Monoclonal antibodies specific for poly(dG) X poly(dC) and poly(dG) X poly(dm5C)

Most duplex DNAs that are in the "B" conformation are not immunogenic. One important exception is poly(dG) X poly(dC), which produces a good immune response even though, by many criteria, it adopts a conventional right-handed helix. In order to investigate what features are being recognized, monoclonal antibodies were prepared against poly(dG) X poly(dC) and the related polymer poly(dG) X poly(dm5C). Jel 72, which is an immunoglobulin G, binds only to poly(dG) X poly(dC), while Jel 68, which is an immunoglobulin M, binds approximately 10-fold more strongly to poly(dG) X poly(dm5C) than to poly(dG) X poly(dC). For both antibodies, no significant interaction could be detected with any other synthetic DNA duplexes including poly[d(Gm5C)] X poly[d(Gm5C)] in both the "B" and "Z" forms, poly[d(Tm5Cm5C)] X poly[d(GGA)], and poly[d(TCC)] X poly[d(GGA)], poly(dI) X poly(dC), or poly(dI) X poly(dm5C). The binding to poly(dG) X poly(dC) was inhibited by ethidium and by disruption of the DNA duplex, confirming that the antibodies were not recognizing single-stranded or multistranded structures. Furthermore, Jel 68 binds significantly to phage XP-12 DNA, which contains only m5C residues and will precipitate this DNA in the absence of a second antibody. The results suggest that (dG)n X (dm5C)n sequences in natural DNA exist in recognizably distinct conformations.     

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.     

Complete disproportionation of duplex poly(dT)*poly(dA) into triplex poly(dT)*poly(dA)*poly(dT) and poly(dA) by coralyne

Coralyne is a small crescent-shaped molecule known to intercalate duplex and triplex DNA. We report that coralyne can cause the complete and irreversible disproportionation of duplex poly(dT)·poly(dA). That is, coralyne causes the strands of duplex poly(dT)·poly(dA) to repartition into equal molar equivalents of triplex poly(dT)·poly(dA)·poly(dT) and poly(dA). Poly(dT)·poly(dA) will remain as a duplex for months after the addition of coralyne, if the sample is maintained at 4°C. However, disproportionation readily occurs upon heating above 35°C and is not reversed by subsequent cooling. A titration of poly(dT)·poly(dA) with coralyne reveals that disproportionation is favored by as little as one molar equivalent of coralyne per eight base pairs of initial duplex. We have also found that poly(dA) forms a self-structure in the presence of coralyne with a melting temperature of 47°C, for the conditions of our study. This poly(dA) self-structure binds coralyne with an affinity that is comparable with that of triplex poly(dT)·poly(dA)·poly(dT). A Job plot analysis reveals that the maximum level of poly(dA) self-structure intercalation is 0.25 coralyne molecules per adenine base. This conforms to the nearest neighbor exclusion principle for a poly(dA) duplex structure with A·A base pairs. We propose that duplex disproportionation by coralyne is promoted by both the triplex and the poly(dA) self-structure having binding constants for coralyne that are greater than that of duplex poly(dT)·poly(dA).     

High pressure fourier transform infrared spectroscopy of poly(dA)poly(dT), poly(dA) and poly(dT)

The effect of hydrostatic pressure upon the DNA duplex, poly(dA)poly(dT), and its component single strands, poly(dA) and poly(dT) has been studied by fourier-transform infrared spectroscopy (FT-IR). The spectral data indicate that at 28 degrees C and pressures up to 12 kbar (1200 MPa) all three polymers retain the B conformation. Pressure causes the band at 967 cm(-1), arising from water-deoxyribose interactions, to shift to higher frequencies, a result consistent with increased hydration at elevated pressures. A larger pressure-induced frequency shift in this band is observed in the single stranded polymers than in the double stranded molecule, suggesting that the effect of pressure on the hydration of single strands may be greater than upon a double stranded complex. A pressure-dependent hypochromicity in the bands attributed to base stacking indicates that pressure facilitates the base stacking in the three polymers, in agreement with previous assessments of the importance of stacking in the stabilization of DNA secondary structure at ambient and high pressures.     

Laser Raman spectroscopic studies of poly(r-cytidylic acid) and poly(r-adenylic acid) complexes with poly(L-lysine) and poly(L-arginine)

Complexes of polyribocytidylic acid and polyriboadenylic acid with poly(L -lysine) and poly(L -arginine) were studied by Raman spectroscopy. The backbones of both polynucleotides are distorted by poly(L -arginine). On the other hand, poly(L -lysine) could distort the backbone of polyriboadenylic acid but not that of polyribocytidylic acid. In general, poly(L -arginine) can increase the order of the base stacking, while poly(L -lysine) causes disordering in the base stacking.     

Plasticization of poly(L-lactide) with poly(propylene glycol)

A new plasticizer for poly(L-lactide) (PLA)-poly(propylene glycol) (PPG) is proposed. The advantage of using PPG is that it does not crystallize, has low glass transition temperature, and is miscible with PLA. PLA was plasticized with PPGs with nominal Mw of 425 and 1000 g/mol. Poly(ethylene glycol) (PEG), long known as a plasticizer for PLA, with nominal Mw of 600 g/mol, was also used to plasticize PLA for comparison. The thermal and tensile properties of PLA and PLA with 5-12.5 wt % of the plasticizers were studied. In blends of PLA with PPGs the glass transition temperature was lower than that of neat PLA. Both PPGs enhanced the crystallizability of PLA albeit less than PEG. All of the plasticizers increased also the ability of PLA to plastic deformation which was reflected in a decrease of yield stress and in an increase of elongation at break. The effect was enhanced by the higher PPG content and also by lower molecular weight of PPG. A phase separation occurred only in the blend containing 12.5 wt % of PPG with higher molecular weight. The evidences of crazing were found in deformed samples of PLA with low plasticizer content, whereas the samples with higher content of plasticizers crystallized due to deformation.     

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.     

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.     

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.     

Helix-coil equilibria of poly (rA) · poly (rU)

Absorbance melting curves of the double-stranded (rA) · (rU) helix, made with fractionated homopolynucleotides of matched length, have been obtained over a 15-fold range of [Na⁺] and 30° range of temperature. An excellent fit of the observed profiles was obtained with theoretical curves calculated on the basis of the simplest interpretation for the occurrence of particular equilibria [1–3]; the complete molecular partition function being evaluated by the power series method developed by Applequist [4–6]. The stability constant was evaluated from literature values for the calorimetric enthalpy. The loop closure exponent was best represented by 2.22 ± 0.04 for the mismatching loop mode of melting and 1.22 for the matching mode and was independent of [Na⁺] and temperature. Assuming the applicability of the nonintersecting random walk value of 1.9 ± 0.1, these results would suggest a slight bias toward matched loop formation during melting of homopolynucleotides that might be expected to form only mismatched loops. The value of the stacking parameter at 60°C was only ~6% higher than that at 30°C, 0.0221 (0.0184 for the matching case). Calculated melting curves indicate the occurrence of a fifth-order phase transition when the mean helix length is only ~13 base-pairs, or about one full turn of the helix.     

Thermodynamics of interactions of TAlPyP4 and AgTAlPyP4 porphyrins with poly(rA)poly(rU) and poly(rI)poly(rC) duplexes

We employed UV light absorption and circular dichroism (CD) spectroscopic measurements to study the binding of novel water-soluble porphyrins meso-tetra-(4N-allylpyridyl)porphyrin [TAlPyP4], and its Ag containing derivative to the poly(rA)poly(rU) and poly(rI)poly(rC) RNA duplexes. Our results suggest that TAlPyP4 associate with the duplexes via intercalation, whereas the conservative CD spectra indicates that AgTAlPyP4 preferably binds via outside self-stacking mode. We used our determined binding isotherms for each ligand-RNA binding event to calculate the binding constant, Kb, and binding free energy, DeltaGb = -RTlnKb. By performing these experiments as a function of temperature, we evaluated the van't Hoff binding enthalpies, DeltaH. The binding entropies, DeltaSb, were calculated as DeltaSb = (DeltaHb - DeltaGb)/T. We interpret our data in terms of specific interactions that stabilize/destabilize each ligand-RNA complex studied in this work. Taken together, our data provide important new information about the thermodynamics of interactions of porphyrins with nucleic acids.     

Solution conformation of poly(L-lysyl-L-glutamic acid) and poly(L-lysyl-L-glutamine)

Solution conformation of poly(L-lysyl-L-glutamic acid) (PLGU) and poly(L-lysyl-L-glutamine) (PLGN) was studied in water as a function of pH, added salt, detergents, methanol and trifluoroethanol (TFE). Both the polypeptides exhibit no ordered conformation in the pH range 1.5-12.5; salts and detergents did not have any marked effect. Replacement of side chain carboxyl by an amide group did not help in inducing PLGN to adopt a helical conformation even at pH as high as 12.0, unlike poly(L-lysine). The helicogenic solvents, methanol and TFE, induce formation of weak helices in PLGU as well as in PLGN. It is not unlikely that H-bonding between the side chains leads to stabilizing an unordered conformations.     

Lattice vibrational modes of poly(rU) and poly(rA)

A normal coordinate analysis has been performed for the polyribonucleotides poly(rU) and poly(rA). The polymers are assumed to be 11-fold infinite helical structures in the A conformation. The hydrogen atoms have been rigidly attached to the appropriate atoms in order to reduce the dimension of the problem. The potential energy is defined in terms of a valence force field initially and a model for the inclusion of nonbonded interactions has been presented. A method for factoring the secular equation for infinite helical polymers in Cartesian coordinates is presented. All of the general features of polyribonucleotide spectra have been reproduced and, in many cases, good quantitative agreement between observed and calculated frequencies is obtained. More specific assignments are offered for some of the observed lines.     

Interaction of poly(I) and poly(I)·poly(C) with chlorodiethylenetriamino platinum(II) chloride

The fixation of dien-Pt on poly(I)·poly(C) leads to only minor changes in the uv and CD spectra at ambient temperature, showing that there is little perturbation of the secondary structure in the r_b range studied (up to 0.30). However, the melting profiles show two steps. The T_m for strand separation increases linearly from 61°C (r_b = 0) to 80°C (r_b = 0.18), after which it declines on further increasing the r_b. The second melting step is not complete at 100°C, and the magnitude of the absorbance change in this second step also appears to be at a maximum at r_b = 0.18. Although dien-Pt can only coordinate to one base, the nmr spectra at 80°C also show a second type of interaction with the adjacent bases, which is only destroyed in the presence of a strong denaturing agent, 5M guanidinium hydrochloride. From these results and the spectrophotometric data, we observe that dien-Pt forms a triple sandwich by hydrogen bonding of the platinum amino groups to the adjacent hypoxanthine bases (N⁷). The presence of these hydrogen bonds accounts for the increased stability (maximal at one Pt to three hypoxanthine bases) and their rupture is seen in the second melting step. No interaction has been observed with poly(C) strand. Reaction of dien-Pt with poly(I) shows the formation of the same triple sandwich structure in the nmr spectra.     

Proton-NMR study of the interaction of trans-dichloro-diamine-platinum(II) with poly(I) and poly(I).poly(C)

The fixation of trans-(NH₃)₂Cl₂ Pt(II) to poly(I)·poly(C) at low r_b (< 0.05) leads to the formation of two complexed species. The major species (ca. 82% of bound platinum) involves coordination of platinum to a single hypoxanthine base, while the other species involves coordination of two hypoxanthine bases, which are either far apart on the same strand or on separate poly(I) strands, to the platinum. These same two species are found after reaction with poly(I), as are two other species throughout the entire r_b range studied (r_b = 0–0.30). The latter two species are assigned to trans-Pt bound to two bases on a poly(I) strand with (a) one or (b) two free bases between the two bound bases. These two species, (a) and (b), account for ca. 35% of the bound platinum, although the 1:1 species remains dominant (ca. 55%). These two additional species are observed at high r_b (>0.075) after reaction with poly(I)·poly(C) but as very minor species. They are formed by reaction with melted poly(I) loops. Also at high r_b, we have observed a shifted cytidine H⁵ resonance arising from interaction of trans-Pt with a melted loop of poly(C). Most probably, this arises from an intramolecular poly(I) to poly(C) crosslink. Results from the reaction of trans-Pt with poly(C) are presented for comparison.     

Study of the interaction of cis-dichloro-(1,2 diethyl-3-aminopyrrolidine)Pt(II) complex with poly(I), poly(C) and poly(I) x poly(C)

The interaction of cis-dichloro-(1,2 diethyl-3-aminopyrrolidine)platinum(II) (Ptpyrr) with the polynucleotides poly(I), poly(C) and poly(I) x poly(C) acids was studied by circular dichroism, molecular fluorescence and (1)H NMR spectroscopies. Multivariate Curve Resolution, a factor analysis method, was applied for the analysis and interpretation of spectroscopic data obtained in mole ratio and kinetics studies. This procedure allows the determination of the number of different interaction complexes present during the experiments and the resolution of both concentration profiles and pure spectra for all of them. Two different interaction complexes were observed at the experimental conditions studied. The first one, at low Ptpyrr:polynucleotide ratio (r(Ptpyrr:poly)) values, corresponds to the interaction of Ptpyrr with hypoxanthine bases in the poly(I) moiety. This interaction leads to the destabilization and dissociation of the double-stranded conformation. The second complex was observed at higher r(Ptpyrr:poly) values and corresponds to the interaction of Ptpyrr to cytosine bases in poly(C) moiety. The formation of both complexes showed that the interaction of Ptpyrr with hypoxanthine bases occurred at the first stages of the reaction and with cytosine bases at longer reaction times. The results obtained show the utility of the Multivariate Curve Resolution approach for the analysis of data obtained by monitoring spectroscopically the interaction equilibria of platinum compounds with nucleic acids.     

Helix-helix transitions in DNA: fibre X-ray study of the particular cases poly(dG-dC) · poly(dG-dC) and poly(dA) · 2poly(dT)

The helix-helix transitions which occur in poly(dG-dC) · poly(dG-dC) and in poly (dG-m⁵dC) · poly(dG-m⁵dC) are commonly assumed to be changes between the right-handed A- or B-DNA double helices and the left-handed Z-DNA structure. The mechanisms for such transconformations are highly improbable, especially when they are supposed to be active in long polynucleotide chains organised in semicrystalline fibres. The present alternative possibility assumes that rather than the Z-DNA it is a right-handed double helix (S-DNA) which actually takes part in these form transitions. Two molecular models of this S form, in good agreement with X-ray measurements, are proposed. They present alternating C(2′)-endo and C(3′)-endo sugar puckering like the “alternating B-DNA” put forward some years ago. Dihedral angles, sets of atomic coordinates and stereo views of the two S-DNA structures are given, together with curves of calculated diffracted intensities. Furthermore, we question the possibility of obtaining semicrystalline fibres with triple helices of poly(dA) · 2poly(dT) in a way which renders X-ray diffraction efficient. It is suggested that, up to now, only double helices of poly(dA) · poly(dT) can actually be observed by fibre X-ray diffraction measurements. Received: 30 March 1999 / Revised version: 30 June 1999 / Accepted: 30 June 1999     

Synthesis and characterization of stereoregular poly(D-methionyl-L-methionine) and poly(L-methionyl-D-methionyl-L-methionine)

By use of a polycondensation procedure free of racemization, stereoregular polymethionines have been synthesized from C-activated D -methionyl-L -methionine and L -methionyl-D -methionyl-L -methionine. The poly(D -methionyl-L -methionine) and poly(L -methionyl-D -methionyl-L -methionine) so prepared are soluble in chloroform and can be purified through dissolution in this solvent and precipitation by ligroin. Poly(D -Met-L -Met)which is obtained in a 25% yield, is about 5000 in average molecular weight. It has no discernible optical activity when examined between 400 and 600 nm in a trifluoroacetic acid solution. Poly(L -Met-D -Met-L -Met) (40% yield, M. W. = 10,000) is an optically active polymer. [α]₄₃₆²⁴ ≈ + 170° for a chloroformic solution (c = 0.2 CHCl₃).     

Study of specific interactions of amino acid esters with the synthetic polynucleotides poly(A) x 2 poly(U), poly(A) x poly(U) and poly(A) by thermal denaturation

The interactions of amino acid esters with poly(A)x2poly(U) and poly(A)xpoly(U) have been investigated by means of thermal denaturation of these polynucleotides. The esters under consideration raised the melting point, revealing the preferable binding to helical polynucleotide structures. The melting point shifts demonstrate the following sequence of the stabilities of these complexes: Arg greater than Lys much greater than His greater than Met greater than Ser greater than Gly. The same stability order is observed when studying the polynucleotide renaturation in the presence of esters. This order coincides with that previously obtained for the nucleotide base--amino acid ester complexes excepting basic amino acid esters. The ester interactions with poly(A) and poly(U) also reveal the specificity of monomer--monomer interactions. Some dynamic contributions into the studied specificity are also discussed.