Interaction of nucleic acids with electrically charged surfaces: II. Conformational changes in double-helical polynucleotides

The influence of adsorption of double-stranded (ds) DNA, ds RNA and homopolymeric pairs at a mercury electrode on conformation of these polynucleotides was studied. Changes in the polarographic reducibility of polynucleotides, which were followed by means of normal pulse polarography and linear sweep peak voltammetry at the dropping mercury electrode were exploited to indicate conformational changes. It was found that, as a consequence of adsorption of ds polynucleotides on the negatively charged electrode conformational changes similar to denaturation take place in a narrow potential region around ?1.2 V (the region U). After sufficiently long time of the contact with the electrode (under our conditions about 10 s) these changes reach limiting values, which can approach total denaturation. Upon adsorption of ds polynucleotides on the electrode charged to more positive potentials than the region U either (1) no conformational changes occur or (2) only a small part of the polynucleotide (probably labile regions of the ds molecule) is very quickly denatured - the remainder of the molecule preserves its ds structure. Conformational changes of adsorbed ds polynucleotides are influenced by factors which change the stability of ds polynucleotides in solution. It is supposed that denaturation of ds polynucleotides in the region U might result from the strains connected with the repulsion of certain segments of the molecule anchored on the electrode from the negatively charged surface.     

Interaction of nucleic acids. VI. Interaction of purine with nucleic acids

The interaction of purine with DNA, tRNA, poly A, poly C, and poly A. poly U complex was investigated. In the presence of purine, the nucleic acids in coil form (such as denatured DNA, poly A and poly C in neutral solutions, or tRNA) have lower optical rotations. In addition, hydrodynamic studies indicate that in purine solutions the denatured DNA has a higher viscosity and a decreased sedimentation coefficient. These findings indicate that through interaction with purine, the bases along the poly-nucleotide chain are unstacked and are separated farther from each other, resulting in increased assymmetry (and possibly volume) of the whole polymer. Thus, the de-naturation effect of purine reported previously can be explained by this preferential interaction of purine with the bases of nucleic acids in coil form through a hydrophobic-costacking mechanism. Results from studies on optical rotation and helix-coil transition show that the interaction of purine is greater with poly A than with poly C. The influence of temperature, Mg⁺⁺ concentration, ionic strength, and purine concentration on the effect of purine on nucleic acid conformation has also been investigated. In all these situations the unraveling of nucleic acid conformation occurs at much lower temperatures (20–40°C lower) in the presence of purine (0.2–0.6M).     

Interaction between hydroxystilbamidine and DNA. I. Binding isotherms and thermodynamics of the association.

Isotherms describing the binding of hydroxystilbamidine to DNA and polydeoxyribonucleotides were obtained by means of sedimentation or dialysis experiments and fluorescence measurements, over a large range of ionic strengths, temperatures and base compositions. Two different sets of binding sites are necessary to explain the shapes of the isotherms. The first one is characterized by a higher binding constant, a topological specificity for the A-T pair, exclusion of four base pairs per bound dye molecule, the involvement of two ion-pairs, an almost purely entropic free energy of binding and a large enhancement of the blue fluorescence (450 nm) when the site corresponds to three adjacent A-T pairs. The latter does not present any specificity nor enhancement of fluorescence and only one ion-pair is formed. From the geometry of the dye and its selective binding to a double stranded structure, the hydroxystilbamidine molecule in the first set of sites is likely to be situated in the small groove astride the two complementary strands and slightly distorting the helical structure. The angle of the dye axis with the helix axis has a value close to 47 degrees. No definite explanation could be given for the specific binding of hydroxystilbamidine but the phenolic hydroxyl group is likely to play a major role. The hydroxystilbamidine molecule can be considered as a useful tool for checking the accessibility of the small groove.     

Interaction of antisense DNA with nucleic acids/proteins.

In order to study interaction of various types of labeled antisense DNAs were prepared. Fluorescein and 2,2,6,6-tetramethypiperidine-N-oxyl were the label molecules, which were introduced to 5'-end of oligonucleotides and their analogs. Interactions of labeled antisense DNAs with nucleic acids or proteins such as HSA, HIG and TF, were studied by UV, fluorescence depolarization spectroscopy, and ESR spectroscopy. Hybrid formation of antisense DNAs with oligonucleotides in solution could be monitored by the increase in fluorescence anisotropy (r) and by intensity change in ESR spectra. When phosphorothioate type antisense molecules anchoring fluorescein (F-OPT) were mixed with proteins, r drastically increased, whereas ODN slightly increased. These results suggest that OPTs have much more affinity for proteins than ODNs.     

Theoretical calculations of base-base interactions in nucleic acids: II. Stacking interactions in polynucleotides.

Base-base interactions were computed for single- and double stranded poly,ucleotides, for all possible base sequences. In each case, both right and left stacking arrangements are energetically possible. The preference of one over the other depends upon the base-sequence and the orientation of the bases with respect to helix-axis. Inverted stacking arrangement is also energetically possible for both single- and double-stranded polynucleotides. Finally, interacting energies of a regular duplex and the alternative structures were compared. It was found that the type II model is energetically more favourable than the rest.     

A single-stranded nucleic acid-binding protein from Artemia salina. II. Interaction with nucleic acids

A helix-destabilizing protein, HD40 (Mr 40,000), isolated from the cytoplasm of Artemia salina (Marvil, D.K., Nowak, L., and Szer, W. (1980) J. Biol. Chem. 255, 6466-6472) stoichiometrically disrupts the secondary structures of synthetic single-stranded and helical polynucleotides (e.g. poly(rA), poly(dA), poly(rC), poly(dC), and poly(rU)) as well as those of natural polynucleotides (e.g. MS2 RNA and phi X174 viral DNA). The conformations of double-stranded DNA and double- or triple-stranded synthetic polynucleotides are not affected by the protein. Formation of duplexes, e.g. poly(rA . rU), is prevented by HD40 at 25 to 50 mM but not at 100 to 140 mM NaCl. The unwinding of the residual secondary structure of RNA and DNA by HD40 is not highly cooperative and has a stoichiometry of one HD40 per 12 to 15 nucleotides. The addition of HD40 in excess of 1 molecule per 12 to 15 nucleotides results in the cooperative formation of distinct bead-like structures along the nucleic acid strand. The beads are about 20 nm in diameter with a center to center distance of about 40 nm. The appearance of the beads is not accompanied by any spectral changes (CD and UV) beyond those obtained at a stoichiometry of one HD40 molecule per 12 to 15 nucleotides.     

Interaction between polyamines and nucleic acids or phospholipids

The binding of polyamines to DNA, RNA, and phospholipids has been studied by gel filtration and sucrose density gradient centrifugation. Spermine was found to bind more to a GC-rich DNA. Among RNAs containing double-stranded region [poly(AU), poly(GC), and ribosomal RNA], the binding of spermine was nearly equal. Among the single-stranded RNAs, the binding of spermine was in the order poly(U) > poly(C) > poly(A). An increase in K⁺ or Mg²⁺ concentration resulted in a great decrease in spermine binding to DNA and in a slight decrease in spermine binding to RNA. Therefore, in the presence of more than 2 mm Mg²⁺ and 100 mm K⁺, the binding of spermine to RNA was greater than that to DNA. No significant difference in spermine binding was observed between 16 S ribosomal RNA and 30 S ribosomal subunits, suggesting that ribosomal proteins did not affect significantly the binding of spermine to ribosomal RNA. The binding of spermine to microsomes was dependent on phospholipids. The binding strength was in the order phosphatidylinositol > phosphatidylethanolamine > phosphatidylcholine.     

Interaction of phenosafranine with nucleic acids and model polyphosphates: II. Characterisation of phenosafranine binding to DNA

The binding of phenosafranine (PS) to DNA was studied by a combination of spectroscopic methods (absorption and fluorescence) together with hydrodynamic measurements (sedimentation and viscosity), Analysis of spectroscopic binding curves revealed that the strength of binding of PS to DNA is generally lower than that of proflavine. These measurements enabled recognition of several modes of interaction between PS and native DNA: strong monomer binding prevailing at high DNA phosphate/dye ratios (p) comprising binding outside the DNA helix as well as intercalation; two modes of dimer binding at lower values of p; and probably also weak surface-binding of monomers as p approaches unity. Longer surface-bound aggregates of PS were not detected because of the low tendency of the dye to form aggregates, though the presence of dimeric species distinct frorn pure surface-stacked PS dimer was indicated by various observations. It occurs over a broad range of p values Starting at p ≈110 for ionic strengths 10^?3–10^?1. Thermal denaturation data indicate that this species is bound more strongly than pure surface-bound stacked dimer. Its dimeric character may be explained in terms of interaction of an intercalated dye molecule with an adjacent outside-bound one as suggested for acridines by Armstrong et al. Various properties of this species are discussed. Both strong and weak modes or binding of PS to DNA are sensitive to the presence of organic solvents. The effectiveness of solvents to destabilise the complexes substantially coincides with their capacity to alter the water activity. Viscometric investigations reveal that in the region of strongest bindins (p ? 15) the elongation of the DNA helix by approximately 0.18 nm per bound PS molecule is accompanied by a strong negative change in persistence length, i.e. bending. Similar bending is also found at higher levels of binding (p ? 15) induced by less lightly bound PS molecules, in which region, however, the unusually high elongation of approximately 0.34 nm per bound PS molecule is observed.     

Interaction of aminoacridines with nucleic acids. A pulse-radiolysis study.

Pulse radiolysis has been used to study the interaction of aminoacridines with nucleic acids. The data confirm that there are two modes of binding. These are: a weak interaction which has a maximum binding ratio of one site per dye; and a strong binding process effected by both electrostatic and Van der Waals interactions. The limit of this latter, strong binding mode is approximately six sites per dye. The radiation-induced transient absorption spectrum of benzoflavine is characterized by a pronounced bleaching at 440 nm, which is quenched by the addition of nucleic acids. Mechanisms have been proposed for the reactions of both eaq-and .OH with benzoflavine which account both for the observed bleaching of benzoflavine solutions and for the protective effect of nucleic acids. It is proposed that eaq-reacts with benzoflavine to form a stable benzoflavine semiquinone radical and that .OH reacts with subsequent formation of a very stable benzoflavine hydroxycyclohexadienyl radical.     

Raman studies of nucleic acids. XII. Conformations of oligonucleotides and deuterated polynucleotides

Laser Raman spectra of the trinucleoside diphoshate ApApA and dinucleoside phosphates ApU, UpA, GpC, CpG, and GpU are reported and discussed. Assignments of conformationally sensitive frequencies are-facilitated by comparison with spectra reported here of poly(rA), poly(rC), and poly(rU) in deuterium oxide solutions. The significant spectral differences between ApU and UpA, and between GpC and CpG, reveal that the sequence isomers have nonidentical conformations in aqueous solution. In UpA at low temperature the bases are stacked and the backbone conformation is similar to that found in ordered polynucleotide structures and RNA. In ApU no base stacking can be detected and the backbone conformation differs from that found in UpA, both in the orientation of phosphodiester linkages and in the internal conformation of ribose. At the conditions employed neither ApU nor UpA exhibits base pairing in aqueous solutions. In both GpC and CpG the bases are stacked and the phosphodiester conformations are similar to those encountered for UpA and RNA. However, major differences between spectra of GpC and CpG indicate that the geometries of stacking and ribosyl conformations are different. In GpC the Raman data favor the formation of hydrogen bonded dimers containing GC pairs. Protonation of C in GpC is sufficient to eliminate the ordered conformation detected by Raman spectroscopy. Despite the ordered backbone conformation evident in GpU, this dinucleoside apparently contains neither stacked nor hydrogen bonded bases at the conditions employed here. The Raman data also confirm the stacking interactions in ApApA, poly(rA), and poly(rC) but suggest that the backbone conformation in poly(rC) differs qualitatively from that found in most ordered polynucleotide structures and is thermally more stable. The present results demonstrate the sensitivity of the Raman technique to sequence-related structural differences in oligonucleotides and provide additional spectra–structure correlations for future conformational studies of RNA by laser Raman spectroscopy.     

Stereochemistry of nucleic acids and their constituents. X. Solid-state base-stacking patterns in nucleic acid constituents and polynucleotides

The base-stacking patterns in over 70 published crystal structures of nucleic acid constituents and polynucleotides were examined. Several recurring stacking patterns were found. Base stacking in the solid state apparently is very specific, with particular modes of interaction persisting in various crystalline environments. The vertical stacking of purities and pyrimidines in polynucleotides is similar to that observed in crystals of nucleic acid constituents. Only partial base overlap was found in the majority of the structures examined. Usually, the base overlap is accomplished by positioning polar substituents over the ring system of an adjacent base. The stacking interactions are similar to those found in the crystal structures of other polar aromatic compounds, but are considerably different from the ring–ring interactions in nonpolar aromatic compounds. Apparently, dipole-induced dipole forces are largely responsible for solid-state base stacking. It is found that halogen substituents affect base-stacking patterns. In general, the presence of a halogen substituent results in a stacking pattern which permits intimate contact between the halogen atom and adjacent purine or pyrimidine rings. Considering differences in the stacking patterns found for halogenated and nonhalogenated pyrimidines, a model is proposed to account for the mutagenic effects of halogenated pyrimidines.     

Stereochemistry of nucleic acids and polynucleotides. V. Conformational energy of a ribose-phosphale unit

The potential energy calculations on the sugar-phosphate unit for different puckerings of the sugar are reported in this paper. The results obtained here essentially confirm our earlier predictions made by using criteria of contact distances (hard-sphere potential) and are also supported by observed conformation in crystal structures. The minimum energy conformations of the sugar phosphate unit, along with the preferred orientations of the base with respect to the sugar given in the previous paper, determine the probable conformations of the monomer unit of a polynucleotide (or nucleic acid) chain.