Formation of triple-helical nucleic acids studied by using antibodies specific for poly(A).poly(U).poly(U)

The formation of the triple helix of poly(A).poly(U).poly(U) was studied by using antibodies specific to poly(A).poly(U).poly(U). the 10-11 base chain length for oligo(A) and the 20-30 base chain length for oligo(U) may be the minimum sizes required to maintain a stable triple helix. Double-stranded poly(A).poly(U) which was the core of triple-stranded poly(A).poly(U).poly(U) could bind poly(U) and produce an analogue of poly(A).poly(U).poly(U) reactive with the antibodies even if the poly(A) or poly(U) was brominated or acetylated to the extent of 35-55%. However, brominated or acetylated poly(U) did not produce a stable triple helix with double-stranded poly(A).poly(U).     

Stabilisation of oxygen-labile nitrilases via co-aggregation with poly(ethyleneimine)

Three different nitrilases lost 50–100% of their activity upon exposure to oxygen for 40 h, whereas their activity was fully retained under an argon atmosphere. This effect is ascribed to a reaction of oxygen, presumably with the catalytic cysteine residue.

Co-aggregates of the nitrilases and high MW poly(ethyleneimine) were prepared by precipitation; these were physically very stable and protein release was not observed. The PEI co-aggregates of the nitrilases were much more oxygen-tolerant than the freely dissolved enzymes. The nitrilase from Pseudomonas fluorescens EBC 191, in particular, retained its full activity upon exposure to oxygen for 40 h. This result is ascribed to a low local oxygen concentration in the biocatalyst, due to the salting-out effect of the polycationic PEI.     

Interaction of poly(A) with different adsorbents for affinity chromatography of nucleic acids

Chromatography on adsorbents for separation of mRNA containing poly(A) has given interesting results, even if the nature of the occurring interaction was not always well understood. In the present study we report the chromatographic behaviour of poly(A) homopolynucleotides on different substituted matrices: poly(U)-: poly(A)-: phenyl-, octyl-, ethanolamine-, acriflarin- and DNA-Sepharose: oligo-dT and MN-cellulose. Using different experimental conditions as ionic strength, neutral salt, pH, temperature, buffer composition it was possible to evaluate the participation of electrostatic, hydrophobic hydrogen-bonding, and/or charge-transfer interaction. Furthermore, it is shown that poly(A) interacts non-specifically with matrices like acriflavin or DNA-Sepharose, as well as with oligo-dT cellulose or poly(U)-Sepharose.     

Storage stabilization of enzyme activity by poly(ethyleneimine)

The effect of mixing penicillin acylase with poly(ethyleneimine) is discussed. The properties of the polymer-enzyme system were evaluated for a wide range of enzyme concentrations (0.3–45.5 mg/cm³) and poly(ethyleneimine) concentrations (0.0001–10% wt). It was shown that addition of poly(ethyleneimine) to crude enzyme preparation caused precipitation of ballast protein and stabilization of the enzyme fraction remaining in the supernatant. The soluble fraction had stable activity for 21 days storage at 37 °C while the native enzyme lost about 80% of its initial activity. Additionally, it was ascertained that the polymer very slightly affected the properties of penicillin acylase in the PEI-enzyme preparations. Finally, possible ways of using the polymer-enzyme preparations in a membrane reactor are suggested.This work was supported by Government Committee of Science: Grant KBN # 3 0321 91 1     

Interaction of aromatic residues of proteins with nucleic acids. Circular dichroism studies of the binding of oligopeptides to poly(adenylic acid).

The binding of peptides containing lysyl and aromatic residues to poly(A) in its single-stranded form at pH 7 leads to a change of its circular dichroism (CD) spectrum, which is mainly due to the stacking of the aromatic amino acid with the bases of poly(A). Comparison is made between the binding of peptides having different primary structures which gives indications on the way the peptides bind to poly(A). A method is described which allows the calculation of the binding parameters from CD data. The magnitude of the association constant depends on the size of the aromatic ring and decreases in the order tryptophan greater than tyrosine greater than phenylalanine. The CD amplitude decreases linearly with the concentration of bound molecules. These results are discussed with respect to the role played by aromatic amino acids in complex formation between nucleic acids and proteins.     

Structure of poly (dT).poly (dA).poly (dT)

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 A 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.     

Structure of poly (I).poly (A).poly (I)

The molecular structure of poly (I).poly (A).poly (I) has been determined and refined using the continuous intensity data on layer lines in the x-ray diffraction pattern obtained from an oriented fiber of this polymorphic RNA complex. The polymer forms a 12-fold right-handed triple-helix of pitch 39.7A and each base-triplet is stabilized by quasi Crick-Watson-Hoogsteen hydrogen bonds. The ribose rings in all the three strands have C3'-endo conformations. The final R-value for this best structure is 0.24 and the x-ray fit is significantly superior to all the alternative structures where the different chains might have different furanose conformations. This all-purine triple-helix, counter-intuitively, has a diameter roughly 3A shorter than that of DNA and RNA triple-helices containing a homopurine and two complementary homopyrimidine strands. Its compact, grooveless cylindrical shape is consistent with the lack of lateral organization.     

Control of electron transfer in DNA by peptide nucleic acids (PNA).

The one-electron oxidation of PNA-DNA hybrid containing G-triplet sequence was examined. In DNA duplex G-triplet was selectively cleaved by oxidation, whereas in PNA-DNA hybrid cleavage efficiency was extremely lowered. These result suggested that cleavage efficiency of PNA-DNA hybrid was different from that of B-form DNA duplex.     

Kinetic behaviour of penicillin acylase stabilized by poly (ethyleneimine)

The paper presents the kinetic evaluations of poly(ethyleneimine)-penicillin acylase preparations. The comparative studies show that the investigated system is much better than the native enzyme, and slightly worse than commercially available Boenringer preparation. Additionally, the high stability of PEI-enzyme system, very easy way of its preparation, high flexibility, and possibility to set the needed enzyme concentration are particularly favourable for use of the membrane bioreactor with PEI-enzyme system immobilized in its volume. Some advantages of the use of such bioreactor are also discussed.List of Symbols C_E IU/m³ activity concentration - C_S mol/m³ substrate concentration - C_P, C_Q mol/m³ products concentration - K_A mol/m³ constant which defines the affinity of a substrate to enzyme - K_iS mol/m³ substrate inhibitory constant - K_iP mol/m³ PhAA inhibitory constant - K_iQ mol/m³ 6-APA inhibitory constant - k₃, mol/IU min constant rate of dissociation of the active complex - mol/m³ min rate of reactionThis work was supported by Government Committee of Science: Grant KBN # 3 0321 92 01     

Molecular recognition of nucleic acids: coralyne binds strongly to poly(A)

The small molecule coralyne was found to bind preferentially and strongly to single-stranded poly(A) with an apparent association constant (Ka) of (1.8+/-0.3) x 10(6)M(-1). Binding of coralyne to poly(A) is predominantly enthalpically driven with a stoichiometry of one coralyne per four adenine bases. Poly(A) forms a coralyne dependent secondary structure with a melting temperature of 60 degrees C, for the conditions of our study.     

Four-stranded DNA. Conformational analysis of regular spirals of poly(dT).poly(dA).poly(dA).poly(dT) with different base binding variations

Conformational analysis of four stranded DNA helices poly(dT).poly(dA).poly(dA).poly(dT) with parallel arrangement of the identical sugar-phosphate chains connected by twofold symmetry has been performed. All possible models of symmetrical base binding were checked. By the potential energy optimization the dihedral angles and helices parameters of stable conformations of four stranded polynucleotides were calculated. The dependences of conformational energy on the base complex structure and mutual orientation of the poly(dA).and poly(dT) chains were studied. Possible biological functions of four stranded helices are discussed.     

Structure of poly d(A).poly d(T).

On the basis of the x-ray data from polycrystalline and well oriented fibers of the sodium salt of poly d(A).poly d(T) (Arnott et al, Nucl. Acids Res. 11, 4141-4155 (1983), a revised B'-DNA model incorporating B-like adenine and thymine strands is shown to give a much better x-ray agreement (R = 0.25) than the previously assigned model consisting of mixed sugar conformations in the two strands. The narrowing of the minor and the widening of the major grooves are promiscuous features of B'-DNA, which are common to all poly d(purine).poly d(pyrimidine) duplexes with two hydrogen bonded base-pairs and are in marked contrast with classical B-DNA. Due to modest propeller (-15 degrees), the cross strand diagonal hydrogen bonds (0.37 nm) in this duplex are not as strong as those in A,T-rich oligonucleotide crystal structures.     

Four-stranded DNA helices: conformational analysis of regular poly(dT).poly(dA).poly(dA).poly(dT) helices with various types of base binding

The paper presents results obtained in conformational analysis of homopolymeric four-stranded poly(dT).poly(dA).poly(dA).poly(dT) DNA helices in which the pairs of strands with identical bases are parallel and have a two-fold symmetry axis. All possible models of base binding to yield a symmetric complex have been considered. The dihedral angles of sugar-phosphate backbones and helix parameters, which are consistent with the minima of conformational energy for four-stranded DNAs, have been determined using the results of optimization of conformational energy calculated at atom-atom approximation. Potential energy is shown to depend on the structure of base complexes and on the mutual orientation of unlike strands. Possible biological functions of four-stranded helices are discussed.     

Structure of Poly (U).poly (A).poly (U)

The molecular structure of poly (U).poly (A).poly (U) 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 RNA. The final R-value for the preferred structure is 0.24, far lower than that for the plausible alternatives. The polymer forms an 11-fold right-handed triple-helix of pitch 33.5A and each base triplet is stabilized by Crick-Watson-Hoogsteen hydrogen bonds. The ribose rings in the three strands have C3'-endo, C2'-endo and C2'-endo conformations, respectively. The helix derives additional stability through systematic interchain hydrogen bonds involving ribose hydroxyls and uracil bases. The relatively grooveless cylindrical shape of the triple-helix is consistent with the lack of lateral organization.     

Nucleosome reconstitution of core-length poly(dG).poly(dC) and poly(rG-dC).poly(rG-dC)

The double-stranded polypurine.polypyrimidines poly(dG).poly(dC) and poly[d(A-G)].poly[d(T-C)] and the mixed ribose-deoxyribose polynucleotide poly(rG-dC).poly(rG-dC) have been successfully reconstituted into nucleosomes. The radioactively labeled particles comigrate in gel electrophoresis and sucrose density gradient experiments with authentic nucleosomes derived from chicken erythrocyte chromatin. These results show that nucleosomes are able to accommodate a wider variety of polynucleotides than was previously believed.     

The propeller DNA conformation of poly(dA).poly(dT).

Physical properties of the DNA duplex, poly(dA).poly(dT) differ considerably from the alternating copolymer poly(dAT). A number of molecular models have been used to describe these structures obtained from fiber X-ray diffraction data. The recent solutions of single crystal DNA dodecamer structures with segments of oligo-A.oligo-T have revealed the presence of a high propeller twist in the AT regions which is stabilized by the formation of bifurcated (three-center) hydrogen bonds on the floor of the major groove, involving the N6 amino group of adenine hydrogen bonding to two O4 atoms of adjacent thymine residues on the opposite strand. Here we show that it is possible to incorporate the features of the single crystal analysis, specifically high propeller twist, bifurcated hydrogen bonds, and a narrow minor groove, as well as the close interstrand NMR signal between adenine HC2 and ribose HC1' of the opposite strand, into a model that is fully compatible with the diffraction data obtained from poly(dA).poly(dT).     

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).     

Poly(dG).poly(dC) at neutral and alkaline pH: the formation of triple stranded poly(dG).poly(dG).poly(dC).

Alkaline titrations of different samples of poly(dG).poly(dC) and of the constituent homopolymers poly(dG) and poly(dC) have been performed in 0.15 M NaCl and their CD spectra followed. Sample I contained a slight excess of poly(dC) (52% C: 48% G) and showed a single reversible transition (pK = 11.9) due to the dissociation of double stranded poly(dG).poly(dC). Sample II, containing an excess of poly(dG) (43% C: 57% G), showed two transitions (pK1 = 11.4, PK2 = 11.9) the first one being only partially reversible. Examination of the CD spectra along the alkaline titrations indicated the presence of another hydrogen-bonded complex of higher G content. Mixing curves performed at pH 8 have confirmed the presence of a 2G: 1C complex, besides the double stranded complex. It can be formed in amounts up to 30% by mixing the two homopolymers, alkali treatment and heating. The CD spectra of the two complexes have been computed from the CD data of the mixing curves. This permitted the determination of the concentrations of both complexes and homopolymers in all samples. The ratio of triple to double stranded complex is not only dependent on the G/C ratio of the sample, but also a function of the previous physico-chemical conditions. These results explain the variability of many properties of different poly(dG).poly(dC) samples observed by other workers.     

Lysozyme association with nucleic acids

Lysozyme is well known for the ability to hydrolyze the cell wall of bacteria. Based on the similarity of structure between lysozyme and histones as seen from the results of X-ray crystal structure determinations, we have postulated that binding to nucleic acids may be another biological function of lysozyme. We have therefore begun a systematic study of the interactions of lysozyme and related molecules with nucleic acids, and present here a preliminary report. Binding to DNA and RNA has been demonstrated from gel electrophoresis, enzyme activity, and coprecipitation studies. We suggest that this function of lysozyme will provide an explanation why Lee-Huang et al. (1999) [Proc. Natl. Acad. Sci. USA 96, 2678-2681] were able to call lysozyme a "killer protein" against the AIDS virus, and may provide a new avenue of research on AIDS therapy.     

Proton exchange and base-pair kinetics of poly(rA).poly(rU) and poly(rI).poly(rC)

Proton exchange of poly(rA).poly(rU) and poly(rI).poly(rC) has been studied by nuclear magnetic resonance line broadening and saturation transfer from H2O. Five exchangeable peaks are observed. They are assigned to the imino, amino and 2'-OH ribose protons. The aromatic spectrum is also assigned. Contrary to previous observations, we find that the exchange of the imino proton is strongly buffer sensitive. This property is used to derive the base-pair lifetime, which is in the range of milliseconds at 27 degrees C, 100 times smaller than published values. The enthalpy for the base-opening reaction (-86 kJ/mol) and the insensitivity of the reaction to magnesium suggest that the open state involves a small number of base-pairs. The similarities in the exchange from the two duplexes indicate that the same open state is responsible for exchange of purine and pyrimidine imino protons. For the lifetime of the open state and for the base-pair dissociation constant, we obtain only lower limits. At 27 degrees C they are three microseconds and 10(-3), respectively. The analysis that yields the much larger values published previously is based on the assumption that amino protons exchange only from open base-pairs. But theory and preliminary experiments indicate that it may occur from the closed duplex. The exchange of amino protons is slower than that of the imino protons. Exchange of the 2'-OH protons from the duplexes is much slower than from single-stranded poly(rU), and it is accelerated by magnesium. This could indicate hydrogen-bonding to backbone phosphate. Discrepancies between our results and those of previous studies are discussed.