Conformational and structural constraints in double-helical polynucleotides

Conformational analysis of antiparallel double-helical polynucleotides with Watson-Crick base pairing was reduced to a four-dimensional problem using original mathematical methods. In the four-dimensional conformational space the family of structures, characterized by the base-pair stacking with the most stable conformations in water solution as well as in the solid state, was localized. For the C′₂-endo sugar pucker, both right-handed and left-handed structures were found; right-handed structures only, however, seem to be allowed for the C′₃-endo pucker, the only possible one for ribonucleotides with base stacking.     

Interaction of nucleic acids with electrically charged surfaces. VI. A comparative study on the electrochemical behaviour of native and denatured DNAs at graphite electrodes.

Adsorption and electrochemical oxidation of deoxyribonucleic acid (DNA) at a pyrolytic graphite electrode (PGE) and a paraffin wax-impregnated spectroscopic graphite electrode (WISGE) were studied using differential pulse voltammetry. DNA is adsorbed at the surface of the graphite electrodes in a broad range of potentials including the potentials of electrochemical oxidation of DNA. Both native and denatured DNAs yield two single, well-defined and separated peaks, G and A, on the differential pulse voltammograms at the PGE and WISGE. The more negative peak, G, corresponds to electrochemical oxidation of adenine residues. Peaks G and A of native DNA occur at the same potentials as peaks G and A of denatured DNA. However, electrochemical oxidation of adenine and guanine residues at graphite electrodes is markedly suppressed in native DNA. The heights of the peaks G and A represent a sensitive indicator of the helix-coil transition of DNA. An analysis of the product of interaction of a sample of native DNA with a large pyrolytic graphite electrode in the presence of formaldehyde at approximately neutral pH did not prove changes in the secondary structure of native DNA due to its interaction with the graphite electrode. It is suggested that the decreased differential pulse-voltammetric activity of native DNA is connected with its decreased flexibility.     

Interaction of nucleic acids with electrically charged surfaces. VI. A comparative study on the electrochemical behaviour of native and denatured DNAs at graphite electrodes

Adsorption and electrochemical oxidation of deoxyribonucleic acid (DNA) at a pyrolytic graphite electrode (PGE) and a paraffin wax-impregnated spectroscopic graphite electrode (WISGE) were studied using differential pulse voltammetry. DNA is adsorbed at the surface of the graphite electrodes in a broad range of potentials including the potentials of electrochemical oxidation of DNA. Both native and denatured DNAs yield two single, well-defined and separated peaks, G and A, on the differential pulse voltammograms at the PGE and WISGE. The more negative peak, G, corresponds to electrochemical oxidation of guanine residues, whereas the more positive peak, A, corresponds to electrochemical oxidation of adenine residues. Peaks G and A of native DNA occur at the same potentials as peaks G and A of denatured DNA. However, electrochemical oxidation of adenine and guanine residues at graphite electrodes is markedly suppressed in native DNA. The heights of the peaks G and A represent a sensitive indicator of the helix-coil transition of DNA. An analysis of the product of interaction of a sample of native DNA with a large pyrolytic graphite electrode in the presence of formaldehyde at approximately neutral pH did not prove changes in the secondary structure of native DNA due to its interaction with the graphite electrode. It is suggested that the decreased differential pulse-voltammetric activity of native DNA is connected with its decreased flexibility.     

Adsorption of peptide nucleic acid and DNA decamers at electrically charged surfaces.

Adsorption behavior of peptide nucleic acid (PNA) and DNA decamers (GTAGATCACT and the complementary sequence) on a mercury surface was studied by means of AC impedance measurements at a hanging mercury drop electrode. The nucleic acid was first attached to the electrode by adsorption from a 5-microliter drop of PNA (or DNA) solution, and the electrode with the adsorbed nucleic acid layer was then washed and immersed in the blank background electrolyte where the differential capacity C of the electrode double layer was measured as a function of the applied potential E. It was found that the adsorption behavior of the PNA with an electrically neutral backbone differs greatly from that of the DNA (with a negatively charged backbone), whereas the DNA-PNA hybrid shows intermediate behavior. At higher surface coverage PNA molecules associate at the surface, and the minimum value of C is shifted to negative potentials because of intermolecular interactions of PNA at the surface. Prolonged exposure of PNA to highly negative potentials does not result in PNA desorption, whereas almost all of the DNA is removed from the surface at these potentials. Adsorption of PNA decreases with increasing NaCl concentration in the range from 0 to 50 mM NaCl, in contrast to DNA, the adsorption of which increases under the same conditions.     

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.     

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 of nucleic acids with electrically charged surfaces. VII. The effect of ionic strength of neutral medium on the conformation of dna adsorbed on the mercury electrode

Triangular-wave direct current (d.c.) voltammetry at a hanging mercury drop electrode and phase-selective alternating current (a.c.) polarography at a dropping mercury electrode were used for the investigation of adsorption of double-helical (ds) DNA at mercury electrode surfaces from neutral solutions of 0.05-0.4 M HCOONH4. It was found for the potential region T (from -0.1 V up to ca. -1.0 V) that the height of voltammetric peaks of ds DNA is markedly influenced by the initial potential only at relatively low ionic strength (mu) (from 0.05 up to ca. 0.3). Also a decrease of differential capacity (measured by means of a.c. polarography) in the region T depended markedly on the electrode potential only at relatively low ionic strength. The following conclusions were made concerning the interaction of ds DNA with a mercury electrode charged to potentials of the region T in neutral medium of relatively low ionic strength mu < 0.3). (i) When ds DNA is adsorbed, a significantly higher number of DNA segments is anchored in the positively charged electrode surface than in the surface bearing a negative charge, (ii) In the region T, especially adsorbed labile regions of ds DNA are opened in the electrode surface, which are present in ds DNA already in the bulk of the solution, (iii) In the narrow region of potentials in the Vicinity of the zero charge potential a higher number of ds DNA segments can be opened, probably as a consequence of the strain which could act on the ds DNA molecule in the course of the segmental adsorption/desorption process.     

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.     

Interaction between hydroxystilbamidine and DNA. II. Temperature jump relaxation study. Dynamics of nucleic acids and polynucleotides.

A temperature-jump relaxation study of the interaction of hydroxystilbamidine with DNA and synthetic polynucleotides has been performed. Two concentration dependent relaxation times tau1 and tau2 have been observed in the submillisecond range when detecting relaxation effects by means of light absorption. The longer of these two times (tau1) is also observed when using "blue" or "red" fluorescence detection. In the longer time scale the "red" fluorescence shows no other relaxation but the blue fluorescence shows two additional relaxation processes (tau3 and tau4) which correspond to an increase of fluorescence with temperature and which are independent of concentration. The experimental results clearly indicate that tau1 and tau2 are associated with the binding of the dye to strong and weak binding sites, respectively. A kinetic model is given to explain the results. It allows the determination of the four rate constants for the two binding reactions and yields equilibrium association constants in good agreement with those obtained from stoichiometric studies. The study of the effect of temperature, nature of the polymer, ionic strength and fraction of bound dye on tau3 and tau4 indicates that the dye acts only as a "blue" fluorescence probe of some processes involving the DNA or polynucleotide alone. These processes appear to be related with the dynamic structure of the polymers.     

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 of electrically charged lipid monolayers with malate dehydrogenase.

Malate dehydrogenase was adsorbed onto monomolecular lipid films, using a multicompartment trough. The quantity of adsorbed protein and its enzymatic activity were studied with monolayers of various electrical charge densities and subphases of various electrolyte compositions. A closely packed layer of enzyme molecules was adsorbed onto negatively charged films, whereas considerably less protein was adsorbed onto neutral and positively charged monolayers. Electrolytes reduce the quantity of adsorbed protein. The adsorption was found to be irreversible even at high ionic strength. When adsorbed to uncharged lipid films the enzyme is nearly inactive, whereas negatively charged lipid headgroups enhance the specific activity of the enzyme.     

Conformational classification of functional nucleic acids.

The kink parameters would provide the tolerant aspect for irregular helical structure of nucleic acid. Using these kink parameters, the classification of conformation space was carried for the functional nucleic acid molecules. The kink parameters could afford us the simple structural aspects about the constructive parts of functional molecules. Local elastic kink phenomena can be classified by rod like models with the combination of kink parameters. The constructive parts, such as the stable tetra nucleotides loop, U-turn conformation and adenosine platform, were selected and the statistical analyses were carried on the parameters calculated by program BIOCON.     

Cross-linking of double-helical nucleic acids with a photoreactive analogue of ethidium

Intercalation of the ethidium analogue 3,5-diazido-5-ethyl-6-phenylphenanthridinium into double helices followed by irradiation with blue or ultraviolet light results in cross-linking between the two strands with an efficiency around 30% for DNA, RNA, and DNA-RNA hybrids. Details of this reaction and a convenient synthesis of the ethidium analogue are described. Stable tertiary structure in RNA impedes intercalation and thus reduces the efficiency of cross-linking. In contrast to the ethidium derivative, various acridine diazides show little or no cross-linking ability.     

Interaction of electrically charged lipid monolayers with malate dehydrogenase

Malate dehydrogenase was adsorbed onto monomolecular lipid films, using a multicompartment trough. The quantity of adsorbed protein and its enzymatic activity were studied with monolayers of various electrical charge densities and subphases of various electrolyte compositions. A closely packed layer of enzyme molecules was adsorbed onto negatively charged films, whereas considerably less protein was adsorbed onto neutral and positively charged monolayers. Electrolytes reduce the quantity of adsorbed protein. The adsorption was found to be irreversible even at high ionic strength. When adsorbed to uncharged lipid films the enzyme is nearly inactive, whereas negatively charged lipid headgroups enhance the specific activity of the enzyme.     

Interaction of electrically charged drug molecules with phospholipid membranes

Model membranes (egg-yolk PC liposomes) were exposed to the cationic form of amphiphilic drugs. Microelectrophoresis was used to measure the change of the electrokinetic potential as a function of the drug concentration. By use of the Gouy-Chapman theory the surface potential and surface charge density were calculated. A theoretical model postulating a simple partition equilibrium of the charged drug molecules between the membrane and the aqueous phase in the vicinity of the membrane failed to describe the experimental results. Modification of the partition law by introducing a mechanism of saturation at high drug concentrations, however, resulted in concordance of model and experiment. Some parameters of the model can be used as a means of evaluating the efficiency of neuroactive drugs.     

Calculations of the circular dichroism of double-helical nucleic acids. II. Effects involving n leads to pi transitions

The circular dichroism of double-helical nucleic acids was calculated as a function of geometry, including terms involving n → π* transitions. The “nonbonding” n or σ orbitals were of the azine type, delocalized, but concentrated at the nitrogen atoms of the purines and pyrimidines. Dynamic coupling of the magnetic moments of the n → π* transitions with the electric moments of π → π* transitions generated important terms. Mixing of electric dipole character into n → π* transitions by the static electric field perturbation of the molecule is of lesser importance. The largest contributions of n → π* transitions to the circular dichroism of double-helical nucleic acids are comparable in magnitude to the sum of π → π* terms only for geometries where the circular dichroism is weak. Using both n → π* and π → π* contributions one is able to match experimental and calculated circular dichroism spectra for DNA's over a much wider range of conditions than was possible previously.