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.     

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.     

An ethidium-induced double helix of poly(dA)-poly(rU).

Equilibrium dialysis, relaxation kinetic, melting, and continuous variation mixing experiments on complexes of poly(dA) and poly(rU) demonstrate that ethidium induces conversion of a 1:1 mixture of these homopolymers (at one molar salt and 19 degrees C) from a three stranded to a two stranded helix. This is the first demonstration of a double helix of poly(dA)-poly(rU) in solution.     

Poly(rA).poly(rU) with Ni(2+) ions at different temperatures: infrared absorption and vibrational circular dichroism spectroscopy

Phase transitions were studied of the sodium salt of poly(rA).poly(rU) induced by elevated temperature without Ni(2+) and with Ni(2+) in 0.07 M concentration in D(2)O (approximately 0.4 [Ni]/[P]). The temperature was varied from 20 degrees C to 90 degrees C. The double-stranded conformation of poly(rA).poly(rU) was observed at room temperature (20 degrees C-23 degrees C) with and without Ni(2+) ions. In the absence of Ni(2+) ions, partial double- to triple-strand transition of poly(rA).poly(rU) occurred at 58 degrees C, whereas only single- stranded molecules existed at 70 degrees C. While poly(rU) did not display significant helical structure, poly(rA) still maintained some helicity at this temperature. Ni(2+) ions significantly stabilized the triple-helical structure. The temperature range of the stable triple-helix was between 45 degrees C and 70 degrees C with maximum stability around 53 degrees C. Triple- to single-stranded transition of poly(rA).poly(rU) occurred around 72 degrees C with loss of base stacking in single-stranded molecules. Stacked or aggregated structures of poly(rA) formed around 86 degrees C. Hysteresis took place in the presence of Ni(2+) during the reverse transition from the triple-stranded to the double-stranded form upon cooling. Reverse Hoogsteen type of hydrogen-bonding of the third strand in the triplex was suggested to be the most probable model for the triple-helical structure. VCD spectroscopy demonstrated significant advantages over infrared absorption or the related electronic CD spectroscopy.     

Base-pair opening and closing reactions in the double helix. A stopped-flow hydrogen exchange study in poly(rA).poly(rU).

The hydrogen-deuterium exchange of AMP, uridine, poly(rA), and poly(rA) · poly(rU) was investigated by a spectral difference method using stopped-flow spectrophotometry. Proton exchange rates were measured as a function of pH, added catalysts, temperature and salt concentration. The results confirm and extend previous conclusions on the H-exchange chemistry of the bases, on the large equilibrium opening of the double helix, and on its slow opening and closing rates, but an alternative conformation for the major open state is considered. Two H-exchange rate classes are found in poly(rA) · poly(rU). The slower class represents the two exocyclic amino protons of A which exchange through a pre-equilibrium opening mechanism, therefore revealing the fraction of time the helix is open. Base-pairs are open 5% of the time at 25 °C. The faster class is assigned to the U-N-3 H proton, the rate of which is limited by helix opening. Both opening and reclosing of the duplex are slow, 2 s^?1 and 40 s^?1, respectively, at 25 °C. Thermodynamic parameters for the equilibrium helix opening and for the rate of opening were determined. These properties may be consistent with a simple opening involving swinging out of the U base while retaining A more or less stacked within the duplex. The results demonstrate that no faster or more populated helix-open state occurs (when structure is stable). It appears that, unlike opening—closing reactions at a helix end or a helix-coil boundary, internal base opening and closing are innately slow. One implication of this is that any chemical or biological process requiring access to sequences in the interior of a closed stable DNA duplex may be constrained to proceed only on a time scale of seconds, and not in milliseconds or microseconds.     

Vibrational CD study of the thermal denaturation of poly(rA) · poly(rU)

The vibrational cd (VCD) of a double-stranded RNA, poly(rA) - poly(rU), at pH 7 and moderate added salt concentration (0.1M) has been measured in both the base-stretching and phosphate-stretching regions of the ir as a function of temperature. The data in both cases show two distinct phase transitions. The first is from double- to a triple-stranded form, and the second is from triple- to single-stranded forms, which still retain substantial local order even up to 80°C. The nature of these transitions has been identified by comparison of the VCD and ir absorption spectra of the initially double-stranded samples with those of single-stranded poly(rA) and poly(rU) and with triple-stranded poly-(rA) -poly-(rU) poly (rU). The large differences in the VCD band shapes allows positive identification of the intermediate and final states. Thus under VCD-concentration conditions, a simple helix-to-coil transition can be eliminated for poly (rA ) - poly (rU) while such a two-step transition can be seen at low salt conditions. All of these observations are consistent with previous studies of the phase transitions of poly (rA) - poly (rU) under various salt conditions. Additionally, the VCD is indicative of premelting for all the triple-, double-, and single-strand complexes studied. The triple-strand complex did not show disproportionation to double strand on heating under these added salt conditions. The unusual VCD pattern for low temperature poly (rA) - poly (rU), as compared to high G? C content RNAs and DNAs, is qualitatively, but not quantitatively, explained using exciton coupling of localized dipolar transitions in each type of base within the strand. © 1993 John Wiley & Sons, Inc.     

Mg2+-induced triplex formation of an equimolar mixture of poly(rA) and poly(rU)

Magnesium ions strongly influence the structure and biochemical activity of RNA. The interaction of Mg²⁺ with an equimolar mixture of poly(rA) and poly(rU) has been investigated by UV spectroscopy, isothermal titration calorimetry, ultrasound velocimetry and densimetry. Measurements in dilute aqueous solutions at 20°C revealed two differ ent processes: (i) Mg²⁺ binding to unfolded poly(rA)·poly(rU) up to [Mg²⁺]/[phosphate] = 0.25; and (ii) poly(rA)·2poly(rU) triplex formation at [Mg²⁺]/[phosphate] between 0.25 and 0.5. The enthalpies of these two different processes are favorable and similar to each other, ~–1.6 kcal mol^–1 of base pairs. Volume and compressibility effects of the first process are positive, 8 cm³ mol^–1 and 24 × 10^–4 cm³ mol^–1 bar^–1, respectively, and correspond to the release of water molecules from the hydration shells of Mg²⁺ and the polynucleotides. The triplex formation is also accompanied by a positive change in compressibility, 14 × 10^–4 cm³ mol^–1 bar^–1, but only a small change in volume, 1 cm³ mol^–1. A phase diagram has been constructed from the melting experiments of poly(rA)·poly(rU) at a constant K⁺ concentration, 140 mM, and various amounts of Mg²⁺. Three discrete regions were observed, corresponding to single-, double- and triple-stranded complexes. The phase boundary corresponding to the transition between double and triple helical conformations lies near physiological salt concentrations and temperature.     

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.     

The binding of poly(rA) and poly(rU) to denatured DNA. I. Model studies with homopolymers.

We have compared the properties of the poly(rA).oligo(dT) complex with those of the poly(rU).oligo(dA)n complex. Three main differences were found. First, poly(rA) and oligo(dT)n do not form a complex in concentrations of CsCl exceeding 2 M because the poly(rA) is insoluble in high salt. If the complex is made in low salt, it is destabilized if the CsCl concentration is raised. Complexes between poly(rU) and oligo(dA)n, on the other hand, can be formed in CsCl concentrations up to 6.6 M. Second, complexes between poly(rA) and oligo(dT)n are more rapidly destabilized with decreasing chain length than complexes between poly(rU) and oligo(dA)n. Third, the density of the complex between poly(rA) and poly(dT) in CsCl is slightly lower than that of poly(dT), whereas the density of the complex between poly(rU) and poly(dA) in CsCl is at least 300 g/cm3 higher than that of poly(dA). These results explain why denatured natural DNAs that bind poly(rU) in a CsCl gradient usually do not bind poly(rA).     

Fluorescence studies on the complex formation between poly(rA) containing 1,N6-ethenoadenosine and poly(rU)

The interaction of the 1,N6-etheno derivatives of poly(rA) (poly(epsilon rA] with poly(rU) has been studied by absorption and fluorescence spectroscopy. The stoichiometry of the interaction is found to be 1 epsilon A:1 rU and 1 epsilon A:2 rU as well as in the case of poly(rA)-poly(rU) interaction. The fluorescence properties, including the intensity and polarization of fluorescence, respond to the conformational transition of poly(epsilon rA)-poly(rU) complexes. The introduction of epsilon A groups into poly(rA) results in a marked decrease in the melting temperature, suggesting that epsilon A may destabilize the helical structure. The three-exponential decay law obtained with poly(epsilon rA)-poly(rU) complexes indicates the existence of at least three different stacked conformational states.     

The vibrational spectra and structure of poly(rA-rU)-poly(rA-rU)

Infrared and Raman spectra of aqueous poly(rA-rU)·poly(rA-rU), the double-helical complex containing strands of alternating riboadenylate and ribouridylate residues, display significant differences from one another and from corresponding spectra of poly(rA)·poly(rU), the double-helical complex of riboadenylate and ribouridylate homopolymers. Parallel studies on the copolymer and homopolymer complexes by cesium sulfate density gradient centrifugation, ultraviolet absorption spectroscopy, hydrogenion titration, 1-N oxidation of adenine residues by monoperphthalic acid and X-ray diffraction reveal, however, that the geometry of base pairing between adenine and uracil is closely similar in each complex and apparently of the Watson-Crick type. Therefore the differences observed between vibrational spectra of poly (rA-rU)·poly (rA-rU) and poly(rA)·poly(rU) are not due to different base-pairing schemes but may be attributed to differences in vibrational coupling between vertically stacked bases. Vibrational coupling may also account for the differences between infrared and Raman spectra of the same complex. Thus, the present results indicate that infrared and Raman frequencies of RNA in the region 1750–1550 cm^?1 should be dependent on the base sequence.     

Three cytoplasmic DNA polymerases that utilize poly(rA).oligo(dT)

Three DNA polymerases that use poly(rA).oligo(dT) were partially purified from cytoplasmic extracts of cultured mouse cells (after removal of mitochondria), and characterized. One is similar to, and may be the same as, the mitochondrial DNA polymerase gamma. The other two enzymes, one 7.5 S and the other 3.6 S, share some properties with DNA polymerases beta and gamma, e.g. their responses to certain inhibitors; however, they are not clearly identified with any previously well-characterized mammalian DNA polymerases. It is also demonstrated that the response of DNA polymerase gamma to N-ethylmaleimide is template dependent, and that DNA polymerase alpha has an authentic (albeit small) activity with poly(rA).oligo(dT).     

Poly(rA) binds poly(rG).poly(rC) to form a triple helix.

Poly(rA) binds poly(rG).poly(rC) to form a triple helix. Evidence for this structure includes ultraviolet absorbance mixing curves and melting curves, and circular dichroism spectroscopy. The formation of the triple helix depends on the length of the poly(rC) strand. Triple helix forms when the average length is around 100 nucleotides but does not form when the average length is about 500 nucleotides.     

Experimental and calculated study of the vibrational modes of poly(dG-dC).poly(dG-dC) in B and Z conformations

Infrared spectra of the B and Z forms of poly(dG-dC).poly(dG-dC) are presented. Experimental assignments relative to certain vibration modes have been confirmed by calculation based on the GF-Wilson method. The calculated results show that only the geometry change between B and Z forms, is responsible for the observed modifications in the vibrational spectra.     

A solution structure for poly(rA).poly(dT) with different furanose pucker and backbone geometry in rA and dT strands and intrastrand hydrogen bonding of adenine 8CH

Equilibrium Raman spectra show that A- and B-form phosphodiester backbone geometries are both present in the solution structure of the RNA.DNA hybrid poly(rA).poly(dT) and that these arise from C3'-endo-rA and C2'-endo-dT nucleosides, respectively. Raman dynamic measurement of deuterium exchange of adenine 8CH groups reveals (i) a single kinetic class of rA conformers and (ii) extraordinary retardation of 8CH exchange in this class--more than 100-fold slower than in canonical DNA structures. The equilibrium and kinetic results, in conjunction with model building, indicate an unusual intrastrand hydrogen bond involving adenosine donor (8C-H) and acceptor (5'O) groups and a double-helical conformation in solution similar to that proposed for fibers at high relative humidity [Zimmerman, S. B., & Pheiffer, B. H. (1981) Proc. Natl. Acad. Sci. U.S.A. 78, 78-82]. In fibers of poly(rA).poly(dT) at low relative humidity, the Raman spectra indicate a conventional A-helix structure.     

Interpretation of DNA vibration modes: IV--A single-helical approach to assign the phosphate-backbone contribution to the vibrational spectra in A and B conformations

A calculated approach based on the Higgs method for assigning the vibration modes of an infinite helicoidal polymeric chain has been performed on the basis of a reliable valence force field. The calculated results allowed the phosphate-backbone marker modes of the A and B forms, to be interpreted. In the dynamic models used, the bases have been omitted and no interchain interaction was considered. The calculation can also interprete quite satisfactorily the characteristic Raman peaks and infrared bands in the 1250-700 cm-1 spectral region arising from the sugar or sugar-phosphate association and reproduce their evolution upon the B----A DNA conformational transition. They clearly show that the phosphate-backbone modes in the above mentioned spectral region constitute the optical branches of the phonon dispersion curves with no detectable variation in the first Brillouin-zone.     

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 (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.