Sté Phane Despax

Alban Debacker

Yunjie Xiang

Quan Li

Fu Li

Xiaofeng Lin

Yuefeng Liu

Jingjie Luo

Xuemei Liao

Xiaojie Liu

Wei Yu

Wen Luo

Yang Zhou

Lei Wang

Sebastien Harlepp

Gilles Versini

Jean-Pierre Vola

Thank

I

Resumé

Dans une premiè re partie, nous mesurons l'affinité de l'interaction entre [Ru(pby) 2 dppz] 2+ et l'ADN en utilisant la luminescence induite lors de la complexation. Nous é tudions l'é volution de l'affinité lorsque la force ionique de la solution augmente. Dans une deuxiè me partie, nous modifions les extrê mité s d'un double brin d'ADN en y greffant des fluorophores. De la mesure de transfer d'é nergie non-radiative entre ces fluorophores, nous é tudions l'é volution de la longueur du complexe. Nous effectuons un dosage d'un double brin de 15 paires de bases d'ADN par le complexe ruthé né . Nous nous servons de la luminescence induite par l'intercalation du groupement dppz. Cependant, l'incré ment de luminescence par groupement intercalé n'est pas connu, et nous ne pouvons pas le mesurer en saturant le brin d'ADN. Nous utilisons alors une technique mise au point par Nishida [Method for Measuring the Binding of Small Molecules to Proteins from Binding-Induced Alterations of Physical-Chemical Properties], dans laquelle deux titrations de deux solutions d'ADN de deux concentrations diffé rentes sont effectué es. En utilisant le fait que, lorsque deux solutions d'ADN complexé par le composé ruthé né , possè dent la mê me luminescence par paire de base , le taux de complexation de ces deux solutions doit ê tre le mê me, nous pouvons alors dé terminer, sans hypothè se supplé mentaire, le taux de complexation de l'ADN. De l'é volution de ce taux en fonction avec la concentration de ligand, nous dé duisons son affinité pour l'ADN. Nous é tudions maintenant le changement de longueur d'un double brin d'ADN de 15 paires de bases, modifié à ses deux extrê mité s par deux fluorophores : Alexa488 et Alexa568. Lorsque Alexa 488 est porté dans un é tat excité , il peut se dé sexciter en transfé rant de l'é nergie de maniè re non-radiative à Alexa568, qui se dé sexcite alors en é mettant des photons de plus faibles é nergie que ceux é mis par Alexa488. L'efficacité de ce transfer d'é nergie peut ê tre quantifié à partir de la mesure des intensité s é mises à II basse et haute é nergie. Elle dé pend a priori de l'efficacité couplage (et en consé quence de la distance) entre les deux fluorophores. Nous effectuons des mesures de temps de vie des é tats excité s de chacun des fluorophores. Nous avons observé que l'addition de ligand a pour consé quence une forte inhibition quenching des fluorophores. De l'analyse de l'é volution du temps de vie du fluorophore donneur d'une part et de celui du fluorophore accepteur d'autre part, nous dé duisons l'é volution de l'efficacité du transfer d'é nergie en fonction de la concentration de ligand. Nous confrontons les ré sultats obtenus par chacune de ces analyses, et en dé duisons finalement, en nous servant de l'analyse de l'é quilibre effectué e dans la premiè re partie, l'é volution de la longueur de la chaî ne en fonction du taux de complexation Abstract This Ph.D thesis is mainly divided in to 2 parts. The first part is luminescence study, we are interested in the affinity constant (Ka) change under different salinity environments when the complexation of [Ru(bpy) 2 dppz] 2+ -DNA arrive equilibrium.

In the second part, we focus our attention on the kinetic study by fluorescence which comes from the fluorophore. The distance change between 2 fluorophores is explored when [Ru(bpy) 2 dppz] 2+ intercalates into DNA, which lead to the variation of DNA conformation. Any changes in DNA conformation will be reflected by the efficiency change of fluorescence resonance energy transfer (FRET). Quantitative analysis on the Ru(bpy) 2 dppz] 2+ -DNA interaction will be built in the second part.

In the first part, the interaction of [Ru(bpy) 2 dppz] 2+ with DNA is studied in a wide range of DNA / [Ru(bpy) 2 dppz] 2+ ratios by using the luminescence signal which comes from complex. The affinity constant (Ka) is explored under different chloride sodium concentration (NaCl=[0, 100 mM]), when the complexation reaches equilibrium. Nishida method is employed to compute the value of Ka without any hypothesis. The value of affinity constant is at the level scale of 10 6 M -1 which is basically identical to the other researcher's results. Ka decreased with increasing the III concentration of NaCl as we expected.

Quantitative analysis on the Ru(bpy) 2 dppz] 2+ -DNA interaction will be done in the second part. DNA was modified by different fluorophores at its extremities, 5' end and 3' end were labeled with alexa488 (seen as donor) and alexa568 (seen as acceptor), respectively. Our goal is to study the efficiency change of FRET and the change of distance between 2 fluorophores with fluorescence technique when one Ruthenium molecule intercalate in to DAN base pair. Two methods will be employed to achieve our idea. One is that the efficiency of FRET can be computed from the donor emission (alexa488), the other is the efficiency of FRET can be calculated from the acceptor emission (acceptor), the efficiency of FRET is highly dependent on the distance of 2 fluorophores ( ), any changes in distance will cause the efficiency change. The FRET efficiency decreased when the [Ru(bpy) 2 dppz] 2+ intercalated into DNA structure, which also meant that the distance between 2 fluorohore increased. IV V Acknowlegements While many of the relationships that I have established during my graduate career were solely professional, some of them transited onto personal level and they have provided a lot more than just learning a particular set of skills or technical information. Nevertheless, I would like to take the opportunity and thank many people, who assisted me at some point along my career in one way or another, and without whom this work would not have been possible. First of all, I would like to pay my deepest thank to my PhD advisor: Jean-Pierre Münch, for the support you provided. You helped and taught me how to solve scientific problems and fight with French administration when needed, your rigorous scientific attitude plays a very important role in my later research life. Many thanks for encouraging me to come to Strasbourg and to begin Ph.D thesis, which turned out to be one of the most challenging

Deoxyribonucleic acid (DNA)

DNA is a very long, threadlike macromolecule built form a large number of dexoyribonucleotides, each composed of a base, a sugar, and a phosphate group. The bases of DNA molecules carry genetic information, whereas their sugar and phosphate group perform a structural role. All living cells on Earth, without any known exception, store their hereditary information in the universal language of DNA sequences. These monomers string together in a long linear sequence that encodes the genetic information. Note that the individual sugar-phosphate units are asymmetric, giving the backbone of the strand a definite directionality, or polarity. The backbone has two important features: it is highly flexible and is highly charged (in water, at room temperature).

Chemical structure of DNA

The negative charge of the backbone is due to the fact that the phosphate groups in water or under physiological pH are fully dissociated. Through template polymerization (Figure 1.2C), the sequence of nucleotides in an existing DNA strand controls the sequence in which nucleotides are joined together in a new DNA strand;

T in one strand pairs with A in the other, and G in one strand pairs with C in the other.

The new strand has a nucleotide sequence complementary to that of the old strand,

Physical properties of DNA

The physical structure of double-stranded DNA is determined by the fact that its character is amphiphilic [START_REF] Bruinsma | Physics of protein-DNA interaction. Physics of bio-molecules and cells[END_REF]. That means that one part of DNA chain (the phosphate backbone) is hydrophilic and another one (bases) is hydrophobic. Along with the flexibility of backbone, this amphiphilic character is a cause of double-helical structure of DNA. Double-stranded DNA occurs as a ladder which is twisted around its axis right-handed. The diameter of such twisted double-helix is 2.37 nm [START_REF] Bruinsma | Physics of protein-DNA interaction. Physics of bio-molecules and cells[END_REF]. The twisting angle between adjacent base pairs is 34.6° and the distance between two neighbor nucleotides is 0.34 nm. The number of base pairs coinciding with the full twist (360°) of DNA double-helix is around 10.4 [START_REF] Bruinsma | Physics of protein-DNA interaction. Physics of bio-molecules and cells[END_REF]. That full twist repeats itself in every 3.4 nm (Figure 1.5). Between two molecules of deoxyribose attached to complementary base pairs, there is a space for creating grooves, which go along the whole DNA chain. Both of the N-glycosidic bonds connecting deoxyribose with base pairs are on the same side of the double helix. Therefore the size of the grooves is not identical. They are 0.22 nm or 0.12 nm wide and are called major and minor groove, respectively [START_REF] Dickerson | Crystal structure analysis of a complete turn of B-DNA[END_REF].

DNA in solution is not rigid but is continually changing its conformation due to thermal fluctuations. Therefore, the bending stiffness of DNA is measured by the persistence length. It is defined as the distance over which the direction of a polymer segment persists, in the time or ensemble average, owing to limited flexibility of the polymer. It means the length of the DNA along which a thermally excited bend of 1 radian typically occurs. For DNA the persistence length is l p ≈ 50 nm, (≈ 150 bp).

Under physiological conditions this value is larger than the persistence length of synthesized polymers: DNA is referred to as semi-flexible. The flexibility of DNA is due to fact that the covalent P-O (phosphate-oxygen) bonds can freely rotate around, so adjacent P-O (phosphate-oxygen), and deoxyribose rings can rotate freely. DNA chain may be described with the Worm-Like Chain model (WLC) [START_REF] Jackson | Molecular and cellular biophysics[END_REF].

Binding modes of ruthenium compound with DNA

DNA association with ruthenium complexes does not involve the formation of a covalent bound, several interactions have been established for these complexes [START_REF] Yang | Study of the interaction between novel ruthenium (II)-polypyridyl complexes and calf thymus DNA[END_REF][START_REF] Hartshorn | Novel dipyridophenazine complexes of ruthenium (II): exploring luminescent reporters of DNA[END_REF][START_REF] Grueso | Thermodynamic and structural study of phenanthroline derivative ruthenium complex/DNA interactions: Probing partial intercalation and binding properties[END_REF][START_REF] Lincoln | DNA binding geometries of ruthenium (II) complexes with 1, 10-phenanthroline and 2, 2'-bipyridine ligands studied with linear dichroism spectroscopy. Borderline cases of intercalation[END_REF][START_REF] Song | Crystal structure of Δ-[Ru (bpy) 2dppz] 2+ bound to mismatched DNA reveals side-by-side metalloinsertion and intercalation[END_REF][START_REF] Niyazi | Crystal structures of Λ-[Ru (phen) 2dppz] 2+ with oligonucleotides containing TA/TA and AT/AT steps show two intercalation modes[END_REF][START_REF] Barton | Binding modes and base specificity of tris (phenanthroline) ruthenium (II) enantiomers with nucleic acids: tuning the stereoselectivity[END_REF][START_REF] Neither | LAMBDA.-Tris (phenanthroline) ruthenium (II) Binds to DNA by Classical Intercalation[END_REF].

Three main different non-covalent modes are induced in this part: groove binding, intercalation and insertion, which are shown in Figure 1.7 and Figure 1.8 [START_REF] Zeglis | Metallo-intercalators and metallo-insertors[END_REF][START_REF] Barton | Enantiomeric selectivity in binding tris(phenanthroline)zinc(II) to DNA[END_REF].

Groove binding:

The earliest work has been performed on the DNA-binding of octahedral metal centers focused on tris(phenanthroline) complexes of ruthenium, chromium, zinc, nickel and cobalt (Figure . 1.9) [START_REF] Barton | Enantiomeric selectivity in binding tris(phenanthroline)zinc(II) to DNA[END_REF][START_REF] Barton | Tris(phenanthroline)ruthenium(II): stereoselectivity in binding to DNA[END_REF][START_REF] Kumar | Photophysics of ruthenium complexes bound to double helical DNA[END_REF][START_REF] Barton | Binding modes and base specificity of tris(phenanthroline)ruthenium(II) enantiomers with nucleic acids: tuning the stereoselectivity[END_REF][START_REF] Rehmann | Proton NMR studies of tris(phenanthroline) metal complexes bound to oligonucleotides: characterization of binding modes[END_REF][START_REF] Rehmann | Proton NMR studies of tris(phenanthroline) metal complexes bound to oligonucleotides: structural characterizations via selective paramagnetic relaxation[END_REF][START_REF]Site-specific cleavage of left-handed DNA in pBR322 by lambda-tris (diphenylphenanthroline) cobalt (III)[END_REF][START_REF]Chiral probes for the handedness of DNA helices: enantiomers of tris (4, 7-diphenylphenanthroline) ruthenium (II)[END_REF]. Extensive photophysical and NMR experiments described that these complexes bind to DNA via two distinct modes: [START_REF]X-ray structure of the major adduct of the anticancer drug cisplatin with DNA: cis[END_REF] hydrophobic interactions in the minor groove and (2) partial intercalation of a phenanthroline ligand into the helix in the major groove. Perhaps more important than the discovery of these dual binding modes, however, was the revelation these complexes provided regarding the importance of chirality in DNA-binding [START_REF]Metals and DNA: molecular left-handed complements[END_REF]. In the case of [Ru(phen) 3 ] 2+ , for instance, the  -enantiomer is found in the intercalative binding mode, while the complementary  -enantiomer is favored in the minor groove binding mode. In subsequent years, it was discovered that metal centers bearing more sterically demanding phenanthroline ligand derivatives, such as diphenylphenanthroline (DIP), display even more dramatic chiral discrimination.

Luminescence and hypochromism assays have revealed enantioselective binding on the part of [Ru(DIP) 3 ] 2+ ; the  -enantiomer binds enantiospecifically to right-handed B-DNA and the  -enantiomer binds only to left-handed Z-DNA [START_REF]Chiral probes for the handedness of DNA helices: enantiomers of tris (4, 7-diphenylphenanthroline) ruthenium (II)[END_REF]. This enantiospecificity has been exploited to map left-handed Z-DNA sites in supercoiled plasmids using [  -Co(phen) 3 ] 3+ [START_REF]Site-specific cleavage of left-handed DNA in pBR322 by lambda-tris (diphenylphenanthroline) cobalt (III)[END_REF]. Indeed, this trend in enantiomeric selectivity for octahedral tris(chelate) complexes, matching the symmetry of the complex to that of the DNA helix, has repeatedly and consistently been observed for non-covalent DNA-binding complexes developed in the years since these initial discoveries [START_REF] Erkkila | Recognition and Reaction of Metallointercalators with DNA[END_REF][START_REF] Satyanarayana | Tris(phenanthroline)ruthenium(II) enantiomer interactions with DNA: Mode and specificity of binding[END_REF][START_REF]Binding of .DELTA.-and[END_REF].

These earliest tris (phenanthroline) complexes do not, of course, represent the only examples of complexes that bind DNA via the minor or major grooves. For instance, the extensively studied [Cu(phen) 2 ] + , has been shown to bind DNA via the minor groove. Indeed, these groove-binding complexes not only bind DNA but also cleave the macromolecule in the presence of hydrogen peroxide [28][START_REF]Nuclease activity of 1,10-phenanthroline-copper ion[END_REF][START_REF] Sigman | Targeted chemical nucleases[END_REF]. Metal complexes that bind in the groove have come a long way since these first studies and are now quite sophisticated. Turro and co-workers, for instance, developed an artificial photonuclease by linking the metallo-groove-binder [Ru(bpy) 3 ] 2+ to an electron-acceptor chain containing two viologen units. Interestingly, the chemistry of metallo-groove-binders also extends to supramolecular self-assembly. Following the initial work of Lehn on the interaction and cleavage of DNA with a cuprous double-helicate [START_REF] Schoentjes | Interaction of Double-Helical Polynuclear Copper (I) complexes with doublestranded DNA[END_REF], Hannon and co-workers designed a triple-helicate capable of recognizing three-way junctions in DNA. This intricate recognition has recently been characterized by single crystal X-ray crystallography [START_REF] Hannon | Supramolecular DNA recognition[END_REF][START_REF] Childs | A DNA-Binding Copper (I) Metallosupramolecular Cylinder that Acts as an Artificial Nuclease[END_REF][START_REF] Oleksi | Molecular Recognition of a Three -Way DNA Junction by a Metallosupramolecular Helicate[END_REF][START_REF] Uerpmann | Design and DNA Binding of an Extended Triple -Stranded Metallosupramolecular Cylinder[END_REF]. As their name suggests, these ligands, oriented parallel to the base pairs and protruding away from the metal center, can readily  -stack in the DNA duplex.

Furthermore, upon binding, the ligands behave as a stable anchor for the metal complex with respect to the double helix and direct the orientation of the ancillary ligands with respect to the DNA duplex. Two well known examples of intercalating ligands are phi (9,10-phenanthrenequinone diimine) and dppz (Figure . 1.10) [START_REF] Erkkila | Recognition and Reaction of Metallointercalators with DNA[END_REF].

Ligand intercalation was first demonstrated by photophysical studies [START_REF] Hartshorn | Novel dipyridophenazine complexes of ruthenium (II): exploring luminescent reporters of DNA[END_REF][START_REF] Barton | Enantiomeric selectivity in binding tris(phenanthroline)zinc(II) to DNA[END_REF][START_REF] Barton | Tris(phenanthroline)ruthenium(II): stereoselectivity in binding to DNA[END_REF][START_REF] Kumar | Photophysics of ruthenium complexes bound to double helical DNA[END_REF][START_REF] Barton | Binding modes and base specificity of tris(phenanthroline)ruthenium(II) enantiomers with nucleic acids: tuning the stereoselectivity[END_REF][START_REF] Rehmann | Proton NMR studies of tris(phenanthroline) metal complexes bound to oligonucleotides: characterization of binding modes[END_REF][START_REF] Rehmann | Proton NMR studies of tris(phenanthroline) metal complexes bound to oligonucleotides: structural characterizations via selective paramagnetic relaxation[END_REF][START_REF]Site-specific cleavage of left-handed DNA in pBR322 by lambda-tris (diphenylphenanthroline) cobalt (III)[END_REF][START_REF]A molecular light switch for DNA[END_REF][START_REF] Jenkins | Characterization of dipyridophenazine complexes of ruthenium (II): the light switch effect as a function of nucleic acid sequence and conformation[END_REF][START_REF] Tuite | Photophysical Evidence That Δ-and Λ-[Ru(phen)2(dppz)]2+ Intercalate DNA from the Minor Groove[END_REF][START_REF]DNA binding of .DELTA.-and .LAMBDA.-[Ru(phen)2DPPZ]2+. C. Hiort, P. Lincoln, B. Norden[END_REF][START_REF]Diastereomeric DNA-Binding Geometries of Intercalated Ruthenium(II) Trischelates Probed by Linear Dichroism: [Ru(phen)2DPPZ]2+ and[END_REF]. However, it was not until extensive NMR studies [START_REF]Differential DNA Recognition by the Enantiomers of 1-Rh(MGP)2phi: A Combination of Shape Selection and Direct Readout[END_REF][START_REF] Hudson | Solution Structure of a Metallointercalator Bound Site Specifically to DNA[END_REF][START_REF]Use of Selective Deuteration and 1H NMR in Demonstrating Major Groove Binding of .DELTA.-[Ru(phen)2dppz]2+ to d(GTCGAC)[END_REF][START_REF]1H-NMR of Rh(NH3)4phi3+ Bound to d(TGGCCA)2: Classical Intercalation by a Nonclassical Octahedral Metallointercalator[END_REF] and high resolution crystal structures had been performed that the structural details of this binding mode were properly illuminated (Figure . 1.11) [START_REF] Kielkopf | Structure of a photoactive rhodium complex intercalated into DNA[END_REF]. Metallo-intercalators intercalate into the double helix from the major groove, with the intercalating ligand acting in effect as a new base pair. No bases are ejected out of the duplex. Further, intercalation leads to a doubling of the rise and a widening of the major groove at the binding site. Importantly, this interaction distorts only minimally the structure of DNA. In the case of B-DNA, for example, the sugars and bases all maintain their original C 2 ' endo and anti conformations, respectively. Indeed, only the opening of the phosphate angles, not any base or sugar perturbations, is necessary for intercalation. -Rh[(R,R)-Me 2 trien](phi) 3+ bound to its target sequence, 5'-TGCA-3' [START_REF] Zeglis | Metallo-intercalators and metallo-insertors[END_REF].

Metallo-intercalators between mismatched DNA:

The vast majority of non-covalent, DNA binding metal complexes are either groove-binders or intercalators. However, the nonexistence of complexes that bind DNA via other ways does not necessarily exclude the existence of alternative modes.

Indeed, L. S. Lerman, in his article proposing intercalation as the DNA-binding mode for organic dyes, presciently proposed a third non-covalent binding mode: insertion [START_REF] Lerman | Structural considerations in the interaction of DNA and acridines[END_REF]. He pointed that a molecule, may bind ''a DNA helix with separation and displacement of a base-pair.'' Metallo-insertors, like metallo-intercalators, contain a planar aromatic ligand that extends into the base-stack upon DNA-binding. However,

Interaction between Ru(bpy)2dppz 2+ and DNA

When the first observation of molecular "light switch" of Ru(bpy) 2 dppz 2+ for DNA was discovered, intense studies were explored on the interaction between them [START_REF] Song | Crystal structure of Δ-[Ru (bpy) 2dppz] 2+ bound to mismatched DNA reveals side-by-side metalloinsertion and intercalation[END_REF][START_REF]A molecular light switch for DNA[END_REF][47][48][START_REF] Hu | Ru (bpy) 2dppz] 2+ electrochemiluminescence switch and its applications for DNA interaction study and label-free ATP aptasensor[END_REF][START_REF] Lim | Sensitivity of Ru (bpy) 2dppz2+ Luminescence to DNA Defects[END_REF][START_REF] Sun | Efficient DNA photocleavage by [Ru (bpy) 2 (dppn)] 2+ with visible light[END_REF]. In this part, we will briefly and mainly introduce some studies of J K Barton because of her excellent work in the developing of probing the structure of DNA.

1). First observation of molecular "light switch" for DNA: Ru(bpy) 2 dppz 2+ [START_REF]A molecular light switch for DNA[END_REF]:

In this study, they find Ru(bpy) 2 dppz 2+ to be a highly sensitive spectroscopic reporter of double-helical DNA by using steady-state emission spectra technique [START_REF]A molecular light switch for DNA[END_REF].

In aqueous solution, luminescence is detectable only when Ru(bpy) 2 dppz 2+

intercalates into the nucleic acid structure. The emission characteristics furthermore sensitively distinguish both in terms of intensity and emission maximum the different helical forms of the polynucleotide (Figure 1.14). Ru(bpy) 2 dppz 2+ was useful as a sensitive, nonradioactive, luminescent DNA probe in both heterogeneous and homogeneous assays. double-helical DNA [START_REF]A molecular light switch for DNA[END_REF].

2). Ru(bpy) 2 dppz 2+ --the light switch effect as a function of nucleic acid sequence and conformation [START_REF] Jenkins | Characterization of dipyridophenazine complexes of ruthenium (II): the light switch effect as a function of nucleic acid sequence and conformation[END_REF]:

The spectroscopic propeties for Ru(bpy) 2 dppz 2+ and Ru(phen) 2 dppz 2+ on binding to nucleic acids of different sequences and conformations have been explored by spectroscopic measurements. Both complexes (Ru(bpy) 2 dppz 2+ and Ru(phen) 2 dppz 2+ ) serve as "molecular light switch" for DNA, luminescing intensely in the presence of DNA but no photoluminescence in aqueous solution. The luminescent enhancement observed upon binding is attributed to the sensitivity of the excited state to quenching by water; the metal complex, upon intercalation into the DNA helix, is protected from the aqueous solvent, thereby preserving the luminescence. Correlations between the extent of protection (depending upon the DNA conformation) and the luminescence parameters are observed. Indeed, the strongest luminescent enhancement is observed for intercalation into DNA conformations which afford the greatest amount of overlap with access from the major groove, such as in triple helices. Differences are observed in the luminescent parameters between the two complexes which also correlate with the level of water protection. In the presence of nucleic acids, these two complexes exhibit biexponential decays in emission. Quenching studies are consistent with two intercalative binding modes for the dppz ligand from the major groove: one in which the metal-phenazine axis lies along the DNA dyad axis and another where the metal-phenazine axis lies almost perpendicular to the DNA dyad axis. Ru(bpy) 2 dppz 2+

and Ru(phen) 2 dppz 2+ can be seen as unique reporters of nucleic acid structures and may become valuable in the design of new diagnostics for DNA.

3). Sensitivity of Ru(bpy) 2 dppz 2+ luminescence to DNA defects [START_REF] Lim | Sensitivity of Ru (bpy) 2dppz2+ Luminescence to DNA Defects[END_REF]:

Ru(bpy) 2 dppz 2+ , upon binding to DNA contained a defect, exhibits significant luminescent enhancements above then well matched DNA. In the presence of a single base mismatch, large luminescent enhancements are evident when ruthenium bound to an oligonucleotide containing an abasic site (Figure 1.15). Titrations with hairpin oligonucleotides containing a variable mismatch site exists correlation between the level of luminescent enhancement and the thermodynamic destabilization associated with the mismatch. This correlation is reminiscent of that found earlier for a bulky rhodium complex that binds mismatched DNA sites through metalloinsertion, where the complex binds the DNA from the minor groove side, ejecting the mismatched bases into the major groove. This metalloinsertion mode for the dppz complex at the defect site is proved by differential quenching studies with minor and major groove quenchers and time-resolved emission studies. For sure, the utility of Ru(bpy) 2 dppz 2+ can be seen as a sensitive reporter of DNA structure with defect site. 4). Crystal structure of  -Ru(bpy) 2 dppz 2+ bound to mismatched DNA reveals side-by-side metalloinsertion and intercalation [START_REF] Song | Crystal structure of Δ-[Ru (bpy) 2dppz] 2+ bound to mismatched DNA reveals side-by-side metalloinsertion and intercalation[END_REF]:

The versatile binding modes attainable for octahedral metal complexes bearing an intercalating ligand are depicted in detail. Here it is shown that two independent views of metalloinsertion in this work, two of intercalation and one of end-capping (Figure 1.16 and 1.17). The metal complex binds with DNA through metalloinsertion in the minor groove at destabilized regions of the DNA, accompanied by extrusion of the mismatched bases. This binding mode has been observed previously with a sterically expansive ligand, but this structure clearly demonstrates that a narrower ligand such as dppz is equally capable of recognizing mismatches by the means of metalloinsertion, pointing to the generality of this binding mode. The smaller size of the dppz ligand also allows the ruthenium complex to bind through classical intercalation between two consecutive well-matched base pairs. Curiously, intercalated complexes are also located in the minor groove, which they hypothesize is stabilized by extensive ancillary interactions. This discrepancy notwithstanding, the crystal structure attests to the remarkable structural flexibility of DNA upon high-density ligand binding, illustrates the nuanced binding geometries sampled by a non-covalently bound small molecule, and highlights the dominance of metalloinsertion as the preferred binding mode to destabilized regions of DNA. These newly obtained structural understandings will help guide the development of future generations of metal complexes as chemical tools and medicinal agents. binds between two well-matched base pairs; (3) end-capping, whereby the complex (yellow) stacks with the terminal Watson-Crick pair of the duplex [START_REF] Song | Crystal structure of Δ-[Ru (bpy) 2dppz] 2+ bound to mismatched DNA reveals side-by-side metalloinsertion and intercalation[END_REF].

Figure 1.17: The end-capping complex. The duplex (dark grey) is end-capped by the ruthenium complex (red), which stacks between an extruded adenosine (blue) and the first complex (yellow) in a crystallographically related duplex (light grey). The last GC base pair (cytidine, cyan; guanosine, green) forms a frayed end [START_REF] Song | Crystal structure of Δ-[Ru (bpy) 2dppz] 2+ bound to mismatched DNA reveals side-by-side metalloinsertion and intercalation[END_REF].

Characterization of Ru(bpy)2dppz 2+ and DNA interaction

Ru(bpy) 2 dppz 2+ exhibits intense luminescence when upon binding to DNA due to the ligand part (dppz) intercalation into intact DNA base pair. There are two very interesting questions which deserve to be determined by their interactions as the interaction between them reaches equilibrium, these will help us to understand the interaction modes and the structural changes of DNA.

Outline of the manuscript

In chapter 2, the interaction between Ru(bpy) 2 dppz 2+ and DNA will be reported. We are going to follow the evolution of the luminescence intensity to compute the affinity constant a K and the number of base pair occupied by one ruthenium molecule with Nishida method. The dependence of a K on ionic strength is also explored. The results we get from chapter 2 will also be used in chapter 3 in order to quantify the dynamic changes.

In chapter 3, we use Fluorescence Resonance Energy Transfer (FRET) in order to monitor the average distance between the extremities of a 15 bp dsDNA modified with 2 fluorophores at its extremities. In this chapter, both ends of dsDNA are modified by two types of fluorophores: Alexa488 and Alexa568. When Alexa488 is excited to an excited state, it can decay by transferring non-radiative energy to Alexa568, which then de-excites by emitting photons of lower energy than those emitted by Alexa488. The efficiency of this energy transfer can be quantified from the measurement of intensities emitted at low and high energy. It depends on a priori of the coupling efficiency (and therefore the distance) between the two fluorophores. We will show that the increase of the average distance between the DNA extremities is incompatible with the assumption of a rigid and straight DNA/Ru(bpy) 2 dppz 2+ complex.

Chapter 2

2 Luminescence study

A brief introduction of luminescence

Since the elucidation of the structure of anti-parallel double helical DNA, intense research has focused on the development of nonradioactive probes for nucleic acids [START_REF] Zeglis | Metallo-intercalators and metallo-insertors[END_REF][START_REF] Langer-Safer | Immunological method for mapping genes on Drosophila polytene chromosomes[END_REF][START_REF]Interaction du bromhydrate d''ethidium avec les acides nucl'eiques (AN). etude spectrofluorim[END_REF][START_REF] Ward | Fluoresence studies of nucleotides and polynucleotides III. diphosphopyridine nucleotide analogues which contain fluorescent purines[END_REF][START_REF] Sharp | Detection of two restriction endonuclease activities in Haemophilus parainfluenzae using analytical agarose-ethidium bromide electrophoresis[END_REF][START_REF] Matthews | Enhanced chemiluminescent method for the detection of DNA dot-hybridization assays[END_REF]. The construction of small molecules that recognize and react at specific DNA sites has been an area of intense interest. In particular, the study of transition metal complexes that bind DNA with specificity has been a burgeoning field. This growth has been due in large part to the useful properties of metal complexes, which possess a wide array of photophysical attributes and allow for the modular assembly of an ensemble of recognition elements [START_REF] Zeglis | Metallo-intercalators and metallo-insertors[END_REF][START_REF]Unusual photophysical properties of a ruthenium (II) complex related to [Ru (bpy) 2 (dppz)[END_REF].

There has been considerable interest in Ru(bpy) 2 dppz 2+ as a luminescent probe of DNA over the past few decades since the first observation of molecular "light switch"

for DNA [START_REF]A molecular light switch for DNA[END_REF]. The chemical structure of the ruthenium complex was shown in Figure 2.1. In particular, this complex is brightly luminescent when bound to DNA, but it is non-emissive in aqueous solution [START_REF]A molecular light switch for DNA[END_REF][START_REF] Jenkins | Characterization of dipyridophenazine complexes of ruthenium (II): the light switch effect as a function of nucleic acid sequence and conformation[END_REF][47][48][START_REF] Hu | Ru (bpy) 2dppz] 2+ electrochemiluminescence switch and its applications for DNA interaction study and label-free ATP aptasensor[END_REF][START_REF] Turro | Proton transfer quenching of the MLCT excited state of [Ru(phen) 2 dppz] 2+ in homogeneous solution and bound to DNA[END_REF]. The "light-switch" effect originates from hydrogen bond formation with water, which quenches the excited-state luminescence and reduces the quantum yield by 2-3 orders of magnitude.

In the bound form, the dppz ligand is intercalated into the DNA strand. Intercalation shields the phenazine nitrogens from the solvent and results in a luminescent excited state [START_REF] Holmlin | Ru(phen) 2 dppz] 2+ luminescence: Dependence on DNA sequences and groove-binding agents[END_REF][START_REF]Emission Spectroscopy of Ru (bpy) 2dppz2+ in Nafion. Probing the Chemical Environment in Cast Films[END_REF][START_REF] Guo | A long-lived, highly luminescent Re (I) metal-ligand complex as a biomolecular probe[END_REF][START_REF] Batista | On the Excited States Involved in the Luminescent Probe [Ru (bpy) 2[END_REF].

Our goal of luminescence study

The goal in this part is to study the interaction of Ru(bpy) 2 dppz 2+ with DNA in a wide range of DNA / Ru(bpy) 2 dppz 2+ ratios by using the luminescence signal which comes from the complex. The interaction of ruthenium-based compound with DNA has been studied in the limit of smaller concentrations of ruthenium derivatives than DNA base pairs. Luminescence studies have shown that ruthenium complexes exhibit high affinity with DNA and can recognize specific DNA sequences. Several tools have been employed to study the interaction between DNA and organometallic compound:

spectroscopic measurements, such as absorption or emission spectroscopy [START_REF] Yang | Study of the interaction between novel ruthenium (II)-polypyridyl complexes and calf thymus DNA[END_REF][START_REF] Hartshorn | Novel dipyridophenazine complexes of ruthenium (II): exploring luminescent reporters of DNA[END_REF][START_REF] Barton | Binding modes and base specificity of tris (phenanthroline) ruthenium (II) enantiomers with nucleic acids: tuning the stereoselectivity[END_REF][START_REF] Klajner | DNA binding to an anticancer organo-ruthenium complex[END_REF], dichroic activity, either circular [START_REF] Grueso | Thermodynamic and structural study of phenanthroline derivative ruthenium complex/DNA interactions: Probing partial intercalation and binding properties[END_REF], that uses the chiral properties of DNA and of studied complexes or linear [START_REF] Lincoln | DNA binding geometries of ruthenium (II) complexes with 1, 10-phenanthroline and 2, 2'-bipyridine ligands studied with linear dichroism spectroscopy. Borderline cases of intercalation[END_REF], as well as single molecule manipulations [START_REF] Vladescu | Quantifying force-dependent and zero-force DNA intercalation by single-molecule stretching[END_REF][START_REF] Mihailovic | Exploring the interaction of ruthenium (II) polypyridyl complexes with DNA using single-molecule techniques[END_REF].

The equilibrium may be described by a single adsorption equilibrium:

 

). How to build the relationship between luminescence intensity and binding intensity ( v )? A linear relationship (  intensity) is often assumed, and the linear coefficient is determined when a plateau of the intensity is observed at high concentration of ligand. Nevertheless these 2 hypothesis ( linearity and at the intensity plateau) have proven not to be correct for similar molecules and we will analyze our titration without any further hypothesis [START_REF] Despax | Spectral properties of polypyridyl ruthenium complexes intercalated in DNA: theoretical insights into the surrounding effects of [Ru (dppz)(bpy) 2[END_REF].

In this study, we follow the evolution of luminescence intensity when we add ruthenium compound into DNA solution, but we do not know the relationship between the change of luminescence intensity and binding intensity ( v ). Our purpose Ru . So if we can successfully build a relationship between a K and v , the question will be answered. Now we reorganize the a K equation with consideration of v :

(1 )[ ]

a f v K v Ru   (2. 4) (1 ) [] a f v Kv Ru  (2. 5)
This model assumes that the size of ligand is very tiny and hence one ruthenium molecule can bind to just one DNA base pair. Therefore, plotting the

() [] f v v Ru
, we expect to obtain the linear plot whose slope would be -1 and intercept with y-axis is

a K . Nevertheless when we plot [] f v Ru
as a function of v , the slopes is different from -1. It means that the number of bound DNA per Ru(bpy) 2 dppz 2+ is not 1 and that slope and intercept cannot be interpreted in that way, even the estimation of their linear part results in errors. In order to compute the affinity constant and comlexation degree of ruthenium with DNA, we used a method to analyze the experiment results without any hypothesis, proposed by Toshiro Nishida and Wlodzimierz Bujalowski [START_REF] Halfman | Measuring the binding of small molecules to protein from binding-induced alterations of physical-chemical properties[END_REF][START_REF]A general method of analysis of ligand-macromolecule equilibria using a spectroscopic signal from the ligand to monitor binding. Application to Escherichia coli single-strand binding protein-nucleic acid interactions[END_REF][START_REF] Szymanski | Full-length Dengue virus RNA-dependent RNA polymerase-RNA/DNA complexes: stoichiometries, intrinsic affinities, cooperativities, base, and conformational specificities[END_REF]. It has been proven that it was very efficient in Nishida's and Bujalowski's study.

We design similar experiments according to their study, three titrations with different ruthenium concentrations are performed in our work. The interaction between Ru(bpy) 2 dppz 2+ and DNA was explored with increasing the concentration of Ru(bpy) 2 dppz 2+ in sodium chloride solvent, the evolution of luminescence intensity as a function of ruthenium concentration is recorded. The salt dependence of a K is also explrored in our study. (Salt concentration varies from 10 mM to 100 mM).

Description of Nishida technique

Any method used to analyze ligand binding to a macromolecule generally related the extent of the complex formation to the free ligand concentration in solution. Binding isotherms of a protein association with a nucleic acid or, in general, of ligand binding to a macromolecule represent a direct relationship between the degree of binding (moles of ligand bound per mole of a macromolecule) and the free ligand concentration [START_REF]A general method of analysis of ligand-macromolecule equilibria using a spectroscopic signal from the ligand to monitor binding. Application to Escherichia coli single-strand binding protein-nucleic acid interactions[END_REF][70][START_REF] Hill | Chapter 6) Cooperativity theory in biochemistry: steady-state and equilibrium systems[END_REF][START_REF]Negative cooperativity in the binding of nucleotides to Escherichia coli replicative helicase DnaB protein. Interactions with fluorescent nucleotide analogs. W. Bujalowski[END_REF]. Analogously, in the case of the protein (ligand) binding to a long one-dimensional nucleic acid lattice, the equilibrium binding isotherm represents the direct relationship between the binding density (moles of ligand bound per mole of bases or base pairs) and the free protein concentration [START_REF]A general method of analysis of ligand-macromolecule equilibria using a spectroscopic signal from the ligand to monitor binding. Application to Escherichia coli single-strand binding protein-nucleic acid interactions[END_REF][START_REF]Negative cooperativity in the binding of nucleotides to Escherichia coli replicative helicase DnaB protein. Interactions with fluorescent nucleotide analogs. W. Bujalowski[END_REF][START_REF] Mcghee | Theoretical aspects of DNA-protein interactions: co-operative and non-co-operative binding of large ligands to a one-dimensional homogeneous lattice[END_REF][START_REF]Cooperative and non-cooperative binding of large ligands to a finite one-dimensional lattice: A model for ligand-ougonucleotide interactions[END_REF][START_REF] Menetski | Interaction of recA protein with single-stranded DNA: quantitative aspects of binding affinity modulation by nucleotide cofactors[END_REF][START_REF] Kowalczykowski | Cooperative and noncooperative binding of protein ligands to nucleic acid lattices: experimental approaches to the determination of thermodynamic parameters[END_REF][START_REF] Effects | Fluorescence Anisotropy Studies on the Ku-DNA Interaction[END_REF][START_REF] Cera | Theory of multivalent binding in one and two-dimensional lattices[END_REF]. A true thermodynamic binding isotherm is model-independent and reflects only this relationship. Only then, when such a relationship is available, can one proceed to extract physically meaningful interaction parameters that characterize the examined interacting systems. This is accomplished by comparing the experimental isotherms to theoretical predictions based on specific statistical thermodynamic models that incorporate known molecular aspects of the system, such as intrinsic binding constants, cooperativity parameters, allosteric equilibrium constants, discrete character of the binding sites or overlap of potential binding sites, etc. Only then, can one make rational molecular interpretations of the nature and mechanisms of the observed phenomena.

Numerous techniques have been developed or applied to study equilibrium properties of specific and nonspecific protein-nucleic acid interactions [START_REF] Garner | A gel electrophoresis method for quantifying the binding of proteins to specific DNA regions: application to components of the Escherichia coli lactose operon regulatory system[END_REF][START_REF] Clore | Theory and applications of the transferred nuclear Overhauser effect to the study of the conformations of small ligands bound to proteins[END_REF][START_REF]A double-filter method for nitrocellulose-filter binding: application to protein-nucleic acid interactions[END_REF][START_REF] Kranz | RNA binding mediates the local cooperativity between the β-sheet and the C-terminal tail of the human U1A RBD1 protein[END_REF][START_REF] Lusetti | The DinI protein stabilizes RecA protein filaments[END_REF][START_REF] Schubert | Comparative Thermodynamic Analysis of DNA-Protein Interactions Using Surface Plasmon Resonance and Fluorescence Correlation Spectroscopy[END_REF][START_REF] Fried | Equilibria and kinetics of lac repressor-operator interactions by polyacrylamide gel electrophoresis[END_REF][START_REF] Draper | Measurement of macromolecular equilibrium binding constants by a sucrose gradient band sedimentation method. Application to protein-nucleic acid interactions[END_REF][START_REF] Wu | DNA Bends in TATA-binding Protein• TATA Complexes in Solution Are DNA Sequence-dependent[END_REF]. In our study, we mainly use a method to analyze our results, performed by Toshiro Nishida. This method is presented for obtaining binding data in any ligand-macromolecule system for which binding induces a measurable change in a physical property of the system. The relationship between the equivalents of ligand bound and the physical property change need not be a known theoretical function, nor need it be empirically determined at an impractically high ligand concentration. Rather, the physical property change is measured at a number of total ligand-macromolecule mole ratios at two or more protein concentrations. The number of equivalents of bound ligand and the concentration of free ligand is computed from data obtained at a minimum of two macromolecule concentrations.

Binding of small ligand to macromolecule is described by the same mass action law (Equation 2.6) that applies to the association of simpler monovalent substances [START_REF] Muralt | The titration constants of multivalent substances[END_REF].

1 af af nK c v Kc   (2. 6)
where v is equal to the average mole ratio of bound ligand to macromolecule (also can be called as bind intensity), f c is the equilibrium free-ligand concentration, n is number of binding sites on a macromolecule, and K a is the intrinsic association constant of the binding sites. This relationship describes the binding when the intrinsic association constants of all n sites are equal and when binding to any one site does not influence binding to any other site.

An ideal analysis of data from binding studies would allow each n and K a , and be determined unambiguously. When all sites are equivalent, n and K a may be determined by plotting 1 v  as a function of 1 f c  as suggested by Klotz [89] or by

plotting f v c
vs. v suggested by Scatchard et al [START_REF]The attractions of proteins for small molecules and ions. G. Scatchard[END_REF][START_REF] Scatchard | Physical Chemistry of Protein Solutions. IV. The Combination of Human Serum Albumin with Chloride Ion1[END_REF].

At a sufficiently low macromolecule concentration (P), the fraction of bound to total ligand is significantly less than 1 and the free concentration of ligand ( f c ) can be determined from the difference between the mole ratio of total ligand to protein and multiplied by the protein concentration, The determination was given by Equation 2.7. from which, using Equation 2.7, and we have:

[( / ) ( / ) ] ab f t a t b ab PP c c P c P PP   (2.9)
Our goal is to study and quantify the association between a double stranded DNA macromolecule and Ru(bpy) 2 dppz 2+ with this method. DNA base pairs and Ru(bpy) 2 dppz 2+ are seen as protein and ligand, respectively. We also study on the salt dependence of affinity constant K a with different concentrations of NaCl (10 mM, 20 mM, 50 mM, 100 mM) in our study. This saturation level is approximately proportional to the DNA strand concentration (see the plateau at high C Ru in Figure 2.3). Nishida's method is applied to compute the affinity constant, the binding intensity and the free concentration of Ru(bpy) 2 dppz 2+ .

Experimental part

Experimental results

Luminescence

Data analysis and results

To obtain reliable results from this treatment of the data, it is necessary that the binding doesn't involve DNA concentration effects and that intercalation-induced luminescence change is not dependent upon DNA concentration, so the luminescence intensity was normalized firstly. Then we plot the normalized intensity versus concentration of ruthenium (C Ru ), one group titration results with 0.01 M of NaCl The determination of  can be described by the following equation:

1 af af nK c v Kc  
Now if c f is very close to 0,  can be written into the following equation:

af v nK c  (2.12)
We then perform an affine curve fitting of  (C [Ru]f ), the slope of the curve the value of nK a . The results are shown in

Discussions: salt dependence of Ka

It is known that simple monovalent counterions (like Na + ) interact with polyelectrolyte such as DNA by direct condensation. This reduces the axial charge density of the polyelectrolyte. According to Manning's theory not all of the charges are neutralized, but only a fraction of them, so that, the unneutralized polyelectrolyte charges are screened from each other [START_REF] Record | Ion effects on ligand-nucleic acid interactions[END_REF][START_REF]Limiting laws and counterion condensation in polyelectrolyte solutions I. Colligative properties[END_REF][START_REF]On the application of polyelectrolyte "limiting laws" to the helix-coil transition of DNA. I. Excess univalent cations[END_REF]. Let us call  is the ratio of neutralized charges along the DNA chain. For dsDNA 0.88

  [95].
We write the equilibrium between [Ru(bpy)

[ ][ ] [ ] [ ] bp a bp f f DNA Ru Na K DNA Na Ru      (2.15)
Our previous analysis of the experimental data leads to an apparent

a K : [] [ ] [ ] bp a bp f f Ru DNA K DNA Ru   so * a K might be then written as: * [] aa K K Na    (2.16) * [] aa K K Na    (2.17) (2.18)
At constant temperature and pressure, under conditions of excess Na  , variations of a K with Na  concentrations are written:

log log[ ] a K Na      (2.19)
In deriving this relationship, we neglected the change in chemical activity of DNA and Ruthenium due to addition of salt into the solution. A general derivation is given in [START_REF] Record | Thermodynamic analysis of ion effects on the binding and conformational equilibria of proteins and nucleic acids: the roles of ion association or release, screening, and ion effects on water activity[END_REF].

It is thus expected that the affinity decreases with salt concentration with a logarithm, slope equals to -0. Lifetimes measurements of excited states of each fluorophore are performed. We observed that the addition of ligand results in a strong quenching of fluorophores.

From the analysis of the evolution of the lifetime of the donor on one hand, and that of the acceptor on the other hand, we deduce the evolution of the efficiency of energy transfer in the concentration of function ligand. We compare these results obtained by each of these analyses, and finally deduce, using our analysis of the balance made in the first part, the evolution of the chain length on the rate of complexation.

Relaxation process of excited fluorophore molecule

Fluorescence

During the past 20 years fluorescence has been widely used in the biological sciences.

Fluorescence spectroscopy and time-resolved fluorescence are considered to be primarily research tools in biochemistry and biophysics. This emphasis has changed, and the use of fluorescence has expanded. Fluorescence now becomes a dominant methodology and is used extensively in biotechnology, such as flow cytometry [START_REF] Nebe-Von-Caron | Analysis of bacterial function by multi-colour fluorescence flow cytometry and single cell sorting[END_REF][START_REF] Rieseberg | Flow cytometry in biotechnology[END_REF], medical diagnostics [START_REF] Andersson-Engels | Laser-induced fluorescence in medical diagnostics[END_REF][START_REF] Kobayashi | New strategies for fluorescent probe design in medical diagnostic imaging[END_REF]

Phenomenon of Fluorescence

Luminescence is the emission of light from any substance, and occurs from electronically excited states. Luminescence is formally divided into two categories, fluorescence and phosphorescence, which depends on the nature of the excited state.

Fluorescence is the emission of the light from the excited singlet states, in which the electron in the excited orbital is paired (by the opposite spin) with the second electron in the ground-state orbital. The process of returning to the ground state of the electron is spin allowed and occurs rapidly by emission of a photon. The emission rates of fluorescence are typically 10 -8 s -1 , so that a typical fluorescence lifetime is near 10 ns (10 -9 s). The lifetime (presented as τ) of a fluorophore is the average time between its excitation and return to the ground state. It is valuable to consider a 1 ns lifetime within the context of the speed of light. Light travels 30 cm, or about one foot, in 1 ns. A lot of fluorophores display sub-nanosecond lifetimes. The measurement of the time-resolved emission requires sophisticated optics and electronics due to the short timescale of fluorescence. In spite of the added complexity, time-resolved fluorescence is widely used because of the increased information available from the data when compared with stationary or steady-state measurements. Additionally, advances in technology have made time-resolved measurements easier, even when using microscopes.

Phosphorescence is emission of light from triplet excited states, in which the electron in the excited orbital has the same spin orientation as the ground-state electron. Transitions to the ground state are forbidden and the emission rates are slow (10 3 to 10 0 s -1 ), so that phosphorescence lifetimes are typically milliseconds to seconds. Even longer lifetimes are possible, as is seen from "glow-in-the-dark" toys.

Following exposure to light, the phosphorescence substances glow for several minutes while the excited phosphors slowly return to the ground state. Phosphorescence is not usually seen in fluid solutions at room temperature. This is because there exist many deactivation processes that compete with emission, such as non-radiative decay and

Fluorescence lifetimes and quantum yields

The fluorescence lifetime and quantum yield (presented by  and  ) are perhaps the most important characteristics of a fluorophore. Quantum yield is the ratio of emitted photons relative to the number of absorbed photons. The lifetime is also important, as it determines the time available for the excited fluorophore to interact with or diffuse in its environment, and hence the information available from its emission.

The lifetime ( ) of the excited state is defined by the average time the molecule spends in the excited state prior to return to the ground state (Equation. 3.1).

1 k nr    (3.1)
 is the emissive rate, and k nr is rate of all possible non-radiative decay, they both depopulate the excited state, and can be easily presented by a very simplified Jablonski diagram (Figure 3   is the frequency of absorbed photon,  F is the frequency of emitted photon when the excited molecule relaxes from the excited states to the ground state,  is the emissive rate, and  nr is rate of all possible non-radiative decay.

Fluorescence quenching

The processes that refer to a decrease of the fluorescence intensity of a molecular can be called quenching. Quenching can occur by different mechanisms. A variety of molecular interactions can result in quenching, for instance, ground-state complex formation, collisional quenching, energy transfer, excited-state reactions and molecular rearrangements. In our study, we will be concerned primarily about two types of quenching:

One is dynamic quenching, which results from collisional encounters between the fluorophore and quencher, the other that will be discussed is static quenching, where of binding between the fluorescent sample and the quencher occurs.

Two simplified sketches are shown in We assume that collisional quenching happens due to the collision of two and nor more than two molecules :one fluorophore and one quencher particle, so the collisional quenching of fluorescence can be described by the Stern-Volmer equation:

0 0 1 [ ] 1 [ ] qD F k Q K Q F      (3.6
* * 0 ( ) [ ] 0 dF f t F dt     (3.7) * * ( ) ( [ ])[ ] 0 q dF f t k Q F dt      (3.8)
where () ft is the constant excitation function,

1 0   
is the decay rate of the fluorophore in the absence of quencher. In the absence of quenching, the excited-state population decays with a rate of nr k     , where  is the radiative rate and nr k represents all the possible way of non-radiative decay rate. In the presence of quencher, there is an additional decay rate [] q kQ. The excited-state population will be a constant under the continuous excitation, so eq. 3.7 and eq. 3.8 can be set to 0.

Division of eq. 3.8 and eq. 3.7 yields:

0 * 0 * [] 1 [ ] 1 [ ] q qD F kQ k Q K Q F          (3.9)
which is the Stern-Volmer equation. Since collisional quenching is a rate process that depopulates the excited state, the lifetimes in the absence and presence of quencher are given by:

1 0    (3.10) 1 ( [ ]) q kQ    (3.11)
Divided by eq. 3.10 with eq. 3.11 yields the same results of 0 F F

0 0 [] 1 [ ] 1 [ ] q qD kQ k Q K Q           (3.12)
It is obvious that 00 F F    which can be seen as an important characteristic of collisional quenching. The decrease in lifetime happens because quenching is an additional rate process that depopulates the excited state. The decrease in yield occurs because quenching depopulates the excited state without fluorescence emission.

b) Theory of static quenching

For static quenching, the fluorescence intensity depends upon the quencher concentration. Now we consider that one fluorophore can complex with one quencher.

S K F Q FQ 
The complexation between fluorophore and quencher reaches equilibrium, the association constant K S is given by equation:

[] [ ][ ] S FQ K FQ   (3.13)
where [] FQ  is the concentration of the complex, [F] is the concentration of uncomplexed fluorophore, and [] Q is the free concentration of quencher. If the complexed specy is non-fluorescent then the fraction of the fluorescence that remains (F/F 0 ) is given by the fraction of the total fluorophores that are not complexed: F/F 0 . The total concentration of fluorophore 0 [] F can be written as Equation 3.14:

0 [ ] [ ] [ ] F F F Q    (3.14)
Substitute eq. 3.14 into eq. 3.13 yields equation 3.15:

00 [ ] [ ] [ ] 1 [ ][ ] [ ][ ] [ ] S F F F K F Q F Q Q     (3.15)
Rearrange Equation 3.15, and given that the fluorescence intensity depends upon the quencher concentration, the relationship of fluorescence intensity and quencher concentration will be obtained by Equation 3.16:

  0 1 S F K Q F  (3.16)
Note that the dependence of 0 F F

versus [] Q is also linear, which is identical to dynamic quenching. K S represents the association constant, which is given by Equation 3.13.

For static quenching, the lifetime does not decrease because only the fluorescent molecules are observed, and the uncomplex fluorophores have the unquenched lifetime τ 0 . c) How to distinguish dynamic quenching and static quenching?

FRET

General description of FRET

Fluorescence resonance energy transfer (FRET) has become widely used in biology and biotechnology [START_REF]FRET tells us about proximities, distances, orientations and dynamic properties[END_REF].This process occurs between a donor (D) molecule in the excited state and an acceptor (A) molecule in the ground state and strongly depends on the distance between them, typically in a range of 5 to 100 Å. A FRET Jablonski diagram is shown in Figure 3.7. The energy will be transferred without the appearance of a photon and is the result of long range dipole-dipole interactions between the donor and acceptor. This process is non-radiative, which means that the energy is not emitted or absorbed as photons [START_REF]FRET tells us about proximities, distances, orientations and dynamic properties[END_REF].The rate of energy transfer depends upon the extent of spectral overlap of the emission spectrum of the donor with the absorption spectrum of the acceptor, the quantum yield of the donor, the relative orientation of the donor and acceptor transition dipoles, and the distance between the donor and acceptor molecules [START_REF]Theory of the energy transfer efficiency and fluorescence lifetime distribution in single-molecule FRET. I. V. Gopich[END_REF][115][116][117]. The distance dependence of FRET allows measurement of the distances between donors and acceptors. So the most common application of FRET is to measure the distances between two sites on a macromolecule by labeling it with a donor and a acceptor.

Measurement of FRET efficiency

The most important factor we care in FRET process is the transfer efficiency, there are two main independent ways of measuring efficiency. Before describing these two methods of measuring efficiency, three assumptions will be introduced in this part.

All of the statements about these two methods assume that these three assumptions are satisfied.

1) The presence of acceptor neither change nor add any relaxation process except FRET process.

2) The number of excited donor molecules at a given excitation wavelength is the same in the absence and presence of the acceptor.

3) The number of excited acceptor molecules at a given excitation wavelength is the same.

The transfer efficiency is the probability that an excited donor fluorophore comes back to its ground state by FRET process. This can be expressed as:

Dynamic measurement-donor emission

Molecular description from the donor point of view

When FRET occurs, at least two mechanisms of donor relaxation coexist. The first one is the emission process with a decay time τ D , the other, relaxation through FRET process, whose rate is presented by T  . We thus have

Evolution of the emitted intensity

We do not measure the number of excited donors, but the emitted intensity. The total number of photons emitted by the donor with two different wave lengths 517 nm

Dynamic measurement-acceptor emission

Molecular description from the point of view of the acceptor

The acceptor can be excited following two different mechanisms. One process is excited through the energy transfer, the other excitation process is attributed to the laser. let us call τ A the overall relaxation time of acceptor in the presence of donor molecule, the existence of FRET doesn't change the relaxation path of an excited acceptor, and τ A is identical to the relaxation time of acceptor excited by the laser in the absence of donor. From the perspective of the acceptor, the FRET process can be determined by the following equation:

Evolution of the emitted intensity

The number of photon emitted ( 600 N AD ) by the acceptor can now be obtained: represent the number of acceptor excited by the FRET process.

Relationship of efficiency with the donor-acceptor distance

In our study, the organometallic compound will form a complex with DNA when we perform the titration, meanwhile, this lead to the change of distance between two fluorophores. If we want to quantify the variation of the amount of energy transfer between the donor and acceptor when complex done. It becomes essential to know the relationship of transfer efficiency and donor-acceptor distance.

The distance at which FRET is 50% efficient is called the Föster distance and the rate of energy transfer from a donor to an acceptor is normally written as a simplified equation:

6 0 1 ( ) ( ) T D R r r    (3.35)
Where D

 is the decay time of the donor in the absence of acceptor, R 0 is the Föster distance, and r is the donor-to-acceptor (D-to-A) distance. Hence, the rate of transfer is equal to the decay rate of the donor ( 1

D 
) when the D-to-A distance (r) is equal to the R 0 , and the transfer efficiency is 50%. At this distance (r = R 0 ) the donor emission would be decreased to half its intensity in the absence of acceptors, R 0 is dependent on the spectroscopic properties of fluorophore in the used environment.

In order to better understand the relationship of transfer efficiency and D-to-A distance, we start back with the equation of transfer rate written in terms of Föster distance R 0 , assuming that the donor and the acceptor separated by a distance r :

4 6 5 4 2 D 0 9000(ln10) ( ) ( ) ( ) 1 κ 28 T D A D r F d r Nn           4 64 D 0 2 0.529 () κ ( ) ( ) DA D Fd r Nn          (3.36)
where D is the quantum yield of the donor in the absence of acceptor, n is the refractive index of the medium, N is Avogadro's number, r is the distance between the donor and acceptor, and D  is the lifetime of the donor in the absence of acceptor. with the total intensity normalized to unity. ()

A
 is the extinction coefficient of the acceptor at  , which is in units of M -1 cm -1 . The term 2 κ is a factor describing the relative orientation in space of the transition dipoles of the donor and acceptor. 2 κ is usually assumed to be equal to 2/3, which is appropriate for dynamic random averaging of the donor and acceptor.

The overlap integral, we call it    , represents the degree of spectral between the donor emission and the acceptor absorption:

  4 0 ( ) ( ) DA Fd          (3.
  D 0 1/6 4 2 κ 0.529 [ ] R Nn   (3.40)
Where the

   is in unit of M -1 cm 3
, the wavelength is expressed in cm.

Once the value of R 0 is known, the transfer rate can be easily calculated by end is labeled with Alexa488; 2): 3' end is labeled with Alexa568; 3): 5' end and 3' end are labeled with alexa488 and Alexa568, respectively. Alexa fluorophore is chemically linked with DNA base. There is no modification on the pairing sequence.

The information of modified DNA is shown in Table 3.1. A sketch that depicts the labeled dsDNA is shown in Figure . 3.9.

Preparation of dsDNA and its titration

Experimental titration

The experimental titration has been carried out in salinity environment [START_REF] Rehmann | Proton NMR studies of tris(phenanthroline) metal complexes bound to oligonucleotides: characterization of binding modes[END_REF]

Fluorescence measurement technique

Time-correlated single photon counting (TCSPC) method

Time-correlated single photon counting (TCSPC) method is often employed to do lifetime measurement because of the simplified data collection and enhanced the quantitative photon counting [START_REF] Becker | Advanced time-correlated single photon counting techniques[END_REF]. In this study, lifetime measurements are performed with the Fluorolog-3 model FL3-22 instruments. All emission decay datas are transferred to a personal computer and fitted with a sum of exponentials convolved with the appropriate instrument response function by employing a nonlinear least-squares weighted-residuals routine. The instrument response function is measured by using a dilute suspension of latex collsional particles to scatter laser pulses in the direction of the detector.

Apparatus acquisition parameters

Every single measurement is performed in the same quartz cuvette. A pulsed laser with a wavelength of 495 nm and a repetition rate of 1MHZ is employed to excite the fluorophore, the photons emitted by the fluorophore are collected by the photon detector with temporal resolution defined by a channel width equal to 0.1 ns. The wavelength collected is at 517 nm (Alexa488) and 600 nm (Alexa568) with 2 nm spectral slit. The acquisition time is chosen so that the number of photons collected in the first channel is equal to 10 4 . One measurement lasts between 10 min and 120 min under these experimental conditions. All measurements are performed at fixed ambient temperature (20 ℃). One typical emission decay curve of Alexa488 is shown in Figure 3.10, and an instrument response function reflects the distribution of photons of the excitation pulse.

Description of photon collection

The fluorophore is excited with a short pulse of laser (pulse width 5 ns) which has a repetition rate equal to 1 MHZ. Photomultiplier detector is used for recording the time-dependent distribution of emitted photons after each pulse. Intensity of the laser is chosen so that the detector collects 0 or 1 photon during one internal, the collected photons are arranged in 2000 channels (the channel width is equal to 0.1 ns). The

Data analysis procedure

One excited population corresponds to monoexponential decay (as we show in Figure 3.10), regardless of how many kinds of decay process, the decay time is the average of all decay processes. But what we observe in our study is that there are several decay exponential when the ratio of ruthenium to DNA bp is over than 1

        
(Figure 3.12), several arguments happen next, if these amplitudes are too small or are too close to each other, how many decay times should be considered? So it is reasonable to find a proper way to get an average of decay time. DAS software is employed to fit the curve in this study, we tried 1 exponential, 2 exponential and 3 exponential function to fit the curve, it is difficult to differentiate these three kinds of exponential function at low concentration of ruthenium [0 0.5 M], Figure 3.13 shows that there is almost no difference among three kinds of exponential fitting functions. To be strict and reasonable, 2 exponential function is employed to fit the experimental curve due to reasonable amplitude and 2  constant which represent the quality of fitting curve result. Two characteristic decay times ( 1  and 2  ) and two amplitudes ( 1  and 2  ) will be directly given by the fitting results with Equation 3.43:  .The total number of photons collected should be the same whatever the analytical description, that is:

1 1 2 2       (3.45) Moreover using that (0) (0) FF   We have 12     (3.46) 1  and 2
 obtained from fitting results will be weighted by the following equation.

1 1 2 2 12          (3.47)
where  will be seen as the weighted characteristic decay time, it will be used for analyzing experiment results. Red dash line: 3 exponential fitting curve.

.

Experimental results

In this part, the lifetime of each kind of fluorophore (Alexa488-donor and The titrations are performed by increasing the concentration of Ru(bpy) 2 dppz 2+ , the alexa fluorophore concentration is kept at 1 M. We characterize the evolution of decay time after each single measurement, the specific information of titration is already shown in Table 3.2.

Definition of 2 regions of experimental results

The intensity of donor (alexa488) emission of single labeled dsDNA and double labeled dsDNA was measured with increasing the concentration of ruthenium, the results of emission intensity are shown in Figure 3.14. It is obvious that the intensity decreases with adding ruthenium. Strong quenching is observed when the concentration of ruthenium is over than 1 M, so the experimental result analysis is divided into two regions. First region corresponds to of ruthenium concentration between 0 and 1 M, second region is determined when the concentration of ruthenium is over than 1 M. The crossover between the two regions corresponds to 1 mol of ruthenium per 1 mol of DNA. As we are interested in the change of the dynamic of DNA upon complexation with Ru(bpy) 2 dppz 2+ , we will consider only the low ruthenium concentration region in the subsequent analysis. C Ru (

Analysis of the emission at 517 nm

Quenching constant computation

Quenching constant is computed with Equation 3.6 and Equation 3.12 (Part 3.1.1.3 ): We now are going to perform a quantitative analysis of the intensity and time changes when Ru(bpy) 2 dppz 2+ complexes with DNA.

Evolution of the transfer rate with CRu

According to the theory part of measurement of FRET efficiency:

1) The presence of acceptor doesn't change nor add any relaxation process except FRET process. 2) The number of excited donor molecules at a given excitation wavelength is the same in the absence and presence of the acceptor.

3) The number of excited acceptor molecules at a given excitation wavelength is the same.

The transfer rate T  and transfer efficiency E are separately computed from Equation 3.18 and Equation 3.19;

11 τ τ T DA D   τ 1 DA DA D T E      
Where τ DA and τ D are already shown in the Table 3.3 and Table 3.4, so the evolution of transfer relaxation time .

We found that the evolution of  F and E with the Ruthenium concentration may be well described by a linear behavior. Nevertheless in order to perform this analysis, we had to compare the intensity emitted by single labeled and double labeled DNA. That is, we assume that the number of excited fluorophores is the same in the presence or absence of the acceptor ( ** 01 02

DD 

). We will see in the next part, by showing the emission at 600 nm, this hypothesis may be avoided.

Analysis of the emission at 600 nm

We now consider the emission at 600 nm of double labeled dsDNA bound with ruthenium complex, 600 N . The evolution of 600 N with C Ru is given in Figure 3. 20.

There are three contributions to this emission. 2) Acceptor emission, the total number of photon at 600 nm emitted by the acceptor will be called 600 AD N , the intensity emitted at 600 nm by the acceptor after a pulse will be called 600 ()

AD It
3) Ru(bpy) 2 dppz 2+ contribution when it is intercalated into DNA base pair, the total number of photon at 600 nm emitted by the bound ruthenium will be called 600 Ru N , the intensity emitted at 600 nm by the bound ruthenium after a pulse will be called 600 ()

Ru

It

So the total number of photons emitted at 600 nm ( 600 N ) can be expressed as: The intensity of emission at 600 nm of bound Ru(bpy) 2 dppz 2+ as a function of C Ru has been measured when the ruthenium complex bound with non-labeled DNA, results are shown in Figure 2.2 (Chapter 2), we observe a linear region up to 0.5 bound ruthenium per DNA base pair, corresponding to the diluted region defined in Figure 3.14 in which the intensity emitted by alexa488 at 517 nm decreased linearly with ruthenium concentration. In order to compare the intensity emitted by bound ruthenium with total intensity at 600 nm, 600 I , we performed 3 measurements at 3 ruthenium concentrations up to saturated region, we observe the contribution of bound ruthenium to the total intensity measured at 600 nm is always lower than 0.005. The results are shown in Table 3.5. So the contribution of bound ruthenium emitted at 600 nm is negligible. In order to obtain the emission at 600 nm from the donor, we will use the intensities emitted by DNA single labeled with Alexa488 at 600 nm and 517 nm, and the intensity emitted by double labeled DNA at 517 nm. All of these 3 quantities can be directly obtained from measurements. Indeed, we have:

Computation of the efficiency of energy transfer

The number of photons emitted by the acceptor is (Equation 3. In order to compute the efficiency, we will determine the three ratios involved:

DA A t AD t T T A A A DA A DA t I t                     (3.55)
That defines 1  and 2  .

We thus obtain the ratio

Discussions of the efficiency

We have shown a decrease of the FRET efficiency due to the complexation of DNA with Ru(bpy) 2 dppz 2+ . At the ruthenium concentration under study, the average number of the ruthenium complex per DNA strand is smaller than 1.

More precisely, we have measured the affinity constant of Ru(bpy) The measured lifetime may be written as:

14 0 1, 1 1 (1 ) 14 n n           (3.57)
Where  is the fraction of DNA strands complexed with one Ru(bpy) 2 dppz 2+ , 0  is the decay time of non-complexed DNA, 1,n  is the decay time of DNA strand intercalated with Ru(bpy) 2 dppz 2+ at the n th position (Figure 30). We have moreover assumed that all the intercalation positions are the same probability. is the distance between the two fluorophores when the intercalation occurs at the n th position.

It is known that the intercalation of the ruthenium compound induces a length increase of the DNA chain equal to the base pair distance. Let us moreover assume that the DNA double strand remains linear and rigid [START_REF] Vladescu | Quantifying force-dependent and zero-force DNA intercalation by single-molecule stretching[END_REF].

Then, the length of the dsDNA, complexed with one Ru(bpy) 2 dppz 2+ doesn't depend on the intercalation position and we have:

1, 0 [START_REF] Barton | Enantiomeric selectivity in binding tris(phenanthroline)zinc(II) to DNA[END_REF] 15

n rr  (3.61)
where r 0 is the length of a 15bp dsDNA chain.

Let us now compute 1 r from the experimental measurements. We can use Equation 3.60, recognizing that: This may be due to the presence of dsDNA complexed with 2 Ru(bpy) 2 dppz 2+ which have not been taken into account in this analysis.

6 1 1 1 1 00 0 () (1 )
As a conclusion, we have observed that the end-to-end distance increase of 15bp dsDNA complexed with Ru(bpy) 2 dppz 2+ is smaller than that would be increase if the DNA would remain rigid upon complexation.

We may thus conclude that DNA bends upon complexation. We have found a bending angle of the order 30 degrees. Nevertheless, our experiments cannot determine whether time-average bending is due to a local dynamic flexibility or a static kink induced by the intercalation.

Chapter 4 4 Conclusion

The interactions between Ru(bpy) 2 dppz 2+ and DNA under different salt

List of figures

.17: The end-capping complex. The duplex (dark grey) is end-capped by the ruthenium complex (red), which stacks between an extruded adenosine (blue) and the first complex (yellow) in a crystallographically related duplex (light grey).

The last GC base pair (cytidine, cyan; guanosine, green) forms a frayed end [START_REF] Song | Crystal structure of Δ-[Ru (bpy) 2dppz] 2+ bound to mismatched DNA reveals side-by-side metalloinsertion and intercalation[END_REF]. ....

1

  Introduction .................................................................................................................................. 1.1 DNA and Ru(bpy) 2 dppz 2+ ....................................................................................................... 1.2 Deoxyribonucleic acid (DNA) ................................................................................................1.2.1 Chemical structure of DNA .............................................................................................. 1.2.2 Physical properties of DNA .............................................................................................. 1.2.3 Brief description of 3 kinds of DNA forms .......................................................................1.3 Binding modes of ruthenium compound with DNA ............................................................... 1.4 Interaction between Ru(bpy) 2 dppz 2+ and DNA .................................................................... 1.5 Characterization of Ru(bpy) 2 dppz 2+ and DNA interaction .................................................... Outline of the manuscript ............................................................................................................. 2 Luminescence study ................................................................................................................... 2.1 A brief introduction of luminescence .................................................................................... Our goal of luminescence study .............................................................................................. 2.2 Description of Nishida technique .......................................................................................... 2.3 Experimental part .................................................................................................................. 2.3.1 Experimental results ....................................................................................................... 2.3.2 Data analysis and results ............................................................................................... 2.3.3 Discussions: salt dependence of K a ................................................................................ 3 Fluorescence resonance energy transfer (FRET) study .......................................................... Outline......................................................................................................................................... 3.1 Relaxation process of excited fluorophore molecule ............................................................ 3.1.1 Fluorescence .................................................................................................................. 3.1.1.1 Phenomenon of Fluorescence ................................................................................. 3.1.1.2 Fluorescence lifetimes and quantum yields............................................................. 3.1.1.3 Fluorescence quenching ..........................................................................................

3. 1

 1 .2 FRET .............................................................................................................................. 3.1.2.1 General description of FRET .................................................................................. 3.1.2.2 Measurement of FRET efficiency ...........................................................................3.1.2.2.1 Dynamic measurement-donor emission ........................................................... VIII 3.1.2.2.2 Dynamic measurement-acceptor emission ....................................................... 3.1.3 Relationship of efficiency with the donor-acceptor distance ......................................... 3.2 Experimental Procedure ........................................................................................................ 3.2.1 Experimental design ....................................................................................................... 3.2.2 Preparation of dsDNA and its titration .......................................................................... 3.2.3 Fluorescence measurement technique ............................................................................3.2.3.1 Time-correlated single photon counting (TCSPC) method ..................................... 3.2.3.2 Apparatus acquisition parameters............................................................................ 3.2.3.3 Description of photon collection ............................................................................. 3.2.3.4 Data analysis procedure .......................................................................................... 3.3 Experimental results .............................................................................................................. 3.3.1 Definition of 2 regions of experimental results .............................................................. 3.3.2 Analysis of the emission at 517 nm ................................................................................ 3.3.2.1 Quenching constant computation ............................................................................ 3.3.2.2 Evolution of the transfer rate with C Ru ....................................................................

3. 3 . 3

 33 Analysis of the emission at 600 nm ................................................................................ 3.3.3.1 Measurement of emission at 600 nm from bound Ru(bpy) 2 dppz 2+ ( 600 Ru N ) .............

Figure 1 . 1 :

 11 Figure 1.1: Chemical structure of ligand (dppz) fragments and [Ru(bpy) 2 dppz] 2+.

Figure 1 . 2 :

 12 Figure 1.2: (A) Building block of DNA. (B) DNA strand. (C) Templated polymerization of new strand. (D) Double-stranded DNA. (E) DNA double helix [1].

Figure 1 . 3 :

 13 Figure 1.3: Chemical structure of four types of the DNA bases.

Figure 1 . 4 :

 14 Figure 1.4:: DNA consists of nucleotides. Single nucleotide is a sugar-phosphate molecule with attached nitrogen-containing base. Here, thymine (T) is presented [1].

Figure 1 . 5 : 1 . 2 . 3

 15123 Figure 1.5: Physical structure of -B form DNA

Figure 1 . 6 :

 16 Figure 1.6: A-form (left), B-form (middle) and Z-DNA (right) .

Figure 1 . 7 :

 17 Figure 1.7: Geometries of (a) groove binder, (b) metallo-intercalator, (c) metallo-insertor [15].

Figure 1 . 8 :

 18 Figure 1.8: The three binding modes of metal complexes with DNA: (a) groove binding, (b) intercalation, and (c) insertion [15].

Figure 1 . 9 :

 19 Figure 1.9:  -and  -enantiomers of [Rh(phen) 3 ] 3+

Figure 1 . 10 :

 110 Figure 1.10: Chemical structure of two common metallo-intercalators:  -[Rh(phen) 2 (phi)] 3+ (left) and  -[Ru(bpy) 2 (dppz)] 2+ (right). The intercalating ligands are highlighted in blue, the ancillary ligands in yellow.

Figure 1 . 11 :

 111 Figure 1.11: Crystal structure of the metallo-intercalator  -

  while metallo-intercalators unwind the DNA and insert their planar ligand between two intact base-pairs, metallo-insertors eject the bases of a single base-pair, with their planar ligand acting as a  -stacking replacement in the DNA base stack. Until recently, no examples of DNA-binding insertors, neither metal-based nor organic, had been reported. However, the research of J Barton's on mismatch-specific DNA-binding agents has led to the discovery of a family of rhodium complexes that bind DNA via this unique mode. These complexes have been dubbed metallo-insertors (Figure.1.12), and the crystal structure of the metallo-insertor (red) bound to a target CA mismatch is given in Figure1.13.

Figure 1 . 12 :

 112 Figure 1.12: Chemical structures of mismatch-specific metallo-insertors.

Figure 1 . 13 :

 113 Figure 1.13: Crystal structure of the metallo-insertor (red) bound to a target CA mismatch [15].

Figure 1 . 14 :

 114 Figure 1.14: Steady-state emission spectra of Ru(bpy) 2 (dppz) 2+ (10 ) in the absence and presence of B-form (top left), Z-form (top right), and A-form (bottom)

Figure 1 . 15 :

 115 Figure 1.15: Titrations of Ru(bpy) 2 dppz 2+ with DNAs containing defects. Top: DNA sequences of matched, mismatched and abasic 27-mer duplex DNA (R denotes a tetrahydrofuranyl abasic site). Bottom: plots of the integrated emission intensity (λ ex = 440 nm) of rac-(left), Δ-(middle), and Λ-Ru(bpy) 2 dppz 2+ (right) (100 nM) upon increasing the concentration of DNA in 50 mMNaCl, 5 mM Tris, pH7.5 [50].

Figure 1 . 16 :

 116 Figure 1.16: Structure of  -[Ru(bpy) 2 dppz] 2+ bound to the mismatched oligonucleotide 5′-CGGAAATTACCG-3′. Front view (left) and view rotated 90 degrees (right) around the helix axis. Three DNA-binding modes are observed: (1) metalloinsertion, whereby the ruthenium complex (red) inserts the dppz ligand into the DNA duplex (grey) at the mismatched sites through the minor groove, extruding the mispaired adenosines (blue); (2) metallointercalation, whereby the complex (green)

Figure 2 . 1 :

 21 Figure 2.1: Chemical structure of ligand (dppz) fragments and [Ru(bpy) 2 dppz] 2+ .

  in this study is thus to find out the relationship between them. Any changes in luminescence intensity stem from the bound ruthenium ( [] b Ru ), and a K has a relationship with bound ruthenium [] b

c

  is total concentration of ligand, P represents the total concentration of macromolecule. Let us perform 2 titrations, a and b, at 2 macromolecule concentrations, the measured intensity per macromolecule is the same, then the binding ratios  a and  b are equal. Let us call c ta and c tb the total ligand concentrations corresponding to identical intensities per macromolecule. We have, from (Equation 2.7) and solving for = a =  b . concentration of ligands per macromolecule added macromolecules concentrations P a and P b , that leads to the same intensity per macromolecule to the same .

  study experiments have been performed in different salinity environments (10 mM, 20 mM, 50 mM, and 100 mM of NaCl). We perform a titration of 15 base pairs dsDNA by adding Ru(bpy) 2 dppz 2+ to that the DNA concentration remains constant. For each salinity, we perform titrations at [DNA] strand = 2.5  5 and 10  The luminescence intensity as a function of salinity concentrations are shown in Figure 2.2. At first glance, the luminescence intensity increases with adding more Ru(bpy) 2 dppz 2+ into the DNA solution. In other words, the complexation of Ru(bpy) 2 dppz 2+ with DNA increases, more and more Ru(bpy) 2 dppz 2+ molecules intercalate into the DNA base pairs lead to the intensity increase up to a saturation.

Figure 2 . 2 :

 22 Figure 2.2: Evolution of luminescence intensity as a function of concentration ratio (C Ru / C DNAbp )under different salinity concentrations. The concentrations of DNA strand are 2.5  (circle), 5  (square) and 10  (triangle), respectively.

CP

  Ru / C DNA bp NaCl = 100 mM were shown in Figure 2.3. In order to correctly apply Nishida technique on our experiment result, valid range for intensity should be chosen between 0 and plateau value which depends on the concentration of Ruthenium and DNA. After choosing the valid range, a linear approximation is done between successive points, the points with the same intensity per DNA strand are picked up (Figure 2.3 circles), but this method creates unphysical changes of curvature when at each experimentally measured value. So we keep only these pair of points where one of the points is a real experimental value. Series of binding intensity ( v ) and free concentration of ruthenium ( f c ) can be obtained from the following equation. The results are shown in Figure 2.4. represent the 2 different concentrations of DNA for the 2 titrations considered, f c is the free concentration of ruthenium, and t c is total concentration of ruthenium (C Ru ) at every single measurement.

Figure 2 . 3 :Figure 2 . 4 :

 2324 Figure 2.3: Evolution of intensity per DNA strand versus C Ru under 3 different concentrations of DNA, 2.5 M (circle), 5 M (square), 10 M (triangle). A linear approximation is performed between consecutive experimental points in order to determine the ruthenium concentration at when the normalized intensities are equal. Inset: the region of unphysical change of curvature between two successive experimental points only the circled pair of points are considered .

Figure 2

 2 Figure 2.5: 1 v as function of [] 1 Ru f c under different salt concentrations. Solid lines

  88. Our experiments indeed show such a decrease, the nK values obtained from the fitting of ( f c ), which gives the more reliable slopes lead to a logarithm slope of -0.82 (Figure 2.6) in good agreement with Manning's theory. The values of K a computed in this chapter will be used in the next chapter in order to analyze the dynamical and structural changes induced by the binding of Ru(bpy) 2 dppz 2+ .

Figure 2 . 6 : 3 3

 263 Figure 2.6: Logarithm of

  , DNA sequencing [100-102], and genetic analysis [103,104], to name a few. Fluorescence detection is highly sensitive, and there is no longer the need for the expense and difficulties of handling radioactive tracers for most biochemical measurements. There has been dramatic growth in the use of fluorescence for cellular and molecular imaging [105-107]. Fluorescence imaging can reveal the location and measurements of intracellular molecules, sometimes at the level of single-molecule detection [108,109].

  Figure3.1. Jablonski diagrams are often used as the starting point for discussing light

Figure 3 . 1 :Figure 3 . 2 :Figure 3 . 3 :

 313233 Figure 3.1: One form of Jablonski diagram, when the molecule is excited from ground state to the excited state, there exists several ways of relaxation processes. As depicted in the figure, fluorescence, quenching, phosphorescence, intersystem crossing and nor-radiative relaxations happen to the excited molecules. They have different relaxation time which can help us to understand the energy state of the molecule.

2 )Figure 3 . 4 :

 234 Figure 3.4: A simple Jablonski diagram to illustrate quantum yields and lifetimes.

Figure 3 .Figure 3 . 5 :

 335 Figure 3.5: Two simplified sketches of quenching. Left is collisional quenching,  is the emissive rate, [Q] is the concentration of the quencher, q is the prefactor of the

)

  In this equation, 0 F and F are the fluorescence intensities in the absence and presence of quencher, respectively; q k is the bimolecular quenching constant;  0 and  are the lifetime of the fluorophore in the absence and presence of quencher, and Q is the concentration of quencher. The Stern-Volmer quenching constant is given by 0 Dq Kk   under the precondition which the quenching is known to be dynamic. Q  (F-Q)* Derivation of the Stern-Volmer equation: This equation can be derived by considering the fluorescence intensities observed in the absence and presence of quencher. The fluorescence intensity observed for a fluorophore is proportional to its concentration in the excited state, [F * ]. Under a continuous illumination, a constant population of excited fluorophores is established, in the absence and presence of quencher, there are two different equations to describe [ * F ]:

  method to distinguish static and dynamic quenching is the measurement of fluorescence lifetimes. Static quenching removes a fraction of the fluorophores from observation. The complexed fluorophores are not fluorescent any more, and the observed fluorescence is only from the uncomplexed fluorophores. The uncomplexed fraction is unperturbed, and hence the lifetime is still τ 0 . Therefore, shown in Figure 3.6. Lastly and importantly, we should note that the above two quenching and the relationships of fluorescence intensity and decay time we discussed are under the low concentration of quencher. In other words, every equation is obtained from the perspective of one fluorophore versus one quencher particle. In the experimental part of this chapter, we will mainly discuss the results at low concentration of [Ru(bpy) 2 dppz] 2+ .

Figure 3 . 6 :

 36 Figure 3.6: Two simple plots of dynamic quenching (left) and static quenching (right).

F 0 

 0 and F represent the fluorescence intensity in the absence and presence of quencher. 0 is and  are the decay times in the absence and presence of quencher.[]

Figure 3 . 7 :

 37 Figure 3.7: FRET Jablonski diagram, S 0 and S 1 represent the ground state and excited state, respectively.   is the frequency of the photon absorbed by donor,  F is the frequency of the photon emitted by the excited acceptor.



  is the transfer rate of FRET, and τ DA is the overall characteristic decay time of the donor molecule in the presence of acceptor. τ DA is directly measured in our experiment: it is the decay time of the fluorophore emission. Under the first assumption, the presence of acceptor doesn't change any relaxation process except for FRET process, thus FRET process is defined with the following equation:Where τ D is the characteristic time in the absence of acceptor, it can be measured directly from measurement in our study. Then the transfer efficiency can be rewritten as:

Equation 3 . 1 /

 31 [START_REF] Kumar | Photophysics of ruthenium complexes bound to double helical DNA[END_REF] is valid if the relaxation process described by τ D doesn't change when the acceptors are present. So the evolution of the number of excited donors at a given excitation wavelength, can be obtained with following equation: is the number of excited donors at time t, * 02 D is the initial population of excited donors in the presence of acceptor, τ DA and τ D are the lifetimes in the presence and absence of acceptor, respectively.

  the characteristic decay times of photon emission at 517 nm and 600 nm by the donor, whatever in the absence or presence of acceptor, the decay times do not change. * 01 D and * 02 D are the initial number of excited donors by the laser in the absence and presence of acceptor.By dividing Equation3.24 with Equation 3.22, we obtain Equation 3and τ DA , are directly obtained from the measurement. Moreover, if the number of excited donors does not change in the presence of acceptor (hypothesis: number 2), that is *is valid, and the transfer efficiency can also be rewritten as:

F

   is the corrected fluorescence intensity of the donor in the wavelength range  to  

Equation 3 . 35 .

 335 The efficiency of energy transfer (E) is the fraction of photons absorbed by donor which are transferred to the acceptor. It is already given by the Equation 3shows that the transfer efficiency is strongly dependent on distance between the donor and acceptor, especially when the distance is close to the Föster distance R 0 , the unambiguous dependence is shown in Figure 3.8. On basis of the spectroscopic properties of fluorophores (Alexa488 and Alexa568) we used in our study and the typical distance of FRET happening, our experiments are built with 15 base pairs dsDNA, which are chemically linked with Alexa488 and Alexa568 at 5' end and 3' end, respectively. The Föster distance of Alexa488-Alexa568 fluorophore pair is around 6.2 nm [118]. Titrations were performed by adding [Ru(bpy) 2 dppz] 2+ in to DNA solution. Any variations in DNA conformation, will cause the change of distance between two fluorophores and consequently on the efficiency of FRET measurements.

Figure 3 . 8 :

 38 Figure 3.8: Dependence of the energy transfer efficiency on distance. R 0 is Föster

Figure 3 . 9 :

 39 Figure 3.9: Three sketches of labeled dsDNA, (a) depicts single labeled dsDNA labeled with Alexa488 at 5' end, (b) draws single labeled dsDNA modified with Alexa568 at 3' end, and (c) represents the double labeled dsDNA modified with Alexa488 at 5' end and Alexa568 at 3' end.

  Both two strands are resuspended to a final strand concentration 20 M (DNA base pairs concentration is 300 M) in NaCl of 20 mM (at 25 ℃). Next they are annealed by heating the DNA to 94 ℃ before cooling down the sample to 16 ℃ for 15 minutes, then DNA is cooled down to 16 ℃ for another 15 minutes. All of the measurements are performed at 20 ℃ to ensure the DNA remains in the double helix structure .

Figure 3 . 10 :

 310 Figure 3.10: Typical emission decay curve obtained from a single labeled dsDNA with Alexa 488. Black: width of a laser pulse. Blue: emission of dsDNA fitted with a single exponential decay (yellow). The channel width is 0.1 ns. The decay time deduced from the single exponential is equal to 4.1 ns.

  computer counts the number of photon in every channel. The experiment duration is stopped when the number of photon in the first channel is equal to 10 4 . The total number of photon distributes along a decay curve that may obey to a multi-exponential function. Total intensity can be obtained by averaging the total number of collected photons per unit time. A simple sketch of photon acquisition is shown in Figure3.11.

Figure 3 . 11 :

 311 Figure 3.11: Photon collection after pulse excitation: 1 photon is collected after each pulse. The lapse time between the pulse and the detected photon,   ,   and   is recorded. The photons are gathered in windows according to their lapse time, and then intensity decay curve is obtained.

Figure 3 . 12 :

 312 Figure 3.12: Typical emission decay curve obtained from a single labeled dsDNA with Alexa 488 with excess ruthenium (C Ru / C DNAbp = 20/3). Black: width of a laser pulse. Blue: emission of dsDNA fitted with 2 exponential decay (yellow). The channel width is 0.1 ns. The decay times deduced from the single exponential are equal to 0.13 ns and 3.8 ns.

1  and 2  represent the amplitude, 1  and 2  44 ) 1  and 2

 12124412 Ft describes the change of the fluorescence intensity with time t, correspond to two decay times. As we discussed in the previous paragraph, one excited population should correspond to one decay time ( ), and the lifetime can be computed from equation 3.44:If we assume that one excited population corresponds to one decay time. This single decay time may be computed from the average of the fitted decay times

Figure 3 . 13 :

 313 Figure 3.13: 1 exponential, 2 exponential and 3 exponential fitting curves from a single labeled dsDNA with Alexa488. Black dash line: width of a laser pulse. Blue solid line: 1 exponential fitting curve. Black dash line: 2 exponential fitting curve.

  Alexa568-acceptor) is measured with TCSPC technique under different experimental conditions. We observe that the addition of Ru(bpy) 2 dppz 2+ results in a strong quenching of fluorophores when the ruthenium concentration is over than 1 M. From the analysis of the evolution of the lifetime of the donor fluorophore and the acceptor fluorophore, we deduce the evolution of the efficiency of energy transfer as a function of ligand concentration. Both donor and acceptor analysis refer to Part 3.1.1.3 (page 49) and Part 3.1.2.2 (page 56) .

Figure 3 . 14 :

 314 Figure 3.14: Evolution of Alexa488 emission intensity as a function of C Ru . Left is the single labeled dsDNA, right is the double labeled dsDNA.

Figure 3 . 15 :

 315 Figure 3.15: Evolution of decay time D  and DA  of donor as a function of C Ru in the absence (left) and presence (right) of acceptor.

Figure 3 . 16 :

 316 Figure 3.16: Number of photons emitted at 517 nm by 1M DNA labeled with donor as a function of C Ru . Left is single labeled dsDNA, right is the double labeled dsDNA.

DI

  the emission intensity of excited fluorophore is proportional to the total number of excited fluorophore with the time evolution, and we can not measure the number of excited fluorophore with time, As a fact of that emission intensity of fluorophore was directly measured in our experiment. So we simply use the ratio 0 to compute the quenching constant, where D I is the intensity emitted by the donor and 0 is the intensity of the donor at 0 ruthenium concentration. of C Ru are shown in Figure3.

17

 17

  . The evolution obeys to a linear behavior the slope of the fitting curve leads to the quenching constant. The slopes are equal to 0.325   and 0.285   , respectively. there is no static quenching at low ruthenium concentration region.

Figure 3 . 17 :I

 317 Figure 3.17: The ratio of 0 D

Figure 3 . 18 :

 318 Figure 3.18: The ratio of 0 DA

F

  (the inverse of T  ) and transfer efficiency E as a function of C Ru can be obtained from Equation 3.18 and Equation3.19, respectively. The evolutions of F  and E as a function of C Ru are shown in Figure 3.19. On the whole, F  increases with the increase of ruthenium concentration. The transfer efficiency decreases with increasing the C Ru .

Figure 3 . 19 :

 319 Figure 3.19: Evolution of F  (left) and E (right) as a function of C Ru . Lines are

1 )

 1 Donor contribution, the total number of photon at 600 nm emitted by the donor will be called 600 DA N , the intensity emitted at 600 nm by the donor after a pulse will be called 600 () DA It

  indirectly, we are going to analyze the emission at 600 nm term by term, the evolution of the efficiency as a function of ruthenium concentration can be finally obtained.

Figure 3 . 20 :

 320 Figure 3. 20: Evolution of the number of photons emitted by complexed double labeled at 600 nm as a function of C Ru . Dashed line is a linear fit of the data:

3

 3 517 N D and 517 N DA as function of C Ru have been shown in Figure depend on the ruthenium concentration, and is measured at the 0 ruthenium concentration. It is equal to 1/36.5. Thus 600 N DA can be calculated as a function of C Ru . The results are given in Figure 3.21.

Figure 3 . 21 : 600 N

 321600 Figure 3.21: Evolution of the number of photons emitted by the donor ( 600 N DA ) at 600

Figure 3 . 22 :

 322 Figure 3.22: Evolution of the number of photons emitted by the acceptor ( 600 N AD ) at



  is the fluorescence lifetime of single labeled DNA with alexa568 and is shown in Figure 3.23, and 600 N AD has been obtained in the previous paragraph.

Figure 3 . 23 :

 323 Figure 3.23: Evolution of decay time of acceptor as a function of C Ru for single labeled system. Dashed line is a linear fit of the data:

1  and 2  3 Figure 3 . 24 :

 123324 Figure 3.24: Evolution of 1  (left ) and 2  (right) as a function of C Ru . Dashed

Figure 3 .

 3 Figure 3.25: Evolution of

Figure 3 . 26 :Figure 3 . 27 :

 326327 Figure 3.26: Evolution of relaxation time as a function of C Ru . F  (black filled circle)

Figure 3 . 28 :

 328 Figure 3.28: Evolution of transfer efficiency (E) as a function of C Ru . A E (blue filled circle) represents the transfer efficiency from acceptor emission, D E (black triangle) represents the transfer efficiency from donor emission. Dashed lines are the linear fits

Figure 3 . 29 :

 329 Figure 3.29: The fraction () of bound dsDNA as a function of C Ru .

Figure 3 . 30 :

 330 Figure 3.30: A sketch of the 14 intercalation positions of dsDNA (blue numbers: from 1 st to 14 th ). The probability of ruthenium molecule intercalation into any positions is the same.

  Figure 3.31 as a function of . At low  , the bound ruthenium fraction is too low and the measurement is not accurate,

Figure 3 . 31 :Equation 3 .

 3313 Figure 3.31: The evolution of 1 0 r r as a function of . Dash line is the average value

Figure 3 . 32 :

 332 Figure 3.32: The values of  as a function of .



  is found to be of the order of 30 degrees, and slightly increases when  increases.

A. 2

 2 concentrations (Ranging from10 mM to 100 mM) have been studied by the change of luminescence intensity when ruthenium molecule intercalates into DNA base pair. Following the evolution of the luminescence intensity, we compute the affinity constant a K and the binding sites (n) of ruthenium molecule to DNA double strand. Our experiments show that the affinity constant a K decrease with the increase of salt concentrations. The nK values obtained from the fitting of (c f ), lead to a logarithm slope of -0.81 in good agreement with Manning's theory. The average binding sites (n) under different salt environments is equal to 3.76, which agrees with the size of sites occupied by similar binding ligand and measure with single molecule force spectroscopy. We have used Fluorescence Resonance Energy Transfer (FRET) to monitor the average distance between the extremities of a 15 bp dsDNA modified with 2 fluorophores at its extremities. The efficiency of this energy transfer is quantified from the measurement of emitted intensities and lifetimes of donor and acceptor when one ruthenium molecule intercalates into DNA base pair. The intensities an lifetimes show a linear decrease behavior with the increase of ruthenium concentration at chosen region [0, 0.5 ]. The efficiencies of energy transfer separately obtained from the point view of donor and the point view of acceptor display in good agreement with each other. The efficiency shows a decrease with the intercalation of Ru(bpy) 2 dppz 2+ into DNA double helix. And given the relationship of efficiency with distance of coupled fluorophores, then we get that the distance between two fluorophores increases due to the intercalation of ruthenium. But in our study, we also have observed that the end to end distance increase of 15bp dsDNA complexed with Ru(bpy) 2 dppz 2+ is smaller than that would be increase if the DNA would remain rigid upon complexation. We may thus conclude that DNA bends upon the intercalation of ruthenium molecule. A bending angle was found with the order of 30 degrees. Nevertheless, our experiments can not determine whether time-average bending is due to a local dynamic flexibility or a static kink induced by the intercalation. resulting red solid was recrystallized from ethanol. Yield 80%. Protocol of preparing dsDNA for our study The measurements were performed with 15 base pair double stranded DNA. Number of DNA base pairs is limited by the range over which the energy transfer can take place that is approximately 10 nm (100 Å). Complementary strands were purchased from IBA NAPS(Gmbh) with sequences: GGA GAC CAG AGG CCT and AGG CCT CTG GTCTCC. The length of 15 base pair DNA equal to 5.1 nm is small enough to stiffen the DNA structure. Thus any unexpected bends are not supposed to appear. The first sequence was 5' labeled with Alexa488 and 3' labeled with Alexa568. The distance at which this fluorophores pair undergoes 50% energy transfer, R 0 , for these pair of fluorophores is R 0 = 62 Å [118]. Both two strands were resuspended to a final stand concentration 20 M (DNA base pairs concentration is 300 M) in NaCl of 20 mM (at 25 ℃). Next they were annealed by heating the DNA to 94 ℃ before cooling down the sample to 16 ℃ for 15 minutes, then DNA was cooled down to 16 ℃ for another 15 minutes. All of the measurements were performed at 20 ℃ to ensure the DNA remain the double helix structure . To perform experiments of salt dependance, DNA was diluted in NaCl. The salinity have been increasing from 10 mM to 100 mM.

Figure 1 . 1 :

 11 Figure 1.1: Chemical structure of ligand (dppz) fragments and [Ru(bpy) 2 dppz] 2+ . .........................

Figure 1 . 2 :

 12 Figure 1.2: (A) Building block of DNA. (B) DNA strand. (C) Templated polymerization of new strand. (D) Double-stranded DNA. (E) DNA double helix. [1] .............................

Figure 1 . 3 :

 13 Figure 1.3: Chemical structure of four types of the DNA bases. .....................................................

Figure 1 . 4 :

 14 Figure 1.4:: DNA consists of nucleotides. Single nucleotide is a sugar-phosphate molecule with attached nitrogen-containing base. Here, thymine (T) is presented [1]. ................

Figure 1 . 5 :

 15 Figure 1.5: Physical structure of -B form DNA ..............................................................................

Figure 1 . 6 :

 16 Figure 1.6: A-form (left), B-form (middle) and Z-DNA (right) . .....................................................

Figure 1 . 7 :

 17 Figure 1.7: Geometries of (a) groove binder, (b) metallo-intercalator, (c) metallo-insertor [15]. ................................................................................................................................

Figure 1 . 8 :

 18 Figure 1.8: The three binding modes of metal complexes with DNA: (a) groove binding, (b) intercalation, and (c) insertion [15]. ...............................................................................

Figure 1 . 9 :

 19 Figure 1.9:  -and  -enantiomers of [Rh(phen) 3 ] 3+ .................................................................

Figure 1 . 10 :

 110 Figure 1.10: Chemical structure of two common metallo-intercalators:  -[Rh(phen) 2 (phi)] 3+ (left) and  -[Ru(bpy) 2 (dppz)] 2+ (right). The intercalating ligands are highlighted in blue, the ancillary ligands in yellow. .......................................................................

Figure 1 . 11 :

 111 Figure 1.11: Crystal structure of the metallo-intercalator  - -Rh[(R,R)-Me 2 trien](phi) 3+ bound to its target sequence, 5'-TGCA-3'[15]. ...........................................................

Figure 1 . 12 :

 112 Figure 1.12: Chemical structures of mismatch-specific metallo-insertors. ....................................

Figure 1 . 13 :

 113 Figure 1.13: Crystal structure of the metallo-insertor (red) bound to a target CA mismatch [15]. ..............................................................................................................................

Figure 1 . 14 :

 114 Figure 1.14: Steady-state emission spectra of Ru(bpy) 2 (dppz) 2+ (10 ) in the absence and presence of B-form (top left), Z-form (top right), and A-form (bottom) double-helical DNA [36]. ............................................................................................

Figure 1 . 15 :

 115 Figure 1.15: Titrations of Ru(bpy) 2 dppz 2+ with DNAs containing defects. Top: DNA sequences of matched, mismatched and abasic 27-mer duplex DNA (R denotes a tetrahydrofuranyl abasic site). Bottom: plots of the integrated emission intensity (λ ex = 440 nm) of rac-(left), Δ-(middle), and Λ-Ru(bpy) 2 dppz 2+ (right) (100 nM) upon increasing the concentration of DNA in 50 mMNaCl, 5 mM Tris, pH7.5 [50]. ..............................................................................................................................

Figure 1 . 16 :

 116 Figure 1.16: Structure of  -[Ru(bpy) 2 dppz] 2+ bound to the mismatched oligonucleotide 5′

Figure 2 . 1 :

 21 Figure 2.1: Chemical structure of ligand (dppz) fragments and [Ru(bpy) 2 dppz] 2+ . .......................

Figure 2 . 2 :

 22 Figure 2.2: Evolution of luminescence intensity as a function of concentration ratio (C Ru / C DNAbp )under different salinity concentrations.The concentrations of DNA strand ................

Figure 2 . 3 :

 23 Figure 2.3: Evolution of intensity per DNA strand versus C Ru under 3 different A linear approximation is performed between consecutive experimental points in order to determine the ruthenium concentration at when the normalized intensities are equal. Inset: the region of unphysical change of curvature between two successive experimental points only the circled pair of points are considered . ...

Figure 2 . 4 :

 24 Figure 2.4: Binding intensity () versus free concentration of ruthenium (C [Ru]f ) ........................

Figure 2

 2 Figure 2.5: 1 v as function of

Figure 2 . 6 :

 26 Figure 2.6: Logarithm of

Figure 3 . 1 :

 31 Figure 3.1: One form of Jablonski diagram, when the molecule is excited from ground state to the excited state, there exists several ways of relaxation processes. As depicted in the figure, fluorescence, quenching, phosphorescence, intersystem crossing and nor-radiative relaxations happen to the excited molecules. They have

Figure 3 . 2 :

 32 Figure 3.2: Chemical structure of alexa fluorophores used in our study. ......................................

Figure 3 . 3 :

 33 Figure 3.3: (a) Excitation and emission spectrum of Alexa 488, (b) Excitation and emission spectrum of Alexa 568. ................................................................................................

Figure 3 . 4 :

 34 Figure 3.4: A simple Jablonski diagram to illustrate quantum yields and lifetimes.  A is the frequency of absorbed photon,  F is the frequency of emitted photon when the excited molecule relaxes from the excited states to the ground state,  is the emissive rate, and  nr is rate of all possible non-radiative decay. ................................

Figure 3 . 5 :

 35 Figure 3.5: Two simplified sketches of quenching. Left is collisional quenching, emissive rate, [Q] is the concentration of the quencher, q is the prefactor of the

Figure 3 . 6 : 0 

 360 Figure 3.6: Two simple plots of dynamic quenching (left) and static quenching (right). F 0 and F represent the fluorescence intensity in the absence and presence of

Figure 3 . 7 :

 37 Figure 3.7: FRET Jablonski diagram, S 0 and S 1 represent the ground state and excited state, respectively.   is the frequency of the photon absorbed by donor,  F is the frequency of the photon emitted by the excited acceptor. ............................................

Figure 3 . 8 :

 38 Figure 3.8: Dependence of the energy transfer efficiency on distance. R 0 is Föster distance, r is the distance between 2 fluorophores (donor-acceptor) .............................................

Figure 3 . 9 :

 39 Figure 3.9: Three sketches of labeled dsDNA, (a) depicts single labeled dsDNA labeled with Alexa488 at 5' end, (b) draws single labeled dsDNA modified with Alexa568 at 3' end, and (c) represents the double labeled dsDNA modified with Alexa488 at 5' end and Alexa568 at 3' end. .........................................................................................

Figure 3 . 10 :

 310 Figure 3.10: Typical emission decay curve obtained from a single labeled dsDNA with Alexa 488. Black: width of a laser pulse. Blue: emission of dsDNA fitted with a single exponential decay (yellow). The channel width is 0.1 ns. The decay time deduced from the single exponential is equal to 4.1 ns. .............................................................

Figure 3 . 11 :

 311 Figure 3.11: Photon collection after pulse excitation: 1 photon is collected after each pulse. The     and

Figure 3 . 12 :

 312 Figure 3.12: Typical emission decay curve obtained from a single labeled dsDNA with Alexa 488 with excess ruthenium (C Ru / C DNAbp = 20/3). Black: width of a laser pulse. Blue: emission of dsDNA fitted with 2 exponential decay (yellow). The channel width is 0.1 ns. The decay times deduced from the single exponential are equal to 0.13 ns and 3.8 ns. ........................................................................................................

Figure 3 . 13 :

 313 Figure 3.13: 1 exponential, 2 exponential and 3 exponential fitting curves from a single labeled dsDNA with Alexa 488. Black dash line: width of a laser pulse. Blue solid line: 1 exponential fitting curve. Black dash line: 2 exponential fitting curve. Red dash line: 3 exponential fitting curve. ...................................................................

Figure 3 . 14 :

 314 Figure 3.14: Evolution of alexa488 emission intensity as a function of C Ru . Left is the single labeled dsDNA, right is the double labeled dsDNA. ...................................................

Figure 3 . 15 :

 315 Figure 3.15: Evolution of decay time D  and DA  of donor as a function of C Ru in the absence (left) and presence (right) of acceptor. ............................................................

Figure 3 . 16 :

 316 Figure 3.16: function of C Ru . Left is single labeled dsDNA, right is the double labeled dsDNA. ...

Figure 3 . 17 :

 317 Figure 3.17: The ratio of 0 D

Figure 3 . 18 :

 318 Figure 3.18: The ratio of 0 DA

Figure 3 . 19 :

 319 Figure 3.19: Evolution of F  (left) and E (right) as a function of C Ru . Lines are linear

Figure 3 . 20 :

 320 Figure 3. 20: Evolution of the number of photons emitted by complexed double labeled at 600 nm as a function of C Ru . Dashed line is a linear fit of the data: 600 1328.5 386.77

Figure 3 . 21 :

 321 Figure 3.21: Evolution of the number of photons emitted by the donor ( 600 N DA ) at 600 nm in

Figure 3 . 22 :

 322 Figure 3.22: Evolution of the number of photons emitted by the acceptor ( 600 N AD ) at 600 nm

Figure 3 . 23 :

 323 Figure 3.23: Evolution of decay time of acceptor as a function of C Ru for single labeled system. Dashed line is a linear fit of the data: 3.4 0.245 A Ru C   . ............................

Figure 3 . 24 :of 1  1 927

 32411 Figure 3.24: Evolution of 1  (left ) and 2  (right) as a function of C Ru . Dashed lines are the linear fits of data: 1

Figure 3

 3 Figure 3.25: Evolution of

Figure 3 . 26 :

 326 Figure 3.26: Evolution of relaxation time as a function of C Ru . F  (black filled circle) and

Figure 3 . 27 :

 327 Figure 3.27: Evolution of transfer rate (K T ) as a function of C Ru .A T K (blue filled circle)

Figure 3 . 28 :

 328 Figure 3.28: Evolution of transfer efficiency (E) as a function of C Ru . A E (blue filled circle) represents the transfer efficiency from acceptor emission, D E (black triangle) represents the transfer efficiency from donor emission. Dashed lines are the linear

Figure 3 . 29 :

 329 Figure 3.29: The fraction () of bound dsDNA as a function of C Ru . ............................................ Figure 3.30: A sketch of the 14 intercalation positions of dsDNA (blue numbers: from 1 st to

Table 1 . 1

 11

		: Comparison of A-, B-, and Z-DNA
			Helix type
		A	B	Z
	Shape	Broadest	Intermediate	Narrowest
	Rise per base pair	2.3 Å	3.4 Å	3.8 Å
	Helix diameter	25.5 Å	23.7 Å	18.4 Å
	Screw sense	Right-handed	Right-handed	Left-handed
	Base pair per turn of	11	10.4	12
	helix
	Pitch per turn	25.3 Å	35.4 Å	45.6 Å
	of helix
	Tilt of base pairs from	19°	1°	9°
	normal to helix
	Major groove	Narrow and	Wide and	Flat
		very deep	quite deep
	Minor groove	Very broad	Narrow and	Very narrow
		and shallow	quite deep	and deep

Table 2 . 1 :

 21 Protocol of titration of luminescence measurementsWhere V DNA,add and V Ru,add are the volume of added DNA and Ru(bpy) 2 dppz 2+ for every single measurement to keep the DNA concentration as a constant and increase the ruthenium concentration, respectively. [DNA] and [Ru(bpy) 2 dppz 2+ ] represent the final concentration of DNA and Ru(bpy) 2 dppz 2+ , respectively

	V total (l)	V DNA,add (l)	V Ru,add (l)	[DNA] (M)	C Ru (M)
	10	0	0	2.5	0
	14	1	3	2.5	0.01
	22	2	6	2.5	0.02
	27	1	4	2.4	0.031
	34	2	5	2.5	0.039
	43	1	8	2.44	0.05
	45	1	1	2.55	0.058
	46	0	1	2.5	0.068
	47	0	1	2.44	0.077
	50	1	2	2.5	0.093
	10	0	0	2.5	0.1
	15	1	4	2.33	0.2
	19	1	3	2.36	0.31
	25	2	4	2.6	0.4
	34	2	7	2.5	0.5
	35	0	1	2.42	0.62
	37	1	1	2.56	0.72
	38	-0	1	2.5	0.842
	39	0	1	2.43	0.94
	41	1	1	2.56	1.02
	47	1	5	2.44	1.95
	10	0	0	2.5	3
	13	1	2	2.69	3.84
	18	1	4	2.5	5
	25	1	6	2.6	6
	26	-0	1	2.5	7.69
	33	1	6	2.87	7.87
	34	-0	1	2.79	9.11
	35	-0	1	2.71	10.28
	39	0	4	2.43	19.48
	49	3	7	2.55	29.79

Table 2

 2

.1. It is unambiguous that a nK value decreases with the increase of the salinity concentration.

Table 2 . 2 :

 22 The values of

	a nK under different salinity environment.

a K are unknown, they can be obtained after reorganizing the Equation 2.6: f c , and perform linear fit (Figure 2.5). a K ) and binding sites ( n ) can be easily obtained by getting reverse of 1 a K and n under different salinity environment are shown in Table 2.2. a K decreased with the increase of salinity concentration. But we have no idea about the variation trend of n .

The n value can't be determined with accuracy, as it is a limit value at 1/c

Table 2 . 3 :

 23 The value of K a and n under different salinity environments.

	[NaCl] (M)	0.01	0.02	0.05	0.1
		2.86±2.02	5.95±3.26	4.59±2.17	1.64±0.41
	a K (M -1 )	7.37*10 5	2.85*10 5	2.44*10 5	2.2*10 5
	a nK (M -1 )	2.11*10 6	1.69*10 6	1.12*10 6	3.6*10 5

Table 3 . 1 :

 31 Specific information of modified DNA

	dsDNA	Length of dsDNA	Modified end	Labeling
		(Base pairs)		fluorophore
	1 labeled dsDNA	15	5'	Alexa 488
	1 labeled dsDNA	15	3'	Alexa 568
	2 labeled dsDNA	15	5' and 3'	5'-Alexa 488
				3'-Alexa 568

  mM of NaCl) with the initial volume of 1000 L. The strand concentration of dsDNA is kept at 1 M, which dilutes from the 20 M of DNA stock solution with 20 mM of NaCl solution, whereas the concentration of Ru(bpy) 2 ddpz 2+ (C Ru ) is being increased for every single measurement. A series of similar titrations are performed in this study: an example of the titration is displayed in table 3.2.

		Table 3.2: Titration of lifetime measurement
	V total	V DNA,add	V Ru,add	[DNA]	C Ru
	(l)	(l)	(l)	(M)	(M)
	1000	0	0	1	0
	1006	1	5	1.004	0.005
	1011	0	5	1	0.01
	1016	1	4	1.004	0.05
	1021	0	5	0.999	0.1
	1033	1	11	0.997	0.2
	1044	1	10	0.996	0.3
	1069	3	22	1.001	0.5
	Where V DNA,add and V Ru,add are the volume of added DNA and Ru(bpy) 2 dppz 2+ for
	every single measurement to keep the DNA concentration as a constant and increase
	the ruthenium concentration, respectively. [DNA] and [Ru(bpy) 2 dppz 2+ ] represent the
	final concentration of DNA and Ru(bpy) 2 dppz 2+ , respectively.

Table 3 . 3 :

 33 Weighted decay time of donor of single labeled dsDNA

	  ns   ns	 	 		C Ru (M)
	2.14	4.13	0.000348	0.0519	4.12	0
	2.13	4.16	0.00235	0.0499	4.07	0.005
	2.14	4.15	0.00177	0.0498	4.08	0.01
	2.11	4.13	0.00188	0.0498	4.06	0.05
	2.07	4.12	0.00297	0.0491	4.00	0.1
	0.58	4.08	0.0087	0.0495	3.56	0.2
	0.91	4.06	0.00708	0.0483	3.66	0.3
	1.49	4.05	0.00937	0.0457	3.61	0.5

Table 3 . 4 :

 34 Decay time of donor of double labeled dsDNA

	  ns   ns	 	 		C Ru (M)
	0.87	2.77	0.0111	0.0519	2.44	0
	1.23	2.83	0.0126	0.0478	2.5	0.005
	1.14	2.83	0.0138	0.0478	2.45	0.01
	1.02	2.8	0.0136	0.0486	2.41	0.05
	1.43	2.89	0.0181	0.0433	2.46	0.1
	1.32	2.86	0.0184	0.0439	2.41	0.2
	1.51	2.93	0.0239	0.0391	2.39	0.3
	1.39	2.87	0.0228	0.039	2.32	0.5

Table 3 . 5 :

 35 Intensity of emission at 600 nm of Ru(bpy) 2 dppz 2+

	Intensity	Ratio of [	I	600 Ru	]	C Ru (M)
			600 ( IC  Ru	0)
	2.1	0.0016			1
	5.6	0.0042			10
	5.5	0.0041			30
	3.3.3.2 Intensity emitted at 600 nm by donor ( 600 DA N )

Many thanks to China Scholarship Council (CSC) for providing the financial support to me, for studying and living at Strasbourg.

Appendix A Sample preparation protocols

A.1 Drugs-Ru(bpy)2dppz 2+ Ruthenium derived compounds were prepared by the group of chemists following their protocols [120,121]. The first step consists in the synthesis of Ru(bpy) 2 Cl 2 .2H 2 O. Commercial RuCl 3 .3H 2 0 (1.56 g, 5.96 mmol), bipyridine (1.87 g, 12 mmol) and LiCl (1.68 g, 0.4 mmol) were refluxed in dimethylformamide (25 mL) for 8 hours. After the reaction mixture was cooled to room temperature, 150 mL of acetone was added and the resultant solution cooled to 0°C overnight. This solution was filtered, a red-violet filtrate and a dark green-black microcrystalline product were obtained. The solid was washed three times with 25 mL portions of water and followed by three 25 mL portions of diethyl ether, and then it was dried by suction. Yield 64% (1.99 g).

The second step is the coordination by dppz. A mixture of Ru(bpy) 2 Cl 2 .2H 2 O (148 mg, 0.28 mmol) and dipyridophenazine (84 mg, 0.3 mmol) in methanol / water (1 / 2, v / v, 40 mL) was refluxed for 4.5 hours. The deep red solution was concentrated to 10%, diluted with water (20 mL), boiled for 10 minutes, cooled in an ice-bath, and filtered.

The tetrafluoroborate salt was precipitated by addition of 10% NaBF 4 (30 mL) to the filtrate. The solid was dried in vacuo, redissolved in CH 3 CN and then it was filtered over Al 2 O 3 using CH 3 CN as eluent. A red fraction was collected and evaporated. The

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