Luminescence of stable stacking aggregates of adenine and uracil in water

Luminescence and excitation spectra of the highly luminescent stacking dimers of adenine and uracil in water solutions are studied. By the luminescence excitation spectra method it is shown that the stacking aggregates of adenine and uracil are formed with participation of rare forms of monomer molecules: N7H tautomers of adenine and the uracil molecules in rare forms of hydratation, for example molecules without H-bonds with water. The study of temperature dependence of luminescence intensity of monomers and stacking dimers of uracil has shown that stacking dimers do not dissociate even at 85 degrees C similarly as described earlier for adenine and adenosine. Stable stacking aggregates of nucleic bases are most likely to be the precursors of RNA molecules in chemical evolution. This hypothesis is supported by new data on their stability.     

Study of spectral and luminescent manifestations of the aggregation of thymine chromophores in aqueous solutions of polythymidylic acid at room temperature in connection with the task of photochemical record of information on thymine

Luminescence and excitation luminescence spectra of water solutions of polythymidylic acid at room temperature were studied. Three luminescence bands at different excitation wavelengths were observed: at 338 nm, which was known earlier, and two new bands, at 320 and 350 nm. The study of excitation luminescence spectra that have not been studied earlier led us to interpret the band at 320 nm as a band of chromophores that do not interact, the band at 338 nm as a band of photochemically most active densely packed stacking dimers (absorption band exciton splitting approximately 4000 cm(-1)), and the band at 350 nm as a band of photochemically inactive big stacking aggregates (n > or = 10, exciton splitting approximately 8000 cm(-1)). Changes in optical density at 270 nm of poly-T water solutions after consecutive irradiations with UV light at 297+302 and 248 nm were studied. The causes of incomplete reversibility are discussed.     

Effect of intramolecular interaction on the electron excitation state of nucleic acid components

A theoretical and experimental investigation of absorption and luminescence features of crystals and aggregates of nucleic acid bases were carried out. The long wavelength low intensity bands in UV-absorption nd excitation spectra, bathochromic shift of fluorescence spectra, the change of correlation between the intensity of fluorescence and phosphorescence spectra were obtained. The interpretation of these experimental results was proposed on the basis of pair interaction calculations (exciton-resonance and charge-resonance) in different conformations of cytosine dimers. The energy transfer after excitation at lambda 280 and 312 nm was investigated in nucleic acid base aggregates.     

Theoretical study of hydrophobicity and hydrophilicity of uracil and its dimers

The influence of hydrophilic and hydrophobic properties of the uracil elementary nucleic acid bases on its solubility and structure in aqueous solution was studied. Complexes of uracil with water molecules (from 1 to 14) were then calculated. The geometrical parameters of the hydrogen bridge of uracil and the changes in the frequency of valence vibrations of the bonds participating directly in hydrogen bond formation were calculated. It is shown that for the hydrogen bonds O_w?HN₁ and O_w?HN₃ the hydrogen atom can tear off, it may lead to tautomeric transformation of uracil. The results obtained having calculated the structure of uracil dimers, formed with the hydrogen bonds, in an isolated state and water solution, energy, dipole moments and the hydrogen bridge parameters made it possible to explain low solubility of uracil in water at room temperature. It is shown that water molecules with increase in their number are located mainly at one side of the plane of a pyrimidine uracil ring, that leads to the formation of stacking. Of two possible variants of stacking formation, the most profitable grouping is when a dipole moment of the formed dimer is equal to zero (anti-parallel stacking).     

A study of the spectral and luminescence manifestations of the aggregation of thymine chromophores in polythymidylic acid aqueous solutions at room temperature in the context of photochemical information recording using thymine

The luminescence and excitation spectra of polythymidylic acid aqueous solutions at room temperature were studied. In addition to the previously described band at 338 nm, two new bands at 320 and 350 nm were recorded at various excitation wavelengths. An examination of the excitation spectra that had not been studied previously, as well as their comparison with the differential absorption spectra previously recorded during photodimerization, allowed us to interpret the band at 320 nm as the band of noninteracting chromophores; the band at 338 nm as the band of the most photochemically active, densely packed stacking dimmers (exciton splitting of absorption band of ～4000 cm^?1); and the band at 350 nm as the band of photochemically inactive large stacking aggregates (n ≥ 10, exciton splitting of ～8000 cm^?1). The changes in the optical density of the polythymidylic acid aqueous solution at γ = 270 nm after successive irradiation of the solution with light at 279 + 302 and 248 nm were studied. The reasons for their incomplete reversibility are discussed.     

Nature of the stacking of nucleic acid bases in water: a Monte Carlo simulation

The results of a Monte Carlo simulation of the hydration of uracil and thymine molecules, their stacked dimers and hydrogen-bonded base pairs are presented. Simulations have been performed in a cluster approximation. The semiempirical atom-atom potential functions have been used (cluster consisting of 200 water molecules). It has been shown that the stacking interactions of uracil and thymine molecules in water arise mainly due to the increase in the water-water interaction during the transition from monomers to dimer. It has been found out that stacked base associates are more preferable than base pairs in water. This preference is mainly due to the energetically more favourable structure of water around the stack.     

Stacking and hydrogen bond interactions between adenine and gallic acid

We have performed DFT and DFT-SAPT calculations on dimers of gallic acid, the model system for plant polyphenols, and the DNA base adenine. These dimers were selected for this study as they exhibit simultaneously hydrogen bonds and stacking interactions and it allows to quantify the relative values of these interactions. We calculate the relationships between the stability of the complexes, the charge transfer between monomers and the properties of the intermolecular bonds including hydrogen bonds and other bonds that do not involve hydrogen atoms. DFT-SAPT calculations were also performed to obtain the participation of the different types of energy and so the resulting physical effects. The results show that the presence of hydrogen bonds is the main stabilizing factor for dimers: the higher number and strength, the lower the dimer energy. The contribution of stacking to the stabilization is related to the strength and number of bonds between non-hydrogen atoms and quantified by the contribution of the dispersion terms to the interaction energy. Dimers I and II are mainly stabilized due to hydrogen bonds whereas dimer III is mainly stabilized by stacking interactions.     

Fluorometric determination of adenine and its derivatives by reaction with glyoxal hydrate trimer

A fluorometric determination of adenine has been devised. It was found that adenine gives strong fluorescence when reacting with glyoxal hydrate trimer in an acidic medium. The maximum wavelengths of excitation and emission spectra were 328 and 382 mμ, respectively. This reaction was successfully applied to determination of 0.2–1.0 or 2.0–10.0 mμmoles of adenine and its derivatives, in which the hydrogen atom at position 9 is substituted with ribose or substituted ribose. Other nucleic acid bases, guanine, uracil, thymine, and cytosine, did not interfere with this fluorometric determination of adenine.     

Nature of the Stacking of Nucleic Acid Bases in Water: a Monte Carlo Simulation

Abstract

The results of a Monte Carlo simulation of the hydration of uracil and thymine molecules, their stacked dimers and hydrogen-bonded base pairs are presented. Simulations have been performed in a cluster approximation. The semiempirical atom-atom potential functions have been used (cluster consisting of 200 water molecules). It has been shown that the stacking interactions of uracil and thymine molecules in water arise mainly due to the increase in the water-water interaction during the transition from monomers to dimer. It has been found out that stacked base associates are more preferable than base pairs in water. This preference is mainly due to the energetically more favourable structure of water around the stack.     

Mechanism of ultraviolet-induced mutagenesis: the coding properties of ultraviolet-irradiated poly(dC) replicated by E. coli DNA polymerase I.

We have identified three lesions rather than cyclobutane dimers which alter the properties of UV-irradiated poly(dC) as a template for E.coli DNA polymerase I, and have characterised these lesions with respect to their coding properties, rates of formation and decay, and their sensitivity to uracil DNA glycosylase. Our results lead us to conclude that these lesions are (1) cytosine hydrates, which code for cytosine and to a lesser extent thymine, (2) uracil hydrates, which code for adenine and are not sensitive to uracil DNA glycosylase, and (3) uracils, which code for adenine and are removed by uracil DNA glycosylase.     

Relationship between absorption and fluorescence excitation spectra of psoralens and coumarins and their concentration

The shape of absorption spectra is changed and their maxima are red shifted with an increase of furocoumarin (psoralen, 8-methoxypsoralen and angelicin) concentration. Fluorescence excitation spectra of psoralen, 7-methoxycoumarin and 7-hydroxy-4-methylocumarin do not depend on the concentration in solutions. They are similar or coincident with absorption spectra of the most concentrated solutions. One may conclude the existence of different forms of furocoumarin and coumarin aggregates in solution. From the coincidence or similarity of fluorescence excitation spectra and absorption spectra in the most concentrated solutions it may be proposed that only the aggregated forms of psoralens and coumarins (the dimers or associates of higher order) are able to emit fluorescence.     

The physical basis of nucleic acid base stacking in water

It has been argued that the stacking of adenyl groups in water must be driven primarily by electrostatic interactions, based upon NMR data showing stacking for two adenyl groups joined by a 3-atom linker but not for two naphthyl groups joined by the same linker. In contrast, theoretical work has suggested that adenine stacking is driven primarily by nonelectrostatic forces, and that electrostatic interactions actually produce a net repulsion between adenines stacking in water. The present study provides evidence that the experimental data for the 3-atom-linked bis-adenyl and bis-naphthyl compounds are consistent with the theory indicating that nonelectrostatic interactions drive adenine stacking. First, a theoretical conformational analysis is found to reproduce the observed ranking of the stacking tendencies of the compounds studied experimentally. A geometric analysis identifies two possible reasons, other than stronger electrostatic interactions, why the 3-atom-linked bis-adenyl compounds should stack more than the bis-naphthyl compounds. First, stacked naphthyl groups tend to lie further apart than stacked adenyl groups, based upon both quantum calculations and crystal structures. This may prevent the bis-naphthyl compound from stacking as extensively as the bis-adenyl compound. Second, geometric analysis shows that more stacked conformations are sterically accessible to the bis-adenyl compound than to the bis-naphthyl compound because the linker is attached to the sides of the adenyl groups, but to the ends of the naphthyl groups. Finally, ab initio quantum mechanics calculations and energy decompositions for relevant conformations of adenine and naphthalene dimers support the view that stacking in these compounds is driven primarily by nonelectrostatic interactions. The present analysis illustrates the importance of considering all aspects of a molecular system when interpreting experimental data, and the value of computer models as an adjunct to chemical intuition.     

Study of the interaction of DNA and acridine orange by various optical methods

The degree of binding of acridine orange to DNA, native or denatured, has been determined by equilibrium dialysis in 0.1M and 0.001M NaCl at 20°. The nature of the binding process has been investigated by studying various optical properties of the dye–DNA complexes and by relating them to the binding ratio. All these properties were found to vary quantitatively and qualitatively according to the successive stages of the process. These stages were assumed to be a strong binding of intercalated monomers followed by formation of bound dimers and finally by external binding of aggregates of native DNA. Absorption spectra of the complexes could be interpreted on that basis. Circular dichroism spectra were resolved into components: one band for intercalated monomers without interactions, two excition splittings for interacting monomers and bound dimers, respectively, weak bands and exciton splitting for external aggregates. The fluorescence intensity was greatly enhanced in intercallated monomers; its quenching at higher binding ratio was quantitatively related to dimer fixation. The value of the anisotropy of fluorescence at low binding ratio suggested a limited mobility of intercalated monomers; the decrease of polarization at higher binding was attributed to energy transfer between monomers. Electric dichroism displayed by the complexes in the dye absorption bands indicated an orientation of the bound molecules quite parallel to the base rings at low binding. In the range of fixation of dimers and external molecules, the dichroism was lower but still indicated an important degree of ordering.     

Migration of electron excitation energy in mixed associates of chlorophyll and its derivatives]

The intracomplex migration is investigated of electron excitation energy between molecules which are a part of a mixed associate. Weakly fluorescent and nonfluorescent associates of chlorophyll and its analogues as well as of bacteriochlorophyll and neoxanthine have been used as donors and acceptors. The migration of excitation energy with variation of quantum yield of donor emission from 5-10(-3) to less than or equal to 10(-4) and also with very weak overlapping of luminescence and absorption spectra for the pair D leads to A is experimentally discovered by luminescence excitation spectra of mixed pigment aggregates. A specific disturbing action of Pd and Cu ions on chlorophyll a luminescence with formation of mixed complexes of chlorophyll with Pd-pheophytin a and Cu-pheophytin b is observed. The presence of Pd and Cu ions in the structure of the mixed complexes gives rise to a considerable reduction of chlorophyll apparently due to increasing intersystem crossing probability in the chlorophyll molecule. The probability of intracomplex energy migration in systems under investigation amounts to approximately 10(12) sec-1 according to the obtained estimations. The probable mechanism of energy migration in mixed associates is discussed. The obtained results may be used for elucidation of a possible role of the mixed aggregates and effective intracomplex migration of excitation energy in photosynthetic apparatus of green plants.     

A proton magnetic resonance study of the interaction of adenylyl (3' is greater than 5') adenosine with polyuridylic acid

The interaction of adenylyl (3′ → 5′) adenosine (ApA) with polyuridylic acid in D₂O solution at neutral pD has been studied by high resolution proton magnetic, resonance spectroscopy. At temperatures above ～32°C, no evidence was obtained for the interaction of ApA with poly U. Below this temperature, a rigid triple-stranded complex involving a stoichiometry of 1 adenine to 2 uracil bases is formed, presumably via specific adenine–uracil base-pairing and cooperative base stacking of the adenine bases in a manner similar to that previously reported for the adenosine–poly U complex.     

Circular dichroism of the nucleic acid monomers.

Circular dichroism spectra of the nucleic acid monomers have been measured in aqueous solution and extended into the vacuum ultraviolet region to about 166 nm. Measurements were made on ribo and deoxyribo derivatives of adenine, guanine, hypoxanthine, cytosine, thymine, and uracil derivatives both with and without the 5′-phosphate (with the exception of ribosyl thymine 5′-phosphate). Absorption spectra of the deoxyribonucleotides measured to about 175 nm are also presented. The results demonstrate that both the circular dichroism and absorption spectra observed below 200 nm are no more complicated than the spectra normally recorded above 200 nm. In most cases, the circular dichroism spectra of the various derivatives of a given base are similar, indicating that the conformations are similar. On the other hand, the differences among the circular dichroism spectra of the various derivatives of a given base are sufficient to identify a particular derivative. The average circular dichroism for the deoxyribonucleotides is compared with the circular dichroism of native E. coli DNA. The comparison reveals that the circular dichroism of DNA below 200 nm is due principally to the interaction between the bases rather than the intrinsic circular dichroism of the monomers. The monomer transitions are discussed in relationship to the absorption and circular dichroism spectra presented.     

Optically detected zero field magnetic resonance characterization of a bromine atom-containing polynucleotide, poly(dA-br5dU)

Phosphorescence and optically detected zero field magnetic resonance ( ODMR ) spectra are reported for a bromine atom-containing polynucleotide, poly(dA- br5dU ). The triplet state luminescence of poly(dA- br5dU ) is dominated by the phosphorescence of the bromouracil base which possesses sub-millisecond triplet lifetimes. Characteristic multiple slow passage ODMR transitions, which are observed in both br5dUrd and poly(dA- br5dU ), are assigned to the triplet state of bromouracil. In addition, an abnormally-perturbed adenine triplet state, which is not apparent in the phosphorescence spectrum of poly(dA- br5dU ), is detected and identified by its slow passage ODMR and amplitude-modulated phosphorescence microwave double resonance spectra. It is proposed that the perturbed adenine is a minor component of the polynucleotide structure which is present in regions of altered stacking induced by the high polarizability of the Br atom.     

Interaction of phycobilisomes with photosystem II dimers and photosystem I monomers and trimers in the cyanobacterium Spirulina platensis.

Distribution of phycobilisomes between photosystem I (PSI) and photosystem II (PSII) complexes in the cyanobacterium Spirulina platensis has been studied by analysis of the action spectra of H2 and O2 photoevolution and by analysis of the 77 K fluorescence excitation and emission spectra of the photosystems. PSI monomers and trimers were spectrally discriminated in the cell by the unique 760 nm low-temperature fluorescence, emitted by the trimers under reductive conditions. The phycobilisome-specific 625 nm peak was observed in the action spectra of both PSI and PSII, as well as in the 77 K fluorescence excitation spectra for chlorophyll emission at 695 nm (PSII), 730 nm (PSI monomers), and 760 nm (PSI trimers). The contributions of phycobilisomes to the absorption, action, and excitation spectra were derived from the in vivo absorption coefficients of phycobiliproteins and of chlorophyll. Analyzing the sum of PSI and PSII action spectra against the absorption spectrum and estimating the P700:P680 reaction center ratio of 5.7 in Spirulina, we calculated that PSII contained only 5% of the total chlorophyll, while PSI carried the greatest part, about 95%. Quantitative analysis of the obtained data showed that about 20% of phycobilisomes in Spirulina cells are bound to PSII, while 60% of phycobilisomes transfer the energy to PSI trimers, and the remaining 20% are associated with PSI monomers. A relevant model of organization of phycobilisomes and chlorophyll pigment-protein complexes in Spirulina is proposed. It is suggested that phycobilisomes are connected with PSII dimers, PSI trimers, and coupled PSI monomers.     

Spectroscopic investigations of the potential-sensitive membrane probe RH421.

The absorbance spectra, fluorescence emission and excitation spectra, and fluorescence anisotropy of the potential-sensitive styryl dye RH421 have been investigated in aqueous solution and bound to the lipid membrane. The potential-sensitive response of the dye has been studied using a preparation of membrane fragments containing a high density of Na+, K(+)-ATPase molecules. In aqueous solution the dye is sensitive both to changes in pH and ionic strength. Evidence has been found that the dye readily aggregates in aqueous solution. Aggregation is enhanced by an increase in ionic strength. The aggregates formed display a low fluorescence intensity. At high pH values (above approx. 8) changes in the dye's fluorescence spectra are observed, which may be due to a reaction of the dye with hydroxide ions. When bound to the membrane the dye also exhibits concentration-dependent fluorescence changes. The potential-sensitive response of the dye in Na(+),K(+)-ATPase membrane fragments after addition of MgATP in the presence of Na+ ions cannot be explained by a purely electrochromic mechanism. The results are consistent with either a potential-dependent equilibrium between membrane-bound dye monomers and membrane-bound dimers, similar to that previously proposed for the dye merocyanine 540, or with a field-induced structural change of the membrane.     

The Structure of Triple Helical Poly(U)·Poly(A)·Poly(U) Studied by Raman Spectroscopy

Abstract

Using Raman spectroscopy, we examined the ribose-phosphate backbone conformation, the hydrogen bonding interactions, and the stacking of the bases of the poly(U)·poly(A) ·poly(U) triple helix. We compared the Raman spectra of poly(U)·poly(A)·poly(U) in H₂O and D₂O with those obtained for single-stranded poly(A) and poly(U) and for double-stranded poly(A)·poly(U). The presence of a Raman band at 863 cm^?1 indicated that the backbone conformations of the two poly(U) chains are different in the triple helix. The sugar conformation of the poly(U) chain held to the poly(A) by Watson-Crick base pairing is C3′ endo; that of the second poly(U) chain may be C2′ endo. Raman hypochromism of the bands associated with base vibrations demonstrated that uracil residues stack to the same extent in double helical poly(A)·poly(U) and in the triple-stranded structure. An increase in the Raman hypochromism of the bands associated with adenine bases indicated that the stacking of adenine residues is greater in the triple helix than in the double helical form. Our data further suggest that the environment of the carbonyls of the uracil residues is different for the different strands.