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.     

The hydration of nucleic acid duplexes as assessed by a combination of volumetric and structural techniques

Using high precision densimetric and ultrasonic measurements, we have determined, at 25°C, the apparent molar volumes ΦV and the apparent molar compressibilities ΦK_S of four nucleic acid duplexes—namely, the DNA duplex, poly(dIdC)poly(dIdC); the RNA duplex, poly(rA)poly(rU); and the two DNA/RNA hybrid duplexes, poly(rA)poly(dT) and poly(dA)poly(rU). Using available fiber diffraction data on these duplexes, we have calculated the molecular volumes as well as the solvent‐accessible surface areas of the constituent charged, polar, and nonpolar atomic groups. We found that the hydration properties of these nucleic acid duplexes do not correlate with the extent and the chemical nature of the solvent‐exposed surfaces, thereby suggesting a more specific set of duplex–water interactions beyond general solvation effects. A comparative analysis of our volumetric data on the four duplexes, in conjunction with available structural information, suggests the following features of duplex hydration: (a) The four duplexes exhibit different degrees of hydration, in the order poly(dIdC)poly(dIdC) > poly(dGdC)poly(dGdC) > poly(dAdT)poly(dAdT) ≈ poly(dA)poly(dT). (b) Repetitive AT and IC sequences within a duplex are solvated beyond general effects by a spine of hydration in the minor groove, with this sequence‐specific water network involving about 8 additional water molecules from the second and, perhaps, even the third hydration layers. (c) Repetitive GC and IC sequences within a duplex are solvated beyond general effects by a “patch of hydration” in the major groove, with this water network involving about 13 additional water molecules from the second and, perhaps, even the third hydration layers. (d) Random sequence, polymeric DNA duplexes, which statistically lack extended regions of repetitive AT, GC, or IC sequences, do not experience such specific enhancements of hydration. Consequently, consistent with our previous observations (T. V. Chalikian, A. P. Sarvazyan, G. E. Plum, and K. J. Breslauer, Biochemistry, 1994, Vol. 33, pp. 2394–2401), duplexes with approximately 50% AT content exhibit the weakest hydration, while an increase or decrease from this AT content causes enhancement of hydration, either due to stronger hydration of the minor groove (an increase in AT content) or due to stronger hydration of the major groove (an increase in GC content). (e) In dilute aqueous solutions, a B‐DNA duplex is more hydrated than an A‐DNA duplex, a volumetric‐based conclusion that is in agreement with previous results obtained on crystals, fibers, and DNA solutions in organic solvent–water mixtures. (f) the A‐like, RNA duplex poly(rA)poly(rU) and the structurally similar A‐like, hybrid duplex poly(rA)poly(dT), exhibit similar hydration properties, while the structurally distinct A‐like, hybrid duplex poly(rA)poly(dT) and non‐A‐like, hybrid duplex poly(dA)poly(rU) exhibit differential hydration properties, consistent with structural features dictating hydration characteristics. We discuss how volumetric characterizations, in conjunction with structural studies, can be used to describe, define, and resolve the general and sequence/conformation‐specific hydration properties of nucleic acid duplexes. © 1999 John Wiley & Sons, Inc. Biopoly 50: 459–471, 1999     

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.     

Thermodynamics-structure relationship of single mismatches in RNA/DNA duplexes

Thermodynamics of 66 RNA/DNA duplexes containing single mismatches were measured by UV melting methods. Stability enhancements for rG. dT mismatches were the largest of all mismatches examined here, while rU.dG mismatches were not as stable. The methyl group on C5 of thymine enhanced the stability by 0.12 approximately 0.53 kcal mol(-)(1) depending on the identity of adjacent Watson-Crick base pairs, whereas the 2'-hydroxyl group in ribouridine stabilized the duplex by approximately 0.6 kcal mol(-)(1) regardless of the adjacent base pairs. Stabilities induced by the methyl group in thymine, the 2'-hydroxyl group of ribouridine, and an nucleotide exchange at rG.dT and rU.dG mismatches were found to be independent of each other. The order for the mismatch stabilities is rG.dT > rU. dG approximately rG.dG > rA.dG approximately rG.dA approximately rA. dC > rA.dA approximately rU.dT approximately rU.dC > rC.dA approximately rC.dT, although the identity of the adjacent base pairs slightly altered the order. The pH dependence stability and structural changes were suggested for the rA.dG but not for rG.dA mismatches. Comparisons of trinucleotide stabilities for G.T and G.U pairs in RNA, DNA, and RNA/DNA duplexes indicate that stable RNA/DNA mismatches exhibit a stability similar to RNA mismatches while unstable RNA/DNA mismatches show a stability similar to that of DNA mismatches. These results would be useful for the design of antisense oligonucleotides.     

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).     

Hydration and conformational transitions in DNA, RNA, and mixed DNA-RNA triplexes studied by gravimetry and FTIR spectroscopy

We have studied by gravimetric measurements and FTIR spectroscopy the hydration of duplexes and triplexes formed by combinations of dA(n), dT(n), rA(n), and rU(n) strands. Results obtained on hydrated films show important differences in their hydration and in the structural transitions which can be induced by varying the water content of the samples. The number of water molecules per nucleotide (w/n) measured at high relative humidity (98% R.H.) is found to be 21 for dA(n).dT(n) and 15 for rA(n).rU(n). Addition of a third rU(n) strand does not change the number of water molecules per nucleotide: w/n=21 for rU(n)*dA(n).dT(n) and w/n=15 for rU(n)*rA(n).rU(n). On the contrary, the addition of a third dT(n) strand changes the water content but in a different way, depending whether the duplex is DNA or RNA. Thus, a loss of four water molecules per nucleotide is measured for dT(n)*dA(n).dT(n) while an increase of two water molecules per nucleotide is observed for dT(n)*rA(n).rU(n). The final hydration is the same for both triplexes (w/n=17). The desorption profiles obtained by gravimetry and FTIR spectroscopy are similar for the rA(n).rU(n) duplex and the rU(n)*rA(n).rU(n) triplex. On the contrary, the desorption profiles of the dA(n).dT(n) duplex and the triplexes formed with it (rU(n)*dA(n).dT(n) and dT(n)*dA(n).dT(n)) are different from each other. This is correlated with conformational transitions induced by varying the hydration content of the different structures, as shown by FTIR spectroscopy. Modifications of the phosphate group hydration and of the sugar conformation (S to N type repuckering) induced by decrease of the water content are observed in the case of triplexes formed on the dA(n).dT(n) duplex.     

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.     

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.     

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.     

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.     

Binding of CC-1065 to poly- and oligonucleotides

The binding of the antitumor agent CC-1065 to a variety of poly- and oligonucleotides was studied by electronic absorption, CD, and resistance to removal by Sephadex column chromatography. Competitive binding experiments between CC-1065 and netropsin were carried out with calf-thymus DNA, poly(dI-dC) · poly(dI-dC), poly(dI) · poly(dC), poly(rA) · poly(dT), poly(dA- dC) · poly(dG-dT), and poly(dA) · 2poly(dT). CC-1065 binds to polynucleotides by three mechanisms. In the first, CC-1065 binds only weakly, as judged by the induction of zero or very weak CD spectra and low resistance to extraction of drug from the polynucleotide by Sephadex chromatography. In the second and third mechanisms, CC-1065 binds strongly, as judged by the induction of two distinct, intense CD spectra and high resistance to extraction of drug from the polynucleotide, by Sephadex chromatography in both cases. The species bound by the second mechanism converts to that bound by the third mechanism with varying kinetics, which depend both on the base-pair sequence and composition of the polynucleotide. Competitive binding experiments with netropsin show that CC-1065 binds strongly in the minor groove of DNA by the second and third mechanisms of binding. Netropsin can displace CC-1065 that is bound by the second mechanism but not that bound by the third mechanism. CC-1065 binds preferentially to B-form duplex DNA and weakly (by the first binding mechanism) or not at all to RNA, DNA, and RNA–DNA polynucleotides which adopt the A-form conformation or to single-strand DNA. This correlation of strong binding of CC-1065 to B-form duplex DNA is consistent with x-ray data, which suggest an anomalous structure for poly(dI) · poly(rC), as compared with poly(rI) · poly(dC) (A-form) and poly(dI) · poly(dC) (B-form). The binding data indicate that poly(rA) · poly(dU) takes the B-form secondary structure like poly(rA) · poly(dT). Triple-stranded poly(dA) · 2poly(dT) and poly(dA) · 2poly(dU), which are considered to adopt the A-form conformation, bind CC-1065 strongly. Netropsin, which also shows a binding preference for B-form polynucleotides, also binds to poly(dA) · 2poly(dT) and occupies the same binding site as CC-1065. These binding studies are consistent with results of x-ray studies, which suggest that A-form triplex DNA retains some structural features of B-form DNA that are not present in A-form duplex DNA; i.e., the axial rise per nucleotide and the base tilt. Triple-stranded poly(dA) · 2poly(rU) does not bind CC-1065 strongly but has nearly the same conformation as poly(dA) · 2poly(dT) based on x-ray analysis. This suggests that the 2′-OH group of the poly(rU) strands interferes with CC-1065 binding to this polynucleotide. The same type of interference may occur for other RNA and DNA–RNA polynucleotides that bind CC-1065 weakly.     

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.     

Thermodynamics of nucleic acid "shape readout" by an aminosugar

Recognition of nucleic acids is important for our understanding of nucleic acid structure as well as for our understanding of nucleic acid-protein interactions. In addition to the direct readout mechanisms of nucleic acids such as H-bonding, shape recognition of nucleic acids is being increasingly recognized as playing an equally important role in DNA recognition. Competition dialysis, UV, flourescent intercalator displacement (FID), computational docking, and calorimetry studies were conducted to study the interaction of neomycin with a variety of nucleic acid conformations (shapes). At pH 5.5, the results suggest the following. (1) Neomycin binds three RNA structures [16S A site rRNA, poly(rA)·poly(rA), and poly(rA)·poly(rU)] with high affinities (K(a) ~ 10(7) M(-1)). (2) The binding of neomycin to A-form GC-rich oligomer d(A(2)G(15)C(15)T(2))(2) has an affinity comparable to those of RNA structures. (3) The binding of neomycin to DNA·RNA hybrids shows a 3-fold variance that can be attributed to their structural differences [for poly(dA)·poly(rU), K(a) = 9.4 × 10(6) M(-1), and for poly(rA)·poly(dT), K(a) = 3.1 × 10(6) M(-1)]. (4) The interaction of neomycin with DNA triplex poly(dA)·2poly(dT) yields a binding affinity (K(a)) of 2.4 × 10(5) M(-1). (5) Poly(dA-dT)(2) shows the lowest association constant for all nucleic acids studied (K(a) < 10(5)). (6) Neomycin binds to G-quadruplexes with K(a) values of ~10(4)-10(5) M(-1). (7) Computational studies show that the decrease in major groove width in the B to A transition correlates with increasing neomycin affinity. Neomycin's affinity for various nucleic acid structures can be ranked as follows: RNAs and GC-rich d(A(2)G(15)C(15)T(2))(2) structures > poly(dA)·poly(rU) > poly(rA)·poly(dT) > T·A-T triplex, G-quadruplex, B-form AT-rich, or GC-rich DNA sequences. The results illustrate the first example of a small molecule-based "shape readout" of different nucleic acid conformations.     

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.     

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).     

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).     

Effect of isonicotinic acid hydrazide-copper complex on Rous sarcoma virus and its genome RNA.

The copper complex of the antituberculous drug, insonicotinic acid hydrazide (INH), inhibits the RNA-dependent DNA polymerase of Rous sarcoma virus and inactivates its ability to malignantly transform chick embryo cells. The INH-copper complex binds to the 70S genome RNA of Rous sarcoma virus (RSV), which may account for its ability to inhibit the RNA-dependent DNA polymerase. The complex binds RNA more effectively than DNA in contrast to M-IBT-copper complexes, which bind both types of nucleic acids equally. The homopolymers, poly rA and poly rU, are bound by the INH-copper complex to a greater extent than poly rC. Isonicotinic acid hydrazide alone and CuSO4 alone bind neither DNA, RNA, poly (rA), poly (rU), nor poly (rC). However, CuSO4 alone binds poly (rI); INH alone does not. In addition to viral DNA synthesis, chick-embryo cell DNA synthesis is inhibited by the INH-copper complex. The extent of inhibition of cellular DNA synthesis is greater than that of cellular RNA and protein synthesis. No selective inhibition of transformation in cells previously infected with Rous sarcoma virus is observed.     

Influence of hydrogen bonding in DNA and polynucleotides on reaction of nitrogens and oxygens toward ethylnitrosourea.

The reactivity of ethylnitrosourea toward hydrogen-bonded sites in double-stranded DNA or oly(rA).poly(rU) was compared with those sites in single-stranded DNA, RNA, or poly(rA). Alkylation of the N-1 of A in poly(rA).poly(rU) was almost suppressed at 5 degrees C but could be markedly increased by raining the reaction temperature to 25 degrees C, well below the Tm of 56 degrees C. In contrast, the N-7 and N-6 of A, which are not hydrogen bonded, reacted to the same extent at temperatures ranging from 5 to 65 degrees C. The extent of reaction at the N-3 of A varied inversely with the reactivity of the N-1 of A, indicating that of these two nitrogens the N-1 of A is the most reactive. The proportion of reaction at the various nitrogens in poly(rA) was not affected by temperature. Hydrogen-bonded oxygens in double-stranded DNA are the O-6 of G, the O-4 of T, and the O2 of C. All are equally reactive at 5, 25, and 51 degrees C. It is concluded that the observed temperature independence is due to these oxygens having an electron pair not involved in hydrogen bonding and, thus, available for reaction. In contrast, the electron pair of the N-1 of A (or the N-3 of C) is involved in hydrogen bonding, and the extent of their reactivity is dependent on thermal fluctuation providing transiently open base pairs at temperatures far below the Tm.     

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.     

Comparative Studies of Duplex and Triplex Formation of 2′-5′ and 3′-5′ Linked Oligoribonucleotides

Abstract

We have studied double and triple helix formation between 2′–5′ or 3–5′ linked oligoriboadenylates and oligoribouridylates with chain length 7 or 10 by CD spectrometry. The complex formation depends on the type of linkage of oligoribonucleotides, chain length, concentration and molar ratio of the strands, temperature and the cationic concentration. Mixture of any linkage isomers of oligo(rA) and oligo(rU) in 1:1 molar ratio form duplex at 0.1 M NaCl. The duplex stability largely depends on the type of the linkages and is in the following order; [35′] oligo(rA)·[3′-5′] oligo(rU) > [2′-5′] oligo(rA)'[3′-5′] oligo(rU) > [3′-5′] oligo(rA)·[2′-5′] oligo(rU) > [2–5′] oligo(rA)*[2′-5′] oligo(rU). The higher cationic concentrations, 0.5 M MgCl₂, stabilize the complex and either duplex or triplex is formed depending on the input strand ratio and the type of linkage. Thermodynamic parameters, DH and DS, for the complex formation between linkage isomers of oligo(rA) and oligo(rU) showed a linear relationship indicating an enthalpy-entropy compensation phenomena. The duplex and triplex composed of [2′-5′] oligo(rA) and [2′-5′] oligo(rU) exhibit different CD spectra compared to those of any others containing 3–5′ linkage, suggesting that the fully 2–5′ duplex and triplex may possess a unique conformation. We describe prebiological significance of the linkage isomers of RNA and selection of the 3–5′ linkage against 2′-5 linkage.