On Conflict Free DNA Codes

DNA storage has emerged as an important area of research. The reliability of DNA storage system depends on designing the DNA strings (called DNA codes) that are sufficiently dissimilar. In this work, we introduce DNA codes that satisfy a special constraint. Each codeword of the DNA code has a specific property that any two consecutive sub-strings of the DNA codeword will not be the same (a generalization of homo-polymers constraint). This is in addition to the usual constraints such as Hamming, reverse, reverse-complement and $GC$-content. We believe that the new constraint will help further in reducing the errors during reading and writing data into the synthetic DNA strings. We also present a construction (based on a variant of stochastic local search algorithm) to calculate the size of the DNA codes with all the above constraints, which improves the lower bounds from the existing literature, for some specific cases. Moreover, a recursive isometric map between binary vectors and DNA strings is proposed. Using the map and the well known binary codes we obtain few classes of DNA codes with all the constraints including the property that the constructed DNA codewords are free from the hairpin-like secondary structures.Comment: 12 pages, Draft (Table VI and Table VII are updated

DNA molecular recognition specificity : pairwise and in competition

Despite its importance in biological systems, the molecular recognition of DNA hybridization within complex, competitive environments is poorly understood. The present thesis investigates DNA hybridization in thermal equilibrium for DNA strands bound to the surface of a microarray as well as in solution in presence of one or more competitors. For the latter we employ fluorescence anisotropy and fluorescence correlation spectroscopy to determine binding affinities of two DNA strands in a pairwise manner and in presence of a single competitor. Our results reveal that there must be a non-trivial interaction between the competing strands that extends beyond simple double helix formation. This is a signature of cooperative behavior, which can lead to more complex binding phenomena than previously thought. Moreover, we find surprising differences between the results of both techniques, which we attribute to differing sensitivities to distinct microstates of double helix formation. The second part of this work is performed with surface-bound DNA and devoted to experimentally determine a sufficient number of differing bases between two sequences to avoid cross-hybridization. We construct a set of 23 non-interacting sequences with a length of 7 bases. We conclude that for systems of increasing complexity a high level of discrimination between many competitors is essential for accurate recognition.Trotz der Relevanz für biologische Systeme sind die Mechanismen molekularer Erkennung bei der Hybridisierung von DNA in komplexen Umgebungen kaum verstanden. Die vorliegende Arbeit untersucht DNA Hybridisierung im thermischen Gleichgewicht mit DNA-Strängen sowohl an die Oberfläche eines Microarrays gebunden als auch in Lösung in Gegenwart von Konkurrenten. Für letztere verwenden wir Fluoreszenzanisotropie sowie -korrelationsspektroskopie, um Bindungsaffinitäten zweier DNA-Stränge paarweise und in Anwesenheit einzelner Konkurrenten zu bestimmen. Unsere Ergebnisse zeigen, dass es nicht triviale Wechselwirkungen zwischen den beteiligten Strängen geben muss, die über die einfache Bildung einer Doppelhelix hinausgehen. Diese Beobachtung deutet auf kooperatives Verhalten hin und zeigt, dass DNA-Hybridisierung komplexer abläuft als bisher angenommen. Außerdem finden wir eine unerwartete Diskrepanz beider Methoden, die auf unterschiedliche Sensitivitäten für bestimmte Mikrozustände der gebundenen DNA zurückgeht. Im zweiten Teil der Arbeit widmen wir uns Experimenten mit oberflächengebundener DNA. Wir bestimmen eine ausreichende Anzahl sich unterscheidender Basenpaare zweier Stränge, um nicht spezifische Hybridisierung zu vermeiden, und zeigen, dass sich damit ein Satz aus 23 nicht interagierenden Strängen á 7 Basen konstruieren lässt. Wir schließen, dass für zunehmend komplexe Systeme ein hoher Diskriminierungsgrad zwischen vielen Konkurrenten unabdingbar für präzise Erkennung ist

Patterns and Signals of Biology: An Emphasis On The Role of Post Translational Modifications in Proteomes for Function and Evolutionary Progression

After synthesis, a protein is still immature until it has been customized for a specific task. Post-translational modifications (PTMs) are steps in biosynthesis to perform this customization of protein for unique functionalities. PTMs are also important to protein survival because they rapidly enable protein adaptation to environmental stress factors by conformation change. The overarching contribution of this thesis is the construction of a computational profiling framework for the study of biological signals stemming from PTMs associated with stressed proteins. In particular, this work has been developed to predict and detect the biological mechanisms involved in types of stress response with PTMs in mitochondrial (Mt) and non-Mt protein. Before any mechanism can be studied, there must first be some evidence of its existence. This evidence takes the form of signals such as biases of biological actors and types of protein interaction. Our framework has been developed to locate these signals, distilled from “Big Data” resources such as public databases and the the entire PubMed literature corpus. We apply this framework to study the signals to learn about protein stress responses involving PTMs, modification sites (MSs). We developed of this framework, and its approach to analysis, according to three main facets: (1) by statistical evaluation to determine patterns of signal dominance throughout large volumes of data, (2) by signal location to track down the regions where the mechanisms must be found according to the types and numbers of associated actors at relevant regions in protein, and (3) by text mining to determine how these signals have been previously investigated by researchers. The results gained from our framework enable us to uncover the PTM actors, MSs and protein domains which are the major components of particular stress response mechanisms and may play roles in protein malfunction and disease

The Evolutionary Feedback between Genetic Conflict and Genome Architecture

The advent of separate sexes set the stage for dramatic evolutionary innovation across a wide range of taxa. Much of this innovation is attributable to divergent evolutionary interests between now distinct sub-populations of males and females. Trade-offs inherent to these divergent life histories, coupled with a common genome, conspire to limit natural selection's ability to simultaneously maximize the fitness of both sexes. Such conflict between the sexes has therefore largely shaped the history of the genomes of sexual taxa. However, various aspects of the genomic environment—including genes' spatial distributions, abilities to regulate their expression, and rates of recombination—also feed back to influence future sex-specific evolutionary trajectories. Using various genomic resources and transcriptome sequences for the lab mouse, I test several theoretical predictions regarding this feedback between genetic conflict and features of genomic organization

Quantification of biomolecular binding dynamics by Fluorescence Correlation Spectroscopy

Diffusion and molecular binding processes are indispensable for biological systems. A vital step towards the understanding of such dynamics and their interplay is a thorough quantification of all parameters involved. This work addresses the characterization of biomolecular diffusion and binding dynamics using fluorescence correlation spectroscopy (FCS). To quantify the reversible surface attachment of fluorescently labeled molecules, a novel method termed surface-integrated FCS (SI-FCS) is developed. Using this technique, the association and dissociation rates of receptor-ligand pairs can be determined over a wide range of time scales, ranging from hundreds of milliseconds to tens of seconds. The surface of interest is exposed to a widefield illumination and a highly sensitive electron-multiplying charge-coupled device (EMCCD) camera is used for detection, not only providing single-molecule sensitivity, but also enabling a parallel detection of the signal, which facilitates multiplexed SI-FCS measurements across the field of view. To validate this approach, we quantify the reversible hybridization of single-stranded deoxyribonucleic acid (DNA) using a standard total internal reflection fluorescence (TIRF) microscope. The nucleotide overlap was systematically varied to demonstrate the sensitivity of SI-FCS. Furthermore, this work extensively employs FCS in its more conventional form using a confocal microscope. The effect of refractive index mismatches on single-focus FCS measurements is thoroughly characterized and a regime in which unbiased experiments are possible is identified. Confocal FCS is used to monitor the filament formation of FtsZ proteins (filamenting temperature-sensitive mutant Z) and their breakage by the protein MipZ in vitro. Potential artifacts are identified and a novel model to analyze diffusing filaments in FCS experiments is derived, applied, and validated. These findings not only demonstrate that filament formation can be efficiently studied using confocal FCS, but also indicate that FtsZ from Caulobacter crescentus may intrinsically form small oligomers. Finally, this work characterizes the diffusion of biomolecules in lipid monolayers at the air-water interface using confocal FCS. A miniaturized fixed area-chamber, which requires only minute amounts of protein, is presented and validated. Using this design, monolayer experiments become accessible to studies where biomolecules can only be purified in small amounts. Moreover, the quantification of diffusion in monolayers using FCS is a major step towards the routine characterization of binding of biomolecules to lipid monolayers.Diffusion und molekulare Bindungsreaktionen sind elementare Prozesse in biologischen Systemen. Für das Verständnis solcher Dynamiken und deren Wechselwirkungen ist es letztlich unabdingbar die beteiligten Parameter exakt zu quantifizieren. Diesem Ziel folgend setzt sich diese Arbeit mit der Quantifizierung von Diffusions- und Bindungsdynamiken unter Nutzung der Fluoreszenzkorrelationsspektroskopie (FCS) auseinander. Um die Assoziations- und Dissoziationsraten von reversiblen Bindungsreaktionen an Oberflächen zu messen, wurde im Rahmen dieser Arbeit eine neuartige Methode namens "surface-integrated FCS" (SI-FCS) entwickelt. Mittels dieser Methode können Bindungsraten zwischen Rezeptoren und fluoreszierenden Liganden in Zeitbereichen von Millisekunden bis über einer Minute gemessen werden. Die zu untersuchende Oberfläche, an der die Bindungsreaktionen stattfinden, wird mit einer Weitfeldausleuchtung beschienen und die daraufhin emittierte Fluoreszenz von den Liganden wird mit einer sehr empfindlichen Kamera (electron-multiplying charge-coupled device) detektiert. Diese Flächendetektion verfügt nicht nur über ausreichende Empfindlichkeit um einzelne Moleküle zu detektieren, sondern ermöglicht auch die parallele Messung mehrerer Autokorrelationskurven im Sichtfeld. Zur Validierung dieses neuartigen Ansatzes wird die reversible Hybridisierung von Desoxyribonukleinsäuren (DNS) mit einem im Rahmen dieser Arbeit konstruierten totalreflexionsbasiertem Fluoreszenzmikroskop (TIRF Mikroskop) quantifiziert. Die Anzahl der hybridisierenden Basenpaare wird in dieser Studie systematisch variiert und drückt sich in klaren Änderungen der gemessenen Bindungsraten aus. Damit wird die Sensitivität der Methode unterstrichen. Darüber hinaus bedient sich diese Arbeit der konventionellen konfokalen FCS. Das Problem von Proben, die einen anderen Brechungsindex als den von Wasser aufweisen, wird intensiv im Kontext von FCS Messungen beleuchtet. Abschließend werden Messbedingungen aufgezeigt unter denen systematische Messfehler und Artefakte, die auf den Brechungsindex zurückzuführen sind, vermieden werden können. In einem Teil dieser Arbeit wird die konfokale FCS genutzt um die Polymerisation von FtsZ Proteinen (Filamenting Temperature-Sensitive Z), sowie deren Zerlegung durch das Protein MipZ, zu untersuchen. Potentielle Fehlerquellen solcher Messungen werden beleuchtet und ein neues Modell für die Analyse von konfokalen FCS Messungen an Filamenten wird hergeleitet. Die präsentierten Ergebnisse zeigen nicht nur, dass FCS eine geeignete Methode ist umWachstum und Zerfall von Filamenten im Allgemeinen zu charakterisieren, sondern liefern auch deutliche Hinweise, dass FtsZ aus dem Bakterium Caulobacter crescentus auch in Abwesenheit von Guanosintriphosphat (GTP) kurze Oligomere bildet. Letzteres ist insbesondere interessant, da typischerweise angenommen wird, dass FtsZ als monomeres Protein vorliegt und erst in Anwesenheit von GTP zu Filamenten polymerisiert. Abschließend quantifiziert diese Arbeit die Diffusion von Biomolekülen in Lipidmonoschichten an der Grenzfläche zwischen Luft und Wasser. Unter Verwendung der konfokalen FCS werden Messungen in Miniaturkammern durchgeführt und validiert. Mithilfe dieser Methode werden Messungen an Biomolekülen ermöglicht, die nur in sehr geringen Mengen aufgereinigt werden können. Die hier präsentierten Diffusionsmessungen stellen einen wichtigen Schritt hin zur FCS basierten Charakterisierung der Bindungskinetiken von Biomolekülen zu Lipidmonoschichten dar

Mitochondrial DNA diversity and origin of human communities from 4th- 11th century Britain.

Neither the archaeological nor the historical data have yet allowed a full understanding of the nature of the Germanic settlement in England. Analysis of the genetic structure of past history has mostly been carried out by inference from extant populations. However, genetic flow through migration over time is likely to have altered the genetic composition of modem samples. Analysis of the genetic composition of ancient populations (provided the authenticity of their DNA is obtained) gives a direct sight into the past. Thus, mitochondrial DNA from pre-Saxon (4th century), early Saxon (5th -7th century) and late Saxon (9th – 11th century) settlements has been analysed to obtain a better understanding of the population history of Britain. A methodology has been optimised, by which, ancient DNA from 1,000-1,800 year old archaeological material was extracted and ~200-bp fragments of the HVS-1, amplified and sequenced. Rigorous controls for work in human ancient DNA were undertaken to prevent and recognise contamination. Established authenticity criteria were followed, including expected ancient DNA behaviour, internal replication of sequences and confirmation by independent labs. The sample size obtained has enabled a population-level study of communities of ancient Britain. In addition, an extensive database of >6500 mitochondrial DNA sequences was compiled for comparisons. Several estimates of haplotype and nucleotide genetic diversity were computed for modem and ancient populations. Counter-intuitively, the modem population of England, encompassing all successive waves of migration to the island, has a lower diversity than the ancient population, suggesting that diversity has been lost over the last millennium. In addition, mtDNA genetic continuity between ancient and modem England seems to have been intermpted. Founder analyses of early (5th -7th century) and late (9th -11th century) periods indicate that, whereas the late period seems to have had Viking genetic influences, the early period has no close relationship with Germanic populations. Instead, the females of the early Anglo-Saxon period seem to represent the native British population. The female contribution of the Anglo-Saxon invasion would have therefore been minor, at least at that time and at these sites. The close genetic affinity between the ancient British population and the northern most populations of Europe suggests they might have shared a common past during pre-history. It is proposed that, after post-glacial times, inhabitants of areas now submerged expanded to northern territories. The early settlements analysed reflect that very early expansion. Some time since then, reduction in diversity seem to have occurred (possibly due to variation in family size after repeated epidemics) leading to the present day mtDNA composition of England

Mutation and selective constraint in the murid genome

A large proportion of the genome of many higher eukaryotes consists of apparently functionless noncoding DNA, the significance of which is a long­standing puzzle in biology. The aim of this work was to quantify the extent to which both mutation and natural selection have influenced molecular evolution in murid noncoding sequence. In particular, the magnitude of and variation in selective constraint within murid noncoding DNA was investigated. Selective constraint is defined as the proportion of all mutations occurring at a locus or site which are strongly deleterious and therefore removed by natural selection. The approach adopted to estimate selective constraint relies on the assumption that we can quantify the past strength of purifying selection in a DNA sequence by comparison with nearby regions which are assumed to be evolving neutrally. To this end, work in this thesis deals both with mutational variation and bias (Chapters 2 and 3) as well as with selective constraint (Chapters 4 and 5) in noncoding DNA.Chapter 2 is concerned with the differential effects of context-dependent mutation (namely, CpG hypermutability) at fourfold synonymous and noncoding sites. Using simulations it was shown that a common method of assigning ancestral CpG status often introduces a substantial level of bias into the estimation of nucleotide substitution rates. The effects of this bias can easily be misconstrued as the action of purifying selection at synonymous sites.Chapter 3 is concerned with mutational variation in the murid genome. Nucleotide substitution rates in murid transposable elements were estimated. It was assumed that the majority of murid transposable elements were evolving neutrally and, therefore, that their molecular evolutionary rate was dictated by mutation alone. Under this assumption, variation in estimated element substitution rates reflects sampling and mutational variation only. The results indicate that greater mutational variation occurs along the length of a chromosome than between individual chromosomes, although the latter has been the primary focus in the literature. This result illustrates the importance of accounting for mutational variation in studies of selective constraint and sequence conservation.In Chapter 4, the level of constraint in intergenic DNA adjacent to coding sequences and a moderate distance inside first introns was estimated in a sample of 300 mouse-rat gene orthologues. The results suggested that whilst selective constraint in intergenic sequence adjacent to the start and stop codons is moderately high, this becomes statistically indistinguishable from zero within 4kb upstream/downstream of the first/last exon. Selective constraint in the 5' end of the first intron was also found to be moderately high. Taking the contributions from noncoding sequence into account, it was estimated that the number of deleterious mutations occurring in murid noncoding DNA was approximately equal to that in protein-coding sequence.Chapter 5 expands on the work done in Chapter 4. The assumption of neutral evolution in non-first introns was addressed by comparing their evolutionary rates with those in transposable elements. In addition the selective constraint in intergenic DNA immediately adjacent to genes with that found large distances from known genes was compared. The results showed that, when repetitive sequence is removed, the selective constraints in intergenic DNA are significantly different from zero. Furthermore, this constraint does not become indistinguishable from zero, even at large distances ( 50kb) from genic regions. The data also showed that a weak correlation between intron length and nucleotide substitution rate exists in murid non-first introns.

Characterization of the genetic structure of the azorean population

Tese de doutoramento em Bioquímica (Genética Molecular), apresentada à Universidade de Lisboa através da Faculdade de Ciências, 200