Analysis methods for single molecule fluorescence spectroscopy

This thesis describes signal analysis methods for single-molecule fluorescence data. The primary factor motivating method development is the need to distinguish single-molecule FRET fluctuations due to conformational dynamics from fluctuations due to distance-independent FRET changes. Single-molecule Förster resonance energy transfer (FRET) promises a distinct advantage compared to alternative biochemical methods in its potential to relate biomolecular structure to function. Standard measurements assume that the mean transfer efficiency between two fluorescent probes, a donor and an acceptor, corresponds to the mean donor-acceptor distance, thus providing structural information. Accordingly, measurement analysis assumes that mean transfer efficiency fluctuations entail mean donor-acceptor distance fluctuations. Detecting such fluctuations is important in resolving molecular dynamics, as molecular function often necessitates structural changes. A problem arises, however, in that factors other than donor-acceptor distance changes may induce mean transfer efficiency fluctuations. We refer to these factors as distance-independent FRET changes. We present analysis methods to detect distance-independent photophysical dynamics and to determine their correlation with distance-dependent FRET dynamics. First, we review a theory of photon statistics and show how we can use the theory to detect FRET fluctuations. Second, we extend the theory to alternating laser excitation (ALEx) measurements and demonstrate how fluorophore stoichiometry, a measure of fluorophore brightness, reports on distance-independent photophysical dynamics. Next, we provide a measure to determine the extent to which stoichiometry fluctuations account for FRET dynamics. Finally, we use a framework similar to the preceding along with recent advances in the theory of total internal reflection fluorescence (TIRF) microscopy FRET measurements to detect TIRF FRET fluctuations which occur on a timescale faster than the measurement temporal resolution. We validate our methods with simulations and demonstrate their utility in delineating RNA polymerase open complex conformational dynamics.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Surfing on a new wave of single-molecule fluorescence methods

Hohlbein J, Gryte K, Heilemann M, Kapanidis AN. Surfing on a new wave of single-molecule fluorescence methods. PHYSICAL BIOLOGY. 2010;7(3): 31001.Single-molecule fluorescence microscopy is currently one of the most popular methods in the single-molecule toolbox. In this review, we discuss recent advances in fluorescence instrumentation and assays: these methods are characterized by a substantial increase in complexity of the instrumentation or biological samples involved. Specifically, we describe new multi-laser and multi-colour fluorescence spectroscopy and imaging techniques, super-resolution microscopy imaging and the development of instruments that combine fluorescence detection with other single-molecule methods such as force spectroscopy. We also highlight two pivotal developments in basic and applied biosciences: the new information available from detection of single molecules in single biological cells and exciting developments in fluorescence-based single-molecule DNA sequencing

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

Sensing DNA Opening in Transcription Using Quenchable Förster Resonance Energy Transfer

Many biological processes, such as gene transcription and replication, involve opening and closing of short regions of double-stranded DNA (dsDNA). Few techniques, however, can study these processes in real time or at the single-molecule level. Here, we present a Förster resonance energy transfer (FRET) assay that monitors the state of DNA (double- vs single-stranded) at a specific region within a DNA fragment, at both the ensemble level and the single-molecule level. The assay utilizes two closely spaced fluorophores: a FRET donor fluorophore (Cy3B) on the first DNA strand and a FRET acceptor fluorophore (ATTO647N) on the complementary strand. Because our assay is based on quenching and dequenching FRET processes, i.e., the presence or absence of contact-induced fluorescence quenching, we have named it a “quenchable FRET” assay or “quFRET”. Using lac promoter DNA fragments, quFRET allowed us to sense transcription bubble expansion and compaction during abortive initiation by bacterial RNA polymerase. We also used quFRET to confirm the mode of action of gp2 (a phage-encoded protein that acts as a potent inhibitor of Escherichia coli transcription) and rifampicin (an antibiotic that blocks transcription initiation). Our results demonstrate that quFRET should find numerous applications in many processes involving DNA opening and closing, as well as in the development of new antibacterial therapies involving transcription.

The Transcription Bubble of the RNA Polymerase–Promoter Open Complex Exhibits Conformational Heterogeneity and Millisecond-Scale Dynamics:Implications for Transcription Start-Site Selection

Bacterial transcription is initiated after RNA polymerase (RNAP) binds to promoter DNA, melts ~14 bp around the transcription start site and forms a single-stranded “transcription bubble” within a catalytically active RNAP–DNA open complex (RPo). There is significant flexibility in the transcription start site, which causes variable spacing between the promoter elements and the start site; this in turn causes differences in the length and sequence at the 5′ end of RNA transcripts and can be important for gene regulation. The start-site variability also implies the presence of some flexibility in the positioning of the DNA relative to the RNAP active site in RPo. The flexibility may occur in the positioning of the transcription bubble prior to RNA synthesis and may reflect bubble expansion (“scrunching”) or bubble contraction (“unscrunching”). Here, we assess the presence of dynamic flexibility in RPo with single-molecule FRET (Förster resonance energy transfer). We obtain experimental evidence for dynamic flexibility in RPo using different FRET rulers and labeling positions. An analysis of FRET distributions of RPo using burst variance analysis reveals conformational fluctuations in RPo in the millisecond timescale. Further experiments using subsets of nucleotides and DNA mutations allowed us to reprogram the transcription start sites, in a way that can be described by repositioning of the single-stranded transcription bubble relative to the RNAP active site within RPo. Our study marks the first experimental observation of conformational dynamics in the transcription bubble of RPo and indicates that DNA dynamics within the bubble affect the search for transcription start sites.