Thermodynamic and kinetic parameters for the triplex-forming reactions between a homopurine-homopyrimidine 22-base-pair duplex (sequence of the purine strand: 5'd[AAAGGAGGAGAAGAAGAAAAAA]3') and the four 22-dN third strands (22 dN: 5'd[TTTCCTCCTCTNCTTCTTTTTT]3', where N = A, C, T, or G) were determined from thermal denaturation and renaturation UV absorbance profiles. Cooling and heating curves were not superimposable and thus allowed us to determine the rate constants of association (k(on)) and dissociation (k(off)) as a function of temperature, assuming a two-state model analogous to that developed for duplex-forming reactions. Experiments were performed in 10 mM cacodylate buffer (pH 6.8) in the presence of NaCl concentrations ranging from 20 to 300 mM. Within experimental accuracy, the main results are the following: (i) The rate constants k(on) and k(off) result in linear Arrhenius plots, consistent with the prediction of two-state association and dissociation (ii) k(on) is independent of the nature of the base N located in the center of the third strand. (iii) k(on) strongly decreases when the NaCl concentration is decreased. (iv) The activation energy, E(on), is always negative and becomes more negative when the NaCl concentration is decreased. (v) k(off) is independent of NaCl concentration but depends on the base N, with its magnitude following the order C greater than G greater than A much greater than T. (vi) The activation energy, E(off), is independent of the base N. All these results are discussed in the light of a nucleation-zipping model similar to that developed for the duplex-coil transitions [Craig, M. E., Crothers, D. M., & Doty, P. (1971) J. Mol. Biol. 62, 383-401; Pörschke, D., Eigen, M. (1971) J. Mol. Biol. 62, 361-381].

A triple helix is formed upon binding of an oligodeoxynucleotide to the major groove of duplex DNA. A benzo[e]pyridoindole derivative (BePI) strongly stabilized this structure and showed preferential binding to a triplex rather than to a duplex. Energy transfer experiments suggest that BePI intercalates within the triple helix. Sequence-specific inhibition of transcription initiation of a specific gene by Escherichia coli RNA polymerase by a triplex-forming oligodeoxynucleotide is strongly enhanced when the triplex is stabilized by BePI. Upon irradiation with ultraviolet light, BePI induces covalent modifications of the target within the triple helix structure.

The specificity of a homopyrimidine oligonucleotide binding to a homopurine-homopyrimidine sequence on double-stranded DNA was investigated by both molecular modeling and thermal dissociation experiments. The presence of a single mismatched triplet at the center of the triplex was shown to destabilize the triple helix, leading to a lower melting temperature and a less favorable energy of interaction. A terminal mismatch was less destabilizing than a central mismatch. The extent of destabilization was shown to be dependent on the nature of the mismatch. Both single base-pair substitution and deletion in the duplex DNA target were investigated. When a homopurine stretch was interrupted by one thymine, guanine was the least destabilizing base on the third strand. However, G in the third strand did not discriminate between a C.G and an A.T base pair. If the stretch of purines was interrupted by a cytosine, the presence of pyrimidines (C or T) in the third strand yielded a less destabilizing effect than purines. This study shows that oligonucleotides forming triple helices can discriminate between duplex DNA sequences that differ by one base pair. It provides a basis for the choice of antigene oligonucleotide sequences targeted to selected sequences on duplex DNA.

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The primary or secondary structure of single-stranded nucleic acids has been investigated with fluorescent oligonucleotides, i.e., oligonucleotides covalently linked to a fluorescent dye. Five different chromophores were used: 2-methoxy-6-chloro-9-amino-acridine, coumarin 500, fluorescein, rhodamine and ethidium. The chemical synthesis of derivatized oligonucleotides is described. Hybridization of two fluorescent oligonucleotides to adjacent nucleic acid sequences led to fluorescence excitation energy transfer between the donor and the acceptor dyes. This phenomenon was used to probe primary and secondary structures of DNA fragments and the orientation of oligodeoxynucleotides synthesized with the alpha-anomers of nucleoside units. Fluorescence energy transfer can be used to reveal the formation of hairpin structures and the translocation of genes between two chromosomes.

From time-resolved measurements of the decay of singlet molecular oxygen phosphorescence at 1270 nm in D,O, direct estimates have been gained for the rate constants of the singlet oxygen reactions with a group of sulphur compounds in the pD range 5 to 13. In the case of most of the thiols, the results are consistent with singlet oxygen reacting exclusively with the thiolate anions. At the normal physiological pH 7, the apparent rate constants (in units of M-' s-') were 8.9 x loh (cysteine), 2.5 x lo6 (N-acetyl cysteine), 2.9 x lo6 (glutathione), 3.0 x lo5 (2-mercaptoethanol), 2.3 x lo7 (ergothioneine) and 2.7 x lo6 (2-mercaptopropionyl glycine). For methionine the rate constant, 1.4 x lo7, was independent of pD in the range studied. These sulphur compounds, in particular Nacetyl cysteine and ergothioneine, or related compounds, might be considered as possible candidates for protection against skin photosensitivity side effects associated with the photodynamic therapy of solid tumours and as observed in the disease erythropoietic protoporphyria.Jori. G . and J. D. Spikes (1984) Photohiochcmistry of

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The binding of carbon monoxide and weak or strong field ligands to deuteroheme has been studied over the widest possible ranges of carbon monoxide and ligand concentrations. The variations of the affinity constants of the monoliganded hemes for carbon monoxide (and of monocarbonylheme for a sixth ligand) have been related to the relative a-donor-rr-acceptor character of the ligands. With respect to the embedded heme group of the 02-carrying hemoproteins, monoimidazoleheme appears in the most favorable situation to bind carbon monoxide. Comparison with T h e hydrophobic environment of the heme group in hemoproteins has led us and others (see, for instance, Caughey et al., 1965;Kassner, 1972Kassner, , 1973 to think that a nonaqueous solution may be a more suitable medium for iron-porphyrin studies. We have previously discussed the preparation of bare, i.e. free of axial ligands, deuteroheme in benzene, and its coordination by weak or strong field ligands (Brault and Rougee, 1974a-c) and by carbon monoxide (Rougee and Brault, 1973). W e present here a more complete study of the coordinating properties of deuteroheme in the presence of both a weak or strong field ligand (L) and carbon monoxide. The purpose of this study is to determine to what extent the fixation of a given ligand (or C O ) alters the reactivity of heme toward CO (or a ligand). Our results give information on the unusual behavior of CO among other ligands and the very high affinity of five-coordinate hemoproteins such as myoglobin and hemoglobin toward CO. We conclude that hemoproteins which bind CO with the maximum affinity hitherto reported (3-5 X IO8 M -' ) are as relaxed, according to the concept of Perutz et al. ( I974a,b), as free, unconstrained monoimidazoleheme in solution. Experimental SectionSolvents and chemicals were of the purest available grade. Chlorodeuterohemin dimethyl ester was synthesized according to routine procedure. The reduced form will be hereafter called heme.Argon, grade U, was purchased from Air-Liquide, and pure carbon monoxide (CO) was from Matheson. Diluted CO in nitrogen, grade U (respectively, 1.08 X 0.98 X lo-', and 1.01 X in volume), was specially supplied by Air-Liquide.Ultraviolet (uv), visible, and near-infrared (ir) (up to 3000 nm) spectra were recorded using a Beckman DKU