The steady-state and time-resolved fluorescence of 2-aminopurine (2AP) have been used to monitor base dynamics and base stacking interactions in DNA single strands and dinucleotides, and to investigate the interactions between DNA and a polymerase, Pfu-Pol. A new class of base analogues has also been investigated using a combination of experiment and quantum chemical computation. In recent years, 2AP has been widely used as a fluorescent probe to study conformational changes and inter-bases interactions in duplex DNA, but the conformational behaviour of DNA in single strands has been far less investigated. In the present work, six 2AP–labelled single strands have been studied by steady-state and time-resolved fluorescence measurements. Single strands were found to show similar conformational heterogeneity (manifested by 4-exponential fluorescence decays) to duplex DNA, but highly stacked conformations, in which 2AP is rapidly quenched by inter-base charge transfer, are less populated in single strands, whereas imperfectly stacked (weakly quenched) conformations are more highly populated. The effect of base pairing in constraining base mobility is evident. To further investigate the influence of base stacking interaction on DNA conformation and the mechanism of inter-base quenching of 2AP, the time-resolved fluorescence of 2AP-containing dinucleotides was measured. The fluorescence decay of 2AP-containing dinucleotides in PBS buffer at room temperature is also multiexponential and the shortest lifetime varies with the identity of the natural base partner, in a manner consistent with quenching by inter-base electron transfer. When the dinucleotides are frozen to 77 K, the quenching of 2AP is almost eliminated, demonstrating the importance of thermal fluctuations of the bases in facilitating inter-base quenching at room temperature. In the frozen dinucleotides, an additional decay component with a lifetime significantly longer than unquenched 2AP is also observed, suggesting the formation of a new, delocalised, inter-base excited-state. Archaeal family-B DNA polymerases bind tightly to uracil and stall replication when they encounter this base in template strands, four bases ahead of the primer-template junction. If the polymerase progresses further towards the uracil, the 3′-5′ proof reading exonuclease becomes stimulated, trimming the primer and re-setting uracil to the +4 position. Uracil sensing prevents copying of the deaminated base and the introduction of mutations into the genome. Time-resolved fluorescence of 2AP has been used to investigate the role played by unwinding of primer-templates in this mechanism. 2AP-labelled primer-templates (2AP positioned next to the terminal 3′ base of the primer strand), with a misincorpated uracil at the +2 position (U+2) or +4 position (U+4) from the replication fork in the complementary template strand, were investigated in complex with the polymerase Pfu-Pol. For the U+2 primer-template, the fluorescence decay parameters show clear evidence for a decrease in the amount of double-stranded DNA on polymerase binding, manifested by marked weakening of inter-base stacking and a large transfer of population from highly stacked to poorly stacked conformations. In contrast, for the U+4 primer-template only a small perturbation to inter-base stacking is seen, together with the persistence of a high population of strongly stacked states. A new class of base analogues with selenium replacing oxygen at the 4 position of thymine and the 6 position of guanine has been investigated experimentally and computationally. These base analogues are interesting because they show a large shift (>80 nm) in their absorption spectrum compared with the natural bases, taking their absorption into the visible region, with minimal change in molecular structure. The potential of two examples of these analogues, 4-selenium thymine-3’-phosphate and 6-selenium-2’-deoxyguanosine-3-phosphate as luminescent probes has been investigated. However, they prove to have very low emission quantum yields for both fluorescence and phosphorescence. The effect of selenium-substitution on the structural and photophysical properties of the bases has been studied by various ab initio computational methods. It has been found that replacement of oxygen by selenium does not affect the ground state structure but changes the structure of the first excited-state from buckled to nearly planar. The shift in the absorption spectrum on introduction of selenium is successfully predicted by the calculations; the red-shifted absorption band of selenium-substituted thymine is due to a new electronic transition that is not present in the natural base, whereas that of selenium-substituted guanine is from red-shifting of a guanine-like transition.

Deoxyribonucleic acid (DNA) forms the basis of all known living organisms. Despite the essential role played by DNA, its dynamic system and functional behaviour are still not completely understood. The work presented in this thesis aims to explore the structural dynamics of DNA systems, using fluorescence-based approaches, and to attempt to develop a technique for the measurement of fluorescence decays of biological molecules on the ultrafast (femtosecond) timescale. Absorption of UV radiation by DNA is known to lead to mutations and damage to DNA structure and functionality. For the majority of absorbed photons, the excitation energy dissipates harmlessly as heat, but in some instances this energy transfers to regions of DNA that are more susceptible to damage. 2-Aminopurine (2AP), a fluorescent analogue of the native DNA base adenine, can be incorporated into DNA with minimal perturbation to the DNA structure, and can be used to investigate inter-base electronic energy transfer. By selectively exciting the native DNA base in 2AP-containing dinucleotides and utilising 2AP fluorescence as an energy acceptor, the mechanism of electronic energy transfer has been investigated. Analysis of the resulting fluorescence lifetimes of 2AP has revealed that energy transfer preferentially excites conformations in which the bases are highly stacked, and the fluorescence of 2AP is highly quenched. This has led to a re-evaluation of energy transfer efficiencies between the natural bases and 2AP, and has shown that transfer efficiencies cannot be determined correctly from steady-state fluorescence measurements. To investigate the influence of base dynamics on the quenching of 2AP fluorescence in DNA, time-resolved fluorescence measurements were carried out on 2AP-containing systems in frozen solution at 77 K. These studies included dinucleotides, single–strand oligonucleotides and their corresponding duplexes. In all cases, comparison of the fluorescence decay parameters measured at room temperature with those measured at 77 K showed that elimination of base dynamics prevented rapid quenching, on the 10s of ps timescale or faster, although quenching on the 100s of ps timescale persisted for 2AP in single strands and duplexes. The multi-exponential fluorescence decay of 2AP in DNA and its high sensitivity to local environment is commonly exploited to investigate DNA-enzyme interactions. Transposases are enzymes involved in the movement of sections of DNA (transposons) within the genome. The Mos1 transposase catalyses the movement of a transposon via a cut-and-paste mechanism involving several intermediate complexes. Understanding the complex mechanism by which the transposase can remove and insert a section of DNA would allow these enzymes to be used as biomolecular tools. The structure of the intermediate Mos1 strand-transfer complex (STC) has been investigated by incorporating 2AP into several regions of the transposon and analysing the fluorescence decay. The involvement of a base-flipping-like mechanism has been identified in the mechanism of strand transfer for the Mos1 transposon. The time-resolved fluorescence measurements performed in this thesis are limited to time resolution of ~20 ps and longer using TSCPC. However, an abundance of photophysical events in DNA occur on the femtosecond timescale. Development of a methodology utilising fluorescence gating techniques (such as sum-frequency generation or diffraction from a transient grating) have been attempted, in order to construct an experimental system that enables the broadband detection of ultrafast fluorescence decays. Despite the lack of immediate success in recording the fluorescence decay from a sample, due to technical issues and time-constraints, initial characterisation of the set-up was performed and the prospect of broadband detection was demonstrated. Overall, this thesis gives insight into some of the dynamic processes taking place in DNA and presents work performed to develop a system that would allow the extension of these studies to processes occurring on the fs timescale.

ABSTRACT Many enzymes require cofactors in order to carry out specific functions. Flavins, which are naturally fluorescent, compose a unique group of redox cofactors because they have the ability to transfer one or two electrons and are therefore found in three different oxidation states. A specific flavin, flavin adenine dinucleotide (FAD), is a crucial cofactor that facilitates electron transfer in many flavoproteins involved in DNA repair, photosynthesis, and regulatory pathways. One example of a FAD-containing DNA repair protein is DNA Photolyase (PL). E. coli PL is a monomeric flavoprotein that facilitates DNA repair via a photoinduced electron transfer reaction. The catalytic cofactor, FAD, transfers an electron to a thymidine dimer lesion, to cleave the cyclobutane ring and restore the DNA strand. Although the mechanism of repair has been partially elucidated by our group, it is still unclear whether or not the electron is transferred directly from the isoalloxazine moiety to the dimer or if the electron hops from the isoalloxazine moiety to the adenine moiety to the dimer. This sequential hopping mechanism should have excited state absorption features for the reduced flavin species, an adenine radical anion, and the semiquinone flavin species. To investigate the mechanistic role of adenine, E. coli PL has been reconstituted with -FAD, an FAD analogue in which the adenine was substituted via chemical means with 1,N6 – ethenoadenine dinucleotide. -FAD was selected due to its ease of synthesis and because its structure changes the thermodynamic driving force for the electron transfer reaction, by lowering the energetic gap (LUMO-LUMO) between the isoalloxazine ring and the modified adenine. In order to characterize the excited state dynamics of the mutant chromophore, the transient absorption measurements were made of each free flavin in solution. These measurements indicate the pathway of electron transfer must be mediated via superexchange rather than a hopping mechanism. This important result shows that the role of adenine in photolyase is to facilitate a superexchange electron transfer mechanism, and a modified flavin can act as a reporter under these experimental conditions. By exploiting Corynebacterium ammoniagenes FAD synthetase adenylation promiscuity, we have enzymatically-synthesized and purified a novel dually fluorescent flavin cofactor. This new flavin adenine dinucleotide (FAD) analogue, flavin 2-aminopurine (2Ap) dinucleotide (F2ApD), can be selectively excited through the 2Ap moiety at 310 nm, a wavelength at which flavins have intrinsically low extinction. The dinucleotide 2Ap emits at 370 nm with high efficiency. This emission has excellent overlap with the absorption spectra of both oxidized and reduced hydroquinone flavin (FlOX and FlHQ respectively), which emit at ~525 and ~505 nm respectively. We have characterized the optical properties of this dually fluorescent flavin, iFAD. Steady state fluorescence excitation and emission spectra were obtained and contrasted with the other flavins. Temperature- and solvent-dependent emission spectra suggest that F2ApD stacking interactions are significantly different compared to FAD and etheno-FAD (FAD). The optical absorption spectra of these dinucleotides were compared with FMN to explore electronic interactions between the flavin and nucleobase moieties. To probe the evolution of the different excited state populations, femtosecond transient absorption measurements were made on the iFADs, revealing that F2ApD exhibited unique transient spectra as compared to either FAD or FAD. The significance of these results to flavins, flavoprotein function, and bioimaging are discussed. The reconstituted -FAD in E.coli photolyase was catalytically active and actually repaired more efficiently than the FAD-reconstituted photolyase. To validate that an enzymatically synthesized iFAD could be reconstituted into a flavoprotein, this work shows a DNA repair assay using F2ApD that was reconstituted into E. coli photolyase, generating the reconstituted analogue, ApPL. Activity assays were compared between FAD-PL and ApPL. This comparison further elucidates the importance of the driving force on the electron transfer reaction in PL. A comparison of fluorescence spectroscopies between the reconstituted PLs highlights their applicability as biosensors and/or mechanistic reporters. / Chemistry

No description available.

La protéine UHRF1 est impliquée dans le maintien et la transmission des modifications épigénétiques. Lors du processus de réplication, elle recrute la méthyltransférase de l’ADN Dnmt1 au niveau des sites CpG hémi-méthylés via son domaine SRA (SET and RING Associated), favorisant la duplication des profils de méthylation. La structure tridimensionnelle du complexe SRA/ADN révèle que la protéine induit un basculement de la méthylcytosine qui permet un ancrage spécifique de la protéine sur les sites hémim éthylés, facilitant le recrutement de la Dnmt1 au niveau de ces positions stratégiques. Dans ce contexte, notre projet vise à comprendre les mécanismes d’interaction du domaine SRA de UHRF1 avec l’ADN hémi-méthylé. Des oligonucléotides doubles brins ont été marqués à la 2-aminopurine, un analogue nucléosidique fluorescent sensible à l’environnement, à différentes positions au voisinage d’un unique site de reconnaissance CpG hémi-méthylé. Les mesures de spectroscopie de fluorescence à l’état stationnaire et résolues en temps de ces duplexes liés au domaine SRA nous ont permis de caractériser de manière site spécifique les changements conformationnels induits par la liaison du domaine SRA. En accord avec la structure tridimensionnelle du complexe SRA/ADN, nos données suggèrent que le domaine SRA est capable de basculer la méthylcytosine tout en préservant la structure des autres bases dans le duplexe. Le domaine SRA semble se lier selon le même mécanisme aux duplexes hémi-méthylés, bi-méthylés et non-méthylés. La protéine UHRF1 jouerait ainsi un rôle de “lecteur“ capable de scanner la séquence d’ADN à la recherche de sites hémi-méthylés. / The UHRF1 protein plays a key role in the maintenance and transmission of epigenetic modifications. Duringthe replication process, it recruits the DNA methyltransferase Dnmt1 to hemi-methylated CpG sites via itsSRA (SET and RING Associated) domain, promoting the duplication of the methylation profiles. Thetridimensional structure of the SRA/DNA complex revealed that the protein induces a base-flipping of themethylcytosine that enables a specific anchoring of the protein to hemi-methylated sites facilitating therecruitment of Dnmt1 to this strategic position. In this context, our project was aimed to further understand themechanism of interaction of the SRA domain with hemi-methylated DNA. To this end, oligonucleotideduplexes were labeled by 2-aminopurine, a fluorescent nucleoside analogue sensitive to environment, atvarious positions close to the single hemi-methylated CpG recognition site. Steady-state and time-resolvedfluorescence spectroscopy measurements of these duplexes bound to the SRA domain enabled us to sitespecificallycharacterize the conformational changes induced by the binding of this domain. In agreement withthe tridimensional structure of the SRA/DNA complex, our data suggest that the SRA domain is able to flip themethylcytosine while preserving the structure of the surrounding bases in the duplex. The SRA domain wasshown to bind with the same mechanism to hemi-methylated, fully-methylated and non-methylated duplexes.Our data suggest the UHRF1 protein plays a role of “reader” that scans the DNA sequence for hemimethylatedsites.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2005. / Härtill 5 uppsatser.

Le but de cette thèse est de sonder la flexibilité de NCp7 et de Δ(-)PBS, deux bio-molécules impliquées dans le second saut de brin de la transcription inverse du VIH. Deux stratégies expérimentales ont été mises en place. Un nouveau montage de spectroscopie ultra-rapide de fluorescence par down-conversion a été construit. Les dynamiques de quenching de la 2-aminopurine (2Ap), insérée en position 6, 8 et 10 de la boucle Δ(-)PBS ont pu être entièrement résolues à une résolution sub-ps. Pour chaque position, 4 temps de vie ont été révélés. Des mesures d'anisotropie confirment que les deux composantes < 5 ps sont liées à un empilement de la 2Ap avec les Guanines avoisinantes. Cet empilement est site-spécifique, prouvé par l'augmentation significative de leurs amplitudes lorsque la 2Ap est située près de la tige (position 10). La faible proportion de conformations reliées à un quenching collisionnel est significative de la faible exposition des 2Ap au solvant et de l'encombrement général de la boucle. La seconde approche avait pour but d'étudier l'effet du repliement du squelette protéique de [35-50] NCp7 autour de son atome de zinc par CID et par LID. Les spectres CID de la protéine nue sont expliqués par le modèle du proton mobile et une description détaillée d'un schéma de fragmentation spécifique autour du Tryptophane (Trp) a été soulignée, attribué une Lysine voisine. Un seul fragment issu de l'excitation à 266 nm a été identifié, son apparition entre en compétition avec les fragments CID du Trp. L'effet général du repliement autour du Zinc se traduit par une augmentation du taux de fragmentation autour du Trp et par une perte de spécificité pour le reste du spectre.Les flexibilités de Δ(-)PBS et NCp7 ont été respectivement évaluées par spectroscopie ultra-rapide de type down-conversion et par spectrométrie en phase gazeuse. La première méthode nécessite l'utilisation d'une sonde fluorescente non invasive, la 2-aminopurine (2Ap), placée en position 6, 8 et 10 de la boucle Δ(-)PBS. Notre résolution temporelle permet de résoudre entièrement les dynamiques locales de quenching et d'anisotropie de la 2Ap. Les composantes liées au quenching statique et quenching collisionnel ont été discriminées et révèlent les degrés d'empilement / encombrement locaux de la boucle. L'effet du repliement de [35-50] NCp7 autour de son atome de zinc a été étudié par CID et par LID à 266 nm. La protéine nue présente un interessant shéma de fragmentation autour du Tryptophane (Trp), exalté par la complexation avec le zinc, au prix une perte de spécificité pour le reste du spectre. Un seul fragment LID a été identifié, un mécanisme de sa formation est proposé. / This thesis aims to probe the flexibility of NCp7 and Δ(-)PBS, two biomolecules involved in the second strand transfer of the HIV's reverse transcription. We brought to the front two original experimental methods. A new ultrafast fluorescence down-conversion setup has been built, suitable for biological chromophore investigations. The quenching dynamics of 2-aminopurine (2Ap), site-mutated at the positions 6, 8 and 10 of Δ(-)PBS loop, were completely resolved under a ps scale. For each location, 4 decay times, were highlighted. Further anisotropy measurements confirmed that the two < 5 ps components correspond to stacking interactions of 2Ap with neighbouring Guanines. The site-specific aspect of the stacking were supported by a significant increase of their relative amplitudes when 2Ap were cloesly located to the stem (position 10). The minor portion of conformations involved with ps to ns collisional quenching suggests a low exposure of 2Ap towards the solvent as well as a general restriction of the loop. The second method planned to investigate the effet of the zinc-folding on [35-50] NCp7's peptidic backbone, thanks to CID and LID. The CID-generated spectra of the bare peptide were explained by the mobile proton model, and an exhaustive tryptophan (Trp) fragmentation pattern was described, mainly due to a neighbouring Lysin effects. Only one LID-fragment has been identified upon 266 nm excitation, probably created through a pathway competing with the generation of Trp fragments by CID. The main aspects related to zinc-folding are a general enhancement of the fragmentation ratios related to Trp and a loss of specificity for the remaining mass spectra parts.Δ(-)PBS et NCp7 has been respectively investigated by ultrafast down-conversion spectroscopy and gas-phase spectrometry. The first method implies the use of a non invasive fluorescent probe, named 2 aminopurine (2Ap), site mutated in position 6, 8 et 10 of the Δ(-)PBS loop. Our time resolution allows to fully depict the local quenching dynamics and anisotropy decays. The component related to static and collisional has been solved, thus describing different stacking degrees as well as local restrictions. The effect of [35-50] NCp7 folding around its zinc atom has been studied by CID and 266 nm LID. The bare protein displays an interesting fragmentation pathway around its Tryptophan (Trp), enhanced with zinc complexation, at the cost of a loss of specificity for the remaining mass spectra parts. Only one LID fragment has been identified, its occurence has been interpreted.

Nucleic acids, such as DNA, play an essential role in all known forms of life; however, despite their fundamental importance, there is still a significant lack of understanding surrounding their functional behaviour. This thesis explores the structure and dynamics of DNA by employing methods based on fluorescence and through the use of computational calculations. Time-resolved fluorescence experiments have been performed on dinucleotides containing 2-aminopurine (2AP) in various alcohol-water mixtures. 2AP, a fluorescent analogue of the nucleobase adenine, has been used extensively to investigate nucleic acids because of its ability to be incorporated into their structures with minimal perturbation and its high sensitivity to its local environment. Direct solvent effects on 2AP were established through measurements on the free fluorophore. Analysis of the complex fluorescence decays associated with the dinucleotides was challenging but has provided insight into their conformational dynamics. Solvent polarity was found to play a significant role in determining both photophysical and conformational properties in these systems. The complicated fluorescence decay of 2AP in nucleic acids highlights the need for accurate and unbiased analysis methods. Various time-resolved fluorescence analysis methods, including iterative reconvolution and the exponential series method, have been investigated with real and simulated data to obtain an overview of their benefits and limitations. The main outcome of the evaluation is that no single method is preferred in all situations and there is likely to be value in using a combination when there is ambiguity in the interpretation of the results. Regardless of the analysis technique used, the parameterised description of the observed fluorescence decay is meaningless if the underlying physical model is unrealistic. The advance of computational methods has provided a new means to rigorously test the viability of proposed models. Calculations have been performed at the M06-2X/6-31+G(d) level of theory to investigate the stability of 2AP-containing dinucleotides in conformations similar to those observed in the double-helical structure of DNA. The results help to explain the similarity of the time-resolved fluorescence behaviour of 2AP in dinucleotide and DNA systems but also bring to light subtle differences that could perhaps account for experimental discrepancies. The recent emergence of advanced optical microscopy techniques has offered the prospect of being able to directly visualise nucleic acid structure at the nanoscale but, unfortunately, limitations of existing labelling methods have hindered delivery of this potential. To address this issue, a novel strategy has been used to introduce reversible fluorescence photoswitching into DNA at high label density. Photophysical studies have implicated aggregation and energy-transfer as possible quenching mechanisms in this system, which could be detrimental to its future application. The reliability of fluorescence photoswitching was investigated at ensemble and single-molecule level and by performing optical lock-in detection imaging. These developments lay the foundations for improved and sequence-specific super-resolution microscopy of DNA, which could offer new insights into the 3D nanoscale structure of this remarkable biopolymer. In summary, the work presented in this thesis outlines important observations and developments that have been made in the study of the structure and dynamics of nucleic acids.

Fluorescent nucleic acid base analogues (FBAs) are an important class of molecule used to study the structure and dynamics of DNA and RNA. These base analogues are molecules with structures that resemble one of the natural bases but which, unlike the natural bases, have high fluorescence quantum yields. 2-Aminopurine (2AP) has long been the most widely used fluorescent base analogue and is one of the few base analogues commercially available. One problem with 2AP is that it undergoes significant quenching when incorporated into DNA: the quantum yield decreases 100 fold from that of the free base, thus becoming too low for use in, for example, single molecule studies. A secondary problem is that the 305 nm absorption peak requires excitation in the UV. A variety of new fluorescent base analogues are being produced, with a view to remedying the deficiencies of 2AP and expanding the current range of use. The first part of this thesis explores the one-photon photophysical properties of several of these novel FBAs. The first of these novel FBAs is the 6-aza-uridine family. These compounds, analogues of uridine, have large Stokes shifts and their absorption and emission spectra are red-shifted in comparison to 2AP; their quantum yields as free bases have been shown to exceed that of 2AP and their environmental sensitivity has been demonstrated. Time-resolved measurements reported in this thesis indicate the presence of multiple emitting species. A density functional theory (DFT) study has been carried out to rationalise these emitting species as rotational isomers. Similar fluorescence lifetime measurements were made on a second class of FBAs, the quadracyclic adenine analogues, qANs; these results also indicated the presence of multiple emitting species. Experimental results show that these FBAs undergo excited-state proton transfer. The final FBA studied in this thesis is pentacyclic adenine, pA. This FBA showed some of the most promising characteristics of all the analogues investigated, such as a high quantum yield in both polar and non-polar solvents. A time-resolved investigation into pA-containing oligonucleotides indicated that in an oligonucleotide pA adopts multiple stacked conformations and its behaviour is highly sequence dependent. Several of these aforementioned fluorescent base analogues have absorption spectra in a region that makes them accessible to two-photon (2P) excitation with a Ti:Sapphire laser. In biological systems, multiphoton excitation has several advantages over one-photon excitation. By avoiding the use of ultraviolet light there is reduced phototoxicity. Out of focus photobleaching and autofluorescence are also minimised which leads to a higher signal-to-background ratio and allows deeper tissue penetration to be achieved. Fluorescent base analogues tend to have small Stokes shifts; this is another problem that can be overcome by using two-photon excitation. To be of potential use in multiphoton microscopy, a FBA must have a high two-photon absorption cross-section and a high two-photon brightness. Previously, the highest two- photon brightness measured for a fluorescent base analogue was less than 2 GM. Amongst the base analogues investigated here, are several that have higher two-photon brightness than ever reported for FBAs; these include pA which is shown to have the highest 2P brightness of a FBA in an oligonucleotide, 1.3 GM, and a member of the 6-azauridine family which as a free base has a 2P brightness of 18 GM. Detection of individual molecules represents the ultimate level of sensitivity and enables details about a molecular system that would otherwise be concealed using conventional ensemble techniques to be revealed. With the improved 2P brightness of the molecules measured in this thesis, it has become feasible to detect single FBA molecules using 2P excitation. To maximise the chance of detection, ultrafast, shaped laser pulses have been used as the excitation source. For the first time, the signal has been high enough and the molecule of interest sufficiently photostable such that 2P fluorescence correlation spectroscopy of a fluorescent base analogue in an oligonucleotide could be measured. In summary, this thesis reports the fluorescence lifetimes and two-photon cross-sections of a series of novel fluorescent base analogues, as well as fluorescence correlation spectroscopy measurements of the most promising candidates.

No description available.