphodiesters in the course of the isomerization of poly(dA-dT)?poly(dA-dT). This excludes that the high-salt structures of poly(dA-dT)?poly(dA-dT) and poly(dG-dC)?poly(dG-dC) are members of the same conformational family. We call the high-salt conformation of poly(dA-dT)?poly(dA-dT) X-DNA.

It furthermore follows from the review that synthetic molecules of DNA with alternating purine-pyrimidine sequences of bases can adopt either the Z form or the X form, or even both, depending on the environmental conditions. This introduces a new dimension into the DNA double helix conformational variability. The possible biological relevance of the X form is suggested by experiments with linear molecules of natural DNA. These indicate that Arich regions in natural DNAs can isomerize into the X form while the bulk of the molecule remains in the B form. The coexistence of both structures in a single DNA molecule may be understood in view of the favourable kinetic and thermodynamic properties with which the X form appears.     

Poly(rA) • Poly(rU) with Ni2+ Ions at Different Temperatures: Infrared Absorption and Vibrational Circular Dichroism Spectroscopy

Abstract

Phase transitions were studied of the sodium salt of poly(rA) ?poly(rU) induced by elevated temperature without Ni²⁺ and with Ni²⁺ in 0.07 M concentration in D₂O (～0.4 [Ni]/[P]). The temperature was varied from 20° C to 90° C. The double-stranded conformation of poly(rA)?poly(rU) was observed at room temperature (20° C—23° C) with and without Ni²⁺ ions. In the absence of Ni²⁺ ions, partial double- to triple-strand transition of poly(rA) ?poly(rU) occurred at 58° C, whereas only single-stranded molecules existed at 70° C. While poly(rU) did not display significant helical structure, poly(rA) still maintained some helicity at this temperature. Ni²⁺ ions significantly stabilized the triple-helical structure. The temperature range of the stable triple-helix was between 45° C and 70° C with maximum stability around 53° C. Triple-to single-stranded transition of poly(rA) ?poly(rU) occurred around 72° C with loss of base stacking in single-stranded molecules. Stacked or aggregated structures of poly(rA) formed around 86° C. Hysteresis took place in the presence of Ni²⁺ during the reverse transition from the triple-stranded to the double-stranded form upon cooling. Reverse Hoogsteen type of hydrogen-bonding of the third strand in the triplex was suggested to be the most probable model for the triple-helical structure. VCD spectroscopy demonstrated significant advantages over infrared absorption or the related electronic CD spectroscopy.     

The Complex of Poly(dG) · Poly(dC) with Arginine: Stabilization of the B Form and Transition to Multistranded Structures

Abstract

We have studied by X-ray diffraction fibers of complexes of poly(dG)·poly(dC) with N-α-acetyl-L-arginine ethylamide. Although these polynucleotides favour the A form of DNA, in this complex it is never found, thus confirming that arginine prevents the appearance of this form of DNA At high relative humidity the B form is present. Upon dehydration two new structures appear. One of them is a triple helix, most likely formed by poly(dC⁺) · poly(dG) · poly(dC). The other structure found also has features which indicate a multistranded conformation.     

Monte-Carlo Simulation of DNA Duplex Hydration. B and B′ Conformations of Poly(dA) · Poly(dT) Have Different Hydration Shells

Abstract

Monte-Carlo simulation of poly(dA) · poly(dT) hydration by 30 water molecules per nucleotide pair has been performed. Two B-family conformations, both with a 36° helical twist but with different minor groove widths, were considered. One conformation is Arnott's standard B form, the other one is specific for poly(dA) · poly(dT) B′ form with a narrowed minor groove. The mean energies and the mean numbers of water-water and water-DNA hydrogen bonds are close for the two conformations. Nevertheless, the hydration shell of the B' form differs drastically from that of the standard B form. The water arrangement in the minor groove of the B′ form resembles the spine of hydration in the central part of Dickerson's dodecamer d(CGCGAATTCGCG). No such spine is formed in the hydration shell of the usual B form with a wider minor groove. In this conformation water bridges between adenine N3 or thymine O2 and oxygen of the sugar ring of the neighbouring nucleotide along the chain can be formed (“strings” in Dickerson's decamer d(CCAAGATTGG)).     

Two Binding Modes of Netropsin are Involved in the Complex Formation with Poly(dA-dT)·Poly(dA-dT) and other Alternating DNA Duplex Polymers

Abstract

Using CD measurements we show that the interaction of netropsin to poly(dA-dT)·poly(dA-dT) involves two binding modes at low ionic strength. The first and second binding modes are distinguished by a defined shift of the CD maximum and the presence of characteristic isodichroic points in the long wavelength range from 313 nm to 325 nm. The first binding mode is independent of ionic strength and is primarily determined by specific interaction to dA·dT base pairs. Employing a netropsin derivative and different salt conditions it is demonstrated that ionic contacts are essential for the second binding mode. Other alternating duplexes and natural DNA also exhibit more or less a second step in the interaction with netropsin observable at high ratio of ligand per nucleotide. The second binding mode is absent for poly(dA)·poly(dT). The presence of a two-step binding mechanism is also demonstrated in the complex formation of poly(dA-dT)·poly(dA-dT) with the distamycin analog consisting of pentamethylpyrrolecarboxamide. While the binding mode I of netropsin is identical with its localization in the minor groove, for binding mode II we consider two alternative interpretations.     

Medium-Chain-Length Poly(β-Hydroxyalkanoate) Synthesis from Triacylglycerols by Pseudomonas saccharophila

Pseudomonas saccharophila NRRL B-628 is capable of utilizing agricultural lipids for growth. The organism exhibited good growth with triacylglycerol substrates that contained saturated fatty acyl moieties such as coconut oil (CO; C_10–12 fatty acids) and tallow (T; C_16–18 fatty acids). Electron micrographs of the triacylglycerol-grown cells showed the presence of intracellular granules indicative of poly(β-hydroxyalkanoate) (PHA) production. Cells grown in a 250-ml CO-containing medium produced ca. 0.2 g of medium-chain-length (mcl)-PHA. Gas chromatographic analysis showed that β-hydroxyoctanoic acid (30%), β-hydroxydecanoic acid (40%), and β-hydroxydodecanoic acid (16%) were the major monomer repeat-units of the CO-derived polymer. The estimated mean molecular mass of the CO-derived mcl-PHA as determined by gel permeation chromatography was 13.1 × 10⁴ g/mol with a polydispersity of 3.16. Received: 15 August 1998 / Accepted: 25 September 1998     

Bh-DNA: Variations of the Poly[d(A)] · Poly[d(T)] Structure within the Framework of the Fibre Diffraction Studies

Abstract

A refinement of the recent results for poly[d(A)] · poly[d(T)] (Alexeev et al., J. Biomol. Struct. Dyn. 4, 989 (1987)) involving additional parameters of the base-pair structure and of the sugar- phosphate backbone expands the conformational potential of this polynucleotide of the B type to include the possibility of bifurcated hydrogen bonds of the kind recently discovered in crystalline deoxyoligonucleotide with lone d(A)_n · d(T)_n stretch (Nelson et al., Nature 330, 221 (1987)).

Still, analysis of the available data and energy calculations do not seem to indicate that the bifurcated H-bonds are a crucial factor responsible for the anomalous structure of the d(A)_n · d(T)_n sequence. The unique structural properties of poly [d(A)] · poly[d(T)] can hardly be explained without taking into account its interactions with the double-layer hydration spine in the minor groove. In view of the hydration mechanism stabilizing poly [d(A)] · poly [d(T)] and of the polynucleotide's heteronomous prehistory (Arnott et al., Nucleic Acids Res. 11, 4141 (1983)) we suggest that this B-type structure be called B_h.     

Poly(β-hydroxybutyrate) production in oilseed leukoplasts of Brassica napus

Polyhydroxyalkanoates (PHAs) comprise a class of biodegradable polymers which offer an environmentally sustainable alternative to petroleum-based plastics. Production of PHAs in plants is attractive since current fermentation technology is prohibitively expensive. The PHA homopolymer poly(β-hydroxybutyrate) (PHB) has previously been produced in leaves of Arabidopsis thaliana (Nawrath et al., 1994, Proc Natl Acad Sci USA 91: 12760–12764). However, Brassica napus oilseed may provide a better system for PHB production because acetyl-CoA, the substrate required in the first step of PHB biosynthesis, is prevalent during fatty acid biosynthesis. Three enzymatic activities are needed to synthesize PHB: a β-ketothiolase, an acetoacetyl-CoA reductase and a PHB synthase. Genes from the bacterium Ralstonia eutropha encoding these enzymes were independently engineered behind the seed-specific Lesquerella fendleri oleate 12-hydroxylase promoter in a modular fashion. The gene cassettes were sequentially transferred into a single, multi-gene vector which was used to transform B. napus. Poly(β-hydroxybutyrate) accumulated in leukoplasts to levels as high as 7.7% fresh seed weight of mature seeds. Electron-microscopy analyses indicated that leukoplasts from these plants were distorted, yet intact, and appeared to expand in response to polymer accumulation. Received: 26 May 1999 / Accepted: 16 June 1999     

Poly(dA-dT)•Poly(dA-dT) in Low Salt Appears to be a Left-Handed B-Helix Combined Use of Chemical Theory,Fiber Diffraction and NMR Spectroscopy

Abstract

Poly(dA-dT)?poly(dA-dT) can adopt the B- and D- forms in the fibrous state. Theoretical energy calculations and fiber diffraction analyses suggest that there can be three structural models of poly(dA-dT)?poly(dA-dT) in each of these two forms viz right and left-handed Watson Crick models and left-handed Hoogsteen—a total of six possible models. Fiber data for the polymer in the B- or the D-form or energy calculations cannot distinguish any one model from the other. However, a comparison of observed proton chemical shifts with the theoretically computed ones and the NOE studies on exchangeable and nonexchangeable protons suggest that poly(dA-dT)?poly(dA-dT) in low salt solution exists predominantly in the left-handed B-conformation.     

Solution Structure of Poly(dA-dT)· Poly(dA-dT) in Low and High Salt: A 500 MHz 1H NMR Study Using One-Dimensional NOE

Abstract

CD spectra of poly(dA-dT)· poly(dA-dT) in low salt (10–100 mM NaCl) and high salt (4–6 M CsF) are different i.e. 275 nm band gets inverted in going from low to high salt (Vorhickova et. al.MarJ. Mol. Biol. 166, 85, 1983). However, from CD spectra alone it is not possible to decipher any structural differences that might exist between the low and high salt forms of poly(dA-dT)? poly(dA-dT). Hence, we took recourse to high resolution NMR spectroscopy to understand the structural properties of poly(dA-dT)? poly(dA-dT) in low and high salt. A detailed analysis of shielding constants and extensive use of NOE studies under minimum spin diffusion conditions using C(8)-deuterated poly(dA-dT)? poly(dA-dT) enabled us to come up with the following conclusions (i) base-pairing is Watson-Crick under low and high salt conditions, (ii) under both the conditions of salt the experimental data can be explained in terms of an equilibrium blend of right and left-handed B-DNA duplexes with the left-handed form 70% and the right-handed 30%. In a 400 base pairs long poly(dA-dT)? polyidA-dT) (as used in this study), equilibrium between right and left-handed helices can also mean the existence of both helical domains in the same molecule with fast interchange between these domains or/and unhindered motion/propagation of these domains along the helix axis, (iii) However, there are other structural differences between the low and high salt forms of poly(dA-dT) ? poly(dA-dT); under the low salt condition, right-and left-handed B-DNA duplexes have mononucleotide as a structural repeat while under the high salt conditions, right-and left-handed B-DNA duplexes have dinucleotide as a structural repeat. In the text we provide the listing of torsion angles for the low and high salt structural forms, (iv) Salt (CsF) induced structural transition in poly(dA-dT)? poly(dA-dT) occurs without any breakage of Watson- Crick pairing, (v) The high salt form of poly(dA-dT)? poly(dA-dT) is not the left-handed Z-helix.

Although the results above from NMR data are quite unambiguous, a question still remains i.e. what does the salt (CsF) induced change in the CD spectra of poly(dA-dT)? poly(dA-dT) really indicate? Interestingly, we could show that the salt (CsF) induced change in poly(dA-dT)? poly(dA-dT) is quite similar to that caused by a basic polypeptide viz. poly-L(Lys₂-Ala)_n i.e. both the agents induced a ψ-structure in DNA. And it was also demonstrated that the changes in poly(dA-dT)? poly(dA-dT) as caused by CsF and poly-L-(Lys₂-Ala)_n could be reverted back by ethidium bromide-a relaxing agent.

To minimize complications from spin diffusion in this study we have used very small presaturation pulse lengths and C(8)-deuterated poly(dA-dT)? poly(dA-dT) of 400 ± 150 bp long. Even though deuteration of a primary site of diffusion such as C(8)H substantially decreases diffusion, in order to make sure that our conclusions are not compromised by possible diffusion in such a long fragment under small presaturation times, we have repeated our experiments using the six base pair long duplex of d(A-T-A-T-A-T) and found the results to be strikingly similar to that from the polymer.     

Dose Responsiveness and Variation Among Inbred Strains of Mice in Production of Interferon After Treatment with Poly (I)· Poly (C) [Poly-d-Lysine] Complexes

Poly-d-lysine forms a stochiometric complex with poly(I) . poly(C) which has a higher T(m) (83 C in 0.15 m NaCl) than the uncomplexed double-stranded polyribo-nucleotide (63 C). The complex was superior to poly(I) . poly(C) alone as an interferon inducer in vivo. Significant serum interferon titers were attained in Swiss mice during a 24-hr period after intraperitoneal injection of 10 to 300 mug of poly(I) . poly(C) [1.0 poly-d-lysine] complex, at concentrations of 100 to 1,000 mug/ml. The serum interferon responses (average and maximum titers) of a series of inbred strains of mice to a single intraperitoneal injection of 100 mug of complex decreased in the order: Swiss > DBA/2 > C3H > BALB/c > CF-1 > AKR, C57Bl/6, NZB > SJL > NZW and varied by a factor of approximately 100 from the most to the least responsive.