Prediction of 1H NMR chemical shifts of DNA oligomers

A set of parameters, devised for the prediction of 1H NMR chemical shifts of heterobase and anomeric protons in the high temperature (greater than 70 degrees C) spectra of RNA oligomers has been found to be applicable to the corresponding DNA oligomers. Fifteen examples of DNA oligomers that have had high temperature spectra recorded and assigned show a mean absolute difference between predicted and assigned shifts of 0.045 ppm. The parameters for uridine H-5 are applied to the calculation of thymidine methyl group shifts and give excellent agreement with experimental assigned shifts. The RNA parameter set is a practical means of assigning heterobase and anomeric protons in DNA oligomers. A programme using the RNA parameter set has been written which enables the sequence of short DNA oligomers to be predicted from their 1H NMR spectra.     

Glutaraldehyde: nature of the reagent

Aqueous solutions of glutaraldehyde used for the chemical modification and stabilization of proteins have been found to consist of free glutaraldehyde (I), the cyclic hemiacetal of its hydrate (II) and oligomers of this (III) in equilibrium with each other. Ultraviolet absorption spectra of these solutions at wavelengths greater than 200 nm should have only a single maximum at 280 nm. Absorption at 235 nm is due to an impurity which can be removed by various means. Reactions of the reagent with proteins involve principally the lysinyl (and hydroxylysinyl) residues in the relative amounts of four moles of glutaraldehyde to one of lysine. Three unstable products can be partially isolated from acid hydrolyzates of glutaraldehyde-treated proteins or from the reaction mixtures of glutaraldehyde and model compounds; two of these products have strong ultraviolet absorption near 265 nm.     

Thermal difference circular dichroism of Pf1 filamentous virus and effects of mercury(II), silver(I), and copper(II)

The circular dichroism (CD) of Pfl filamentous virus has been examined over the temperature range 0-40 degrees C, in the absence and presence of Hg(II), Ag(I), and Cu(II). Thermal difference CD spectra were obtained by subtraction of spectra recorded above and below a thermally induced structure transition near 12 degrees C. The thermal difference spectra look like they arise from shifts in two exciton bands, one centered at 230 nm and the other at 290 nm. The amplitudes on either side of a crossover at 230 nm are 10 times those of a crossover at 290 nm. It is proposed that the difference spectra result from thermally induced shifts in coupled oscillator interactions between Tyr40 residues of the coat protein and the guanine and cytosine bases of the DNA. Metal ions can reduce or block these shifts. The changes in ellipticities at 220, 237, and 270 nm induced by changing the temperature have inflections near 12 degrees C. Ag(I) and Hg(II), which are known to bind to the DNA bases in Pfl, reduce or eliminate the inflections in the thermal profiles, depending on the metal ion type and concentration. Cu(II) ions do not affect the profiles. The spectral changes and the effects of the metal ions indicate intimate contact between the DNA bases and the protein subunits in the virion.     

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.     

Interaction of ethidium bromide with DNA. Optical and electrooptical study

The binding of ethidium bromide to DNA has been studied by various optical methods. From fluorescence polarization studies, and film, electric linear dichroism, and circular dichroism spectra, we propose assignments of the absorption bands of the dye, which are discussed in connection with wave-mechanical calculations recently reported. The optical activity induced in the dye absorption bands upon binding to DNA was attributed to various origins depending on the electronic transition considered. The visible absorption band displayed a circular dichroism due to the asymmetry of the binding site and independent of the amount of binding. The transition identified at 378 nm from the circular dichroism and electric dichroism observations was thought to be due to a magnetic-dipole transition. It remained constant with increasing amounts of dye bound. The main ultraviolet band showed circular dichroism characteristics corresponding to exciton interactions between dye molecules bound to neighboring sites. The electric dichroism observed for the strongly bound dye molecules indicated that the phenanthridinium ring of ethidium bromide was probably not perfectly parallel to the DNA base planes. When the amount of dye bound to DNA exceeded the maximum amount compatible with the exclusion of adjacent binding sites, the electric dichroism decreased owing to the appearance of externally bound dye molecules with no contribution to the dichroism. Sonicated DNA was used to study the lengthening of the DNA molecule upon complexation. Although the viscosity of the complexes increased with the amount of binding, the rotational diffusion coefficient measured by the electric birefringence relaxation was not detectably affected. The absence of variation in the electric birefringence with the binding indicated that the DNA base stacking remained unaltered.     

Analysis of exciton parameters in DNA. Exciton waves in DNA as one of the reasons of mutagenesis

Formulae were obtained for the quantitative analysis of the following parameters of excitons in DNA: 1) the lifetime of electronic excitation; 2) the numbers of exciton runs along the DNA sequence; 3) the energy loss by an exciton for one run; 4) the maximum length of the DNA sequence capable of deactivating an exciton for one run. The maximum and minimum ranges for the constant of electronic excitation migration was determined to meet the requirement of inductive-resonance energy transfer for the case of strong interaction. The constant of exciton energy migration was shown to depend on the activation energy, which is equal to the energy of absorbed quantum. An analytical formula was derived to determine the number of quanta the DNA molecule is able to absorb, depending on its length, without nonlinear effects (without overlapping of spatial areas of electronic excitation). By this formula, DNA sequences consisting of only identical AT and GC nucleotide pairs and aggregate AT + GC (in the ratio 1:1) DNA sequences ranging from 1 up to 10(10) base pairs were analyzed. The results of the analysis suggest that the overlapping of spatial areas of electronic excitation induced by a single ultraviolet quantum occurs in short DNA sequences characteristic of prokaryotes. To achieve the same effects on long DNA sequences specific for eukaryotes, DNA must synchronously absorb a great number of ultraviolet quanta. Based on the above results, the following conclusions were made: 1) disturbances in the normal activity of DNA and RNA polymerases may be due to electromagnetic field, which is caused by the oscillatory relaxation of vibronic levels of nucleotides. The energy enters the vibronic levels of nucleotides from an exciton running along the DNA sequence; 2) the increase in the noncoding DNA sequences in eukaryotes due to evolution is a way of DNA protection from undesirable mutations; 3) prokaryotes must possess a greater potentiality and a higher rate of mutagenesis in comparison with eukaryotes, which is proved by their greater diversity in nature.     

Band Structure and Local Dynamics of Excitons in Bacterial Light-harvesting Complexes Revealed by Spectrally Selective Spectroscopy

Hole-burned absorption and line-narrowed fluorescence spectra are studied at 5 K in wild type and mutant LH1 and LH2 antenna preparations from the photosynthetic purple bacterium Rhodobacter sphaeroides. Evidence was found in all samples, even in intact membranes, of the presence of a broad distribution of bacteriochlorophyll species that are unable to communicate energy between each other and to the exciton states of functional antenna complexes. The distribution maximum of these localized species determined by zero phonon hole action spectroscopy is at 783.5 nm in purified LH1 complexes and at 786.8 nm in B850-only mutant LH2 complexes. A well-resolved peak at 807 nm in LH1 complexes is assigned to the exciton band structure of functional core antenna complexes. Similar structure in LH2 complexes overlaps with the distribution of localized species. Off-diagonal (structural) disorder may be responsible for this exciton band structure. Our data also imply that pair-wise inter-chlorophyll couplings determine the resonance fluorescence lineshape of excitonic polarons.     

Phenylalanine oligopeptides: Synthesis,polarimetric and ultraviolet absorption studies

A series of l-phenylalanine oligomers was synthesized by the dicyclohexyl-carbodiimide and acylazide methods. The compounds prepared in this manner were chemically and optically pure and were obtained in good yield. A preliminary stereo-chemical analysis of these oligopeptides by means of polarimetry and ultraviolet absorption spectroscopy is reported. The vibrational fine structure observed in the ultraviolet spectra is also discussed.     

Ultraviolet circular dichroism of polyproline and oriented collagen

The ultraviolet absorption, linear dichroism, circular dichroism, and oriented circular dichroism of collagen are reported and the spectra are resolved into a self-consistent set of bands in accord with exciton theory. The parallel band at 200 nm has 40% of the π → π* intensity; the perpendicular band is placed at 189 nm yielding a splitting of 2700 cm^?1. The circular dichroism is resolved into two Gaussians at λ_∥ and λ_τ (rotational strengths +14 × 10^?40 and ?32 × 10^?40 esu². cm²) plus a large non-Gaussian (“helix”) band with ampplitude ?25,000° at 201 nm. These data appear to be in reasonably good accord with recent calculations. Measurements of the absorption, linear dichroism and circular dichroism of polyproline I and II are also reported and are resolved into their component bands. Polyproline I is in good accord with exciton theory, whereas polyproline II remains unsatisfactory.     

The circular dichroism in the ultraviolet of aminoacridines and ethidium bromide bound to DNA

The circular dicrosim (CD) spectra of complexes of DNA with ethidiun bromnide, profiavine, 9-aminoacridine and 4-etliyl-9-amino-acridine have been determined between 220 and 450 nm, the range lieing extended to 600 nm for ethidiufm bromide. The variation of the magnitude of the visible and near—ultraviolet CD spectra of ethidium bromide—DNA complexes with the amount of ligand bound (r) suggests a common binding position with profiavine. On the other hand, 4-ethyl-9-aminoacndine complexed to DNA shows CD spectra not distinguishable from those of 9-aminnoacnidmc in both the visible and ultraviolet. The interpretation of these results with respect to the stereochemistry of the DNA-ligand complexes is discussed.     

Thermal difference spectra: a specific signature for nucleic acid structures

We show that nucleic acid structures may be conveniently and inexpensively characterized by their UV thermal difference spectra. A thermal difference spectrum (TDS) is obtained for a nucleic acid by simply recording the ultraviolet absorbance spectra of the unfolded and folded states at temperatures above and below its melting temperature (T_m). The difference between these two spectra is the TDS. The TDS has a specific shape that is unique for each type of nucleic acid structure, a conclusion that is based on a comparison of >900 spectra from 200 different sequences. The shape of the TDS reflects the subtleties of base stacking interactions that occur uniquely within each type of nucleic acid structure. TDS provides a simple, inexpensive and rapid method to obtain structural insight into nucleic acid structures, which is applicable to both DNA and RNA from short oligomers to polynucleotides. TDS complements circular dichroism as a tool for the structural characterization of nucleic acids in solution.     

DNA-templated formation and luminescence of diphenylacetylene dimeric and trimeric complexes

We report on spectral features for two and three diphenylacetylene chromophores aligned in close proximity in aqueous solution by self assembly of attached oligonucleotide arms. Two duplex systems were examined in detail. One was formed by hybridization (Watson-Crick base pairing) of two oligonucleotide 10-mers, each containing the diphenylacetylene insert. The other was generated by self-folding of a 36-mer oligonucleotide containing two diphenylacetylene inserts. The triplex system was obtained by hybridization (Hoogsteen base pairing) of a 16-mer oligonucleotide diphenylacetylene conjugate to the folded 36-mer hairpin. Formation of duplex and triplex entities from these conjugates was demonstrated experimentally by thermal dissociation and spectroscopic studies. The UV and CD spectra for the duplex systems exhibit bands in the 300-350 nm region attributable to exciton coupling between the two chromophores, and the emission spectra show a strong band centered at 410 nm assigned to excimer fluorescence. Addition of the third strand to the hairpin duplex has little effect on the CD spectrum in the 300-350 nm region, but leads to a negative band at short wavelengths characteristic of a triplex and to a strongly enhanced band at 410 nm in the fluorescence spectrum. The third strand alone shows a broad fluorescence band at approximately 345-365 nm, but this band is virtually absent in the triplex system. A model for the triplex system is proposed in which two of the three aligned diphenylacetylenes function as a ground state dimer that on excitation gives rise to the exciton coupling observed in the UV and CD spectra and to the excimer emission observed in the fluorescence spectrum. Excitation of the third chromophore results in enhanced excimer fluorescence, as a consequence of energy transfer from the locally excited singlet of one chromophore to the ground state dimer formed by the other two chromophores.     

Blue and near ultraviolet reversible photoreaction with intracellular particulate fraction of the fungus, Alternaria tomato

In studies of the fungus, Alternaria tomato, we found the blueand near ultraviolet reversible photoreaction, which plays animportant role in photocontrol of conidial development, in thelight-minus-dark difference spectrum of the intracellular particulatefraction isolated from dark-grown mycelia. That is, after irradiationwith near ultraviolet, the difference spectrum of the particulatefraction showed a dip at 300 nm and a peak at 400 nm. When irradiatedwith blue light after exposure to near ultraviolet, both thepeak at 400 nm and the dip at 300 nm partially disappeared.This change was reversibly repeated by alternating doses ofblue and near ultraviolet. Action spectra for the light-mediatedchanges in the difference spectrum showed the peaks at 300 nmand 410 nm. (Received March 19, 1973; )     

Mutagenesis and cytotoxicity in human epithelial cells by far- and near-ultraviolet radiations: action spectra

Action spectra were determined for cell killing and mutation by monochromatic ultraviolet and visible radiations (254-434 nm) in cultured human epithelial P3 cells. Cell killing was more efficient following radiation at the shorter wavelengths (254-434 nm) than at longer wavelengths (365-434 nm). At 254 nm, for example, a fluence of 11 Jm-2 gave 37% cell survival, while at 365 nm, 17 X 10(5) Jm-2 gave equivalent survival. At 434 nm little killing was observed with fluences up to 3 X 10(6) Jm-2. Mutant induction, determined at the hypoxanthine-guanine phosphoribosyltransferase locus, was caused by radiation at 254, 313, and 365 nm. There was no mutant induction at 334 nm although this wavelength was highly cytotoxic. Mutagenesis was not induced by 434 nm radiation, either. There was a weak response at 405 nm; the mutant frequencies were only slightly increased above background levels. For the mutagenic wavelengths, log-log plots of the mutation frequency against fluence showed linear regressions with positive slopes of 2.5, consistent with data from a previous study using Escherichia coli. The data points of the action spectra for lethality and mutagenesis were similar to the spectrum for DNA damage at wavelengths shorter than 313 nm, whereas at longer wavelengths the lethality spectrum had a shoulder, and the mutagenesis spectrum had a secondary peak at 365 nm. No correlation was observed for the P3 cells between the spectra for cell killing and mutagenesis caused by wavelengths longer than 313 nm and the induction of DNA breakage or the formation of DNA-to-protein covalent bonds in these cells.     

Mapping the interactions of the single-stranded DNA binding protein of bacteriophage T4 (gp32) with DNA lattices at single nucleotide resolution: gp32 monomer binding

Combining biophysical measurements on T4 bacteriophage replication complexes with detailed structural information can illuminate the molecular mechanisms of these ‘macromolecular machines’. Here we use the low energy circular dichroism (CD) and fluorescent properties of site-specifically introduced base analogues to map and quantify the equilibrium binding interactions of short (8 nts) ssDNA oligomers with gp32 monomers at single nucleotide resolution. We show that single gp32 molecules interact most directly and specifically near the 3′-end of these ssDNA oligomers, thus defining the polarity of gp32 binding with respect to the ssDNA lattice, and that only 2–3 nts are directly involved in this tight binding interaction. The loss of exciton coupling in the CD spectra of dimer 2-AP (2-aminopurine) probes at various positions in the ssDNA constructs, together with increases in fluorescence intensity, suggest that gp32 binding directly extends the sugar-phosphate backbone of this ssDNA oligomer, particularly at the 3′-end and facilitates base unstacking along the entire 8-mer lattice. These results provide a model (and ‘DNA map’) for the isolated gp32 binding to ssDNA targets, which serves as the nucleation step for the cooperative binding that occurs at transiently exposed ssDNA sequences within the functioning T4 DNA replication complex.     

Predicting ultraviolet spectrum of single stranded and double stranded deoxyribonucleic acids

Synthetic oligodeoxynucleotides are widely used in many biological, biochemical and biophysical applications. The concentration, composition and structure of DNA are often determined from its ultraviolet spectrum. Although parameters for use with the nearest-neighbor model for prediction of extinction coefficients of single stranded DNAs at 260 nm were published some time ago, similar parameters for other wavelengths or for use with DNA duplexes have not been reported. Practical formulae and parameters for prediction of UV spectra, hypochromism and peak wavelengths were experimentally determined for both single stranded and double stranded oligodeoxynucleotides in the range from 215 to 310 nm. The accuracy of predictions made using the nearest-neighbor model and the base composition model was determined and compared. The spectrum of any DNA oligomer can be calculated using a Microsoft Excel application that is available in the Appendix A.     

Photochemical revelation of the stacking aggregation of thymine chromophores in water solutions of polythymidylic acid

The structure heterogeneity of water solutions of polyribothymidylic acid at T(room) was studied from changes caused in their absorption spectra by the photodimerization reaction. Three fractions of thymine chromophores were revealed from the differential absorption spectra: (a) the main fraction consisting of weakly interacting (isolated chromophores) chromophores with the absorption spectrum maximum at approximately 270 nm; (b) pair chromophores of the first type with the absorption spectrum maxima at 260 and 290 nm (exciton splitting 4000 cm(-1)); and (c) pair chromophores of the second type with the absorption spectrum maxima at 250 and 280 nm (exciton splitting 4300 cm(-1)). The revealed aggregates have a relatively high photochemical activity in the photodimerization reaction in comparison with the isolated chromophores. They contribute little to the total absorption spectrum of solutions but make a great contribution to its changes at the initial stages of the UV irradiation of solutions.     

Spectroscopic properties of pyrene-containing DNA mimics

DNA mimics containing non-nucleosidic pyrene building blocks are described. The modified oligomers form stable hybrids, although a slight reduction in hybrid stability is observed in comparison to the unmodified DNA duplex. The nature of the interaction between the pyrene residues in single and double stranded oligomers is analyzed spectroscopically. Intra- and interstrand stacking interactions of pyrenes are monitored by UV-absorbance as well as fluorescence spectroscopy. Excimer formation is observed in both single and double strands. In general, intrastrand excimers show fluorescence emission at shorter wavelengths (approx. 5-10 nm) than excimers formed by interstrand interactions. The existence of two different forms of excimers (intra- vs. interstrand) is also revealed in temperature dependent UV-absorbance spectra.     

Photophysical analysis of class I major histocompatibility complex protein assembly using a xanthene-derivatized beta2-microglobulin

Spectral changes and a sixfold increase in the emission intensity were observed in the fluorescence of a single xanthene probe (Texas red) attached to beta2m-microglobulin (beta2m) upon assembly of beta2m into a ternary complex with mouse H-2Kd heavy chain and influenza nuclear protein peptide. Dissociation of the labeled beta2m from the ternary complex restored the probe's fluorescence and absorption spectra and reduced the emission intensity. Thus changes in xanthene probe fluorescence upon association/dissociation of the labeled beta2m molecule with/from the ternary complex provide a simple and convenient method for studying the assembly/dissociation mechanism of the class I major histocompatibility complex (MHC-I) encoded molecule. The photophysical changes in the probe can be accounted for by the oligomerization of free labeled beta2m molecules. The fluorescence at 610 nm is due to beta2m dimers, where the probes are significantly separated spatially so that their emission and excitation properties are close to those of xanthene monomers. Fluorescence around 630 nm is due to beta2m oligomers where xanthene probes interact. Minima in the steady-state excitation (550 nm) and emission (630 nm) anisotropy spectra correlate with the maxima of the high-order oligomer excitation and emission spectra, showing that their fluorescence is more depolarized. These photophysical features are explained by splitting of the first singlet excited state of interacting xanthene probes that can be modeled by exciton theory.