Dark quenchers and novel fluorescent thymidine monomers for single-molecule detection

Constant improvements of single-molecule techniques over the past two decades have lead to multiple new insights in various biological processes. In particular, single- molecule Forster Resonance Energy Transfer (smFRET), a fluorescence-based method capable of measuring distances in the 2-10nm range, is extensively used in the study of diverse biomolecular interactions. In this thesis, I discuss novel assays for single-molecule fluorescence detection. In the first part, the main single-molecule experimental setup built in the course of my doctorate is described. In the second part, the use of dark quenchers is investigated. One of the main hurdles in single-molecule fluorescence experiments is the difficulty of working at high concentrations of labelled molecules, thus preventing the study of biomolecular interactions with high dissociation constants. Dark quenchers, used as acceptors in a smFRET pair, can circumvent this problem as they can be used at high concentration. However, very few studies using dark quenchers at the single- molecule level have been reported in the literature and little is known about their photophysical properties. This thesis demonstrates that dark quenchers, used in combination with Alternating Laser Excitation (ALEX) spectroscopy, can be used in a 3-probe system (two fluorophores and one dark quencher) to monitor multiple distances and interactions. Their basic photophysical properties are also investigated. Finally, dark quencher detection is applied to study interactions of DNA Polymerase I (Klenow Fragment) with DNA. In the third part of this thesis, fluorescent properties of novel thymidine monomers labelled with Cyanine dyes (CydT monomers) that were used as building blocks for DNA synthesis are characterised. CydT monomers open the possibility of attaching many Cy3 and Cy5 fluorophores site specifically on a DNA strand, thus allowing more complex labelling schemes than currently possible. Their performance as FRET pairs is also investigated. The methods and assays discussed in this thesis can be used to study a broad range of biomolecular interactions, including protein-protein interactions and protein-DNA complexes, and have potential applications in real-time DNA sequencing.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Efeito das propriedades segmentais em tarefas de consciência segmental, de leitura e de escrita

Tese de doutoramento, Linguística (Psicolinguística), Universidade de Lisboa, Faculdade de Letras, 2013O principal objectivo deste estudo é o de contribuir para o conhecimento do efeito das propriedades segmentais no desempenho de operações metassegmentais. Apesar de este tipo de operações constituírem um recorrente foco de investigação, tanto na área da saúde como na do ensino, actualmente, pouco se sabe sobre o impacto das propriedades intrínsecas aos segmentos em tarefas de consciência segmental, de leitura e de escrita, na medida em que a maioria dos estudos desenvolvidos se debruça holisticamente sobre a interacção entre consciência fonológica e alfabetização. A fim de preencher parte das lacunas observadas nesta área, estabeleceu-se como objectivo geral a identificação do efeito de propriedades fonológicas, especialmente das relativas ao vozeamento, ao modo e ao ponto de articulação, inerentes ao sistema consonântico do Português Europeu, no desempenho de tarefas de consciência segmental e de escrita e leitura emergentes. Definiram-se dois objectivos específicos: (i) descrever o efeito das propriedades segmentais numa tarefa de consciência segmental, realizada por crianças com baixo conhecimento ortográfico; (ii) descrever o efeito das propriedades segmentais em tarefas de escrita e de leitura de palavras e de pseudopalavras, realizadas pelos mesmos sujeitos. Com vista à concretização dos objectivos propostos, foram aplicadas provas de consciência segmental, de leitura e de escrita (de palavras e de pseudopalavras) a um grupo de 48 crianças com fraco domínio, embora não patológico, do princípio alfabético. Globalmente, a amostra revelou melhores resultados na prova de consciência segmental (Prova do Intruso) do que nas provas de escrita e de leitura (Provas B). Verificaram-se efeitos das propriedades segmentais que caracterizam as consoantes testadas (efeitos fonológicos e fonéticos), em particular das relativas ao modo de articulação, como também efeitos das propriedades ortográficas, tanto na Prova do Intruso como nas Provas B. Por último, observou-se uma assimetria entre a emergência de propriedades segmentais no desenvolvimento fonológico (conhecimento fonológico implícito) e o processamento metassegmental destas propriedades (conhecimento fonológico analítico), na medida em que, dependendo do tipo de tarefa a executar, as crianças parecem explorar de forma diferente o conhecimento (meta)linguístico de que dispõem.The main goal of this study is to contribute to the knowledge of the effect of segmental properties performing metassegmental operations. Although they are a recurrent focus of investigation, both in health and in education, at present, little is known about the impact of these properties when children perform segmental awareness tasks or reading and writing tasks, because most of the studies focus holistically on the interaction between phonological awareness and literacy. In order to fill some of the gaps observed in this area, the goal defined is to verify and describe the effect of phonological properties, especially those relating to voicing, manner and place of articulation inherent to the consonantal system of European Portuguese, performing segmental awareness and reading/writing tasks, considering two general goals: (i) to describe the effect of segmental properties in a segmental awareness taskheld performed by children with low reading and writing knowledge; (ii) to describe the effect of segmental properties in writing and reading tasks of words and pseudowords, carried out by the same children. To achieve the goals proposed, some tasks of segmental awareness, reading and writing (of words and pseudowords) were applied to a group of 48 children with low knowledge, although not pathological, of alphabetic principle. Overall, the sample showed better results on the segmental awareness task (Oddity Task) than on the reading and writing tasks (Tasks B). Effects of segmental properties that characterize the tested consonants were observed (phonetic and phonological effects), in particular those concerning the manner of articulation, as well as the effects of orthographic properties, in all tasks applied (Oddity Task and Tasks B). Finally, it was verified an asymmetry between the emergence of segmental properties on phonological development (implicit phonological knowledge) and metassegmental processing of these properties (analytic phonological knowledge) because, depending on the kind of task to perform, children seem to explore their (meta)linguistic knowledge differently.Fundação para a Ciência e a Tecnologia (FCT, SFRH/BD/47271/2008

Defining the Limits of Single-Molecule FRET Resolution in TIRF Microscopy

Single-molecule FRET (smFRET) has long been used as a molecular ruler for the study of biology on the nanoscale (∼2–10 nm); smFRET in total-internal reflection fluorescence (TIRF) Förster resonance energy transfer (TIRF-FRET) microscopy allows multiple biomolecules to be simultaneously studied with high temporal and spatial resolution. To operate at the limits of resolution of the technique, it is essential to investigate and rigorously quantify the major sources of noise and error; we used theoretical predictions, simulations, advanced image analysis, and detailed characterization of DNA standards to quantify the limits of TIRF-FRET resolution. We present a theoretical description of the major sources of noise, which was in excellent agreement with results for short-timescale smFRET measurements (<200 ms) on individual molecules (as opposed to measurements on an ensemble of single molecules). For longer timescales (>200 ms) on individual molecules, and for FRET distributions obtained from an ensemble of single molecules, we observed significant broadening beyond theoretical predictions; we investigated the causes of this broadening. For measurements on individual molecules, analysis of the experimental noise allows us to predict a maximum resolution of a FRET change of 0.08 with 20-ms temporal resolution, sufficient to directly resolve distance differences equivalent to one DNA basepair separation (0.34 nm). For measurements on ensembles of single molecules, we demonstrate resolution of distance differences of one basepair with 1000-ms temporal resolution, and differences of two basepairs with 80-ms temporal resolution. Our work paves the way for ultra-high-resolution TIRF-FRET studies on many biomolecules, including DNA processing machinery (DNA and RNA polymerases, helicases, etc.), the mechanisms of which are often characterized by distance changes on the scale of one DNA basepair

Monitoring multiple distances within a single molecule using switchable FRET

Uphoff S, Holden SJ, Le Reste L, et al. Monitoring multiple distances within a single molecule using switchable FRET. NATURE METHODS. 2010;7(10):831-836.The analysis of structure and dynamics of biomolecules is important for understanding their function. Toward this aim, we introduce a method called 'switchable FRET', which combines single-molecule fluorescence resonance energy transfer (FRET) with reversible photoswitching of fluorophores. Typically, single-molecule FRET is measured within a single donor-acceptor pair and reports on only one distance. Although multipair FRET approaches that monitor multiple distances have been developed, they are technically challenging and difficult to extend, mainly because of their reliance on spectrally distinct acceptors. In contrast, switchable FRET sequentially probes FRET between a single donor and spectrally identical photoswitchable acceptors, dramatically reducing the experimental and analytical complexity and enabling direct monitoring of multiple distances. Our experiments on DNA molecules, a protein-DNA complex and dynamic Holliday junctions demonstrate the potential of switchable FRET for studying dynamic, multicomponent biomolecules

New technologies for DNA analysis – a review of the READNA Project

International audienceThe REvolutionary Approaches and Devices for Nucleic Acid analysis (READNA) project received 12 million s funding under the European Union Framework Programme 7 from 1st June 2008 to 30th November 2012. The 19 project partners from both academia and industry from in total 7 countries had a project budget of 16 Ms with which they have discovered, created and developed a huge body of insights into nucleic acid analysis. Results have been presented widely in publications and in innumerous public presentations. Results have been moved to spin-offs such as the Olink enrichment kits (now sold by Agilent as Haloplex) and are findingtheir way to the market, such as the Oxford Nanopore MinIon sequencer that was first released to early-access user sites in 2014