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.     

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.     

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).     

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.     

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.     

Acoustical investigation of poly(dA).poly(dT), poly[d(A-T)], poly(A).poly(U) and DNA hydration in dilute aqueous solutions.

Apparent molar adiabatic compressibilities and apparent molar volumes of poly[d(A-T)].poly[d(A-T)], poly(dA).poly(dT), DNA and poly(A).poly(U) in aqueous solutions were determined at 1 degree C. The change of concentration increment of the ultrasonic velocity upon replacing counter ion Cs+ by the Mg2+ ion was also determined for these polymers. The following conclusions have been made: (1) the hydration of the double helix of poly(dA).poly(dT) is remarkably larger than that of other polynucleotides; (2) the hydration of the AT pair in the B-form DNA is larger than that of the GC pair; (3) the substitution of Cs+ for Mg2+ ions as counter ions results in a decrease of hydration of the system polynucleotide plus Mg2+, and (4) the magnitude of this dehydration depends on the nucleotide sequence; the following rule is true: the lesser is a polynucleotide hydration, the larger dehydration upon changing Cs+ for Mg2+ ions in the ionic atmosphere of polynucleotide.     

Energetics of Z-DNA formation in poly d(A-T), poly d(G-C), and poly d(A-C) poly d(G-T).

The conformational change for the alternating purine-pyrimidine polydeoxyribonucleotides i.e. poly d(A-T), poly d(G-C), and poly d(A-C) poly d(G-T) from a right-handed conformation at room temperature to the left-handed Z-DNA like double helix at elevated temperatures has been studied by UV spectroscopy, Raman spectroscopy, and by adiabatic differential scanning microcalorimetry (DSC) in the presence of Na+ and Mg2+ or Ni2+ respectively as counterions. The differential UV spectra reveal through a hyperchromic shift at around 280nm and a hypochromic shift at 260nm that a conformational change to the left-handed conformation occurs. The Raman spectra clearly show characteristic changes, a drastic decrease of the band at 680cm-1 and the appearance of a new band at 628cm-1, due to the change of the purine bases to the syn conformation upon inversion of the helix-handedness. The course of the transition as function of temperature can be followed quantitatively by plotting the change in the excess heat capacity vs. temperature. The transition enthalpy delta H for the B- to Z-DNA transition per mole base pairs (mbp) amounts to 2.0 +/- 0.2kcal for poly d(G-C), to 4.0 +/- 0.4kcal for poly d(A-T), and to 3.1 +/- 0.3kcal for poly d(A-C) poly d(G-T). The enthalpy change due to the Z-DNA to coil transitions (per mole base pairs) amounts to 11kcal for poly d(G-C), 10.5kcal for poly d(A-T) and 11.3kcal for poly d(A-C) poly d(G-T).     

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.     

Structural transitions in poly(A), poly(C), poly(U), and poly(G) and their possible biological roles

The homopolynucleotide (homo-oligonucleotide) tracts function as regulatory elements at various stages of mRNAs life cycle. Numerous cellular proteins specifically bind to these tracts. Among them are the different poly(A)-binding proteins, poly(C)-binding proteins, multifunctional fragile X mental retardation protein which binds specifically both to poly(G) and poly(U) and others. Molecular mechanisms of regulation of gene expression mediated by homopolynucleotide tracts in RNAs are not fully understood and the structural diversity of these tracts can contribute substantially to this regulation.

This review summarizes current knowledge on different forms of homoribopolynucleotides, in particular, neutral and acidic forms of poly(A) and poly(C), and also biological relevance of homoribopolynucleotide (homoribo-oligonucleotide) tracts is discussed. Under physiological conditions, the acidic forms of poly(A) and poly(C) can be induced by proton transfer from acidic amino acids of proteins to adenine and cytosine bases. Finally, we present potential mechanisms for the regulation of some biological processes through the formation of intramolecular poly(A) duplexes.     

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.     

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.     

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.     

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.     

Raman spectroscopic elucidation of DNA backbone conformations for poly(dG-dT).poly(dA-dC) and poly(dA-dT).poly(dA-dT) in CsF solution

Raman spectra have been recorded for poly(dG-dT) · poly(dA-dC) and poly(dA-dT) · poly(dA-dT) in low salt and at high concentrations of CsF. Poly(dG-dT) · poly(dA-dC) shows no change in the 682-cm^?1 guanine mode, demonstrating the absence of the Z-structure at high salt. The 790-cm^?1 phosphodiester symmetric stretch, however, shifts up 5 cm^?1 in 4.3M CsF, suggesting a slight conformational change, associated with ion binding or hydration changes. Poly(dA-dT) · poly(dA-dT) shows an additional broad band at 816 cm^?1, attributed to the phosphodiester modes associated with the C3′-endo deoxyribose units in the alternating B-structure. In this case, both the 841- and the 816-cm^?1 asymmetric phosphodiester stretches, associated with the C2′- and C3′-endo units, shift down on addition of CsF in a sequential manner. Correlation of this sequence with that previously observed for the two ³¹P-nmr resonances, establishes that the phosphodiester stretching frequencies depend on the conformation of the 5′-sugar, and not on the 3′-sugar.     

Vacuum UV CD of the low-salt Z-forms of poly(rG-dC).poly(rG-dC), and poly(dG-m5dC).poly(dG-m5dC)

The vacuum CD spectra of poly(rG-dC)·poly(rG-dC) and poly(dG-m⁵dC)·poly(dG-m⁵dC) have been obtained for the low-salt Z-conformations of both polymers. The spectra are very similar to those for the high-salt Z-forms. This behavior is consistent with the suggestion that the low- and high-salt Z-forms are comprised of different proportions of Z_I- and Z_II-conformations.     

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.     

Binding mode of 4',6-diamidino-2-phenylindole and ethidium to poly(dG).poly(dC).poly(dC)(+) triplex and poly(dG).poly(dC) duplex

Optical spectroscopic properties of 4',6-diamidino-2-phenylindole (DAPI) and ethidium bromide complexed with poly(dG).poly(dC).poly(dC)(+) triplex and poly(dG).poly(dC) duplex were compared in this study. When complexed with both duplex and triplex, ethidium is characterized by hypochromism and a red shift in the absorption spectrum, a complicate induced circular dichroism (CD) band in the polynucleotide absorption region, and a negative reduced linear dichroism signal in both polynucleotide and drug absorption regions. The spectral properties for both duplex- and triplex-bound ethidium are identical and both can be understood by the intercalation binding mode. In contrast, the absorption and CD spectra of DAPI complexed with triplex differ from those of the DAPI-duplex complex, although both complexes can be understood by the intercalation binding mode. Considering that the third strand runs along the major groove of the template duplex, we conclude that the DAPI molecule partially intercalates near the major groove of the duplex, where the third strand can affect its spectroscopic properties.     

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.