Nucleic acid (NA) purification from clinical samples is commonly achieved using silica solid phase extraction in the presence of a chaotropic salt. Versions of these protocols have been adapted for point of care (POC) diagnostic devices in miniaturized platforms. Most such protocols require a high net amount of input NA, which is often achieved by adding exogenous carrier NA to the clinical sample. As a result, for samples containing less than 1 μg of total NA, NA recovery is low in the absence of carrier NA. Clinical samples used in POC diagnostics may contain very low NA concentrations (~1 ng/ml), which result in NA-limited interactions with the solid phase that are outside the dynamic range of POC diagnostics. This work is a study of DNA-silica interactions in the DNA-limiting regime to gain fundamental understanding of the mechanisms at play in order to increase the dynamic range and sensitivity of miniaturized NA based POC diagnostics. DNA adsorption and recovery from silica surfaces for concentrations less than 1 μg/ml are studied. A protocol was designed and developed to systematically quantify the adsorption of DNA onto a silica surface and the amount of DNA recovered by elution at very low concentrations. Various adsorption conditions were examined including a range of pH, different chaotropes, and DNA concentrations down to 2.5 pg/ml. DNA recovery was further optimized for low concentration samples by varying elution buffers. DNA-silica adsorption was enhanced by low pH and was further improved by the presence of a chaotrope. Different adsorption conditions had little effect on DNA recovery using low salt, high pH elution buffers, but DNA recovery did exceed 40% when adsorbed initially with 5 M guanidinium thiocyanate at pH 5.2. Recovery was enhanced by eluting with 95 °C formamide or 1 M NaOH, supporting the hypothesis that DNA-silica interactions are dominated by hydrophobic forces and hydrogen bonding. While heated formamide and NaOH are non-ideal elution buffers for practical POC devices, these results are important for engineering a set of optimized reagents and conditions that could maximize DNA recovery from a microfluidic POC silica system. / 2017-06-21T00:00:00Z

A toxoplasmose é uma protozoose altamente prevalente que atinge pelo menos um bilhão de indivíduos no mundo. A infecção causada pelo Toxoplasma gondii é benigna e assintomática, mas pode causar perdas visuais ou morte em fetos e pacientes imunossuprimidos. Isto pode ser controlado com diagnóstico e instituição de tratamento, mas depende da determinação de infecção ativa ou recente. O diagnóstico parasitológico é complexo, demorado e só executado em poucos centros, sendo a sorologia específica essencial no diagnóstico da doença. A avidez de anticorpos IgG tem sido utilizada para determinação da infecção recente, porém os testes convencionais de avidez só permitem uma estimativa indireta destes anticorpos a partir dos anticorpos totais e os de alta avidez. A quantificação destes anticorpos de baixa avidez seria interessante devido aos altos títulos na fase aguda da infecção ou como marcadores da atividade da doença. Padronizamos um ensaio imunoenzimático (ELISA), utilizando o pH como agente caotrópico, para permitir a determinação e quantificação dos anticorpos de baixa avidez. Na padronização utilizamos amostras de soro de coelhos experimentalmente infectados ou amostras do banco de material biológico do Laboratório de Protozoologia do IMTSP. Nossos resultados mostraram que pH 3,5 apresentou poder caotrópico semelhante a uréia 6M (r2= 0,9909), e que nos soros experimentais, os anticorpos de alta avidez foram resistentes aos dois caotrópicos associados. Os anticorpos recuperados na eluição com pH 3.5 ou Uréia eram semelhantes quanto a especificidade antigênica por imunomarcação ou Western Blot. A neutralização do anticorpo eluído por pH permitiu seu reensaio por ELISA após 1 hora de renaturação, com a quantificação direta dos anticorpos de baixa avidez.. A reprodutibilidade intra e inter teste foi superior a 95%, embora com resultados piores para o pH 3,5. Uma vez padronizada a reação, foram analisadas 150 amostras de soros humanos com sorologia e avidez conhecidas, composta por grande maioria de soros de alta avidez. As medidas de avidez por porcentagem mostraram um resultado errático, atribuído ao uso de grande maioria de anticorpos de alta avidez, embora a medida dos anticorpos recuperados mantivesse correlação com estimativa a partir da medida indireta (r2= 0.48). Esta abordagem permite a determinação direta dos anticorpos de baixa avidez, que são os anticorpos inicialmente produzidos em um desafio antigênico. Nosso ensaio é semelhante ao imunológico, já que a apresentação de antígenos por exossomos ácidos de células dendríticas foliculares no centro germinativo parece ser o sistema de seleção de clones produtores de anticorpos de alta avidez. As perspectivas futuras de uso da medida dos anticorpos de baixa avidez na toxoplasmose são imensas, desde a relação com a gravidade da doença, pela sua quantidade, ou da presença de infecção recente, principalmente em infecção congênita ou e em imunossuprimidos, ou a reatividade da doença crônica, como na toxoplasmose ocular. / Toxoplasmosis is a highly prevalent protozoosis, affecting at least one billion people worldwide. The infection caused by Toxoplasma gondii is asymptomatic and benign but it can cause visual losses in addition to death in fetuses and immunocompromised patients. The agent can be controlled by early diagnosis and treatment, but this therapy depends on the determination of active or recent infection. The parasitological diagnosis is complex, time consuming and only performed in few centers, so specific serology is essential for diagnosis. The IgG avidity tests has been used to determine recent infection, but avidity conventional tests only provide an indirect estimate of low avidity antibodies from the total and high avidity antibodies. The quantification of low avidity antibodies would be interesting due to high titers in the acute phase of infection or as markers of disease activity. Using reversible chaotrope such as pH, we standardized an enzyme immunoassay (ELISA) to allow the determination and quantification of low avidity antibodies. For standardization we used serum samples from experimentally infected rabbits or samples of biological material bank of the Laboratory of Protozoology, IMTSP. Our results showed that pH 3.5 is a chaotrope similar to 6M urea (r2 = 0.9909) in avidity ELISA, and high avidity antibodies had similar resistance to two associated chaotrope in experimental sera. The antibodies recovered on elution with pH 3.5 or urea had similar antigen specificity by immunostaining or Western blot. The neutralizing antibody eluted by pH allowed retest by ELISA after 1 hour of refolding, with direct quantification of antibodies of low avidity. The reproducibility inter and intra test were above 95%, but with worse results for pH 3.5. After standardization, we analyzed 150 samples of human sera with known serology and avidity, composed by a large majority of high avidity samples. Avidity as percent of high avidity antibodies showed erratic results in chaotrope comparison, attributed to the majority of high avidity samples, although the direct measure of low avidity IgG kept correlation with the indirect estimate (r2 = 0.48). This approach allows the direct determination of low avidity antibodies that are early produced in an antigen challenge. Our test is similar to the biology of antibody selection, since antigen presentation by acid exosomes of follicular dendritic cells in germinal center seems to be the system of selection of clones that produce high avidity antibodies. The prospective use of the quantification of low avidity antibodies in toxoplasmosis are attractive, either by the quantitative relationship with the severity of the disease; or the increased presence in recent infections, especially in congenital infection and in immunosuppressed patients, or their relative increase in reactivated chronic disease, such as ocular toxoplasmosis.

A toxoplasmose é uma protozoose altamente prevalente que atinge pelo menos um bilhão de indivíduos no mundo. A infecção causada pelo Toxoplasma gondii é benigna e assintomática, mas pode causar perdas visuais ou morte em fetos e pacientes imunossuprimidos. Isto pode ser controlado com diagnóstico e instituição de tratamento, mas depende da determinação de infecção ativa ou recente. O diagnóstico parasitológico é complexo, demorado e só executado em poucos centros, sendo a sorologia específica essencial no diagnóstico da doença. A avidez de anticorpos IgG tem sido utilizada para determinação da infecção recente, porém os testes convencionais de avidez só permitem uma estimativa indireta destes anticorpos a partir dos anticorpos totais e os de alta avidez. A quantificação destes anticorpos de baixa avidez seria interessante devido aos altos títulos na fase aguda da infecção ou como marcadores da atividade da doença. Padronizamos um ensaio imunoenzimático (ELISA), utilizando o pH como agente caotrópico, para permitir a determinação e quantificação dos anticorpos de baixa avidez. Na padronização utilizamos amostras de soro de coelhos experimentalmente infectados ou amostras do banco de material biológico do Laboratório de Protozoologia do IMTSP. Nossos resultados mostraram que pH 3,5 apresentou poder caotrópico semelhante a uréia 6M (r2= 0,9909), e que nos soros experimentais, os anticorpos de alta avidez foram resistentes aos dois caotrópicos associados. Os anticorpos recuperados na eluição com pH 3.5 ou Uréia eram semelhantes quanto a especificidade antigênica por imunomarcação ou Western Blot. A neutralização do anticorpo eluído por pH permitiu seu reensaio por ELISA após 1 hora de renaturação, com a quantificação direta dos anticorpos de baixa avidez.. A reprodutibilidade intra e inter teste foi superior a 95%, embora com resultados piores para o pH 3,5. Uma vez padronizada a reação, foram analisadas 150 amostras de soros humanos com sorologia e avidez conhecidas, composta por grande maioria de soros de alta avidez. As medidas de avidez por porcentagem mostraram um resultado errático, atribuído ao uso de grande maioria de anticorpos de alta avidez, embora a medida dos anticorpos recuperados mantivesse correlação com estimativa a partir da medida indireta (r2= 0.48). Esta abordagem permite a determinação direta dos anticorpos de baixa avidez, que são os anticorpos inicialmente produzidos em um desafio antigênico. Nosso ensaio é semelhante ao imunológico, já que a apresentação de antígenos por exossomos ácidos de células dendríticas foliculares no centro germinativo parece ser o sistema de seleção de clones produtores de anticorpos de alta avidez. As perspectivas futuras de uso da medida dos anticorpos de baixa avidez na toxoplasmose são imensas, desde a relação com a gravidade da doença, pela sua quantidade, ou da presença de infecção recente, principalmente em infecção congênita ou e em imunossuprimidos, ou a reatividade da doença crônica, como na toxoplasmose ocular. / Toxoplasmosis is a highly prevalent protozoosis, affecting at least one billion people worldwide. The infection caused by Toxoplasma gondii is asymptomatic and benign but it can cause visual losses in addition to death in fetuses and immunocompromised patients. The agent can be controlled by early diagnosis and treatment, but this therapy depends on the determination of active or recent infection. The parasitological diagnosis is complex, time consuming and only performed in few centers, so specific serology is essential for diagnosis. The IgG avidity tests has been used to determine recent infection, but avidity conventional tests only provide an indirect estimate of low avidity antibodies from the total and high avidity antibodies. The quantification of low avidity antibodies would be interesting due to high titers in the acute phase of infection or as markers of disease activity. Using reversible chaotrope such as pH, we standardized an enzyme immunoassay (ELISA) to allow the determination and quantification of low avidity antibodies. For standardization we used serum samples from experimentally infected rabbits or samples of biological material bank of the Laboratory of Protozoology, IMTSP. Our results showed that pH 3.5 is a chaotrope similar to 6M urea (r2 = 0.9909) in avidity ELISA, and high avidity antibodies had similar resistance to two associated chaotrope in experimental sera. The antibodies recovered on elution with pH 3.5 or urea had similar antigen specificity by immunostaining or Western blot. The neutralizing antibody eluted by pH allowed retest by ELISA after 1 hour of refolding, with direct quantification of antibodies of low avidity. The reproducibility inter and intra test were above 95%, but with worse results for pH 3.5. After standardization, we analyzed 150 samples of human sera with known serology and avidity, composed by a large majority of high avidity samples. Avidity as percent of high avidity antibodies showed erratic results in chaotrope comparison, attributed to the majority of high avidity samples, although the direct measure of low avidity IgG kept correlation with the indirect estimate (r2 = 0.48). This approach allows the direct determination of low avidity antibodies that are early produced in an antigen challenge. Our test is similar to the biology of antibody selection, since antigen presentation by acid exosomes of follicular dendritic cells in germinal center seems to be the system of selection of clones that produce high avidity antibodies. The prospective use of the quantification of low avidity antibodies in toxoplasmosis are attractive, either by the quantitative relationship with the severity of the disease; or the increased presence in recent infections, especially in congenital infection and in immunosuppressed patients, or their relative increase in reactivated chronic disease, such as ocular toxoplasmosis.

<p>Smart windows that modulate solar radiation by changing their optical state in response to temperature stimulus are developing as promising solutions towards reducing the energy consumption of buildings. The market adoption of such systems has been slow due to the barriers in scalability, cost, as well as complexity in their integration into existing systems. Aiming these features, we have proposed a retrofit smart window design based on the temperature-responsive polymer Methyl Cellulose (MC). The system utilizes a sustainable, earth abundant and cost-effective cellulose based thermo-responsive material to transform existing windows to a thermally dynamic smart window system. The observed optical change of MC from transparent to opaque state is dependent on temperature and is triggered by the thermodynamic mechanism of reversible coil-globule transition, which results in a stable performance of the proposed device. Its solar modulation ability was studied using ultraviolet-visible- spectroscopy. Effect of MC concentration and various salts on the optical performance were investigated. It was found that the transition temperature the polymer can be tuned by varying MC concentration and by adding salts to the system. The tunability of transition temperature is a function of the concentration of salt and the type of anion in the salt. It was observed that the transition temperature of the window can be tuned between to , allowing a wide range of control over switching temperature. Controllable LCST, low freezing point, sustainable base material, scalable production, low cost, retrofit system makes them ideal candidates for smart window applications. </p>

In dieser Arbeit werden systematisch die bisher unerforschten grundlegenden physikalischen und chemischen Eigenschaften von DNA-Origami untersucht, die die Stabilität dieser aus doppelsträngiger DNA aufgebauten nanoskopischen Suprastrukturen bestimmen. In Analogie zu den zahlreichen Studien, die sich mit der Stabilität von Proteinen durch kontrollierte Denaturierung beschäftigen, spielen auch in dieser Arbeit die Denaturierungsbedingungen eine zentrale Rolle. Unter Verwendung von Guanidinium (Gdm+) als teilweise DNA-stabilisierendes, aber auch potentiell denaturierendes Kation steht dessen Wirkung auf DNA-Origami-Dreiecke im Mittelpunkt der Untersuchungen, wobei insbesondere die unerwartete Modulation der nanoskopischen Schädigung von DNA-Origami durch die begleitenden Gegenanionen zu Gdm+ im Vordergrund steht. Die Experimente zielen darauf ab, atomistische, molekulare, nanoskopische und thermodynamische Eigenschaften von DNA-Origami zu korrelieren und zu klären, wie diese vom Design des DNA-Origami selbst abhängen können. Die Ergebnisse zeigen einen unerwarteten Zusammenhang zwischen den spezifischen Gegenanionen des Denaturierungsmittels und der Stabilität der DNA-Origami-Dreiecke: Sulfat wirkt stabilisierend, während Chlorid die Superstruktur bereits unterhalb der globalen Schmelztemperatur destabilisiert. Statistische Analysen von Rasterkraftmikroskop (AFM)-Bildern und Zirkulardichroismus (CD)-Spektren zeigen Strukturübergänge auf nano-skopischer bzw. molekularer Ebene. Werden diese Techniken mit thermischer Denaturierung in Gegenwart von schwacher bis starker chemischer Denaturierung kombiniert, so zeigt sich, dass Änderungen der Wärmekapazität (ΔCp) während der strukturellen Veränderungen der DNA-Originale eine Schlüsselrolle bei der Bestimmung ihrer Empfindlichkeit gegenüber Temperatur und Denaturierungsmitteln spielen. Die Daten deuten darauf hin, dass Wasser auf apolaren DNA-Origami-Oberflächen der molekulare Ursprung der abgeleiteten Wärme-kapazitätsänderungen ist. Diese Hypothese wird durch Molekulardynamik-Simulationen (MD) unterstützt, die die Modulation von ΔCp durch die Hydratationshüllen der Anionen zeigen. Ihr unterschiedliches Potential, stabile Ionenpaare mit Gdm+ in konzentrierten Salzlösungen zu bilden, kann die experimentell beobachteten Variationen der strukturellen Stabilität erklären. Die Kopplung von strukturellen Übergängen an ΔCp wird somit als Schlüsselfaktor für die Destabilisierung von DNA-Origami sowohl bei höheren als auch bei niedrigeren Temperaturen identifiziert. Darüber hinaus weisen DNA-Origami nicht nur diese Eigenschaft auf, sondern ermöglichen auch die Beobachtung von kalten Denaturierungsprozessen auf nanoskopischer Ebene, bei denen kälteinduzierte Spannungen innerhalb der Superstruktur bei einem Bruch an vorherbestimmten lokalen Stellen freigesetzt werden, die in AFM-Bildern sichtbar sind. Dies ist die erste Beobachtung der kälteinduzierten Denaturierung von Nukleinsäuren bei Temperaturen über 0 °C sowie von DNA-basierten Superstrukturen. In dieser Arbeit wird die strukturelle Stabilität von sechs verschiedenen 2D- und 3D-DNA-Origami-Nanostrukturen in unterschiedlichen chemischen Umgebungen untersucht. Drei chaotrope Salze - Guanidiniumsulfat (Gdm2SO4), Guanidiniumchlorid (GdmCl) und Tetrapropylammoniumchlorid (TPACl) - werden als Denaturierungsmittel verwendet. Mittels Rasterkraftmikroskopie wird die Integrität der Nanostrukturen quantifiziert, wobei sich Gdm2SO4 als das schwächste und TPACl als das stärkste Denaturierungsmittel für DNA-Origami erweist, was sich auch in den Schmelztemperaturen widerspiegelt. Die Abhängigkeit der DNA-Origami-Stabilität von der Superstruktur wird besonders bei 3D-Nanostrukturen deutlich. Hier zeigen mechanisch flexible Designs sowohl in GdmCl als auch in TPACl eine höhere Stabilität als ihre starren Gegenstücke. Die Abhängigkeit der DNA-Origami-Stabilität von der Superstruktur wird besonders in 3D-Nanostrukturen deutlich, in denen mechanisch flexible Strukturen sowohl in GdmCl als auch in TPACl eine höhere Stabilität aufweisen als ihre steifen Gegenstücke. Dies begünstigt die Bildung von intramolekularen Verformungen, die sich entweder in 'weichen' Architekturen über die gesamte Superstruktur verteilen oder in ansonsten 'steifen' Strukturen in den weniger stabilen Regionen konzentrieren.:Table of contents Questions addressed in this thesis .................................................................................... I Abstract ................................................................................................................................ I Englisch .................................................................................................................................................. I Deutsch .................................................................................................................................................. II Acronyms ........................................................................................................................... III Substances .......................................................................................................................................... IV Physical and Chemical abbreviations ............................................................................................... IV Mathematical abbreviations ................................................................................................................ V 1 Introduction ................................................................................................................. 1 1.1 Deoxyribonucleic acid ................................................................................................................. 1 1.1.1 The structure of DNA ............................................................................................................ 1 1.1.2 Hydrogen bonds .................................................................................................................... 1 1.1.3 Base stacking ........................................................................................................................ 2 1.1.4 Water DNA interactions: A complex dance of stability and dynamics .................................. 5 1.1.5 The effect of ionic strength on DNA conformation ................................................................ 9 1.1.6 Conformational changes ....................................................................................................... 9 1.1.7 Forms of DNA ..................................................................................................................... 10 1.1.8 The role of apolar groups in DNA unfolding ........................................................................ 13 1.1.9 Energetics of DNA structural transitions ............................................................................. 14 1.1.10 Melting temperature ............................................................................................................ 15 1.2 Hofmeister series ....................................................................................................................... 17 1.2.1 Probing the Hofmeister series: Salt effects biomolecules ................................................... 17 1.2.2 Specific ion effects in electrolyte solutions .......................................................................... 19 1.3 DNA nanostructures................................................................................................................... 20 1.3.1 DNA origami ........................................................................................................................ 22 1.3.2 Challenges in DNA origami stability .................................................................................... 26 1.3.3 DNA origami in single molecule studies .............................................................................. 27 1.4 Circular dichroism ...................................................................................................................... 28 1.4.1 Circular dichroism spectroscopy for analyzing DNA conformations ................................... 30 1.4.2 Wavelength-dependent spectroscopic signatures of DNA conformation ........................... 32 1.5 Atomic force microscopy .......................................................................................................... 34 1.6 2D correlation spectroscopy ..................................................................................................... 36 1.6.1 2D correlation spectroscopy: Synchronous and asynchronous spectra analysis ............... 39 1.6.2 Perturbation-correlation moving-window 2D correlation spectroscopy ............................... 40 1.7 Multivariate analysis of spectral data using PCA and ITTFA ................................................ 41 1.8 Cold denaturation ....................................................................................................................... 42 2 Results and Discussion ............................................................................................ 44 2.1 Cold denaturation of the Rothemund DNA origami triangle .................................................. 45 2.2 Heat denaturation of the Rothemund DNA origami triangle .................................................. 50 2.2.1 Investigations by atomic force microscopy ......................................................................... 51 2.2.2 Circular dichroism spectroscopy and thermodynamic modelling ........................................ 56 2.2.3 Divergent effects of Cl- and SO42- on DNA origami stability ................................................ 62 2.3 Magnesium concentration modulation of DNA Origami heat denaturation ......................... 65 2.4 Assessing DNA origami stability in different chaotropic environments .............................. 66 2.4.1 DNA origami integrity influenced by Gdm2SO4 ................................................................... 67 2.4.2 DNA origami integrity influenced by GdmCl ........................................................................ 73 2.4.3 DNA origami integrity influenced by TPACl ........................................................................ 75 2.4.4 Quantitative comparison ..................................................................................................... 77 3 Critics ......................................................................................................................... 78 4 Conclusion ................................................................................................................. 79 5 Outlook ...................................................................................................................... 81 6 Material and Methods ................................................................................................ 82 6.1 DNA origami synthesis .............................................................................................................. 82 6.2 Sample preparation and AFM imaging ..................................................................................... 82 6.2.1 Anion-specific structure and stability of guanidinium-bound DNA origami & Cold denaturation of DNA origami nanostructures ...................................................................... 82 6.2.2 Superstructure-dependent stability of DNA origami nanostructures in the presence of chaotropic denaturants ........................................................................................................ 83 6.2.3 Cold denaturation of DNA origami nanostructures ............................................................. 83 6.3 CD spectroscopy and analysis ................................................................................................. 84 6.3.1 Anion-specific structure and stability of guanidinium-bound DNA origami ......................... 84 6.3.2 Pre-treatment of the CD data and calculation of melting temperatures .............................. 84 6.3.3 Cold denaturation of DNA origami nanostructures ............................................................. 84 6.3.4 Superstructure-dependent stability of DNA origami nanostructures in the presence of chaotropic denaturants ........................................................................................................ 84 6.4 Principal component analysis and iterative target test factor analysis ............................... 85 6.5 Thermodynamic modelling ........................................................................................................ 85 6.6 Molecular dynamics modelling ................................................................................................. 85 Appendix ........................................................................................................................... 88 Acknowledgment ............................................................................................................ 100 Bibliography .................................................................................................................... 101 List of Figures ................................................................................................................. 116 List of Tables ................................................................................................................... 118 Declaration of independence – Selbstständigkeitserklärung ...................................... 119 / This thesis undertakes the systematic study of hitherto unexplored fundamental physical and chemical properties of DNA origami that determine the stability of these designed nanoscopic superstructural assemblies of double-stranded DNA. In analogy to the vast number of studies addressing protein stability by controlled denaturation, denaturing conditions play a central role in this thesis as well. Using guanidinium (Gdm+) as a partly DNA-stabilizing but also potentially denaturing cation, its effect on DNA origami triangles is central to the study which particularly addressed the unexpected modulation of nanoscopic damage of DNA origami by the accompanying counter-anions to Gdm+. The experiments aim at correlating atomistic, molecular, nanoscopic and thermodynamic properties of DNA origami and at elucidating how these may depend on the DNA origami design itself. The results demonstrate an unexpected relationship between the specific counter-anions of the denaturant and the stability of DNA origami triangles: sulphate exhibits stabilizing effects and chloride induces destabilization of the superstructure already below the global melting temperature. Statistical analyses of both atomic force microscopy (AFM) images and circular dichroism (CD) spectra reveal structural transitions at the nanoscopic and molecular level, respectively. Combining these techniques with thermal denaturation in the presence of mild to strong chemical denaturation, changes in heat capacity (ΔCp) during DNA origami structural changes are shown to play the key role in determining their sensitivity to temperature and denaturants. The data suggest that water at apolar DNA origami surfaces is the molecular origin of the derived heat capacity changes. This hypothesis is substantiated by Molecular Dynamics (MD) simulations which shed light on the modulation of ΔCp by the hydration shells of anions. Their different potential to form stable ion pairs with Gdm+ in concentrated salt solutions can explain the experimentally observed variations of structural stability. The coupling of structural transitions to ΔCp is thus identified as a key factor in the destabilization of DNA origami at both elevated and lowered temperatures. Furthermore, DNA origami not only exhibit this property, but also enable the observation of cold denaturation processes at the nanoscopic level, where cold-induced strain within the superstructure is released upon breakage at predisposed local sites, visible in AFM images. This is the first observation of cold-induced denaturation of nucleic acids at temperatures above 0 °C, as well of DNA-based superstructures. Extending the scope, the work evaluates the structural stability of six different 2D and 3D DNA origami nanostructures in different chemical environments. Three chaotropic salts - guanidinium sulfate (Gdm2SO4), guanidinium chloride (GdmCl), and tetrapropylammonium chloride (TPACl) - are used as denaturants. Atomic force microscopy quantifies the nanostructural integrity, revealing Gdm2SO4 as the weakest and TPACl as the strongest DNA origami denaturant, which is also reflected in the melting temperatures. The dependence of DNA origami stability on its superstructure is particularly evident in 3D nanostructures, where mechanically flexible designs exhibit higher stability in both GdmCl and TPACl than rigid counterparts. This supports the buildup of intramolecular strain, which becomes either partitioned among the entire superstructure in “soft” architectures or accumulates at the least stable regions in otherwise “rigid” designs.:Table of contents Questions addressed in this thesis .................................................................................... I Abstract ................................................................................................................................ I Englisch .................................................................................................................................................. I Deutsch .................................................................................................................................................. II Acronyms ........................................................................................................................... III Substances .......................................................................................................................................... IV Physical and Chemical abbreviations ............................................................................................... IV Mathematical abbreviations ................................................................................................................ V 1 Introduction ................................................................................................................. 1 1.1 Deoxyribonucleic acid ................................................................................................................. 1 1.1.1 The structure of DNA ............................................................................................................ 1 1.1.2 Hydrogen bonds .................................................................................................................... 1 1.1.3 Base stacking ........................................................................................................................ 2 1.1.4 Water DNA interactions: A complex dance of stability and dynamics .................................. 5 1.1.5 The effect of ionic strength on DNA conformation ................................................................ 9 1.1.6 Conformational changes ....................................................................................................... 9 1.1.7 Forms of DNA ..................................................................................................................... 10 1.1.8 The role of apolar groups in DNA unfolding ........................................................................ 13 1.1.9 Energetics of DNA structural transitions ............................................................................. 14 1.1.10 Melting temperature ............................................................................................................ 15 1.2 Hofmeister series ....................................................................................................................... 17 1.2.1 Probing the Hofmeister series: Salt effects biomolecules ................................................... 17 1.2.2 Specific ion effects in electrolyte solutions .......................................................................... 19 1.3 DNA nanostructures................................................................................................................... 20 1.3.1 DNA origami ........................................................................................................................ 22 1.3.2 Challenges in DNA origami stability .................................................................................... 26 1.3.3 DNA origami in single molecule studies .............................................................................. 27 1.4 Circular dichroism ...................................................................................................................... 28 1.4.1 Circular dichroism spectroscopy for analyzing DNA conformations ................................... 30 1.4.2 Wavelength-dependent spectroscopic signatures of DNA conformation ........................... 32 1.5 Atomic force microscopy .......................................................................................................... 34 1.6 2D correlation spectroscopy ..................................................................................................... 36 1.6.1 2D correlation spectroscopy: Synchronous and asynchronous spectra analysis ............... 39 1.6.2 Perturbation-correlation moving-window 2D correlation spectroscopy ............................... 40 1.7 Multivariate analysis of spectral data using PCA and ITTFA ................................................ 41 1.8 Cold denaturation ....................................................................................................................... 42 2 Results and Discussion ............................................................................................ 44 2.1 Cold denaturation of the Rothemund DNA origami triangle .................................................. 45 2.2 Heat denaturation of the Rothemund DNA origami triangle .................................................. 50 2.2.1 Investigations by atomic force microscopy ......................................................................... 51 2.2.2 Circular dichroism spectroscopy and thermodynamic modelling ........................................ 56 2.2.3 Divergent effects of Cl- and SO42- on DNA origami stability ................................................ 62 2.3 Magnesium concentration modulation of DNA Origami heat denaturation ......................... 65 2.4 Assessing DNA origami stability in different chaotropic environments .............................. 66 2.4.1 DNA origami integrity influenced by Gdm2SO4 ................................................................... 67 2.4.2 DNA origami integrity influenced by GdmCl ........................................................................ 73 2.4.3 DNA origami integrity influenced by TPACl ........................................................................ 75 2.4.4 Quantitative comparison ..................................................................................................... 77 3 Critics ......................................................................................................................... 78 4 Conclusion ................................................................................................................. 79 5 Outlook ...................................................................................................................... 81 6 Material and Methods ................................................................................................ 82 6.1 DNA origami synthesis .............................................................................................................. 82 6.2 Sample preparation and AFM imaging ..................................................................................... 82 6.2.1 Anion-specific structure and stability of guanidinium-bound DNA origami & Cold denaturation of DNA origami nanostructures ...................................................................... 82 6.2.2 Superstructure-dependent stability of DNA origami nanostructures in the presence of chaotropic denaturants ........................................................................................................ 83 6.2.3 Cold denaturation of DNA origami nanostructures ............................................................. 83 6.3 CD spectroscopy and analysis ................................................................................................. 84 6.3.1 Anion-specific structure and stability of guanidinium-bound DNA origami ......................... 84 6.3.2 Pre-treatment of the CD data and calculation of melting temperatures .............................. 84 6.3.3 Cold denaturation of DNA origami nanostructures ............................................................. 84 6.3.4 Superstructure-dependent stability of DNA origami nanostructures in the presence of chaotropic denaturants ........................................................................................................ 84 6.4 Principal component analysis and iterative target test factor analysis ............................... 85 6.5 Thermodynamic modelling ........................................................................................................ 85 6.6 Molecular dynamics modelling ................................................................................................. 85 Appendix ........................................................................................................................... 88 Acknowledgment ............................................................................................................ 100 Bibliography .................................................................................................................... 101 List of Figures ................................................................................................................. 116 List of Tables ................................................................................................................... 118 Declaration of independence – Selbstständigkeitserklärung ...................................... 119