Temperature-dependent ultraviolet resonance Raman spectroscopy of the premelting state of dA.dT DNA.

Poly(dA).poly(dT) and DNA duplex with four or more adenine bases in a row exhibits a broad, solid-state structural premelting transition at about 35 degrees C. The low-temperature structure is correlated with the phenomena of "bent DNA." We have conducted temperature-dependent ultraviolet resonance Raman measurements of the structural transition using poly(dA).poly(dT) at physiological salt conditions, and are able to identify, between the high and low temperature limits, changes in the vibrational frequencies associated with the C4 carbonyl stretching mode in the thymine ring and the N6 scissors mode of the amine in the adenine ring of poly(dA).poly(dT). This work supports the model that the oligo-dA tracts' solid-state structural premelting transition is due to a set of cross-stand bifurcated hydrogen bonds between consecutive dA. dT pairs.     

Raman scattering of laser radiation in magnetoactive plasma

Raman scattering in a subcritical plasma is simulated numerically for the case where the laser radiation propagates transversely to an external magnetic field. It is shown that, in the linear stage of Raman instability, the magnetic field decreases the instability growth rate for forward scattering and increases it for backward scattering. As the value of the magnetic field increases, the stochastic heating of plasma electrons is enhanced in the region of backscattering.     

Characterization of DNA structures by laser Raman spectroscopy

Oriented fibers drawn from aqueous gels of calf-thymus DNA were maintained at constant relative humidites of 75 and 92% to yield canonical A-DNA and B-DNA structures, respectively. Raman spectra of the two forms of DNA were recorded over the spectral range 300–4000 cm^?1. The authenticated DNA fibers were deuterated in hygrostatic cells containing D₂O at appropriate relative humidities, and the corresponding spectra of deuterated DNAs were also obtained. The spectra reveal all of the Raman scattering frequencies and intensities characteristic of A- and B-DNA structures in both nondeuterated and deuterated froms, as well as the frequencies and intensities of adsorbed solvent molecules from which the hydration content of DNA fibers can be calculated. Numerous conformation-sensitive vibrational modes of DNA bases and phosphate groups have been identified throughout the 300–1700-cm^?1 interval. Evidence has also been obtained for conformation sensitivity of deoxyribosyl CH stretching modes in the 2800–3000-cm^?1 region. Raman lines of both the backbone and the bases are proposed as convenient indicators of A- and B-DNA structures. The results are extended to Z-DNA models investigated previously. Some implications of these findings for the determination of DNA or RNA structure from Raman spectra of nucleoproteins and viruses are considered.     

Raman and resonance-Raman scattering by erythrocyte ghosts.

1. We present the laser-Raman spectra of human erythrocyte ghosts, isolated by standard conditions and compare these with the spectra of lecithin liposomes and fat-free serum albumin. 2. The hydrocarbon stretching modes of membrane lipids are temperature sensitive and may serve as a index of hydrocarbon chain motion. 3. The Amide I and Amide III bands of ghosts in H-2O and 2-H-2O, indicate a mixture of alpha-helical and unordered conformation, but do not allow a quantitative estimate of secondary structure. 4. Strong, scattering bands at 1530 and 1165 cm-1 are attributable to conjugated double bond systems, probably of membrane-associated carotenoids. Their high intensity is due to resonance enhancement.     

Raman and surface-enhanced Raman scattering spectroscopy of bis-netropsins and their DNA complexes

The interactions of three bis-netropsins (bis-Nts), which are potent catalytic inhibitors of DNA-binding enzymes, with three double-stranded oligonucleotides (OLIGs), which contain sites of different specific affinities for each bis-Nt, were analyzed. Raman spectroscopy was performed for selective monitoring of modifications of the bis-Nt or the OLIG structure upon bis-Nt-DNA binding, and surface-enhanced Raman scattering spectroscopy (SERS) was an additional tool for topology studies of ligand-DNA complexes. The spectral data showed conformational changes of both partners (bis-Nt and OLIG) upon complexation. Structural variations of bis-Nts appeared to be dependent on a bis-Nt-OLIG binding constant and were found to be small in the specific DNA binding and highest for nonspecific binding of bis-Nt with the corresponding OLIG. The conformational changes of the OLIGs were varied with a bis-Nt-OLIG binding constant in the same manner. The bis-Nts seemed to induce a perturbation in the OLIG's structure, as well as in the positions of their direct binding. These DNA structural modification effects may explain the inhibition of DNA-binding enzymes in the variety of very distinct DNA-enzyme binding sites by bis-Nts reported previously.     

Detection and identification of labeled DNA by surface enhanced resonance Raman scattering

The detection of specific sequences of DNA bases in a single strand can be achieved by hybridization of a known sequence of synthetic DNA. Due to the low concentrations usually used, a fluorescent label is required to detect the probe. Surface enhanced resonance Raman scattering (SERRS) also has the required sensitivity and provides a specific set of signals that are more applicable to discrimination of a number of probes without separation. A reliable SERRS method is reported here using two probes specifically designed for SERRS. It was possible to detect a 2 x 10(-12)M solution of labeled DNA, which illustrated the sensitive nature of SERRS for DNA analysis.     

Large-angle stimulated Raman scattering of short laser pulses in plasma

The features of the large-angle stimulated Raman scattering of short laser pulses in a homogeneous underdense plasma are studied analytically. It is found that, for scattering angles that are not too close to zero, a steady-state regime of the convective amplification of unstable waves is established in the frame of reference comoving with the laser pulse. The problem of convective amplification in a two-dimensional region is solved in both weak-and strong-coupling regimes. It is shown that the steady-state envelopes of the scattered radiation and scattering plasma waves are two-dimensional in nature. It is found that, for a given scattering angle, the maximum possible spatial amplification at the trailing edge of the pulse is achieved if the ratio of the transverse to longitudinal size of the pulse is larger than the cotangent of one-half of the scattering angle.     

DNA condensation by polyamines: a laser light scattering study of structural effects.

Polyamines such as spermidine and spermine are abundant in living cells and are believed to aid in the dense packaging of cellular DNA. DNA condensation is a prerequisite for the transport of gene vectors in living cells. To elucidate the structural features of polyamines governing DNA condensation, we studied the collapse of lambda-DNA by spermine and a series of its homologues, H2N(CH2)3NH(CH2)n=2-12NH(CH2)3NH2 (n = 4 for spermine), using static and dynamic light scattering techniques. All polyamines provoked DNA condensation; however, their efficacy varied with the structural geometry of the polyamine. In 10 mM sodium cacodylate buffer, the EC50 values for DNA condensation were comparable (4 +/- 1 microM) for spermine homologues with n = 4-8, whereas the lower and higher homologues provoked DNA condensation at higher EC50 values. The EC50 values increased with an increase in the monovalent ion (Na+) concentration in the buffer. The slope of a plot of log [EC50(polyamine4+)] against log [Na+] was approximately 1.5 for polyamines with even number values of n, whereas the slope value was approximately 1 for compounds with odd number values of n. Dynamic light scattering measurements showed the presence of compact particles with hydrodynamic radii (Rh) of about 40-50 nm for compounds with n = 3-6. Rh increased with further increase in methylene chain length separating the secondary amino groups of the polyamines (Rh = 60-70 nm for n = 7-10 and >100 nm for n = 11 and 12). Determination of the relative binding affinity of polyamines to DNA using an ethidium bromide displacement assay showed that homologues with n = 2 and 3 as well as those with n > 7 had significantly lower DNA binding affinity compared to spermine and homologues with n = 5 and 6. These data suggest that the chemical structure of isovalent polyamines exerts a profound influence on their ability to recognize and condense DNA, and on the size of the DNA condensates formed in aqueous solution.     

Characterization of the A in equilibrium B transition of DNA in fibers and gels by laser Raman spectroscopy.

Both Raman spectra and X-ray diffraction patterns have been obtained from oriented fibers of sodium deoxyribonucleic acid (Na-DNA) as a function of salt content and relative humidity. We have confirmed the previously reported X-ray results that, for oriented fibers, the A-form always exists between 75 and 92% relative humidity and that the conformation will change to the B-form at 92% relative humidity only if an excess (3–5%) of added salt is present. Oriented fibers containing low amounts of added salt remain in the A-type conformation at 92% relative humidity and higher. An exact correlation has been found between the familiar A- and B-type X-ray diffraction patterns of DNA fibers and the Raman spectra previously reported without X-ray verification from this laboratory for the A- and B-forms. In particular, a band at 807 cm^?1 was always present when a fiber showed the A-type diffraction pattern, and this band shifts to 790 cm^?1 in the B-form. Using the Raman spectrum to determine the specific conformation of DNA in samples less amenable to X-ray analysis, we have studied the A ? Btransformation in unoriented fibrous masses of DNA and in concentrated, oriented gels. We find that in unoriented fibrous masses, the A ? B transition always occurs at 92% relative humidity even at very low salt concentration (0–4%). However, in oriented DNA gels at low salt, the A-form can persist as a metastable state to concentration as low as 20% DNA. The origin of the bands at 807 and 790 cm^?1 and the possible biological implications of these findings are discussed.     

Depolarization of Raman scattering from some nucleotides of RNA and DNA

Depolarization ratios of Raman bands, excited at 488.0 nm, of guanosine-5′-monophosphoric 4 acid, cytidine-5′-monophosphoric acid, adenosine-5′-monophosphoric acid, thymidine-5′-monophosphoric acid, and uridine-5′-monophosphoric acid have been measured in their H₂O and D₂O solutions in the spectral region from 300 to 1800 cm^?1. For comparison, the disodium salt of 2′-deoxyadenosine-5′-monophosphoric acid was also subjected to the depolarization measurement in its H₂O solution. The results have been correlated with possible orientations of the principal axes of the Raman scattering tensors as well as with the relative magnitudes of the tensor components. Results should be useful for future polarized Raman studies of synthetic and natural DNA. © 1993 John Wiley & Sons, Inc.     

Cross-correlation laser scattering.

The dependence of phospholipid vesicle size on lipid composition is investigated by photon correlation spectroscopy. For each lipid composition prolonged ultracentrifugation was used to isolate a nearly uniform population of minimum-sized vesicles. The residual size variations in the samples were sufficient to cause polydispersity that made comparisons between samples difficult. Analyses of the data by the method of cumulants and by a method for approximating the particle size distributions directly are presented. The latter method made possible unambiguous comparisons that revealed small but systematic dependences of vesicle size on composition in vesicles containing mixtures of egg phosphatidylcholine and phosphatidylethanolamine, egg phosphatidylcholine and beef brain sphingomyelin, and in single lipid vesicles of egg phosphatidylcholine, dioleylphosphatidylcholine, and beef brain sphingomyelin. These size dependences are quantified within the resolution limits of the technique and their implications are discussed.     

Determination of beta-turn conformation by laser Raman spectroscopy.

To correlate the Raman frequencies of the amide I and III bands to beta-turn structures, three peptides shown to contain beta-turn structure by x-ray diffraction and NMR were examined. The compounds examined were tertiary (formula: see text). The amide I band of these compounds is seen at 1,668, 1,665, and 1,677 cm-1, and the amide III band appears at 1,267, 1,265, and 1,286 cm-1, respectively. Thus, it is concluded that the amide I band for type III beta-turn structure appears in the range between 1,665 and 1,677 cm-1 and the amide III band between 1,265 and 1,286 cm-1.     

Cytoplasmic streaming in Chara corallina studied by laser light scattering.

An apparatus is described by means of which the power versus frequency spectrum of the photomultiplier current can be obtained for laser light scattered by streaming cytoplasm in the algal cell Chara corallina. A Doppler peak is noted in the spectrum which is abolished when cytoplasmic streaming is arrested by electrical stimulation. For 5 cells of Chara, this simple laser-Doppler velocimeter gave streaming velocities (46-7 mum s-1, S.D. +/- 4-8 at 20 degrees C) similar to those obtained for the same cells using the light microscope (44-3 mum s-1, S.D. +/- 5-3 at 20 degrees C). A narrow distribution of streaming velocities is indicated. The technique described provides a rapid, quantitative assay of the in vivo rheological properties of cytoplasm.     

Studies of myosin and its proteolytic fragments by laser Raman spectroscopy.

Two bands in the Raman spectrum of myosin, at 1,304 cm-1 and 1,270 cm-1, are attributable to alpha-helical structure. The first of these, also present in the spectrum of light meromyosin (LMM) but not in that of subfragment-1 (S-1), is assigned to the coiled-coil tail region of myosin; the second, seen in spectra of S-1 or heavy meromyosin (HMM), is largely absent from the spectrum of light meromyosin and is likely to correspond to the alpha-helical segments of the head region. When myosin or LMM aggregates, spectral bands attributable to backbone and sidechain groups sharpen suggesting a reduction in motional freedom. This sharpening is particularly apparent in the 902 cm-1 C--C stretching mode. Mg2+ broadens and shifts the peak at 1,244 cm-1 to 1,237 cm-1 and diminishes the intensity from 1,230 to 1,240 cm-1, changes which appear to be associated the S-1 region. MgPPi produces changes in the 1,300 cm-1 region attributable to alpha-helical regions in coiled-coil structures suggesting that MgPPi affects not only S-1, but also some part of the myosin rod.     

Quasi-elastic light scattering by calf thymus DNA and lamdaDNA.

The quasi-elastic light-scattering (homodyne) time-correlation functions of calf thymus and λDNA are shown to contain contributions from at least two relaxation processes. A method of asymptotic analysis is described and used to obtain an estimate of the longest relaxation time as well as the “average” relaxation time and the mean-squared dispersion in this average. Most theories of scattering from macromolecules in the limit of inifinite dilution predict that the longest relaxation time is due to translational self-diffusion. The data obtained, however, indicate that the longest time is not simply related to the translational self-diffusion coefficient of unaggregated macromolecules. It is also shown that the longest relaxation time of λDNA decreases in the later stages of the denaturation transition region. Some possible mechanisms for the origin of this long time are discussed, including a model of restricted motion of a molecule by its neighbors.