Raman spectral studies of nucleic acids. XVI. Structures of polyribocytidylic acid in aqueous solution

Raman spectra of polyribocytidylic acid show the formation of an ordered single-stranded structure [poly(rC)] at neutral pH and an ordered double-stranded structure containing hemiprotonated bases [poly(rC)·poly(rC⁺)] in the range 5.5 > pH > 3.7. Below 40°C, poly(rC) contains stacked bases and a backbone geometry of the A-type, both of which are gradually eliminated by increasing the temperature to 90°C. Below 80°C, poly(rC)·poly(rC⁺) contains bases which are hydrogen bonded and stacked and a backbone geometry also of the A-type. In this structure the bases of each strand are shown to be structurally identical, i.e., hemiprotonated, and therefore distinct from both neutral and protonated cytosines. Infrared and Raman spectra indicate the existence of a center of symmetry with respect to the paired cytosine residues, which suggests that the additional proton per base pair is shared equally by the two hydrogen-bonded bases. Denaturation of poly(rC)·poly(rC⁺) occurs cooperatively (t_m ≈ 80°C) with elimination of base stacking, base pairing, and the A-helix geometry. Each of the separated strands of the denatured complex is shown to contain comparable amounts of both neutral and protonated cytosines, most likely in alternating sequence [poly(rC, rC⁺)]. In both poly(rC, rC⁺) and poly(rC), at 90°C, the backbones do not exhibit the phosphodiester Raman frequencies characteristic of other disordered polyribonucleotide chains. This is interpreted to mean that the single strands, though devoid of base stacking and A-type structure, contain uniformly ordered backbones of a specific type. Fully protonated poly(rC⁺), on the other hand, forms no ordered structure and may be characterized as a disordered (random chain) polynucleotide at all temperatures. Several Raman lines of poly(rC) are absent from the spectrum of poly(rC)·poly(rC⁺) and vice versa. These frequencies, assigned mainly to vibrations of the ribose groups, suggest that the furanose ring conformations are different in the single-stranded and double-stranded structures of polyribocytidylic acid. Several other Raman group frequencies have been identified and correlated with the polymer secondary structures.     

Raman studies of nucleic acids. XII. Conformations of oligonucleotides and deuterated polynucleotides

Laser Raman spectra of the trinucleoside diphoshate ApApA and dinucleoside phosphates ApU, UpA, GpC, CpG, and GpU are reported and discussed. Assignments of conformationally sensitive frequencies are-facilitated by comparison with spectra reported here of poly(rA), poly(rC), and poly(rU) in deuterium oxide solutions. The significant spectral differences between ApU and UpA, and between GpC and CpG, reveal that the sequence isomers have nonidentical conformations in aqueous solution. In UpA at low temperature the bases are stacked and the backbone conformation is similar to that found in ordered polynucleotide structures and RNA. In ApU no base stacking can be detected and the backbone conformation differs from that found in UpA, both in the orientation of phosphodiester linkages and in the internal conformation of ribose. At the conditions employed neither ApU nor UpA exhibits base pairing in aqueous solutions. In both GpC and CpG the bases are stacked and the phosphodiester conformations are similar to those encountered for UpA and RNA. However, major differences between spectra of GpC and CpG indicate that the geometries of stacking and ribosyl conformations are different. In GpC the Raman data favor the formation of hydrogen bonded dimers containing GC pairs. Protonation of C in GpC is sufficient to eliminate the ordered conformation detected by Raman spectroscopy. Despite the ordered backbone conformation evident in GpU, this dinucleoside apparently contains neither stacked nor hydrogen bonded bases at the conditions employed here. The Raman data also confirm the stacking interactions in ApApA, poly(rA), and poly(rC) but suggest that the backbone conformation in poly(rC) differs qualitatively from that found in most ordered polynucleotide structures and is thermally more stable. The present results demonstrate the sensitivity of the Raman technique to sequence-related structural differences in oligonucleotides and provide additional spectra–structure correlations for future conformational studies of RNA by laser Raman spectroscopy.     

Copper insertion facilitates water-soluble porphyrin binding to rA.rU and rA.dT base pairs in duplex RNA and RNA.DNA hybrids

The binding of the copper(II) complex of water-soluble meso-tetrakis(N-methylpyridinium-4-yl)porphyrin (TMPyP) to double-helical polynucleotides has been studied by optical absorption, circular dichroism (CD), and resonance Raman spectroscopic methods. The target polymers were RNA and RNA.DNA hybrids consisting of rA.rU, rI.rC, rA.dT, and rI.dC base pairs. Relative to the metal-free H(2)TMPyP [Uno, T., Hamasaki, K., Tanigawa, M., and Shimabayashi, S. (1997) Inorg. Chem. 36, 1676-1683], CuTMPyP binds to poly(rA).poly(dT) and poly(rA).poly(rU) with a greatly increased binding constant. The external self-stacking of the porphyrin on the surface of the polymers was evident from the strong conservative-type induced CD signals. The signal intensity correlated almost linearly with the number of stacking sites on the polymer except for poly(rA).poly(dT), which showed extraordinarily strong CD signals. Thus, the bound porphyrin may impose an ordered architecture on the polymer surface, the stacking being facilitated by the more planar nature of the CuTMPyP than the nonmetal counterpart. Resonance Raman spectra of the stacked CuTMPyP were indistinguishable from those of the intercalated one with positive delta(Cbeta-H) and negative delta(Cm-Py) bending shifts, and hence the stacked porphyrins are suggested to adopt a similar structure to that of intercalated ones. Porphyrin flattening by copper insertion opens a new avenue for medical applications of porphyrins, blocking biological events related to RNA and hybrids in malignant cells.     

Interaction of sanguinarine with double stranded RNA structures

Interaction of sanguinarine with A-form RNA structures of poly(rI)poly(rC) and poly(rA).poly(rU) has been studied by spectrophotometric, spectrofluorimetric, UV melting profiles, circular dichroism and viscometric analysis. The binding of sanguinarine to A-form duplex RNA structures is characterised by the typical bathochromic and hypochromic effects in the absorption spectrum, increasing steady state fluorescence intensity, an increase in fluorescence quantum yield of sanguinarine, an increase in fluorescence polarization anisotropy, an increase of thermal transition temperature, an increase in the contour length of sonicated rod-like RNA structure and perturbation in circular dichroic spectrum. Scatchard analysis indicates that sanguinarine binds to each polymer in a non-cooperative manner. Comparative binding parameters determined from absorbance titration by Scatchard analysis, employing the excluded site model, indicate a higher binding affinity of sanguinarine to poly(rI).poly(rC) structure than to poly(rA).poly(rU) structure. On the basis of these observations, it is concluded that the alkaloid binds to both the RNA structures by a mechanism of intercalation.     

Nucleic acid vibrational circular dichroism, absorption, and linear dichroism spectra. II. A DeVoe theory approach.

The DeVoe polarizability theory is used to calculate vibrational circular dichroism (VCD) and infrared (IR) absorption spectra of four polyribonucleotides: poly(rA) x poly(rU), poly(rU) x poly(rA) x poly(rU), poly(rG) x poly(rC), and poly(rC+) x poly(rI) x poly(rC). This is the first report on the use of the DeVoe theory to calculate VCD, oriented VCD, IR absorption, and IR linear dichroism (LD) spectra of double- and triple-stranded polyribonucleotides. Results are reported for DeVoe theory calculations--within the base-stretching 1750-1550 cm(-1) spectral region--on several proposed multistranded polyribonucleotide geometries. The calculated spectra obtained from these proposed geometries are compared with previously reported measured and calculated VCD and IR spectral results. Base-base hydrogen-bonding effects on the frequencies and magnitudes of the base carbonyl stretching modes are explicitly considered. The good agreements found between calculated and measured spectra are proposed to be further evidence of the usefulness of the DeVoe theory in drawing three-dimensional structural conclusions from measured polyribonucleotide VCD and IR spectra.     

Induced circular dichroism in nucleic acid-acridine derivative complexes

Visible absorption and circular dichroism (CD) spectra have been measured for complexes formed between nucleic acids (calf thymus DNA, poly(rA).poly(rU) and poly(rI).poly(rC)) and 9-aminoacridines (quinacrine, acranil and 9-amino-6-chloro-2-methoxy acridine). With poly(rA).poly(rU), a new absorption band was observed at longer wavelengths. The nucleic acid-drug complexes showed considerable different induced CD spectra. Analysis of these CD spectra suggests that the cationic side chains of quinacrine and acranil play an important role on the binding properties to DNA and poly(rA).poly(rU).     

A proton NMR spin-lattice relaxation study of the imino proton exchange kinetics in calf-thymus DNA

The results of semiselective ¹H-nmr inversion recovery experiments on sonicated calf thymus DNA fragments are reported. The measurements were conducted in aqueous solutions containing 85% D₂O, in order to reduce the dipolar contribution to the observed relaxation rates. In solutions containing 0.2M NaCl, 0.4 mM EDTA, and 10 mM cacodylate at pH = 7.0, the exchange rates of the imino protons in A-T base pairs confirm values published earlier in the literature, extrapolating to 0.25 s^?1 at 25°C. Corresponding values for the G-C base pairs are published for the first time, and are about sixfold slower. The addition of up to 0.1M Tris buffer (pH = 7.3 at 25°C), caused a striking increase in the measured exchange rates for both the A-T and G-C imino protons, resembling the effect recently observed for poly(rA)-poly(rU) and poly(rI)-poly(rC), and suggesting that the exchange rates measured for nucleic acid duplexes in low buffer concentrations at neutral pH do not reflect base-pair opening rates as assumed in the past. Lower limits to the base-pair opening rates could be estimated from extrapolation of the experimental data to infinite buffer concentration, and are 1 × 10³ s^?1 for the A-T, and 50 s^?1 for the G-C, base paris at 62°C.     

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.     

Conformational changes induced in ionized poly(L-arginine) and poly(L-histidine) by sodium dodecyl sulfate

Circular dichroism spectra have been obtained for cationic poly(L -arginine) and poly(L -histidine) in aqueous solutions containing varying amounts of sodium dodecyl sulfate. The detergent induces a disorder-order transition in both polypeptides. In each case the transition is cooperative and occurs when the ratio of detergent to amino acid residue is near unity. The ordered structure formed by poly(L -arginine) is readily identifiable as an α helix. Poly(L -histidine) appears to form a β structure in which the 211-nm electronic absorption band of the imidazole group exhibits significant rotatory strength.     

Ionic strength dependence of the hysteresis in the polyriboadenylate-polyribouridylate system.

The hysteresis observed in cyclic acid-base titrations of the three-standed polyribonucleotide helix poly (A)-2 POLY (U) strongly depends on ionic strength. For NaCl and at 25 degrees C, hysteresis occurs in the limited concentration range between 0.03 M and 1.0 M(NaCl). The transition points associated with the cyclic conversions between the triple helix and the poly (A)-poly (A) double helix and (free) poly (U) constitute a (pH ionic strength) phase diagram covering the ranges of stability and metastability of the hysteresis system. Variations with NaCl concentration of some hysteresis parameters can be quantitatively described in terms of polyelectrolyte theories based on the cylinder-cell model for rodlike polyions. The results of this analysis suggest that the metastability is predominantly due to dlectrostatic energy barriers preventing the equilibrium transition of the partially protonated triple helix above a critical pH value. Ultraviolet absorbance and potentiometric titration data of poly (A)in the acidic pH range can be analyzed in terms of two types of double-helical structures. Spectrophotometric titrations reveal isosbestic wavelengths for structural transitions of poly (A). "Time effects" commonly observed in poly (A) titrations are suggested to reflect helix transitions between the two acidic structures.     

Triple helical polynucleotidic structures: sugar conformations determined by FTIR spectroscopy.

Fourier Transform Infrared Spectra of triple stranded polynucleotides containing homopurine dA or rA and homopyrimidine dT or rU strands have been obtained in H2O and D2O solutions as well as in hydrated films at various relative humidities. The spectra are interpreted by comparison with those of double stranded helixes with identical base and sugar composition. The study of the spectral domain corresponding to in-plane double bond stretching vibrations of the bases shows that whatever the initial duplex characterized by a different IR spectrum (A family form poly rA.poly rU, heternomous form poly rA.poly dT, B family form poly dA.poly dT), the triplexes present a similar IR spectrum reflecting similar base interactions. A particular attention is devoted to the 950-800 cm-1 region which contains marker bands of the sugar conformation in the nucleic acids. In solution the existence of only N (C3'endo-A family form) type of sugar pucker is detected in poly rU.poly rA.poly rU and poly dt.poly rA.poly rU. On the contrary absorption bands characteristic of both N (C3'endo-A family form) and S (C2'endo-B family form) type sugars are detected for poly rU.poly rA.poly dT, poly rU.poly dA.poly dT and poly dT.poly rA.poly dT. Finally mainly S (C2'endo-B family form) type sugars are observed in poly dT.poly dA.poly dT.     

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.     

Probing hydration of monovalent cations condensed around polymeric nucleic acids

We report the relative molar sound velocity increments, [U], partial molar volumes, V(o), and partial molar adiabatic compressibilities, K(S)(o), of the Li(+), Na(+), K(+), Rb(+), Cs(+), NH(4)(+), and N(CH(3))(4)(+) salts of poly(dAdT)poly(dAdT), poly(dGdC)poly(dGdC), poly(dIdC)poly(dIdC), poly(rA)poly(rU), poly(rG)poly(rC), poly(rI)poly(rC), and poly(rU) at 25 degrees C. When analyzing these data, we take into account the Donnan membrane equilibrium effect. Comparison between the values of [U], V(o), and K(S)(o) exhibited by the nucleic acid salts and respective chlorides (LiCl, NaCl, KCl, RbCl, CsCl, NH(4)Cl, and N(CH(3))(4)Cl) yields information about the state of counterion hydration in the vicinity of each nucleic acid structure studied here. Our analysis reveals that the poly(dGdC)poly(dGdC), poly(dIdC)poly(dIdC), and poly(rI)poly(rC) duplexes and single-stranded poly(rU) do not significantly influence the hydration properties of their condensed counterions. In the vicinity of these polymers, counterions retain their full hydration shells (within +/-15%). By contrast, counterions condensed around the poly(dAdT)poly(dAdT), poly(rA)poly(rU), and poly(rG)poly(rC) duplexes are significantly dehydrated and retain, respectively, only 65(+/-18)%, 34(+/-21)%, and 33(+/-9)% of their original hydration shells. Taken together, the volumetric data reported here provide important new information that ultimately may help us understand the central role that hydration and counterions play in modulating the conformational preferences of nucleic acids and the energetics of DNA recognition events.     

The binding of echinomycin to deoxyribonucleic acid.

Echinomycin is a peptide antibiotic which binds strongly to double-helical DNA up to a limit of approximately one molecule per five base-pairs. There is no detectable interaction with rRNA and only extremely feeble non-specific interaction with poly(rA)-poly(rU). Heat denaturation of DNA greatly decreases the binding, and similarly limited interaction is observed with naturally occurring single-stranded DNA. Association constants for binding to nine double-helical DNA species from different sources are presented; they vary by a factor of approximately 10, but are not simply related to the gross base composition. The interaction with DNA is ionic-strength-dependent, the binding constant falling by a factor of 4 when the ionic strength is raised from 0.01 to 0.10mol/litre. From the effect of temperature on the association constant for calf thymus DNA, the enthalpy of interaction is calculated to be about -13kJ/mol (-3kcal/mol). Binding of echinomycin persists in CsCl gradients and the buoyant density of nicked bacteriophage PM2 DNA is decreased by 25 mg/ml. Echinomycin interacts strongly with certain synthetic poly-deoxynucleotides, the binding constant decreasing in the order poly(dG)-poly(dC) greater than poly(dG-dC) greater than poly(dA-dT). For the latter two polymers the number of base-pairs occluded per bound antibiotic molecule is calculated to be three, whereas for poly(dG)-poly(dC) it is estimated to be four to five. Poly(dA)-poly(dT) and poly(dI)-poly(dC) interact only very weakly with the antibiotic. Poly(dI-dC) interacts to a slightly greater extent, but the binding curve is quite unlike that seen with the three strongly binding synthetic polynucleotides. Echinomycin affects the supercoiling of closed circular duplex bacteriophage PM2 DNA in the characteristic fashion of intercalating drugs. At low ionic strength the unwinding angle is almost twice that of ethidium. Likewise the extension of the helix, determined from changes in the viscosity of rod-like sonicated DNA fragments, is nearly double that expected for a simple (monofunctional) intercalation process. On this basis the interaction process is characterized as bifunctional intercalation. At higher ionic strength the unwinding angle relative to that of ethidium and the helix extension per bound echinomycin molecule fall, indicating a smooth progression towards more nearly monofunctional intercalation. Two simpler compounds which act as analogues of the quinoxaline chromophores of echinomycin, quinoxaline-2-carboxamide and the trypanocidal drug Bayer 7602, interact with DNA very much more weakly than does echinomycin, showing that the peptide portion of the antibiotic plays an essential role in determining the strength and specificity of the interaction.     

Raman spectra of single crystals of r(GCG)d(CGC) and d(CCCCGGGG) as models for A DNA, their structure transitions in aqueous solution, and comparison with double-helical poly(dG).poly(dC)

The self-complementary oligonucleotides [r(CGC)d(CGC)]2 and [d(CCCCGGGG)]2 in single-crystal and solution forms have been investigated by Raman spectroscopy. Comparison of the Raman spectra with results of single-crystal X-ray diffraction and with data from polynucleotides permits the identification of a number of Raman frequencies diagnostic of the A-helix structure for GC sequences. The guanine ring frequency characteristic of C3'-endo pucker and anti base orientation is assigned at 668 +/- 2 cm-1 for both dG and rG residues of the DNA/RNA hybrid [r(GCG)d(CGC)]2. The A-helix backbone of crystalline [r(GCG)d(CGC)]2 is altered slightly in the aqueous structure, consistent with the conversion of at least two residues to the C2'-endo/anti conformation. For crystalline [d(CCCCGGGG)]2, the Raman and X-ray data indicate nucleosides of alternating 2'-endo-3'-endo pucker sandwiched between terminal and penultimate pairs of C3'-endo pucker. The A-A-B-A-B-A-A-A backbone of the crystalline octamer is converted completely to a B-DNA fragment in aqueous solution with Raman markers characteristic of C2'-endo/anti-G (682 +/- 2) and the B backbone (826 +/- 2 cm-1). In the case of poly(dG).poly(dC), considerable structural variability is detected. A 4% solution of the duplex is largely A DNA, but a 2% solution is predominantly B DNA. On the other hand, an oriented fiber drawn at 75% relative humidity reveals Raman markers characteristic of both A DNA and a modified B DNA, not unlike the [d-(CCCCGGGG)]2 crystal. A comparison of Raman and CD spectra of the aqueous [d(CCCCGGGG)]2 and poly(dG).poly(dC) structures suggests the need for caution in the interpretation of CD data from G clusters in DNA.     

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.     

Hydrogen exchange study of some polynucleotides and transfer RNA

The apparent disagreement between published transfer RNA hydrogen exchange results and the tRNA cloverleaf model, prompted a re-investigation of the relationship between hydrogen exchange data and nucleic acid structure. Hydrogen-tritium exchange experiments were carried out with samples of pure and mixed tRNA and with the synthetic polynucleotide bihelices: poly(rA) · poly(rU), poly(rI) · poly(rC), poly(rG) · poly(rC) and poly(dG) · poly (dC).     

The solution conformation of poly(L-lysine). A Raman and infrared spectroscopic study.

The Raman and infrared spectra of poly(L -lysine) and poly(DL -lysine) in solution are reported and the effects of various salts are investigated. The results demonstrate that α-helix formation in solution is induced by specific salts and the spectral data support the hypothesis of regions of local order for poly(L -lysine) in aqueous solutions of low ionic strength.