Kinetic and hydrodynamic studies of the complex of proflavine with poly A·poly U

Relaxation kinetic experiments reveal general similarity between the mechanism of binding of proflavine to poly A·poly U and DNA. There are differences in detail, however. For example, the rate constants are roughly an order of magnitude smaller for the former, and the thermodynamic parameters of the individual steps are also different. The total heat and free energy for intercalation of free dye are quite similar in the two cases. As was the case with DNA, considerable dye (up to 25% of the bound form) is attached externally to the double helix, even in the strong binding region of the isotherm. Sedimentation measurements on small, rodlike fragments of poly A·poly U reveal a length increase on binding proflavine of a magnitude similar to that found with DNA. This length increase seems to become smaller under conditions (high temperature) where the relaxation measurements indicate a higher fraction of externally bound dye.     

Raman studies of nucleic acids. VII. Poly A-poly U and poly G-poly C

Laser-excited Raman spectra of the double-helical complexes poly A·poly U and poly G·poly C are reported for ²H₂O and H₂O solutions. The spectra are discussed in relation to their use as quantitative reference spectra for determining the dependence of the Raman scattering of RNA on secondary structure. The Raman line at 815 cm^?1, due to the phosphodiester group, exhibits the same intrinsic intensity in spectra of poly A·poly U and poly G·poly C and is thus dependent only upon the amount of ordering of the helix and not on the kinds of nucleotides involved. The hypochromic Raman lines in spectra of poly A·poly U are identified and their intensity changes are determined quantitatively over the temperature range 32–85°C. Comparison of the spectra in the 1500–1750 cm^?1 region reveals that the Raman lines from carbonyl group vibrations of uracil are about sevenfold more intense than those of guanine and cytosine for both paired and unpaired states and will thus dominate the spectra of RNA. The Raman frequencies in this region are also compared with previously reported infrared frequencies and give evidence of being strongly perturbed by base-stacking interactions in the helices.     

The interaction of Na ions with synthetic polynucleotides

The interaction of Na⁺ with poly A, poly U, poly A·poly U, and Poly A·2 poly U has been investigated by means of potentiometry, by means of potentiometry, by means of a linked-function analysis of its effect on the binding of Mg⁺⁺ ions, and of K⁺ by means of an analysis of its effect on the sedimentation coefficients of the polymers. The last method was found to be inapplicable. The results of the other two methods were found to be consistent, except in the case of poly A where the existence of base stacking, influenced by the binding of Mg⁺⁺, significantly affects the linked-function analysis. The results are also consistent with the effects of the concentration of Na⁺ ions on the thermally induced conformational transitions of poly A·poly U and poly A·2 poly U, and with the extents of “binding” of Na⁺ to DNA measured by equilibrium and by transport methods. The interaction of Na⁺ with polynucleotides appears to be physically quite specific, although its thermodynamic basis is not clear. The extent of binding of Na⁺, Ψ, was found to be independent of the total Na⁺ concentration but a quadratic function of the extent of Mg⁺⁺ binding, θ. In the absence of Mg⁺⁺, Ψ = 0.35–0.38 for poly U, 0.40 for poly A, 0.59 for poly A·poly U, and 0.66 for poly A·2 poly U.     

Hydrogen exchange kinetics of nucleic acids: Double and triple helices with Hoogsteen-type basepairs

The kinetics of hydrogen-tritium exchange reaction have been followed by a Sephadex technique of a double-helical poly(ribo-2-methylthio-adenylic acid)·poly(ribouridylic acid) complex with the Hoogsteen-type basepair. Only one hydrogen in every 2-methylthio-adenine·uracil basepair has been found to exchange at a measurably slow rate, 0.023 s^?1 (at 0°C), which is, however, much greater than that for a double-helix with the Watson-Crick type A·U pair. The kinetics of hydrogen-tritium exchange were also examined by triple-helical poly(rU)·poly(rA)·poly(rU) which involves both the Watson-Crick and Hoogsteen basepairings. Here, three hydrogens in every U·A·U base triplet have been found to exchange at a relatively slow rate, 0.0116 s^?1 (at 0°C). The kinetics of hydrogen-deuterium exchange reactions of these polynucleotide helices have also been followed by a stopped-flow ultraviolet absorption spectrophotometry at various temperatures. On the basis of these experimental results, the mechanism of the hydrogen exchange reactions in these helical polynucleotides was discussed. In the triple helix, the rate-determining process of the slow exchange of the three (one uracil-imide and two adenine-amino) hydrogens is considered to be the opening of the Watson-Crick part of the U·A·U triplet. This opening is considered to take place only after the opening of the Hoogsteen part of the triplet.     

Extrinsic Cotton effects of proflavine bound to polynucleotides

The magnitude of the Cotton effect of proflavine which is bound to RNA or to denatured DNA depends on the ratio of bound proflavine to nucleic acid base. A statistical treatment which explains this behavior has been fitted to the experimental curves and indicates that optical activity arises through interaction between two or more bound proflavine molecules. The corresponding requirement with double helical DNA is for interaction between 3–4 proflavine molecules. Although proflavine binds to denatured DNA at pH 2.8, as shown by the shift of the proflavine spectrum, the strong binding process is absent, and to this is attributed the absence of the Cotton effect at low pH. Studies on the Cotton effects of proflavine bound to poly A and poly U at neutral pH, to poly A at acid pH and to poly (A + U) allow the generalization that a relatively rigid configuration of the binding macromolecule is required for the induction of these extrinsic Cotton effects.     

Circular dichroism calculations for double-stranded polynucleotides of repeating sequence

Optical property calculations are presented for poly(A·U), poly[(A-U)·(A-U)], poly(G·C), and poly[(G-C)·(G-C)] in RNA, B-DNA, and C-DNA conformations. An all-order classical coupled oscillator polarizability theory was used, and an effective dielectric constant of 2 was assumed. The calculated CD spectra were found to be sensitive to both geometry and sequence. Agreement with the measured CD spectra of poly(A·U), poly(G·C), and poly(dG·dC) is very good. Calculations for other sequences and geometries are less satisfactory and are particularly poor for poly[(G-C)·(G-C)] in RNA geometry and poly(A·T) in B-DNA geometry. Attempts to improve agreement with measured spectra by varying monomer properties have been only partially successful for these calculations, but they illustrate the types of changes that may prove to be necessary. Calculations using other published X-ray coordinates for certain deoxypolynucleotides of simple sequence, some of which are quite different from B-DNA coordinates, did not result in better agreement with measured spectra. Finally, the dependence of the calculated CD on chain length is examined. Results show that non-nearest neighbor interactions can be important when runs of 3 or more identical base pairs appear in a given sequence.     

Sequence and conformational effects on imino proton exchange in A.T- and A.U-containing DNA and RNA duplexes

¹H-nmr relaxation has been used to study the effect of sequence and conformation on imino proton exchange in adenine–thymine (A · T) and adenine–uracil (A · U) containing DNA and RNA duplexes. At low temperature, relaxation is caused by dipolar interactions between the imino and the adenine amino and AH2 protons, and at higher temperature, by exchange with the solvent protons. Although room temperature exchange rates vary between 3 and 12s^?1, the exchange activation energies (E_α) are insensitive to changes in the duplex sequence (alternating vs homopolymer duplexes), the conformation (B-form DNA vs A-form RNA), and the identity of the pyrimidine base (thymine vs uracil). The average value of the activation energy for the five duplexes studied, poly[d(A-T)], poly[d(A) · d(T)], poly[d(A-U)], Poly[d(A) · d(U)], and poly[r(A) · r(U)], was 16.8 ± 1.3 kcal/mol. In addition, we find that the average E_α for the A.T base pairs in a 43-base-pair restriction fragment is 16.4 ± 1.0 kcal/mol. This result is to be contrasted with the observation that the E_α of cytosine-containing duplexes depends on the sequence, conformation, and substituent groups on the purine and pyrimidine bases. Taken together, the data indicate that there is a common low-energy pathway for the escape of the thymine (uracil) imino protons from the double helix. The absolute values of the exchange rates in the simple sequence polymers are typically 3–10 times faster than in DNAs containing both A · T and G · C base pairs.     

The physicochemical essence of the purine·pyrimidine transition mismatches with Watson-Crick geometry in DNA: A·C* versa A*·C. A QM and QTAIM atomistic understanding

It was established for the first time by DFT and MP2 quantum-mechanical (QM) methods either in vacuum, so in the continuum with a low dielectric constant (ε = 4), typical for hydrophobic interfaces of specific protein-nucleic acid interactions, that the repertoire for the tautomerisation of the biologically important adenine·cytosine* (A·C*) mismatched DNA base pair, formed by the amino tautomer of the A and the imino mutagenic tautomer of the C, into the A*·C base mispair (?G = 2.72 kcal?mol^?1 obtained at the MP2 level of QM theory in the continuum with ε = 4), formed by the imino mutagenic tautomer of the A and the amino tautomer of the C, proceeds via the asynchronous concerted double proton transfer along two antiparallel H-bonds through the transition state (TS_A·C*?A*·C). The limiting stage of the A·C*→A*·C tautomerisation is the final proton transfer along the intermolecular N6H···N4 H-bond. It was found that the A·C*/A*·C DNA base mispairs with Watson–Crick geometry are associated by the N6H?N4/N4H?N6, N3H?N1/N1H?N3 and C2H?O2 H-bonds, respectively, while the TS_A·C*?A*·C is joined by the N6–H–N4 covalent bridge and the N1H?N3 and C2H?O2 H-bonds. It was revealed that the A·C*?A*·C tautomerisation is assisted by the true C2H?O2 H-bond, that in contrast to the two others conventional H-bonds exists along the entire intrinsic reaction coordinate (IRC) range herewith becoming stronger at the transition from vacuum to the continuum with ε = 4. To better understand the behavior of the intermolecular H-bonds and base mispairs along the IRC of the A·C*?A*·C tautomerisation, the profiles of their electron-topological, energetical, geometrical, polar and charge characteristics are reported in this study. It was established based on the profiles of the H-bond energies that all three H-bonds are cooperative, mutually strengthening each other. The nine key points, providing a detailed physicochemical picture of the A·C*?A*·C tautomerisation, were revealed and thoroughly examined along the IRC. It was shown that the A*·C base mispair with the population ~1 % obtained at the MP2 level of QM theory in the continuum with ε = 4 is thermodynamically and dynamically stable structure. Its lifetime was calculated to be 5.76·10^?10 s at the MP2 level of QM theory in the continuum with ε = 4. This lifetime, from the one side, enables all six low-frequency intermolecular vibrations to develop, but, from the other side, it is by order less than the time (several ns) required for the replication machinery to forcibly dissociate a base pair into the monomers during DNA replication. This means that the A*·C base mispair “slips away from the hands” of the replication machinery into the A·C* mismatched base pair. Consequently, the authors came to the conclusion that exactly the A·C* base mispair is an active player of the point mutational events and is effectively dissociated by the replication machinery into the A and C* monomers in contrast to the A*·C base mispair, playing the mediated role of a provider of the A·C* base mispair in DNA that is synthesised.     

Study of thermal and acid denaturation of DNA by means of voltammetry at graphite electrodes

Conformational changes in guanine–cytosine (G·C) and adenine–thymine (A·T) pairs in DNA were investigated by means of differential pulse voltammetry at a pyrolytic graphite electrode (PGE). As a monitor of these conformational changes, two separated voltammetric peaks, G and A, which correspond to electrochemical oxidation at the PGE of guanine and adenine residues, respectively, were used. It was found that peak A was first increased in the course of thermal denaturation of DNA. This indicates that, on heating a native DNA sample, regions rich in A·T pairs melt first. In the course of acid denaturation of a native DNA sample, the height of peak A was changed just before the denaturation. It is suggested that protonation of adenine residues in DNA regions rich in A·T pairs was responsible for these changes.     

Fluorescence analysis of G·C versus A·T binding of quinacrine to DNA

The affinity of quinacrine for native DNA has been determined from fluorescence measurements and equilibrium dialysis in Tris-HC10.05 m, NaCl0.1 m, EDTA 10^?3m, pH 7.5. When considering M. lysodeiktikus, E. coli calf thymus and C. perfringens the affinities of DNA for quaniactive have been found to change by a factor of two and the fluorescence intensities to change by a factor of 25. The varying affinities and fluoroescence intensities of bound quinacrine are attributed to heterogeneous binding. For all DNAs we have assumed that there exist three classes of intercalation sites: I, A·T-A·T; 2, G·C-G·C; and 3, A·T-G·C, assuming that base pair ordering is less relevant than base composition of sites. By fitting the affinities of native DNAs with this model it was found that quinacrine binds to site 2 three times more strongly than it does to site 1. When flucrescence intensity is studied, triplets of A·T pairs appear to be responsible for the high quantum yield of A·T rich DNA whereas the quenching properties of a G·C base pair adjacent to an intercalated quinacrine are well known.     

Kinetic analysis of the annealing period in the formation of the poly(A)·2poly(U) triple helix

The slow kinetics of annealing processes in multistranded nucleic acids is spectrophotometrically investigated using poly(A)·2poly(U) as a model system. The absorbance changes at specific wavelengths show that double-helical (A·U) base pairs appear as transient intermediates. The annealing process is identified by the enlargement of triple-helical sequences at the cost of (A·U) base pairs and unpaired (U) residues. A large time range in the reorganization of mismatched chain configurations is characterized by a logarithmic dependence on time. This observation is quantitatively described by a kinetic model developed by Jackson. In Jackson's model the rate-limiting process in the slow annealing phase of maximizing triple-helical sequences, is the removal of strand entanglements, knots, and hairpin loops by complete unwinding of those helical stretches which stabilize the mismatched configurations. The results of the present study are briefly discussed in terms of optimum conditions for hybridization experiments and for the preparation of polynucleotide complexes commonly used to produce interferons.     

Mutagen–nucleic acid complexes at the polynucleotide duplex level in solution: Intercalation of proflavine into poly(dA-dT) and the melting transition of the complex

The nmr chemical shifts and line widths of the nucleic acid base and sugar proton resonances and the proflavine ring protons can be monitored through the melting transition of the proflavine + poly(dA-dT) complex, phosphate/dye (P/D) ratio = 24 and 8 in 1M salt solution. The nucleic acid and mutagen protons in the complex are in fast exchange between duplex and strand states with the midpoint of the melting transition monitored at the nucleic acid resonances increasing from 72.6°C for poly(dA-dT) to 78.1°C for the P/D = 24 complex and 83.4°C for the P/D = 8 complex in 1M salt solution. The melting transition monitored by the proflavine resonances were 80.0°C for the P/D = 24 complex and 84.3°C for the P/D = 8 complex in 1M salt solution. Since the nucleic acid is in excess at high P/D ratios, the nucleic acid transitions are an average for the opening of mutagen-free and mutagen-bound base-pair regions, while the proflavine transitions monitor the melting of mutagen-bound base-pair regions. The observed 0.75 to 0.95 ppm unfield shift at all four proflavine protons on formation of the complex with poly(dA-dT) provides direct evidence for intercalation of the mutagen between base pairs of the nucleic acid duplex. We have deduced the approximate overlap geometry between the proflavine ring and nearest-neighbor base pairs at the intercalation site from a comparison between experimental proflavine complexation shifts and those calculated for various stacking orientations. The experimental chemical shift of the poly(dA-dT) adenine H-2 resonance in the duplex state in the absence and presence of proflavine suggests that intercalation occurs preferentially at dT-dA sites. The selective chemical shift changes at the sugar H-2′,2″ and H-3′ resonances of the poly(dA-dT) duplex on complex formation demonstrates changes in the sugar pucker and/or torsion angles of the sugar phosphate backbone at the intercalation site.     

New information content in RNA base pairing deduced from quantitative analysis of high-resolution structures

Non-canonical base pairs play important roles in organizing the complex three-dimensional folding of RNA. Here, we outline methodology developed both to analyze the spatial patterns of interacting base pairs in known RNA structures and to reconstruct models from the collective experimental information. We focus attention on the structural context and deformability of the seven pairing patterns found in greatest abundance in the helical segments in a set of well-resolved crystal structures, including (i–ii) the canonical A·U and G·C Watson–Crick base pairs, (iii) the G·U wobble pair, (iv) the sheared G·A pair, (v) the A·U Hoogsteen pair, (vi) the U·U wobble pair, and (vii) the G·A Watson–Crick-like pair. The non-canonical pairs stand out from the canonical associations in terms of apparent deformability, spanning a broader range of conformational states as measured by the six rigid-body parameters used to describe the spatial arrangements of the interacting bases, the root-mean-square deviations of the base-pair atoms, and the fluctuations in hydrogen-bonding geometry. The deformabilties, the modes of base-pair deformation, and the preferred sites of occurrence depend on sequence. We also characterize the positioning and overlap of the base pairs with respect to the base pairs that stack immediately above and below them in double-helical fragments. We incorporate the observed positions of the bases, base pairs, and intervening phosphorus atoms in models to predict the effects of the non-canonical interactions on overall helical structure.     

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).     

Crystallographic studies of drug-nucleic acid interactions: Proflavine intercalation between the non-complementary base-pairs of cytidilyl-3′,5′-adenosine

In order to get insights into the binding of dyes and mutagens with denatured and single-stranded nucleic acids and the possible implications in frameshift mutagenesis, a 1:1 complex between the non-self-complementary dinucleoside monophosphate cytidilyl-3′,5′-adenosine (CpA) and proflavine was crystallized. The crystals belong to the tetragonal space group P4₂2₁2 with cell constants $\text{a = b = 19.38(1)}\text{A}\text{?}\text{and}\text{c = 27.10(1)}\text{A}\text{?}$. The asymmetric unit contains one CpA, one proflavine and nine water molecules by weight. The structure was determined using Patterson and direct methods and refined to an R-value of 11% using 2454 diffractometer intensities.The non-self-complementary dinucleoside monophosphate CpA forms a selfpaired parallel chain dimer with a proflavine molecule intercalated between the protonated cytosine-cytosine (C · C) pair and the neutral adenine-adenine (A · A) pair. The dimer complex exhibits a right-handed helical twist and an irregular girth. The neutral A · A pair is doubly hydrogen-bonded through the N(6) and N(7) sites (C(1′)C(1′) distance: 10.97(2) Å) and the protonated C · C pair is triply hydrogen-bonded with a proton shared between the N(3) sites (C(1′)C(1′) distance: 9.59(2) Å). To accommodate the intercalating dye, the sugars of successive nucleotide residues adopt the two fundamental conformations (5′ end: 3′-endo, 3′ end: 2′-endo), the backbone adopts torsion angle values that fluctuate within their preferred conformational domains: the PO bonds (ω, ω′) adopt the characteristic helical (gauche^?-gauche^?) conformation, the CO bonds (φ, φ′) are both in the trans domain and the C(4′)C(5′) bonds (ψ) are in the gauche⁺ region. The bases of both residues are disposed in the preferred anti domain with the glycosyl torsion angles (χ) correlated to the puckering mode of the sugar so that the cytidine residue is C(3′)-endo, low χ (12 dg), and the adenosine residue is C(2′)-endo, high χ (84 °). The intercalated proflavine stacks more extensively with the C · C pair than the A · A pair. Between 4₂-related CpA proflavine units there is a second proflavine which stacks well with both the A · A and the C · C pairs sandwiching it. Both proflavine molecules are positionally disordered. In each of its two disordered sites, the intercalated proflavine forms hydrogen-bonded interactions with only one sugar-phosphate backbone. A total of 26 water sites has been characterized of which only two are fully occupied. These hydration sites are involved in an intricate network of hydrogen bonds with both the dye and CpA and provide insights on the various modes of interactions between water molecules and between water molecules and nucleic acids.The structure of the proflavine-CpA complex shows that intercalation of planar drugs can occur between non-complementary base-pairs. This result can be relevant for understanding the strong binding of acridine dyes to denatured DNA, single-stranded RNA, and single-stranded polynucleotides. Also, the ability of proflayine to promote self-pairs of adenine and cytosine bases could provide a chemical basis for an alternative mechanism of frameshift mutagenesis.     

Induced circular dichroism of DNA-dye complexes

The binding of methylene blue, proflavine, and ethidium bromide with DNA has been studied by spectrophotometric titration. Methylene blue and proflavine or methylene blue and ethidium bromide were simultaneously titrated by DNA. The results indicate that all of these dyes compete for the same bindine sites. The binding properties are discussed in terms of symmetry. The optical properties of the dye–DNA complexes have been studied as a function of DNA/dye ratio. The induced circular dichriosm due to dye–dye interaction was measured at low dye/DNA ratios for cases involving both the same dye and different dyes. A positive Cotton effect for DNA–proflavine complex may be induced at 465 mμ by eithr proflavine or ethidium bromide, whereas a netgative Cotton effect at 465 mμ may be induced by methylene blue. The limiting circular dichroism, with no dye–dye interaction, and the induced circular dichroism spectra are discussed in terms of symmetry rules.     

Fluorescence of proflavine--DNA complexes: heterogeneity of binding sites

Measurements of the relative quantum yield of fluorescence of proflavine bound to DNA as a function of the number of bound dyes per nucleotide and the ionic strength allow the determination of the binding constants and respective number of the two types of sites previously postulated. It is demonstrated that 2–3% of the base pairs form sites where the dye is strongly bound and fluoresces normally while in the other set of sites the binding constant is 3–4 times weaker and the fluorescence completely quenched. Comparison with complexes of Pro with double stranded polynucleotides poly (A + U), poly (I + C), poly(G + C), confirm that the strong binding sites correspond to A-T-rich regions of the DNA while the quenched sites seem to require the presence of a neighboring guanine. The role of charge transfer in quenching of fluorescence and mutagnic action is considered. An original method for the determination of free dye and bound dye, based upon the use of an external quencher is described in the Appendix.     

Effect of DNA base composition on the intercalation of proflavine. A kinetic study.

The effect of DNA base composition on the kinetics of the association between DNA and proflavine has been investigated using the temperature jump relaxation method. It is found that, regardless of the G + C base composition the results fit a two step mechanism, the second of which exhibits characteristics of intercalation of proflavine into DNA. However, they two equilibrium constants corresponding to these steps, KI and KII, depend on the nature of the DNAs. The constant KI is found to be an order of magnitude greater for M. lysodeikticus DNA (72% G + C) than for calf thymus DNA (48% G + C). Increasing G-C content thus appears to favor the intermediate non-intercalated complex of proflavine with DNA. Methylation of M. lysodeikticus DNA with dimethyl sulfate, preferentially yielding N7 methyl guanine as the modified base, again leads to an apparent two step mechanism, with the value of KI unchanged with respect to untreated DNA, while the affinity of proflavine for the intercalated complex measured by the value of KII increases for methylated DNA.     

The structure of triple-stranded G-2C polynucleotide helices.

The thermal stability of a new polynucleotide complex has been used to establish the hydrogen-bonding structure of three-stranded C-G·CH⁺ helices. In the Hoogsteen structure, the 8NH₂ group of 8NH₂GMP can form a third hydrogen bond to the CH⁺ strand, but in the alternative structure, the 8NH₂ group can form no interbase hydrogen bonds. For the new complex, 8NH₂GMP·2 poly(C), a transition temperature of 80°C is observed under conditions in which the corresponding complex formed with 5′-GMP has a T_m of 20°C. We conclude from this 60° elevation of transition temperature that a third hydrogen bond is formed by the 8NH₂ group and that the structure must have Hoogsteen bonding. In order to be compatible with this structure in regular helices formed by U,C copolymers, A·2U bonding would also have to have a Hoogsteen structure.