Alain Laigle

Excited state geometry of uracil from the resonant Raman overtone spectrum using a Kramers–Kronig technique

The Kramers–Kronig transform technique of Blazej and Peticolas is extended to permit the calculation of overtone and combination bands in a resonant Raman spectrum relative to the fundamentals. The method is applied to the ultraviolet (257 nm) resonant Raman spectrum of uracil which exhibits five fundamental bands and numerous overtone and combination bands. The method permits the determination of Δej the shift of the excited state potential energy minimum along the jth normal mode for each of the five resonant Raman active modes. The Δej values obtained are in reasonable agreement with those obtained earlier from the absorption spectrum and excitation profiles of the fundamentals. However, no internal standard for the experimental determination of Raman intensities is required nor is it necessary to assume any value for Γ the vibronic linewidth. The shape of the absorption band and relative intensities of the harmonics to the fundamental are sufficient to obtain the value of each Δej.

The Z-Conformation of Poly(dA-dT) · Poly(dA-dT) in Solution as Studied by Ultraviolet Resonance Raman Spectroscopy

Poly(dA-dT).poly(dA-dT) structures in aqueous solutions with high NaCl concentrations and in the presence of Ni2+ ions have been studied with resonance Raman spectroscopy (RRS). In low water activity the effects of added 95 mM NiCl2 in solution stabilize the syn geometry of the purines and reorganize the water distribution via local interactions of Ni-water charged complexes with the adenine N7 position. It is shown that RRS provides good marker bands for a left-handed helix: i) a purine ring breathing mode around 630 cm-1 coupled to the deoxyribose vibration in the syn geometry, ii) a 1300-1340 cm-1 region characterizing local chemical interactions of the Ni2+ ions with the adenine N7 position, iii) lines at about 1483- and 1582 cm-1 correlated to the anti/syn reorientation of the adenine residues on B-Z structure transition, iv) marker bands of the thymidine carbonyl group couplings at 1680- and 1733 cm-1 due to the disposition of the thymidine residues in the Z helix specific geometry. Hence poly(dA-dT).poly(dA-dT) can adopt a Z form in solution. The Z form observed in alternate purine-pyrimidine sequences does not require G-C base pairs.

Contribution of the Resonance Raman Spectroscopy to the Identification of Z DNA

Poly(dG-dC).poly(dG-dC) at low salt concentration (0.1 M NaCl) and at high salt concentration (4.5 M NaCl) has been studied by Raman resonance spectroscopy using two excitation wavelengths: 257 nm and 295 nm. As resonance enhances the intensity of the lines in a proportion corresponding to the square of the molar absorption coefficient, the intensities of the lines with 295 nm wavelength excitation are enhanced about sevenfold during the B to Z transition. With 257 nm excitation wavelength the 1580 cm-1 line of guanosine is greatly enhanced in the Z form whereas with 295 nm excitation several lines are sensitive to the modifications of the conformation: the guanine band around 650 cm-1 and at 1193 cm-1 and the bands of the cytosines at 780 cm-1, 1242 cm-1 and 1268 cm-1. By comparison with the U.V. resonance Raman spectra of DNA, we conclude that resonance Raman spectroscopy allows one to characterize the B to Z transition from one line with 257 nm excitation wavelength and from three lines with 295 nm excitation. The conjoined study of these four lines should permit to observe a few base pairs being in Z form in a DNA.

Thermodynamic and Structural Properties of r(ACC) as Revealed by Ultraviolet Electronic Absorption, Circular Dichroism,1H-NMR Spectroscopy and Monte Carlo Simulations

UV absorption, circular dichroism (CD) and 1H NMR, associated with Monte Carlo (MC) molecular structure simulations have been applied to the study of the trinucleoside diphosphate: r(ACC). The MC study which has been conducted as a function of temperature, is based on random variations of the nucleotide conformational angles, i.e. phosphodiester chain torsional angles and sugar pucker pseudorotational angles. All of the chemical bond lengths and valence angles remained fixed during the structural simulation, except those of the sugar pucker. Six different initial structures have been selected in order to explore the molecular conformational space as completely as possible. This simulation procedure led to distinct families of equilibrium conformations at 283, 298 and 318 K. The thermodynamical parameters such as variations in entropy, enthalpy and also melting temperature (delta SX0, delta HX0 and Tm) of the stacking (X) equilibrium were obtained from UV absorption and circular dichroism (CD) spectra recorded over a 80K temperature range. Chemical shifts (delta), vicinal coupling constants (3Jk,l), and cross-relaxation rate (sigma k,l) of trimers were measured at 400.13 MHz over a range of concentrations (2-13 mM) and temperatures (283-333K). Least-squares fitting of the experimental chemical shifts to simple models of association (A) and stacking equilibria allowed separation of the variations in the delta values (delta delta X and delta delta A) due to either phenomenon. The three NMR data sets (delta delta X, 3Jk,l, and sigma k,l) were then evaluated for the minima conformers obtained with the MC stimulations. Theoretical values of delta delta X were estimated using the results of an ab initio study while the coupling constant data were simulated with Karplus-type equations. Finally, the relaxation data were simulated from the distance matrices using treatment for cases of both slow conformational exchange accompanied by rapid small-amplitude fluctuations about the minima structures. A consistent picture of the large amplitude deformations (torsional angle variation) of these trimers has emerged from the present study. Optimized conformational blends at 283,296 and 318K were obtained by least-squares fitting of the experimental data to the theoretical ones, while considering the populations as adjustable parameters. As it would be expected, the right-handed helical conformation (A-RNA type) is found to be the major stacked species, in the temperature range of 283 to 318K. Limited evidence for bulged structures has been obtained, whereas novel reverse-stacked and half-stacked conformers also presented theoretical data compatible with the NMR observables of aqueous r(ACC).

Associations DNA-ribonucléase: II — Etude conformationnelle

Summary In a well defined environment of saline concentration and pH, ribonuclease induces DNA destabilisation. The extent of this destabilisation depends on the quantity of bound RNase, on the pH, on the saline concentration and on the DNA base composition. When destabilisation occurs, optical rotatory dispersion and circular dichroism studies show that RNase conformational changes are not responsible for this destabilisation but, on the contrary, that RNase conformation is protected : it maintains in this case the conformation it had before mixing. Structural modifications of each constituent suggest a model to interpret the destabilisation phenomena.

Associations DNA-Ribonucléase: I - Etude physico-chimique

Summary By physical-chemical study of the DNA-RNase interactions, the conditions of their association have been determined, the influence of the parameters (saline concentration, pH, temperature) has been specified, and the preponderant part of the electrostatic forces has been emphasized. For a given pH value, the association has been proven possible only in a well defined environment of saline concentration. By changing the composition of the DNA, it appears that there is no specificity of association.

Intracellular fate of oligodeoxynucleotides: A microspectro FRET study

Fluorescence resonance energy transfer (FRET) under confocal microscopy allows direct intracellular measurements of both localisation and interactions of tagged molecules to be performed. In the antigene and antisense strategies, short oligonucleotide sequences are used to inhibit RNA translation in cell. Numerous techniques (Crook, R.M. “In Vitro Cellular Uptake, Distribution, and Metabolism of Oligonucleotides” Handbook of Experimental Pharmacology, Vol. 131 Antisense Research and Application : 103-140 (1998) were used to follow oligonucleotide penetration and distribution in cultured cells, but no in situ study of the oligonucleotide stability was performed. Therefore, microspectroFRET was chosen as a non destructive method to study in differents cell lines the fate of short oligonucleotides, fluorescently labeled at both ends. When an oligonucleotide is intact, FRET occurs, so, in this study, intramolecular FRET permits us to know the ratio of intact oligonucleotides during time, i. e. the ratio of potentially active oligonucleotides ; single fluorescent labeling is not sufficient : the observed fluorescence may be due to fluorophores alone or still grafted on the oligonucleotide. The oligonucleotides, labeled with fluorescein at the 3’ end and rhodamine at the 5’ end, were added to the cells and subcellular microvolumes (-10-18 cubic meter) were analysed with the confocal microspectrofluorometer (Vigny, P. Amirand, C. Ballini, J.P. Chinsky, L. Duquesne, M. Laigle, A. Manfait, A. Sureau, F. Turpin, P.Y. “Microspectrofluorometry of Single Living Cells” Spectroscopy of Biological Molecules - State of the Art : 345-348 (1989) developed in the laboratory. Different wavelengths of an Argon LASER were used for excitation of the samples. The spectra were recorded on an optical multichannels analyser after separation of the different emission wavelengths by a monochromator. After FFT smoothing and background subtraction, comparisons of the spectra were achieved and analysed in term of : Stability of the oligonucleotides, which is directly related to the transfer efficiency, as a function of time Quantitative localisation of the oligonucleotides, as fluorescence intensity is proportional to concentration Surrounding of the oligonucleotides, as the quatum yield of fluorescein is pH dependent. As an example of granulocytic cell model, we used K562 cells. But these cells have a large nucleus, preventing to distinguish subcellular compartments. So we also used Hela cells as an example of epithelial-like cells with a real differentiation between cytoplasm and nucleus. 3e Colloque SFp, Paris-Sud ‘99 icole Polytechnique, Palaiseau, 28 juin-2 juillet 1999

The Quadruplex Form of Polyriboinosinic Acid as Studied by UV Resonance Raman Spectroscopy

Four-stranded forms of DNA have recently become of great interest due to the discovery of four stranded segments of DNA in such significant structures as telomers[l]. Polyriboinosinic acid (Poly[rI]) forms a self-associated quadruplex in high concentrations of salt, but also exists as a single stranded form at low ionic strength[2], thus forms a useful model for studying such four-stranded forms of polynucleotides, and many studies have been made of this system using a wide range of experimental techniques such as CD, ORD, UV absorption, IR and Raman spectroscopies, and X-ray crystallography. We have studied this system using CD and Resonance Raman Spectroscopy with excitation wavelengths of 257 and 280 nm, thereby allowing us to obtain vibrational spectra with low concentrations of polynucleotide (0.4mg/ml) such as those used for UV and CD spectroscopy. Thus we have been able to follow certain processes which are not readily observed at the higher concentrations required by IR and classical Raman spectroscopies. We also performed a trial experiment with Terbium Tb3+ ions, a fluorescent marker normally used as a structural probe for guanine residues.