Understanding the interaction between pDNA and different chromatographic supports

Gene Therapy and DNA vaccines are recent therapies that take advantage of the potential of plasmid DNA as a therapeutic molecule for the treatment and cure of several diseases. In the last decade both techniques have received a great deal of attention by pharmaceutical companies due to their simplicity, versatility and safe profile. Hence, the usage of pDNA as biopharmaceutical molecule requires its production at the gram scale, with a high purity and homogeneity level as a vital parameter to ensure a good response and the patient safety. Anion-exchange chromatography and hydrophobic interaction chromatography have been successfully used in pDNA purification. Nevertheless, the mechanism of pDNA separation for both purification techniques is still not completely understood. A better understanding of the driving forces and mechanisms underlying both purification processes are of great interest to help optimize chromatographic systems. Flow Microcalorimetry (FMC) has proven its ability to provide an improved understanding of the driving forces, mechanisms and kinetics involved in the interaction process during biomolecules adsorption onto several chromatographic systems]. Thus, using Flow Microcalorimetry as a central technique, this study aims to understand and compare the interaction between different pDNA isoforms (pVAX1-LacZ) and the anion-exchange support (Q-sepharose Fast Flow) or the hydrophobic support (Phenyl Sepharose 6 Fast Flow), considering only the isotherm linear conditions, showing the role of nonspecific effects on the adsorptive process. The results obtained in the binding capacity studies revealed that the ln pDNA adsorption process follows a Langmuir isotherm up to a specific ln pDNA equilibrium concentration. In all cases, the binding capacity increases with the plasmid concentration in equilibrium until it reaches a level at which saturation of the chromatographic medium is achieved. The anion-exchange support was found to have a higher binding capacity for ln pDNA adsorption than the hydrophobic interaction support, as expected. FMC results reveled that for both processes the endothermic heat major contributor was suggested to be the desolvation and changes in the solvation shell processes while exothermic heats were related to the interaction (electrostatic attraction) between pDNA and support and also to the secondary adsorption of already adsorbed pDNA molecules. The enthalpies of adsorption showed that the overall adsorption process is mainly enthalpically driven for the adsorption of ln pDNA onto Phenyl Sepharose and entropically driven for the sc pDNA-Q-sepharose system.A terapia génica e as vacinas de DNA são terapias recentes que aproveitam o potencial do DNA plasmídeo (pDNA) como uma molécula terapêutica para o tratamento e cura de muitas doenças. Na última década, as empresas farmacêuticas deram muita atenção a ambas as técnicas devido à sua simplicidade, versatilidade e segurança. O uso do pDNA como uma molécula com um propósito farmacêutico requer a sua produção em grande escala, com um alto grau de pureza e homogeneidade. Estes parâmetros são fundamentais para garantir uma boa resposta e a segurança do paciente. São utilizados vários processos na purificação de plasmídeos, tanto na eliminação de impurezas como restos celulares, proteínas, DNA genómico, etc., como para separar as diferentes isoformas em que um plasmídeo se pode encontrar (linear, circular aberto ou superenrolado). As propriedades únicas do pDNA fazem com que a sua purificação seja a parte mais complicada de todo o processo de fabrico. A cromatografia de troca aniónica e de interação hidrofóbica são as técnicas utilizadas com mais sucesso na purificação de plasmídeos. Contudo, o mecanismo de separação de plasmídeos por estas técnicas não é totalmente compreendido. É difícil prever o comportamento das moléculas durante a separação, e ainda, os métodos usados na escala laboratorial são economicamente e ambientalmente incompatíveis com a escala industrial. Assim sendo, uma melhor compreensão dos mecanismos subjacentes à cromatografia terá um elevado interesse prático. A análise dos eventos termodinâmicos dos processos de adsorção através da Microcalorimetria de Fluxo (FMC) tem vindo a provar a sua boa utilidade na obtenção de uma melhor compreensão das forças motrizes, dos mecanismos e das cinéticas envolvidas no processo de adsorção de biomoléculas em diferentes sistemas cromatográficos. Por isso, neste trabalho, através da utilização da microcalorimetria de fluxo, pretende-se compreender e comparar o mecanismo de adsorção de um plasmídeo na sua forma linear (ln pDNA) a um suporte cromatográfico de troca aniónica (Q-sepharose Fast Flow) e a um suporte cromatográfico de interação hidrofóbica (Phenyl-sepharose 6 Fast Flow), e ainda a adsorção de um plasmídeo na sua forma linear e superenrolada (sc pDNA) a um suporte cromatográfico de troca aniónica (Q-sepharose Fast Flow). Os resultados serão analisados dando enfase às diferenças entre cada sistema. Para além da microcalorimetria de fluxo, foram também realizados estudos da capacidade de ligação em modo estático (static binding capacity) para obter uma melhor compreensão do mecanismo de adsorção. Os resultados obtidos nos estudos de capacidade de ligação revelaram que o processo de adsorção do plasmídeo na forma linear segue uma isotérmica de Langmuir até uma certa concentração de plasmídeo em equilíbrio. Em todos os casos testados, a capacidade de ligação do suporte aumenta com o aumento da concentração de plasmídeo em equilíbrio até se atingir um patamar em que a saturação do suporte cromatográfico é alcançada. O suporte de troca iónica revelou ter uma capacidade de ligação maior para o ln pDNA do que o suporte de interação hidrofóbica, como esperado. Não foi possível fazer os mesmos testes com o sc pDNA dado a sua instabilidade nas condições em que os testes são realizados. Não obstante, conseguiu-se de forma teórica chegar a uma previsão da capacidade máxima de ligação do sc pDNA ao suporte de troca aniónica que revelou ser inferior à capacidade teórica atingida na adsorção de ln pDNA. Os estudos realizados utilizando a microcalorimetria de fluxo foram efetuados na zona linear das isotérmicas. Dois modos diferentes de injeção foram utilizados através da introdução de um pulso de amostra na célula do microcalorímetro ou através da alimentação contínua de amostra à célula. Estes modos de injeção foram conseguidos usando loops de diferentes volumes. Considerando os resultados do FMC obtidos com a adsorção do ln pDNA à Phenyl Sepharose, todos os termogramas são compostos por um pico endotérmico seguido de um ou dois picos exotérmicos, dependendo do modo de injeção, e um último pico endotérmico. Os termogramas foram deconvoluidos e as áreas e o timing dos picos considerados na comparação com o processo de adsorção do ln pDNA a Q-Sepharose. O primeiro pico endotérmico relaciona-se com o processo de desolvatação. O primeiro pico exotérmico está associado à interação entre o ln pDNA e o suporte. Visto que este apresenta valores energéticos similares aos da interação do ln pDNA com a Q-Sepharose, verificou-se que a Phenyl Sepharose também interage electrostaticamente com o DNA plasmídeo através de interações anião-?? Utilizando a alimentação contínua da coluna (loop de 430 µL), encontraram-se diferenças não só na magnitude e no timing de cada sinal, como também no perfil do termograma. A presença de diferentes picos num termograma indica a existência de diferentes eventos durante o processo de adsorção. O segundo pico exotérmico, encontrado apenas utilizando o modo de alimentação contínua, está relacionado com adsorção secundária. Este processo também acontece na adsorção do ln pDNA à Q-Sepharose. Considerou-se que o último pico endotérmico estivesse relacionado com a reorganização das moléculas de água e iões na interface do ln pDNA com a solução. Acredita-se que este processo seja causado pela alta concentração de sulfato de amónio na solução. As experiencias realizadas no FMC com sc pDNA e Q-Sepharose resultaram em termogramas compostos por um pico endotérmico seguido de um exotérmico. O pico endotérmico obtido com o loop de 430 µL foi deconvoluidos, posteriormente, em dois sinais endotérmicos. Tal como no caso anterior, as intensidades, áreas e o timing dos sinais foram considerados na comparação com a adsorção do ln pDNA à Q-Sepharose. Acredita-se que o primeiro sinal endotérmico esteja relacionado com processo de desolvatação, tal como na adsorção do ln pDNA. Contudo, verificou-se que o processo é mais energético no caso do sc pDNA dado que este tem uma maior densidade de carga que é conferida pela isoforma superenrola, conduzindo a um maior gasto energético na desolvatação. Ao utilizar o loop de 430 µL conseguiu-se promover uma sobrecarga no volume de amostra que passa na coluna. Este método resultou no aparecimento de um segundo sinal endotérmico. Este sinal relaciona-se com as repulsões entre as moléculas de sc pDNA livres na solução. Por último, a área e o timing do pico exotérmico leva-nos a acreditar que este está relacionado com a reorientação da molécula e com adsorção secundária. As entalpias de adsorção revelaram que o processo global de adsorção é exotérmico (perto de zero) na adsorção do ln pDNA à Phenyl Sepharose. Alguns estudos mostram que os processos cromatográficos que envolvem interações hidrofóbicas são fortemente influenciados pela variação da entropia, e são muitas vezes considerados maioritariamente endotérmicos. Neste caso particular, não temos um processo só com interações hidrofóbicas o que explica a divergência dos resultados. Na adsorção do sc pDNA à Q-Sepharose, o processo global é conduzido entropicamente visto que este é endotérmico para todos os casos estudados, este resultado é similar aos obtidos anteriormente com o estudo da adsorção pela análise de van´t Hoff [24]

Plasmonic DNA: Towards Genetic Diagnosis Chips

"Plasmonics and DNA" special issueInternational audienc

RNA structure prediction using high-throughput chemical modification techniques

Functional RNA molecules require the formation of defined structures in order to perform their critical tasks in biology. Complete understanding of this structure-function relationship in RNA requires the elucidation of accurate RNA structural models. RNA chemical modification has proven to be an invaluable tool in the characterization of RNA structure. Recently, the throughput of RNA chemical modification approaches has increased significantly through the adaptation of chemical modification techniques to next-generation sequencing platforms. In this work, I create several new methodologies for the generation of accurate RNA structural models based on high-throughput RNA chemical modification analysis. First, I create a general methodology for predicting three-dimensional RNA structures based on RNA interactions implicated by biochemical and bioinformatic approaches. In this work, I develop a three-dimensional model for the hepatitis C virus internal ribosome entry site (HCV IRES) pseudoknot domain. This methodology is then applied to a new high-throughput chemical modification approach called RING-MaP (RNA interaction groups identified by mutational profiling). Implicated interactions from RING-MaP analysis allow for accurate prediction of RNA tertiary folds. Second, I create an algorithm for the comparison of high-throughput chemical modification data from related RNA sequences. Using SHAPE chemical modification alone, this approach allows recapitulation of ribosomal RNA alignments made using sequence identity. Chemical modification data for three HIV-related viral RNA genomes are then compared. Following creation of chemical modification-dependent alignments, statistically related RNA structures are found across the three viral genomes. Consensus secondary structures considering both chemical modification data and covariation are then made, recapitulating all known RNA structures in the HIV genome and suggesting previously undescribed functional elements.Doctor of Philosoph

Computational Modeling of Protein Kinases: Molecular Basis for Inhibition and Catalysis

Protein kinases catalyze protein phosphorylation reactions, i.e. the transfer of the γ-phosphoryl group of ATP to tyrosine, serine and threonine residues of protein substrates. This phosphorylation plays an important role in regulating various cellular processes. Deregulation of many kinases is directly linked to cancer development and the protein kinase family is one of the most important targets in current cancer therapy regimens. This relevance to disease has stimulated intensive efforts in the biomedical research community to understand their catalytic mechanisms, discern their cellular functions, and discover inhibitors. With the advantage of being able to simultaneously define structural as well as dynamic properties for complex systems, computational studies at the atomic level has been recognized as a powerful complement to experimental studies. In this work, we employed a suite of computational and molecular simulation methods to (1) explore the catalytic mechanism of a particular protein kinase, namely, epidermal growth factor receptor (EGFR); (2) study the interaction between EGFR and one of its inhibitors, namely erlotinib (Tarceva); (3) discern the effects of molecular alterations (somatic mutations) of EGFR to differential downstream signaling response; and (4) model the interactions of a novel class of kinase inhibitors with a common ruthenium based organometallic scaffold with different protein kinases. Our simulations established some important molecular rules in operation in the contexts of inhibitor-binding, substrate-recognition, catalytic landscapes, and signaling in the EGFR tyrosine kinase. Our results also shed insights on the mechanisms of inhibition and phosphorylation commonly employed by many kinases

Human platelet adhesion to heparinized silica surfaces

Heparin was modified and immobilized to surface-activated silica surfaces using two different reaction schemes. End-aminated heparin was reacted with 2-iminothiolane to produce free thiol groups at the terminal ends of the heparin chains. The end-thiolated heparin was immobilized by reaction with a pyridyl disulfide activated poly[ethylene oxide]-poly[propylene oxide]-poly[ethylene oxide] triblock copolymer that was non-covalently adsorbed to hydrophobic silica. In addition, heparin was modified with adipic dihydrazide and covalently immobilized to silica treated with of 3-aminopropyl triethoxy-silane and succinic anhydride to favor a side-on-orientation of heparin at the surface. X-ray photoelectron spectroscopy (XPS) and contact angle analysis were performed at each stage of surface treatment to determine if successful immobilization had taken place. XPS results indicated that successful immobilization of adipic dihydrazide modified heparin had taken place. However, for end-thiolated heparin, XPS results were not entirely consistent with successful heparin immobilization and determination of surface-bound heparin was thus inconclusive. Platelet adhesion under dynamic conditions was investigated on each surface during the immobilization of heparin. Scanning electron microscopy (SEM) was used to characterize platelet adhesion from human platelet-rich plasma on each surface. The images recorded from SEM were used to determine the number and morphology of adherent platelets. For both heparin immobilization methods, there was a significantly lower number of adherent platelets on heparinized surfaces in comparison to the non-heparinized surfaces. Also, the platelets that did adhere to the heparinized surfaces showed less aggregation and spreading in comparison to the non-heparinized surfaces, which is consistent with a lower extent of platelet activation on these surfaces

Supramolecular Optical Chemosensors and Assays for Sensing of Bioactive Analytes in Water and Biofluids

The recognition and detection of biologically important analytes, especially small biomolecules, is of prime relevance and has become an upsurging area of research in chemistry and biology. Consequently, the development of robust chemical molecular sensors (“chemosensors”) based on artificial recognition elements with the potential to detect molecules with high sensitivity and selectivity and coupled with a sensitive signal transduction strategy continues to attract considerable attention. Optical methods based on fluorescence are highly desirable for signal transduction because of their versatility, high sensitivity, low cost with readily available instrumentation, and potential for real-time analysis. Thus, optical/fluorescent chemosensors, in combination with innovative assay protocols, find broad application potential in many disciplines, such as biochemistry and clinical and medical diagnostics. They offer a cost efficient alternative to conventional instrumental analytical methods, such as HPLC-MS, GC-MS, and NMR, and are superior to biosensors in terms of stability, equilibration time, price, and scope for small molecule detection. However, developing chemosensors that fully meet the requirements for practical applications is still challenging. The low binding affinity or selectivity of chemosensors for most biomolecules or their metabolites in biofluids, as well as the low stability of the chemosensor\u27s guest-host ensemble (e.g., upon dilution), are main reasons why the practical application potential of artificial chemosensors has not yet been fully realized. In this work, artificial chemosensors based on supramolecular host guest chemistry coupled with optical signal transduction are utilized to realize both detection and chirality sensing of biologically relevant analytes in aqueous media and complex biofluids. In addition, the various aspects of realizing their practical diagnostic applications are addressed. The first research project involves the development of electronic circular dichroism (ECD) based chemosensors for the detection and chirality sensing of diverse chiral organic analytes in water. Chemosensors that can detect molecular chirality are crucial due to the significance of chiral bio-relevant molecules and the influence of chirality on their related biological activity, e.g., in drug production. However, only a few chirality-based chemosensors are available to date for the detection of compounds in aqueous media. My thesis utilized achiral chromophoric hosts, i.e., acyclic cucurbit[n]urils and molecular tweezers as recognition elements in the chemosensor. The achiral chromophoric hosts were found to respond with information-rich induced ECD signals to the presence of micromolar concentrations of chiral small molecule guests, such as chiral hydrocarbons, terpenes, amino acids and their derivatives, steroids, and drugs in water. In favorable cases, this also allowed for analyte identification and reaction monitoring. In the second research project, fluorescence-detected circular dichroism (FDCD) spectroscopy is applied for the first time for the chiroptical analysis of supramolecular host guest and host protein systems and compared to the widely utilized electronic circular dichroism (ECD). The main goal was to explore the utility of FDCD to improve the sensitivity and selectivity of chiroptical supramolecular assays. The comprehensive investigations demonstrate that FDCD is an excellent choice for common supramolecular applications, e.g., the detection and chirality sensing of chiral organic analytes and label free reaction monitoring. FDCD can be conducted in favorable circumstances at much lower concentrations than ECD measurements, even in chromophoric and auto-emissive biofluids such as blood serum, overcoming the sensitivity limitation of absorbance-based chiroptical spectroscopy. Furthermore, the combined use of FDCD and ECD provided additional valuable information about the system, e.g., the chemical identity of an analyte or hidden aggregation phenomena. The third research project addresses the importance of thermodynamic and kinetic investigations to properly analyze the association and dissociation processes of supramolecular host-guest recognition interactions, which are crucial to designing host guest systems with improved properties and advancing their practical applications. However, kinetic descriptions of supramolecular systems are scarce in the literature, mainly due to the lack of suitable experimental protocols. Thus, three novel fluorescence-based time resolved approaches are introduced that allowed the convenient determination of kinetic rate constants of spectroscopically silent and even insoluble guests with the macrocyclic cucurbit[n]uril and human serum albumin as representative hosts. Furthermore, a new kinetic method is adopted to achieve selective analyte sensing even in situations of poor thermodynamic selectivity due to the host’s often observed similar binding affinities for structurally similar analytes. The method allowed a selective identification and quantification of analytes without the need to modify the parent host synthetically. The fourth research project involves the development of a novel fluorescent chemosensor for the detection of biogenic polyamines, which serve as health indicators in the human body. The fluorescent chemosensor self-assembled from sulfonated pillar[n]arene host in combination with suitable dicationic indicator dyes responds instantly with a fluorescence “turn on” signal to the presence of biogenic polyamines. The photophysical and binding properties of the new fluorescent chemosensor explored in detail in both saline buffers and biologically relevant media display their excellent functionality for polyamine sensing with no salt interferences on the sensing assay. Moreover, the chemosensor allowed the detection of biogenic polyamines down to the low micromolar concentration range in biofluids, such as urine and saliva, with good selectivity even in the presence of potential interferents present in the media. Thus, because of its simplicity, cost-effectiveness, and fast detection capabilities, the newly developed fluorescent chemosensor for polyamines will assist the future development of rapid diagnostic tests for home-use and point-of-care applications. In summary, this doctoral thesis highlights the different strategies for developing supramolecular optical chemosensors for sensitive and selective analyte detection, which are also applicable in biologically relevant media. Future research and development of sensors with improved practical applicability will contribute significantly to the advancement of analytical chemistry and biochemical/medical research

The Use of Protein Modification and Ion Mobility-Mass Spectrometry to Probe Protein Structure

Mass spectrometry (MS) is considered to be indispensable technology for the use in modern pharmaceutical drug discovery and development processes. However, MS is rarely used as a screening technology for protein structure. In this project, ion mobility-mass spectrometry (IM-MS) methods are developed to investigate protein structure with the use of chemical modification and genetic modification. Collision induced unfolding (CIU) method was optimised for measuring the mobility of ubiquitin (Ub) drift traces and the collision cross section (CCS) was calculated. The mobility was measured in the trap by acquiring several voltages and monitoring the drift trace of the lower state ion ([M+6H]6+ and [M+5H]5+. By combining the CIU method and chemical modification of proteins we can enhance the understanding of protein structure in the gas phase. Acetylation was carried out first on ubiquitin, the results showed a difference in the drift trace for ubiquitin after acetylation. This led to inspection of the MS/MS spectrum of intact Ub. The b-ion, corresponding to fragmentation at lysine residue K6, showed this residue to have importance for the structural integrity of the protein. Therefore, different K6 mutant were obtained and their CIU were acquired. The results confirmed that the K6 reside is indeed crucial in the ubiquitin unfolding pathway. Acetylation of this residue, or its replacement with alanine (K6A Ub) produced a conformationally destabilised form of the protein, which unfolded at lower collision energies. Wild type Ub and its mutant K6O mutant shared the fact the K6 is present, and the result showed they have the same CIU unfolding profile, In contrast the NoK and K6R mutant where the K6 has been modified to R, resulted in a more stable compact structure as evidenced by the CIU profile. Diethylpyrocarbonate modification of the single the histidine residue in Ub, which was postulated to interact with K6 in the gas phase structure resulted in modest destabilisation of compact Ub, while succinylation of the N-terminus had no clear effect on stability of the protein structure. Studies of molecular dynamics and charge distribution support the experimental data by rationalising the importance of protonated K6 and H68 interaction in the gas-phase stabilization of the native folding of Ub. Finally, the ubiquitin associating domain UBA2 was destabilised by adding an acetyl group to the N-terminus of the protein. The observation was interpreted by the breaking of a key intramolecular interaction between the N-terminus and the glutamate residue E22. Moreover, the behavious of different in charge states showed the important of addition of charge on the structure of proteins