Fluorescence of actinomycin D and its DNA complex.

In order to show that naturally occurring actinomycin D can be used as a fluorescent cytochemical probe, the fluorescence spectra of 50 micronM solution of actinomycin D in several solvents and in its DNA complex are reported. The excitation spectra of both bands observed are compared to the absorption spectrum, and the fluorescence quantum yields is estimated to be 0.65-10(-4), taking rhodamine B as a reference. Strong solvent-induced spectrum modifications are evidenced, interpreted as a solvent shell reorientation about the molecule chromophore in the excited state. The changes in the fluorescence spectrum of actinomycin due to an interaction with DNA (marked blue shift and decreased quantum yield) cannot be interpreted as a solvent effect; they express the properties of the DNA-chromophore eta-complex involving an orbital overlapping of actinomycin and guanine in DNA.

Interactions of iron-anthracycline complexes with living cells: a microspectrofluorometric study.

The interaction of iron-anthracycline complexes with tumor cells has been studied using microspectrofluorometry. The anthracyclines used were adriamycin, 4'-O-tetrahydropyranyladriamycin and daunorubicin. In every case, a 1:3 Fe(III)-anthracycline complex is formed. The three daunorubicin molecules that bind to one Fe(III) are not chemically modified through complexation with iron. In the case of the Fe(III)-adriamycin and Fe(III)-4'-O-tetrahydropyranyladriamycin complexes, about one of the three anthracycline molecules is chemically modified, yielding a highly lipophilic derivative, the 7,8-dehydro-9,10-desacetyladriamycin. The others molecules remain unchanged, i.e., highly hydrophilic in the case of adriamycin. These two species have a different fluorescent spectrum and can be identified inside the cell, using microspectrofluorometry. In the case of the Fe(III)-adriamycin complex, the lipophilic derivative is more rapidly internalized in the cell than the hydrophilic one. Diffusion into the plasmic membrane is the limiting step for the uptake of anthracycline by cells; this means that the plasmic membrane speeds up the dissociation of the Fe(III)-anthracycline complex.

[Resonance Raman spectroscopy of polynucleotides]. / Spectroscopie Raman de résonance des polynucléotides.

Resonance Raman Spectroscopy allows a selective study of the bases of DNA and therefore of the interactions of these bases with ligands. This technique is also sensitive to structural modifications. We show here that, first, the structures of native poly(dA-dT).poly(dA-dT) and poly(dA).poly(dT) are not the same and that, secondly, it is possible to characterize the B----Z transition of poly(dG-dC).poly(dG-dC). The study of the Raman hypochromism during the thermal denaturation of the polynucleotides reveals that the stacking of the adenines in poly(dA).poly(dT) is near that observed in poly(rA) but differs of this stacking in poly(dA-dT).poly(dA-dT). The enhancement of the intensity of the guanine line at 1193 cm-1 and of the cytosine lines at 780 cm-1, 1 242 cm-1 and 1268 cm-1 as well as the shift of the guanine line at low frequency should allow to characterize a small proportion of base pairs in Z form in any DNA.

[Specificity of DNA-peptide and DNA-histone interactions in ultraviolet Raman resonance spectroscopy]. / Spécificité des interactions DNA-peptides et DNA-histones en spectroscopie de résonance Raman dans l'ultraviolet.

Resonance Raman spectra of complexes between DNA and peptides on one hand, DNA and histones on the other, were obtained. Our work has clearly shown a DNA-base recognition by various protides. The basic residues selectively modify the environment of DNA bases: G-C bases for the lysine residues, A-T bases for the arginine residues, wether the residues are alone or included in a protein. This selectivity of interaction has allowed us to confirm the particular role played by H4 in the structure of nucleosomes and specially of the arginine residues.

Evaluation of the structural modifications induced by mitomycin C on nucleic acids.

The interaction of poly(dG-dC).poly(dG-dC) with mitomycin C, an antitumor antibiotic, has been studied by various spectroscopic methods: circular dichroism, Fourier transform infrared resonance Raman scattering and using fluorescence emission of terbium bound to unpaired guanines as local conformation probe. The results allowed us to confirm the lack of long range modification of the DNA secondary structure upon binding. They also brought first information concerning the modification of the local structure of the nucleic acid at the level of mono- or bifunctional adducts.

Subcellular distribution of hypericin in human cancer cells.

Confocal laser microspectrofluorometric measurements on human T47D mammary tumor cells have been performed to assess the intracellular distribution of hypericin within the various cell compartments: cytoplasmic membrane, cytoplasm and nucleus. Confocal fluorescence measurements obtained from microvolumes (approximately 1 micron3) located within the three sites of interest show that, while being primarily located in the cell membrane and cytoplasm after a short-term incubation in a 10(-6) M hypericin-containing culture medium, hypericin actually reaches the inside of the cell nucleus after a long-term incubation (210 min). Moreover, owing to the relative fluorescence quantum yields of hypericin determined in vitro when the molecule interacts with DNA, membrane and protein model systems, it is assumed that there is a significant accumulation of the drug into the cell nucleus. Consequently, the nucleus has to be considered as a possible target for the toxic action of hypericin.

A porphyrin-DNA exciplex: resonance Raman spectra of electronically excited Cu(TMpy-P4) bound to poly(dA-dT).poly(dA-dT).

The resonance Raman spectra of water-soluble porphyrins, Cu(TMpy-P4) and Ni(TMpy-P4), and their mixtures with DNA, Poly(dG-dC).Poly(dG-dC), and Poly(dA-dT).Poly(dA-dT) were measured using 426 nm pulsed laser excitation (and 556 nm for some applications). At high laser power, the solution of Cu(TMpy-P4) mixed with DNA or Poly(dA-dT).Poly(dA-dT) exhibits new bands at 1550 and 1349 cm-1 that are not observed for Cu(TMpy-P4) alone or for Cu(TMpy-P4) mixed with Poly(dG-dC).Poly(dG-dC). These extra bands do not appear when the resonance Raman spectra are measured by a cw laser or by a pulsed laser with low power. Similar mixtures of M(TMpy-P4) (where M = Ni, Zn, Co, Mn, and H2) with these nucleic acids exhibit no such bands even by high power pulsed laser excitation. We attribute the new resonance Raman bands to an electronically excited Cu(TMpy-P4), stabilized by forming an exciplex with the A-T site of the nucleic acid. The minimum lifetime value of such an exciplex was estimated to be on the order of 10 ps.

Interaction of hypericin with serum albumins: surface-enhanced Raman spectroscopy, resonance Raman spectroscopy and molecular modeling study.

Surface-enhanced Raman spectroscopy, resonance Raman spectroscopy and molecular modeling were employed to study the interaction of hypericin (Hyp) with human (HSA), rat (RSA) and bovine (BSA) serum albumins. The identification of the binding site of Hyp in serum albumins as well as the structural model for Hyp/HSA complex are presented. The interactions mainly reflect: (1) a change of the strength of H bonding at the N1-H site of Trp; (2) a change of the Trp side-chain conformation; (3) a change of the hydrophobicity of the Trp environment; and (4) a formation of an H-bond between the carbonyl group of Hyp and a proton donor in HSA and RSA which leads to a protonated-like carbonyl in Hyp. Our results indicate that Hyp is rigidly bound in IIA subdomain of HSA close to Trp214 (distance 5.12 A between the centers of masses). In the model presented the carbonyl group of Hyp is hydrogen bonded to Asn458. Two other candidates for hydrogen bonds have been identified between the bay-region hydroxyl group of Hyp and the carbonyl group of the Trp214 peptidic link and between the peri-region hydroxyl group of Hyp and the Asn458 carbonyl group. It is shown that the structures of the Hyp/HSA and Hyp/RSA complexes are similar to, and in some aspects different from, those found for the Hyp/BSA complex. The role of aminoacid sequence in the IIA subdomains of HSA, RSA and BSA is discussed to explain the observed differences.

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.

Sequence specific interaction of the antiretrovirally active drug hypericin with 5'ATGGCAGGATAT3' oligonucleotide: a resonance Raman spectroscopy study.

The resonance Raman spectra of two oligonucleotides and their complexes with potent antiretrovirally and antineoplastic active photochemical drug hypericin are reported. The Raman spectra of two oligonucleotides containing twelve base pairs on addition of hypericin (one and two molecules per one oligonucleotide) were compared. The first one contains the first nine base pairs of the "rev" gene coming from HIV genome with three base pairs added to stabilize the duplex (5'ATGGCAGGATAT3') and the second one consists of the same content of the nucleotide bases but in changed sequence order which serves as a control sequence (5'ACGTGATGATGA3'). Differences in the spectra of the "rev" gene sequence and control sequence in interaction with the drug indicate that: i) the AG and GA nucleotide doublets are structurally specific targets for hypericin and ii) the hypericin interaction with 5'AG3' target is stronger than with 5'GA3' one.

Sequence specific interaction of the photoactive drug hypericin depends on the structural arrangement and the stability of the structure containing its specific 5AG3 target: a resonance Raman spectroscopy study.

The resonance Raman spectra of three oligonucleotides with different lengths containing a specific 5'AG3' target doublet for hypericin - a potent antiretroviral and anticancer photoactive agent, and their 1:1 and 1:2 (oligonucleotide: hypericin) complexes are reported. It is shown that the structural arrangement of the oligonucleotides, their structural stability and the local structural arrangement around the 5'AG3' hypericin target, are the factors which determine the formation of a stable, specifically bounded DNA-hypericin complexes.

Internal DNA modes below 25 cm-1: a resonance Raman spectroscopy observation.

The first resonance Raman scattering observation of the low-frequency (LF) region (below 40 up to 12 cm-1) of DNA motions is presented. Since the concentration of the studied DNA solution was very low (1 mg/ml), the spectra features reflect internal vibrations of the macromolecule. The decomposition of the spectra into Lorentzians clearly indicate three intrahelical DNA modes: the corresponding peaks are located at the frequencies 16, 19, and 23 (+/- 1) cm-1. This result is in agreement with our quasi-continuity model of the LF B-form DNA dynamics (V. Lisy, P. Miskovsky and P. Schreiber, J. Biomol. Struct. Dyn. 13, 707 (1996)). The fit of the experimental frequencies to the theory, using the Genetic Algorithms approach, allowed us to make some conclusions about the model force constants which could be found by independent conformational energy calculations. Possible positions of five lowest-frequency DNA peaks, predicted by the model, are discussed.

Fluorescence resonance energy transfer analysis of the degradation of an oligonucleotide protected by a very stable hairpin.

In vitro degradation of antisense oligonucleotides protected or not on their 3' side against enzymatic attack by a naturally forming hairpin has been studied by fluorescence resonance energy transfer (FRET). The two oligonucleotides d(5"TTCTCGCGAAGC3') forming the hairpin and d(5"TTCTCCGGAAGC3') as a control were labeled on their 5' side by tetramethylrhodamine and on their 3' side by fluorescein. Fluorescein has been shown not to hinder the hairpin formation and to give an additional protection against nucleases. The FRET technique proved adequate for an in situ study of these protected antisense oligonucleotides in living cells.

A study of metalloporphyrin-polynucleotide interactions by microcalorimetry and circular dichroism.

In this paper we examine the interactions of Calf Thymus DNA and the model polynucleotides poly(dA).poly(dT), poly(dAdT)2 and poly(dG.dC)2 with a group of metalloporphyrins derived from the freebase porphyrin tetrakis(4-N-methylpyridyl)porphine, H2(TMpy-P4), by means of ultraviolet absorption spectroscopy, circular dichroism spectroscopy and microcalorimetry. We have studied the interactions of the copper, cobalt, nickel and zinc derivatives of H2(TMpy-P4) in addition to the free base porphyrin itself. We have found strong evidence for an external self-stacking interaction of the Cu(TMpy-P4) and Zn(TMpy-P4) derivatives with poly(dA).poly(dT) and poly(dAdT)2 even at low concentrations of porphyrin, and all of the porphyrin derivatives studied appear to display such a self-stacking in interaction with poly(dA.dT)2 at sufficiently high ratios of porphyrin to polynucleotide.

A UV resonant Raman spectroscopic study of the interaction of metallic derivatives of tetrakis(4-N-methylpyridyl)porphine with polynucleotides.

Resonance Raman spectra excited at 257 nm are reported for the complexes of the Nickel, Cobalt and Zinc derivatives of Tetrakis(4-N-methylpyridyl)porphine with poly(dA.dT)2, poly(dA).poly(dT), poly(dG.dC)2 and poly(dG).poly(dC). These spectra are interpreted as evidence of multiple outside binding modes with poly(dA).poly(dT), and of evidence for an outside binding mode with Poly(dG.dC). Some results obtained for the zinc derivative with poly(dA).poly(dT) suggest a binding mode peculiar to this derivative.

A resonance Raman spectroscopic study of the quadruplex form of polyriboinosinic acid.

The four stranded form of polyriboinosinic acid, or poly(rl), formed under conditions of high ionic strength, has been studied principally by resonance Raman spectroscopy excited in the ultraviolet absorbent band of the hypoxanthine residues. UV Absorption and circular dichroism studies were made, principally in order to verify the presence of the quadruplex form at the low concentrations of poly(rl) used, and a trial experiment with the structural probe Tb3+ was also performed. Experimental evidence is found for highly stacked metastable forms present at low concentrations of polynucleotide, which are destroyed by heating in favor of the two well known forms.

Distortion after monofunctional alkylation by mitomycin C of a dodecamer containing its major binding site.

The structural distortions of the duplex dodecamer d(ATTAACGTTAAT)2 monofunctionally alkylated by mitomycin C have been studied by the use of chemical probes reactivity and resonance Raman spectroscopy. This sequence contains the 5'-ACGT sequence for which mitomycin C was determined to present the best affinity (S. Kumar, R. Lipman, and M. Tomasz, Biochemistry 31, 1399 (1992)). Raman spectroscopy as well as osmium tetroxyde reactivity indicate that the distortion of the double helix structure is located around the central CG bases. Mitomycin C reacts exclusively with the 2-amino group of guanine and this binding does not disrupt the inter bases H-bonds, as indicated by chloroacetaldehyde reactivity. Although resonance Raman spectroscopy does not allow the handedness of the monoalkylated CG/GC sequence to be determined, it indicates a similarity between the base stacking and that which would be observed for alternating purine/pyrimidine sequences at high salt concentration.

The concentration dependence of the right to left conformational transition in natural DNA identified by Raman spectroscopy.

The classical and resonance Raman spectra of DNA from Chicken Erythrocytes have been obtained for different DNA concentrations in solution with low and high ionic strengths. The classical Raman spectra of 30 mg/ml DNA solutions were measured in varying the sodium chloride concentration from 0.1 to 4.5 M NaCl. An increase in the salt content of the solution leads to spectral changes in the 600-700 cm-1 region, indicating a C2' endo/anti to C3' endo/syn conformational transition of the purine residues. Other changes around 840 cm-1, due to the antisymmetrical stretching vibration of the PO2 group, are also detected: they were characteristic for the B----Z transition in model systems such as poly(dG-dC).poly(dG-dC). The resonance Raman spectra of low (1 mg/ml) and high (30 mg/ml) concentrated DNA solutions were obtained with low (0.1 M) and high (4.5 M) NaCl contents, in using a 284 nm excitation wavelength. No change was observed in the intensities and band positions in the low and high salt solutions of low concentrated DNA. Thus it is assumed that the DNA structure remains unchanged whatever the salt concentration for low concentrated DNA. In contrast, great modifications of the intensities and positions of some lines were found in the spectra of high DNA concentration solution when the NaCl content is increased up to 4.5 M: these changes resemble to some extent those observed in the study of B----Z transition of several polynucleotide model compounds. It is assumed that the right-handed to left-handed conformational transition may occur in certain sections of natural DNA, likely containing alternating purine-pyrimidine sequences, when the DNA concentration is sufficiently important.

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.

Ultraviolet resonance Raman marker bands of the right to left helix structure transitions in DNA and polynucleotide model compounds.

The right to left helix structural transition in purine-pyrimidine alternating copolymers has been extensively studied by vibrational spectroscopies, amongst many other experimental approaches. Here, the use of resonance Raman spectroscopy in the ultraviolet region (223-, 257- and 281 nm excitation wavelengths) to monitor such structural changes is reviewed in the light of new results obtained on poly(dA-dC).poly(dG-dT) on one hand, and the previous results obtained on poly(dG-dC)2, poly(dA-dT)2 and natural DNA (Chicken erythrocytes) on the other. It is now possible to define B----Z transition marker bands involving the proper bases, which show a similar behaviour on structural transition whatever the composition of alternating purine-pyrimidine sequences: the 1580- and 1487 cm-1 lines of the purines, the 1486- and 1294 cm-1 lines of the pyrimidines are good markers in the vibrational spectra recorded at various UV excitation wavelengths.