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.     

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).     

The binding of poly (rA) and poly (rU) to denatured DNA. II. Studies with natural DNAs.

We have studied the interaction of poly(rA) and poly(rU) with natural DNAs containing (dA.dT)n sequences. The results indicate that hybridization of poly(rA) to denatured DNA can be used to estimate the size and frequency of large (dA.dT)n tracts, whereas hybridization with poly(rU) does not give reliable information on these points. In 6.6 M CsCl, poly(rU) can form stable complexes with denatured DNA containing short (dA)n tracts (n less than or equal to 6), whereas binding of poly(rA) to denatured DNA under these conditions requires much larger (dT)n tracts (estimated n greater than 13). Moreover, binding of poly(rA) requires pre-hybridization in low salt, because free poly(rA) precipitates in 6.6 M CsCl.     

Bleomycin mediated degradation of DNA-RNA hybrids does not involve C-1' chemistry.

Incubation of Fe(II) bleomycin and O2 with a number of 'A'-like DNA-RNA hybrid homopolymers at 4 atm O2 results in formation of base propenal and base in a ratio of approximately 1.0:1.0. This ratio differs dramatically from the corresponding ratio of approximately 10:1.0 observed when activated BLM degrades 'B'-like DNA homopolymers. Experiments were undertaken to determine if the shift to enhanced base production observed in the A-like hybrids is the result of C-1' chemistry in addition to the C-4' chemistry normally observed with B-like DNA under identical conditions. Increased accessibility of the 1'-hydrogen might be anticipated due to widening of the minor groove in the A-like conformers. Experiments using poly([1'-3H]dA) poly(rU) and poly([U-14C]dA) poly(rU) indicated that neither 3H2O nor deoxyribonolactone accompanied adenine release. In addition, studies using poly([4'-2H]dA) poly(rU) and poly([1'-2H]dA) poly(rU) unambiguously establish that the altered base to base propenal ratio is not the result of C-1' chemistry, but a direct consequence of C-4' chemistry.     

A Polydeoxythymidylic acid-specific deoxyribonuclease from rat ascites hepatoma cells.

A deoxyribonuclease has been purified 950-fold from rat ascites hepatoma cells and has been separated from another deoxyribonuclease that appears to have DNase III type activity. The enzyme preferentially degrades single stranded poly(dT), requires Mg²⁺ for maximum activity and has a pH optimum at 8.5 in Tris-HCl buffer. Poly(dA), poly(dC), poly(rA), and poly(rU) are not effective substrates. The hydrolysis of poly(dT) is strongly inhibited when poly(dA) or poly(rA) is annealed with poly(dT). Poly(dT) is degraded ultimately into 5′-deoxythymidylic acid via the formation of oligodeoxythymidylate intermediates.     

The poly dA strand of poly dA.poly dT adopts an A-form in solution: a UV resonance Raman study.

The study by resonance Raman spectroscopy with a 257 nm excitation wave-length of adenine in two single-stranded polynucleotides, poly rA and poly dA, and in three double-stranded polynucleotides, poly dA.poly dT, poly(dA-dT).poly(dA-dT) and poly rA.poly rU, allows one to characterize the A-genus conformation of polynucleotides containing adenine and thymine bases. The characteristic spectrum of the A-form of the adenine strand is observed, except small differences, for poly rA, poly rA.poly rU and poly dA.poly dT. Our results prove that it is the adenine strand which adopts the A-family conformation in poly dA.poly dT.     

Counterion association with native and denatured nucleic acids: an experimental approach

The melting temperature of the poly(dA) . poly(dT) double helix is exquisitely sensitive to salt concentration, and the helix-to-coil transition is sharp. Modern calorimetric instrumentation allows this transition to be detected and characterized with high precision at extremely low duplex concentrations. We have taken advantage of these properties to show that this duplex can be used as a sensitive probe to detect and to characterize the influence of other solutes on solution properties. We demonstrate how the temperature associated with poly(dA) . poly(dT) melting can be used to define the change in bulk solution cation concentration imparted by the presence of other duplex and triplex solutes, in both their native and denatured states. We use this information to critically evaluate features of counterion condensation theory, as well as to illustrate "crosstalk" between different, non-contacting solute molecules. Specifically, we probe the melting of a synthetic homopolymer, poly(dA) . poly(dT), in the presence of excess genomic salmon sperm DNA, or in the presence of one of two synthetic RNA polymers (the poly(rA) . poly(rU) duplex or the poly(rU) . poly(rA) . poly(rU) triplex). We find that these additions cause a shift in the melting temperature of poly(dA) . poly(dT), which is proportional to the concentration of the added polymer and dependent on its conformational state (B versus A, native versus denatured, and triplex versus duplex). To a first approximation, the magnitude of the observed tm shift does not depend significantly on whether the added polymer is RNA or DNA, but it does depend on the number of strands making up the helix of the added polymer. We ascribe the observed changes in melting temperature of poly(dA) . poly(dT) to the increase in ionic strength of the bulk solution brought about by the presence of the added nucleic acid and its associated counterions. We refer to this communication between non-contacting biopolymers in solution as solvent-mediated crosstalk. By comparison with a known standard curve of tm versus log[Na+] for poly(dA) . poly(dT), we estimate the magnitude of the apparent change in ionic strength resulting from the presence of the bulk nucleic acid, and we compare these results with predictions from theory. We find that current theoretical considerations correctly predict the direction of the t(m) shift (the melting temperature increases), while overestimating its magnitude. Specifically, we observe an apparent increase in ionic strength equal to 5% of the concentration of the added duplex DNA or RNA (in mol phosphate), and an additional apparent increase of about 9.5 % of the nucleic acid concentration (mol phosphate) upon denaturation of the added DNA or RNA, yielding a total apparent increase of 14.5 %. For the poly(rU) . poly(rA) . poly(rU) triplex, the total apparent increase in ionic strength corresponds to about 13.6% of the amount of added triplex (moles phosphate). The effect we observe is due to coupled equilibria between the solute molecules mediated by modulations in cation concentration induced by the presence and/or the transition of one of the solute molecules. We note that our results are general, so one can use a different solute probe sensitive to proton binding to characterize subtle changes in solution pH induced by the presence of another solute in solution. We discuss some of the broader implications of these measurements/results in terms of nucleic acid melting in multicomponent systems, in terms of probing counterion environments, and in terms of potential regulatory mechanisms.     

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.     

Determination of the kinetics of deuteration of DNA.RNA hybrids by ultraviolet spectroscopy

The kinetics of the hydrogen-deuterium exchange reactions of poly(dA).poly(rU) and poly(rA).poly(dT) has been examined, at pH 7.0 and at various temperatures in the 15-35 degrees C range, by stopped-flow ultraviolet spectrophotometry. For comparison, the deuteration kinetics of poly[d(A-T)].poly[d(A-T)] and poly(rA).poly(rU) has been reexamined. At 20 degrees C, the imino deuteration (NH----ND) rates of the two hybrid duplexes were found to be 1.5 and 1.8 s-1, respectively. These are nearly equal to the imino deuteration rates of poly[d(A-T)].poly[d(A-T)] (1.1 s-1) and poly(rA).poly(rU) (1.5 s-1) but appreciably higher than that of poly(dA).poly(dT) (0.35 s-1). It has been suggested that a DNA.RNA hybrid, an RNA duplex, and the AT-alternating DNA duplex have in general higher base-pair-opening reaction rates than the ordinary DNA duplex. The amino deuteration (NH2----ND2) rates, on the other hand, have been found to be 0.25, 0.28, and 0.33 s-1, respectively, for poly(dA).poly(rU), poly(rA).poly(dT), and poly[d(A-T)].poly[d(A-T)], at 20 degrees C. These are appreciably higher than that for poly(rA).poly(rU) (0.10 s-1). In general, the equilibrium constants (K) of the base-pair opening are considered to be greatest for the DNA.RNA hybrid duplex (0.05 at 20 degrees C), second greatest for the RNA duplex (0.02 at 20 degrees C), and smallest for the DNA duplex (0.005 at 20 degrees C), although the AT-alternating DNA duplex has an exceptionally great K (0.07 at 20 degrees C). From the temperature effect on the K value, the enthalpy of the base-pair opening was estimated to be 3.0 kcal/mol for the DNA.RNA hybrid duplex.     

Comparison of base inclination in ribo-AU and deoxyribo-AT polymers

The inclination angle between the base normal and the helix axis is measured for ribo-AU polymers by using flow linear dichroism (LD), and compared to measurements for deoxyribo-AT polymers under dehydrating conditions. The CD of the DNA polymers under the dehydrating conditions is not the same as the corresponding RNA polymers, which are presumed to be in the A form. However, the LD indicates that poly(dAdT)-poly(dAdT) can assume the A form in 80% 2,2,2-trifluoroethanol, although poly(dA)-poly(dT) retains B form structure in this dehydrating solvent. The inclination angles are similar for B form poly(dAdT)-poly(dAdT) and poly(dA)-poly(dT), and these parameters are also similar for A form poly(rArU) -poly(rArU) and poly(rA) -poly(rU). All the inclination axes are similar. © 1995 John Wiley & Sons, Inc.     

The thermodynamic contribution of the 5-methyl group of thymine in the two- and three-stranded complexes formed by poly(dU) and poly(dT) with poly(dA)

To assess the thermodynamic contribution of the 5-methyl group of thymine, we have studied the two-stranded helical complexes poly(dA).poly(dU) and poly(dA).poly(dT) and the three-stranded complexes--poly(dA).2poly(dU), poly(dA).poly(dT).poly(dU) and poly(dA).2poly(dT)--by differential scanning calorimetry, and uv optical melting experiments. The thermodynamic quantities associated with the 3 --> 2, 2 --> 1, and 3 --> 1 melting transitions are found to vary with salt concentration and temperature in a more complex manner than commonly believed. The transition temperatures, T(m), are generally not linear in the logarithm of concentration or activity of NaCl. The change in enthalpy and in entropy upon melting varies with salt concentration and temperature, and a change in heat capacity accompanies each transition. The poly(dA).2poly(dU) triple helix is markedly different from poly(dA).2poly(dT) in both its CD spectrum and thermodynamic behavior, while the poly(dA).poly(dT).poly(dU) triple helix resembles poly(dA).2poly(dT) in these properties. In comparing poly(dA).2poly(dT) with either the poly(dA).poly(dT).poly(dU) or the poly(dA).2poly(dU) triplexes, the substitution of thymine for uracil in the third strand results in an enhancement of stability against the 3 --> 2 dissociation of deltadeltaG degrees = -135 +/- 85 cal (mol A)(-1) at 37 degrees C. This represents a doubling of the absolute stability toward dissociation compared to the triplexes with poly(dU) as the third strand. The poly (dA).poly (dT) duplex is more stable than poly(dA).poly(dU) by deltadeltaG degrees = -350 +/- 60 cal (mol base pair)(-1) at 37 degrees C. Poly(dA).poly(dT) has 50% greater stability than poly(dA).poly(dU) as a result of the dT for dU substitution in the duplex.     

Parallel DNA double helices. II. Conformational analysis of regular helices having a second order axis of symmetry

Conformational analysis of double helices of DNA with parallel arranged sugar-phosphate chains connected by twofold symmetry has been performed. Homopolymers poly(dA).poly(dA), poly(dC).poly(dC), poly(dG).poly(dG) and poly(dT).poly(dT) were studied. For each of the homopolymers all variants of H-bond pairing were checked. The maps of closing of sugar-phosphate backbone were previously computed. By the optimization of potential energy the dihedral angles and helix parameters of relatively stable conformations of parallel stranded polynucleotides were calculated. The dependence of conformational energy on the nucleic base character and the base pair type were studied. Two main conformational regions for favourable "parallel" helix of polynucleotides were found. The former of these two regions coincide with the region of typical conformational parameters of B-DNA. On an average the conformational energy of "parallel" DNA is close to the energy of canonic "antiparallel" B-DNA.     

Destabilization of the secondary structure of RNA by ribosomal protein S1 from escherichia coli

S1 is an acidic protein associated with the 3′ end of 16S RNA; it is indispensable for ribosomal binding of natural mRNA. We find that S1 unfolds single stranded stacked or helical polynucleotides (poly rA, poly rC, poly rU). It prevents the formation of poly (rA + rU) and poly (rI + rC) duplexes at 10–25 mM NaCl but not at 50–100 mM NaCl. Partial, salt reversible denaturation is also seen with coliphage MS2 RNA, $\text{E. coli}$ rRNA and tRNA. Generally, only duplex structures with a Tm greater than about 55° are formed in the presence of S1. The protein unfolds single stranded DNA but not poly d(A·T).     

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.     

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.     

Binding of nonintercalative antitumor drugs to DNA-polymers: structural effects of bisquaternary ammonium heterocycles

The binding of the antitumor agents SN-16814 nd SN-13232 to various DNA's in solution was monitored by CD and UV absorption measurements. In addition comparative studies with dA.dT containing duplex DNA of the related ligands SN-6136 and SN-6324 were included with respect to effects of structural variations. In general all four ligands show a dA.dT preference in their binding affinity to DNA. Differences were observed for the reaction of SN-16814 which contains bicyclic ring system: it has a lower base pair selectivity, shows some affinity to poly(dG-dC).poly(dG-dC), poly(rA).poly(rU) and poly(rU). The binding mechanism of SN-16814 is associated with a significant time dependent binding effect in CD spectra and UV absorption in case of reaction with poly(dA).poly(dT) and poly(dI).poly(dC) indicating a slow kinetics. The preferred binding to dA.dT base pairs in DNA decreases in the order from SN-61367 greater than SN-13232 greater than SN-6324,SN-16814 as judged from CD titration studies, salt dissociation and melting temperature data. Competitive binding experiments with netropsin (Nt) or distamycin-5 revealed that SN-16814 and SN-13232 are displaced from poly(dA.dT).poly(dA-dT) suggesting that both ligands are less strongly bound than Nt and Dst-5 within the minor groove of B-DNA. These studies are consistent with results of the DNAse I cleavage of poly(dA-dT).poly(dA-dT) which show the same relative order of inhibition of the cleavage reaction due to ligand binding. The results suggest that the variability of the DNA binding and dA.dT sequence specificity may reside in the adaptability of benzamide-type ligands in the helical groove which is influenced by distinct structural modifications of the ligand conformation.     

Binding of Mg2+ to single-stranded polynucleotides: hydration and optical studies

The binding of Mg(2+) to single-stranded ribo- and deoxy-polynucleotides, poly(rA), poly(rU), poly(dA) and poly(dT), has been investigated in dilute aqueous solutions at pH 7.5 and 20 degrees C. A combination of ultrasound velocimetry, density, UV and CD spectroscopy have been employed to study hydration and spectral effects of Mg(2+) binding to the polynucleotides. Volume and compressibility effects of Mg(2+) binding to random-coiled poly(rU) and poly(dT) correspond to two coordination bonds probably between the adjacent phosphate groups. The same parameters for poly(rA)+Mg(2+) correspond to an inner-sphere complex with three-four direct contacts. However, almost no hydration effects are arising in binding to its deoxy analog, poly(dA), indicating mostly a delocalized binding mode. In agreement with hydration studies, optical investigations revealed almost no influence of Mg(2+) on poly(dA) properties, while it stabilizes and aggregates poly(rA) single-helix. The evidence presented here indicates that Mg(2+) are able to bind specifically to single-stranded polynucleotides, and recognize their composition and backbone conformation.     

A Raman spectroscopic study of the interaction between nucleotides and the DNA binding protein gp32 of bacteriophage T4

Raman spectra of the bacteriophage T4 denaturing protein gp32, its complex with the polynucleotides poly(rA), poly(dA), poly(dT), poly(rU), and poly(rC), and with the oligonucleotides (dA)₈ and (dA)₂, were recorded and interpreted. According to an analysis of the gp32 spectra with the reference intensity profiles of Alix and co-workers [M. Berjot, L. Marx, and A. J. P. Alix (1985) J. Ramanspectrosc., submitted; A. J. P. Alix, M. Berjot, and J. Marx (1985) in Spectroscopy of Biological Molecules, A. J. P. Alix, L. Bernard, and M. Manfait, Eds., pp. 149–154], 1 gp32 contains ≈ 45% helix, ≈ 40% β-sheet, and 15% undefined structure. Aggregation of gp32 at concentrations higher than 40 mg/mL leads to a coordination of the phenolic OH groups of 4–6 tyrosines and of all the sulfhydryl (SH) groups present in the protein with the COO^? groups of protein. The latter coordination persists even at concentrations as low as 1 mg/mL. In polynucleotide–protein complexes the nucleotide shields the 4–6 tyrosine residues from coordination by the COO^? groups even at high protein concentration. The presence of the nucleotide causes no shielding of the SH groups. With Raman difference spectroscopy it is shown that binding of the protein to a single-stranded nucleotide involves both tyrosine and trytophan residues. A change in the secondary structure of the protein upon binding is observed. In the complex, gp32 contains more β-sheet structure than when uncomplexed. A comparison of the spectra of complexed poly(rA) and poly(dA) with the spectra of their solution conformations at 15°C reveals that in both polynucleotides the phosphodiester vibration changes upon complex formation in the same way as upon a transition from a regular to a more disordered conformation. Distortion of the phosphate–sugar–base conformation occurs upon complex formation, so that the spectra of poly(rA) and poly(dA) are more alike in the complex than they are in the free polynucleotides. The decrease in intensity of the Raman bands at 1304 cm^?1 in poly(rA), at 1230 cm^?1 in poly(rU), and at 1240 and 1378 cm^?1 of poly(dT) may be indicative of increased stacking interactions in the complex. No influence of the nucleotide chain length upon the Raman spectrum of gp32² in the complex was detected. Both the nucleotide lines and the protein lines in the spectrum of a complex are identical in poly(dA) and (dA)₈.