Dissertations / Theses on the topic 'Non-specific binding'

Accurate determination of the in vitro kinetic parameters Km (Michaelis constant) and Ki (inhibition constant) is critical for the quantitative prediction of in vivo drug clearance and the magnitude of inhibitory drug interactions. A cause of inaccuracy in vitro arises from the assumption that all drug added to an incubation mixture is available for metabolism or inhibition. Many drugs bind non-specifically to the membrane of the in vitro enzyme source. The aims of this thesis were to: 1) investigate the comparative importance of lipophilicity (as log P), and pKa as determinants of the non-specific binding of drugs to human liver microsomes; 2) develop and validate an ANS fluorescence technique for measuring the non-specific binding of drugs to human liver microsomes; 3) characterise the non-specific binding of a large dataset of physicochemically diverse drugs using the ANS fluorescence procedure; 4) evaluate relationships between selected physicochemical characteristics and the extent of non-specific binding of drugs to human liver microsomes and; 5) computationally model the non-specific binding of drugs to discriminate between high binding (fu(mic) less than 0.5) and low binding (fu(mic) greater than 0.5) drugs. The comparative binding of the basic drugs atenolol (log P = 0.1; fu(mic) = 1.00), of propranolol (log P = 3.1; fu(mic) = 0.36 - 0.84), and imipramine (log P = 4.8; fu(mic) = 0.42 - 0.82) suggested that lipophilicity is a major determinant of non-specific binding. In contrast, the comparative binding of diazepam (pKa = 3.3; fu(mic) = 0.69 - 0.80), a neutral compound; and the bases propranolol (pKa = 9.5; fu(mic) = 0.36 - 0.84) and lignocaine (pKa = 9.5; fu(mic) = 0.98), indicated that pKa was not a determinant of the extent of non-specific binding. The non-binding of lignocaine, a relatively lipophilic base, was unexpected and confirmed by the non-binding of the structurally related compounds bupivacaine and ropivacaine. These results implicated physicochemical characteristics other than lipophilicity and charge as important for the non-specific binding of drugs to human liver microsomes. An assay based on 1-anilinonaphthalene-8-sulfonate (ANS) fluorescence was developed using the seven drugs employed in the initial study. Non-specific binding data from equilibrium dialysis and the ANS fluorescence methods were compared and a linear correlation (r2 = 0.92, p less than 0.01) was observed at drug concentrations of 100 and 200 micromolar. The approach was further validated by characterising the microsomal binding of nine compounds (bupropion, chloroquine, chlorpromazine, diflunisal, flufenamic acid, meclofenamic acid, mianserine, triflupromazine, and verapamil) using both binding methods (i.e. equilibrium dialysis and ANS fluorescence). A significant logarithmic relationship (r2 greater than or equal to 0.90) was demonstrated between fu(mic) and the modulus of ANS fluorescence for all drugs and for basic drugs alone at concentrations of 100 and 200 micromolar, while the acidic/neutral drugs showed a significant linear relationship (r2 greater than or equal to 0.84) at these two concentrations (p less than 0.01). The non binding of bupropion provided further evidence that physicochemical properties other than log P and charge were important for non-specific binding of drugs to human liver microsomes. The ANS fluorescence technique was then used to characterise the non-specific binding of 88 physicochemically diverse compounds. In general, acids and neutrals bound to a ‘low’ extent (fu(mic) greater than 0.5) whereas bases bound the full fu(mic) range (0.0001 to 1). Statistically significant relationships were observed between the non-specific binding of bases and log P, the number of hydrogen bond donors and hydrogen bond acceptors per molecule, and molecular mass. Preliminary in silico modeling of the dataset generated by the ANS fluorescence technique, using the program ROCS, provided discrimination of all but one (itraconazole) of the ‘high’ binding bases. However, there were 14 false positives, resulting in low overall prediction accuracy. Taken together, the studies conducted in this thesis provide important insights into the physicochemical factors that determine the non-specific binding of drugs to human liver microsomes.

MicroRNAs are known to be upregulated or downregulated in various types of cancer, leading to changes in the expression of genes involved in cellular proliferation, anti-apoptosis, migration, and invasion. To study the effects of microRNA loss or gain in different neoplasms, numerous models have been described to decrease or increase expression of microRNAs, but the off-target effects of different methods have not been well investigated. I investigated the possibility of off-target effects in a model of miR-143 knockdown in myeloid leukemia cell lines that implemented a microRNA sponge, or decoy, as a method to reduce microRNA expression. The high expression of a sponge with repetitive sequence elements and low expression of the intended microRNA for knockdown, miR-143, created conditions with increased potential for non-specific microRNAs to bind to the sponge. Therefore, I investigated the potential binding sites present in the sponge and whether any novel microRNAs could bind to these sites. I found a number of potential candidates and eliminated them based on their likelihood of regulating protein targets and their resemblance to a microRNA in structure, leaving one potential candidate. I found genomic evidence of the existence of this novel microRNA, evolutionary conservation of function, and performed assays that confirmed the biological activity. Next, the original sponge was redesigned to inhibit the binding of the potential non-specific microRNA; miR-X, or the miR-143 binding sites were mutated to inhibit the binding of miR-143 and capture miR-X instead. This demonstrated that binding of non-specific microRNA could be abrogated and differential protein abundance specific to the knockdown of each microRNA separately was verified. I conclude that non-specific binding to the sponge is a distinct possibility in experiments using this method of microRNA knockdown, which needs to be taken into account when designing sponges in the future. This work also demonstrates that there remain novel microRNAs awaiting discovery.
Medicine, Faculty of
Graduate

The non-invasive imaging modality positron emission tomography (PET) is used extensively in clinical settings and is increasingly being used by the pharmaceutical industry in drug development. Molecules of biological interest are labelled with positron emitting isotopes e.g. 11C, allowing their biodistribution and kinetics to be followed in vivo. A major factor in the failure of radioligands is the magnitude of unwanted background signal, non-specific binding (NSB) obscuring binding to the desired target. Assumptions have previously been made as to the physiochemical and pharmacological properties of radioligands that can affect NSB. However, little work has been carried out to quantify NSB with regard to determining structure-activity relationships (SARs) in order to optimise efficient radiotracer discovery. Non-specific binding is a poorly understood process but is believed to be related to the non-saturable binding of labelled molecules with tissue membranes. In this work the synthesis of novel radiolabelled molecular libraries has been conducted, their physicochemical properties determined and their non-specific binding measured in vitro using autoradiographical and cell based mass spectrometry assay methods. Structure-activity relationships have been formed between partition coefficient properties, acid dissociation constants, interaction energies and molecular weight in order to determine the effect each of these properties has on non-specific binding. Traditionally lipophilicity, log P, of a radioligand is the main predictor to its non-specific binding properties. However from this work it has been shown that a single physicochemical property cannot be relied on to predict the NSB of a radioligand but multiple properties must be considered.

Fragment based lead discovery (FBLD), where libraries of small fragments are screened andlater on developed to lead compounds, is an alternative to the classical drug discovery methods such as high trough-put screening. Weak affinity chromatography (WAC) is a new promising approach to the screening process of FBLD. WAC is performed by injections of fragments onto a high performance liquid chromatography (HPLC) column in which a protein is immobilized to a silica support. The retention of the injected fragments is correlated to the binding affinity of the fragments towards the immobilized protein. Immobilization capacity of three different silica materials with varying pore size (Kromasil240 Å, Nucleosil 1000 Å and Kromasil 300 Å) was evaluated by immobilization of trypsin. Retention of benzamidines on the trypsin columns was evaluated with different mobile phases. Contribution of non-specific binding in the interaction between the 4-aminobenzamidine and thrombin was estimated by frontal chromatography on a capillary columnusing PBS and PBS/acetonitrile as mobile phases. This study showed that the Kromasil 300 Å had a superior immobilization capacity of trypsin compared to the Kromasil 240 Å andthe Nucleosil 1000 Å (100 mg compared to 87.4 mg and 15.1 mg trypsin/g silica, respectively). However, the Nucleosil 1000 Å might be a more suitable support for the immobilization of larger proteins. Adding 5 % methanol or acetonitrile to the mobile phase resulted in a significant (p < 0.05) decreased retention of benzamidine fragments on the trypsin 240 Å column. Non-specific binding between thrombin and 4-ABA was not statistically significantly altered when 5 % acetonitrile was added to the mobile phase.

Pregnancy tests and related immunoassays are heavily dependent on specific and non-specific protein adsorption. These interfacial processes are affected by many factors that influence the in situ conformations of interfacially immobilised antibodies. This thesis examines a number of representative features with dual polarisation interferometry (DPI) and neutron reflection (NR), thus combining real-time dynamic monitoring with high interfacial structural resolution. Bovine serum albumin (BSA) was initially used as a model system to compare the surface coverage and thickness measurements of DPI and NR. The results show that DPI and NR provided similar surface coverage data but the measured thicknesses differed at BSA concentrations above 0.1 mg/ml. This discrepancy arose from the adoption of the uniform-layer model used by DPI for data analysis and the greater thickness sensitivity of NR. A model pregnancy immunoassay was built in steps on a silica surface so that the adsorption of each protein could be accurately monitored. Both DPI and NR provided evidence of BSA insertion into the gaps on the surface between the antibody molecules. This suggests that BSA adsorption is an excellent method to block the non-specific adsorption of target antigens to the immunoassay test surface. A magnetic tweezer system was designed and built in order to measure the specific antibody/antigen binding force. The antibodies and antigens were used to immuno-link magnetic beads to the experimental surface before the immuno-links were broken by increasing the attractive force between the magnetic tweezers and beads. The force per antibody/antigen immuno-link was estimated to lie between the values of 13.6 pN and 43.8 pN.Immuno-link detachment as a function of time was investigated. It was found that the immuno-link comprised both a strong and a weak interaction. The dissociation constant of the strong antibody/antigen interaction was found to equal 3E-4 /s and had an interaction length of 0.06 nm. The low population of beads bound by the second, weaker interaction meant that it was not possible to obtain accurate values of the dissociation constant and bond length of the second weaker interaction.

The objective of this dissertation is to improve the performance of surface acoustic wave (SAW) biosensors for use in point-of-care-testing (POCT) applications. SAW biosensors have the ability to perform fast, accurate detection of an analyte in real time without the use of labels. However, the technology suffers from the inability to differentiate between specific and non-specific binding. Due to this limitation, direct testing of bodily fluids using SAW sensors to accurately determine an analyte's concentration is difficult. In addition, these sensors are challenged by the need to detect small concentrations of a biomarker that are typically required to give a clinical diagnosis. Sensitivity, selectivity and reliability are three critical aspects for any sensing platform. To improve sensitivity the delay path of a SAW sensor has been modified with microcavities filled with various materials. These filled cavities increased sensitivity by confining wave energy to the surface by way of constructive interference and waveguiding. Thus, the improved sensitivity will result in a lower limit of detection. In addition, insertion loss is decreased as a consequence of increased wave confinement to the surface. Sensor selectivity and reliability are adversely affected by non-specific binding of unwanted species present in a sample. To address this issue a multifunctional SAW sensor is presented. The sensor consists of two SAW delay lines oriented orthogonal to each on ST-quartz in order to generate two distinct wave modes. One wave mode is used for sensing while the other is used to remove loosely bound material. By using the same transduction mechanism for both removal and sensing, the sensor chip is simplified and complex electronics are avoided. The findings of this research involve the technological advances for SAW biosensors that make their use in POCT possible.

The hepatitis C virus (HCV) is a positive-strand RNA virus that belongs to the Flaviviridae family. HCV expresses the non-structural protein, NS5B, an RNA-dependent RNA polymerase required for genome replication. Here, we describe the allosteric properties of both the GTP-specific binding site (G-site) of the HCV polymerase and VIR-1327, a benzimidazole-derivative non-nucleoside inhibitor (NNI). Using biochemical assays, we identified certain G-site residues that affect NS5B activity, including serine-29, proline-495 and valine-499. We also demonstrated that VIR-1327 is a potent inhibitor with a resistance profile consisting of P495A and V499A mutants, but is non-competitive with regards to GTP. Moreover, biophysical results suggest that the mechanism of action of VIR-1327 is to prevent the formation of a stable enzyme-template complex. Further analysis of the G-site and NNIs will not only provide a better understanding of HCV replication, but also validate different allosteric sites as virus-specific targets for the development of anti-HCV therapeutics.
Le virus de l’hépatite C (VHC) est un virus d’ARN de polarité positive qui appartient à la famille Flaviviridae. Le VHC exprime la protéine non-structurelle NS5B, une ARN polymérase dépendante de l’ARN indispensable à la réplication du génome virale. Nous décrivons ici les propriétés allostériques du site GTP-spécifique (site-G) du VHC polymérase, ainsi que les propriétés allostériques de VIR-1327, un inhibiteur non-nucléoside (INN) qui est dérivé du benzimidazole. En utilisant des tests biochimiques, nous avons identifié certains résidus du site-G qui influencent l’activité du NS5B, incluant sérine-29, proline-495 et valine-499. Nous avons également démontré que VIR-1327 est un puissant inhibiteur avec un profil de résistance comprenant les mutants P495A et V499A, mais qu’il est non-compétitif avec le GTP. De plus, des résultats biophysiques suggèrent que le méchanisme d’action de VIR-1327 est de prévenir la formation d’un complexe enzyme-matrice stable. D’autres analyses du site-G et des INN vont non seulement fournir une meilleure compréhension de la réplication du VHC, mais aussi valider différents sites allostériques comme étant des cibles virus-spécifiques pour le développement de thérapeutiques contre le VHC.

The purpose of this investigation was to create and optimise a capture assay for the detectionof anti-drug antibodies (ADA) in human plasma, using Biacore. We also dealt with the nonspecificplasma binding to mouse-derived anti-biotin which may occur in the capture assay.By paying attention to these things we aimed at reaching as high sensitivity as possible for theADA detection. The capture assay also benefited and gained flexibility from using the same regenerationsolution irrespective of drug and from having a composition that minimises the risk ofdamaging drug epitopes.

Many irreversible antagonists are known to bind irreversibly to pharmacological receptors. However, few studies suggest that these irreversible antagonists may also display irreversible non-specific antagonism by binding irreversibly to non-syntopic binding sites on the receptor macromolecule, whereby they modulate the signal transduction of these receptors or reduce the agonist binding affmity. The aim of this study was to investigate whether the classical irreversible antagonists phenoxybenzamine, benextramine and 4-DAMP mustard display irreversible nonspecific antagonism at various G protein-coupled receptor (GPCR) types. In addition, the subcellular mechanism whereby benextramine displays irreversible non-specific antagonism was investigated. Three cell lines were employed to investigate the antagonism by these irreversible antagonists: Chinese hamster ovary (CHO-K1) cells transfected to express the porcine a2A-adrenoceptor (a2A-AR) at higher (a2A-H) or lower (a2A-L) numbers, human neuroblastoma (SH-SY5Y) cells that endogenously express muscarinic acetylcholine receptors (mACh-Rs), and SH-SY5Y cells transfected (5HT2A-SH-SY5Y)o express the human 5HT2A-serotonirne ceptor (5HTZA-R).C ells of the appropriate cell line were pre-treated at the appropriate concentrations and incubation times with an appropriate irreversible antagonist, with or without an appropriate reversible competitive antagonist at a sufficient concentration to protect the specific receptors. This was followed by washing procedures with drug-free media to rinse any unbound or reversibly bound drugs from the cells. When appropriate, cell membranes were prepared. Receptor function was evaluated by measuring whole-cell [3H]-cAMP or [3H]-IPx acumulation, or the binding of [35S]-GTPyS to membraness. Receptor concentrations were determined from radioligand-binding assays. In addition, the constitutive [35S]-GTPyS binding to Go protein before and after pre-treatment with benextramine was investigated. Results suggest that phenoxybenzamine (100 uM, 20 minutes) and benextramine (10 uM, 20 minutes) display irreversible non-specific antagonism at a2A-ARs when measuring Gi-mediated effects in a2A-L cells, but the affinity for a2A-ARs in a2A-H cells was not changed. In addition, it was found that the observed irreversible nonspecific antagonism by benextramine appears to be time- and concentration-dependent. When the mechanism of irreversible antagonism by benextramine was further investigated, benextramine reduced the binding of [35S]-GTPyS to a2A-H membranes with protected a2A-ARs, but did not modulate the constitutive binding of [35S]-GTPyS to Go. In addition, benextramine displays irreversible non-specific antagonism by inhibiting the G,-mediated effects of a2A-ARs in a2A-H cells and the Gq-mediated effects of mACh-Rs or 5HT2A-Rs in SH-SY5Y or 5HT2A-SH-SY5Y cells respectively. 4-DAMP mustard (100 uM, 20 minutes) did not display irreversible non-specific antagonism at mACh-Rs in SH-SY5Y cells, but irreversible non-specific antagonism was observed when the incubation time was increased (100 uM, 60 minutes). In conclusion it was found that phenoxybenzamine, benextramine and 4-DAMP mustard display irreversible non-specific antagonism at typical experimental conditions. These findings confirm concerns in literature and supports the possibility that more irreversible antagonists could display irreversible non-specific antagonism, and that could influence the interpretation of data obtained with such drugs. In addition, benextramine may prove to be a useful experimental drug in studying GPCR signalling.
Thesis (Ph.D. (Pharmacology))--North-West University, Potchefstroom Campus, 2004.

碩士
國立中正大學
機械系
99
In biochemistry detection, the analysis usually includes two kinds of particles, one are specific absorption particles which will react with the detection area, another are non-specific absorption particles which will not react with the detection area. However, non-specific absorption particles will bind the detection area with Van der Waal Force, causing the result of detection error. Apply high frequency electric field on PZT, which is the piezoelectric material, will produce an ultrasonic wave. Apply ultrasonic wave on the detection area can remove non-specific absorption particles, makes specific absorption particles react with the detection area. Besides, apply ultrasonic wave on the detection area under the condition that the analysis only contain specific absorption particles, can increase the number of specific absorption particles that react with the detection area.

During the past decade the effects of macromolecular crowding on reaction pathways is gaining in prominence. The stress is to move out of the realms of ideal solution studies and make conceptual modifications that consider non-ideality as a variable in our calculations. In recent years it has been shown that molecular crowding exerts significant effects on all in vivo processes, from DNA conformational changes, protein folding to DNA-protein interactions, enzyme pathways and signalling pathways. Both thermodynamic as well as kinetic parameters vary by orders of magnitude in uncrowded buffer system as compared to those in the crowded cellular milieu. Ignoring these differences will restrict our knowledge of biology to a “model system” with few practical understandings. The recent expansion of the genome database has stimulated a study on numerous previously unknown proteins. This has whetted our thirst to model the cellular determinants in a more comprehensive manner. Intracellular extract would have been the ideal solution to re-create the cellular environment. However, studies conducted in this solution will be contaminated by interference with other biologically active molecule and relevant statistical data cannot be extracted out from it. Recent advances in methodologies to mimic the cellular crowding include use of inert macromolecules to reduce the volume occupancy of target molecules and the use of immobilization techniques to increase the surface density of molecules in a small volumetric region. The use of crowding agents often results in non-specific interaction and side-reactions like aggregation of the target molecules with the crowding agents themselves. Immobilization of one of the interacting partners reduces the probability of aggregation and precipitation of bio-macromolecules by restricting their degrees of freedom. Covalent linkage of molecules on solid support is used extensively in research for creating a homogeneous surface of bound molecules which can be interrogated for their reactivity. However, when it comes to biomolecules, direct immobilization on solid support or use of organic linkers often results in denaturation. The use of bio-affinity immobilization techniques can help us overcome this problem. Since mild conditions are needed to regenerate such a surface, it finds universal applicability as bio-memory chips. This thesis focuses on our attempts to design a physiologically viable immobilization technique for following rotein-protein/protein-DNA interactions. The work explores the mechanism for biological interactions related to transcription process in E. coli. Chapter 1 deals with the literary survey of the importance and effects of molecular crowding on biological reactions. It gives a brief history of the efforts been made so far by experimentalists, to mimic macromolecular crowding and the methods applied. The chapter tries to project an all-round perspective of the pros and cons of different immobilization techniques as a means to achieve a high surface density of molecules and the advancements so far. Chapter 2 deals with the detailed technicality and applicability of the Langmuir-Blodgett method. It discusses the rationale behind our developing this technique as an alternate means of bio-affinity immobilization, under physiologically compatible conditions. It then goes on to describe our efforts to follow the sequence-specific and sequential assembly process of a functional RNA polymerase enzyme with one immobilized partner and also explore the role of omega subunit of RNAP in the reconstitution pathway. This chapter uses the assembly process of a multi-subunit enzyme to evaluate the efficiency of the LB system as a universal two-dimensional scaffold to follow sequence-specific protein-ligand interaction. Chapter 3 discusses the application of LB technique to quantitatively evaluate the kinetics and thermodynamics of promoter-RNA polymerase interaction under conditions of reduced dimensionality. Here, we follow the interaction of T7A1 phage promoter with Escherichia coli RNA polymerase using our Langmuir-Blodgett technique. The changes in mechanistic pathway and trapping of kinetic intermediates are discussed in detail due to the imposed restriction in the degrees of freedom of the system. The sensitivity of this detection method is compared vis-a-vis conventional immobilization methods like SPR. This chapter firmly establishes the universal application of LB technique as a means to emulate molecular crowding and as a sensitive assay for studying the effects of such crowding on vital biological reaction pathway. Chapter 4 describes the mechanistic pathway for the physical binding of MsDps1 protein with long dsDNA in order to physically protect DNA during oxidative stress. The chapter describes in detail the mechanism of physical sequestering of non-specific DNA strands and compaction of the genome under conditions where a kinetic bottleneck has been applied. The data obtained is compared with results obtained in the previous chapter for the sequence-specific DNA-protein interaction in order to understand the difference in recognition process between regulatory and structural proteins binding to DNA. Chapter 5 deals with the evaluation of the σ-competition model in E. coli for three different sigma factors (all belonging to the σ-70 family). Here again, we have evaluated the kinetic and thermodynamic parameters governing the binding of core RNAP with its different sigma factors (σ70, σ32and σ38) and performed a comparative study for the binding of each sigma factor to its core using two different non-homogeneous immobilization techniques. The data has been analyzed globally to resolve the discrepancies associated with establishing the relative affinity of the different sigma factors for the same core RNA polymerase under physiological conditions. Chapter 6 summarizes the work presented in this thesis. In the Appendix section we have followed the unzipping of promoter DNA sequence using Optical Tweezers in an attempt to follow the temporal fluctuations occurring in biological reactions in real time and at a single molecule level.

碩士
國立臺灣大學
工程科學及海洋工程學研究所
97
Protein-DNA interactions are essential for fundamental biochemical activities including DNA transcription, replication, packaging, repair and rearrangement. Proteins interacting with DNA can be classified into two modes distinguished by sequence-specific and non-specific binding respectively. Protein-DNA specific binding provides a mechanism to recognize correct nucleotide base pairs namely sequence-specific identification. On the other hand, protein-DNA non-specific binding shows relatively little base-sequence preference and interacts with DNA backbone. In this thesis, we present a two stage Protein-DNA binding prediction. In the first stage of DNA-binding residues prediction, the predictor for DNA specific binding residues achieves 96.45% accuracy with 50.14% sensitivity, 99.31% specificity, 81.70% precision, and 62.15% F-measure. The predictor for DNA non-specific binding residues achieves 89.14% accuracy with 53.06% sensitivity, 95.25% specificity, 65.47% precision, and 58.62% F-measure. In addition, we combine the results of sequence-specific and non-specific binding residues predicted in previous stage with OR operation, and the predictor achieves 89.26% accuracy with 56.86% sensitivity, 95.63% specificity, 71.92% precision, and 63.51% F-measure. In the second stage, a protein-DNA interaction mode predictor is proposed. It can achieve 75.83% accuracy while using support vector machine with multi-class prediction. This article presents the design of a sequence-based predictor aiming to identify the sequence-specific and non-specific DNA-binding residues in a transcription factor with DNA binding-mechanism concerned. The protein-DNA interaction mode prediction was introduced to provide biochemist more structural hint and help improve previous DNA-binding residues prediction. In addition, we will exploit the experiences learned in this study to design binding-mechanism concerned predictors for other types of DNA-contacted proteins.

abstract: Protein-surface interactions, no matter structured or unstructured, are important in both biological and man-made systems. Unstructured interactions are more difficult to study with conventional techniques due to the lack of a specific binding structure. In this dissertation, a novel approach is employed to study the unstructured interactions between proteins and heterogonous surfaces, by looking at a large number of different binding partners at surfaces and using the binding information to understand the chemistry of binding. In this regard, surface-bound peptide arrays are used as a model for the study. Specifically, in Chapter 2, the effects of charge, hydrophobicity and length of surface-bound peptides on binding affinity for specific globular proteins (&beta-galactosidase and &alpha1-antitrypsin) and relative binding of different proteins were examined with LC Sciences peptide array platform. While the general charge and hydrophobicity of the peptides are certainly important, more surprising is that &beta-galactosidase affinity for the surface does not simply increase with the length of the peptide. Another interesting observation that leads to the next part of the study is that even very short surface-bound peptides can have both strong and selective interactions with proteins. Hence, in Chapter 3, selected tetrapeptide sequences with known binding characteristics to &beta-galactosidase are used as building blocks to create longer sequences to see if the binding function can be added together. The conclusion is that while adding two component sequences together can either greatly increase or decrease overall binding and specificity, the contribution to the binding affinity and specificity of the individual binding components is strongly dependent on their position in the peptide. Finally, in Chapter 4, another array platform is utilized to overcome the limitations associated with LC Sciences. It is found that effects of peptide sequence properties on IgG binding with HealthTell array are quiet similar to what was observed with &beta-galactosidase on LC Science array surface. In summary, the approach presented in this dissertation can provide binding information for both structured and unstructured interactions taking place at complex surfaces and has the potential to help develop surfaces covered with specific short peptide sequences with relatively specific protein interaction profiles.
Dissertation/Thesis
Doctoral Dissertation Biochemistry 2014

Thesis (Ph. D.)--University of Virginia, 1997.
Spine title: M-CAT motifs of the SM [alpha]-actin gene. Includes bibliographical references (90-100). Also available online through Digital Dissertations.

A series of studies from our lab have investigated the threading polyintercalator approach to sequence specific DNA binding using a 1,4,5,8-naphthalene tetracarboxylic diimide (NDI) intercalating unit connected by flexible peptide linkers. Herein is a report of the sequence specificity, as well as a detailed kinetic analysis, of a threading NDI tetraintercalator. DNase I footprinting using two ~500 base pair DNA fragments containing one designed binding site for the tetraintercalator confirmed highly sequence specific binding. Kinetic analyses include 1H NMR, gel mobility-shift assays, and stopped-flow UV measurements to reveal a polyintercalation binding mode that demonstrates significant similarities between association rate profiles and rate constants for the tetraintercalator binding to its preferred versus a random oligonucleotide sequence. Sequence specificity was found to derive almost entirely from large differences in dissociation rates from the preferred versus random oligonucleotide sequences. Interestingly, the dissociation rate constant of the tetraintercalator complex dissociating from its preferred binding site was extremely slow, corresponding to a 16 day half-life at a benchmark 100 mM [Na+]. This dissociation result for the tetraintercalator is one of the longest bound half-lives yet measured, and to the best of our knowledge, the longest for a DNA binding small molecule. Such a long-lived complex raises the possibility of using threading polyintercalators to disrupt biological processes for extended periods. Current focus is given to deciphering a mechanism for the molecular recognition of the tetraintercalator preferred binding site within a long sequence of DNA. Initial DNase I footprinting results on an approximate 500mer DNA sequence containing three sequential preferred binding sites reveal that the tetraintercalator likely locates its designed binding site by a macro- or microscopic dissociation/re-association type of mechanism. Cooperativity is a possible ally to binding, leaving future studies to distinguish the mechanism for molecular recognition in a manner that is capable of circumventing cooperative binding. Taken together, the threading polyintercalation binding mode presents an interesting topology to sequence specific DNA binding. Extraordinarily long dissociation rates from preferred binding sites offers many future possibilities to disrupt biological processes in vivo.
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Cette thèse porte sur le développement de biocapteurs basés sur la technique de résonance des plasmons de surface (SPR) pour effectuer des analyses directement dans un fluide sanguin n’ayant subi aucune purification ou dilution. L’ensemble des biocapteurs discutés exploiteront un instrument SPR portable développé dans le groupe du professeur Masson. Le premier volet de la thèse portera sur le processus d’interférence lié à l’adsorption non spécifique du sérum à la surface du capteur. L’analyse des biomolécules adsorbées sera effectuée en combinant la SPR à la spectrométrie de masse. Les informations obtenues seront exploitées pour la construction de biocapteurs adaptés à l’analyse en milieu sanguin. Un premier biocapteur développé ciblera la protéine antigène prostatique spécifique (APS) contenue dans le sérum servant de biomarqueur pour dépister le cancer de la prostate. Pour détecter les faibles concentrations de cette protéine directement dans le sérum, un matériel plasmonique microstructuré sera utilisé pour amplifier les signaux obtenus et sera recouvert d’une monocouche peptidique minimisant l’adsorption non spécifique du sérum. L’instrument SPR aura été adapté pour permettre également la détection simultanée de fluorescence. Un test ELISA sera ainsi effectué en parallèle du test SPR. Chacune des techniques fournira un contrôle pour la deuxième, tout en permettant de détecter le biomarqueur au niveau requis pour dépister la maladie. La combinaison des deux méthodes permettra aussi d’élargir la gamme dynamique du test de dépistage. Pour terminer, l’instrument SPR portable sera utilisé dans le cadre de détection de petites biomolécules ayant un potentiel thérapeutique directement dans un échantillon de sang. Des peptides ayant une activité anti-athérosclérotique pourront ainsi être détectés à même un échantillon de sang ni purifié ni dilué, et ce à des concentrations de l’ordre du micromolaire. Une modification de la microfluidique via l’introduction d’une membrane poreuse au cœur de celle-ci sera la clé permettant d’effectuer de telles analyses. La présente thèse met de l’avant de nouvelles stratégies et des modifications instrumentales permettant d’analyser des protéines et des petites molécules directement dans un échantillon non purifié de sérum ou de sang. Les modifications apportées au système fluidique, à l’instrument SPR et au niveau du biocapteur employé permettront d’effectuer des biodétections dans des matrices aussi complexes que les fluides sanguins. Les présents travaux mettent en lumière la capacité d’un instrument SPR/fluorescence portable à faire en 12 minutes la biodétection d’un marqueur du cancer de la prostate directement dans un échantillon de sérum. Finalement, on rapporte ici un des premiers articles où un biocapteur SPR est utilisé à même un échantillon de sang non-purifié pour faire des biodétections.
This thesis discusses the development of surface plasmon resonnance (SPR) biosensors to perform detection directly on unpurified and undiluted blood based fluids such as serum or blood. Every biosensor discussed in the following chapters rely on a home-built portable SPR device developed in Professor Masson’s research laboratories. Non-specific adsorption, which greatly hinders biosensing in crude fluids, will be the first topic of the thesis. Serum adsorption was performed on the SPR sensor surface and then characterized by SPR and mass spectrometry. This study provided useful information for biosensing directly in blood-based fluids. It also provided a better fundamental understanding of the nonspecific adsorption process on surfaces. The first biosensor was developed to detect prostate specific antigen (PSA), a protein normally contained in serum, which is a known biomarker for prostate cancer. In order to detect low concentrations of this protein directly in serum, a microstructured gold film was used to amplify the signal generated by the binding event on the biosensor. A peptide monolayer covered the metallic surface of the sensor to reduce non-specific protein adsorption. The SPR portable instrument was modified to simultaneous detect fluorescence in order to perform a SPR and ELISA test in a single instrumental platform. Each technique provided a control for the other for detection of the prostate cancer biomarker at concentration levels required for the screening of the disease. The SPR and ELISA combination also extended the dynamic range of the biosensing assay. Finally, the portable SPR device was used to detect small biomolecules with potential therapeutic activity directly in a sample of blood. Peptides with an anti-atherosclerotic activity were thus detected in an unpurified and undiluted blood sample at micromolar concentration. The addition of a porous membrane to the microfluidic used for the biosensing assay facilitated the successful detection of these molecules in whole blood. The present thesis describes novel strategies and instrumental modifications to unlock the possibility of performing biosensing directly on unpurified and undiluted blood-based fluids. Modifications of the fluidic system, the SPR instrument and biosensor used will allow detection in fluids with high complexity such as serum or blood. The work described herein reports a prostate cancer screening assay performed in 12 minutes directly in serum using a portable SPR/fluorescence instrument. Finally, this thesis reports one of the first scientific papers where a SPR biosensor is used to perform analysis directly in blood.

Research Doctorate - Doctor of Philosophy (PhD)
Specific ion effects (SIEs) encompass any phenomenon induced by ions that is dependent on the identity of the ions, and not just their charge or concentration. These occur in salts, electrolyte solutions, ionic liquids, acids and bases and have been known for over 130 years, from which the Hofmeister series originated. They are important in biology, nutrition, electrochemistry and various interfacial or geophysico-phenomena. It is perhaps harder to find a “real-world” system in which specific ion effects don’t occur, than systems where they do. Nonetheless, despite such ubiquity and effect on our daily lives, our understanding of these salty solutions is limited. This thesis addresses the knowledge gap surrounding the lack of parameters (for both ion and solvent) for quantifying SIEs in aqueous and nonaqueous environments. This thesis begins with a deeper introduction to the topic of SIEs and highlights the current state-of-play. The theories underlying quantum mechanics and computational chemistry are discussed to highlight how they may be applied to elucidate some of the fundamental origins of SIEs. These methods were subsequently used to investigate possible energetic origins of counterion and solvent induced reversals to the Hofmeister series, and highlights that the Lewis acidity and basicity (collectively Lewis strength) indices of the cations and anions respectively, can quantify SIEs. Following this revelation, these empirical parameters were recast in terms of intermolecular forces. Electrostatics appeared to govern the Lewis strength indices, so these were replaced with an electrostatic parameter, ϸ (“sho”), that originates from Coulomb’s Law. For anions, ϸ is shown to quantify SIE trends observed in enthalpies of hydration, polymer lower critical solution temperatures, enzyme and viral activities, SN2 reaction rates and Gibbs free energies of transfer from water to nonaqueous solvents highlighting the versatility of ϸ as a new SIE parameter. Cation interactions are more prone to deviations from ϸ correlations. In the absence of any cosolute (i.e., pure ion-solvent interactions) however, cation solvent interactions follow a strong trend with Coulomb’s Law for ~15 different solvents. This supports a conclusion that competing electrostatic interactions between the solvent and a cosolute for the cation may mask each other allowing non-electrostatic contributions to play a dominant role. Furthermore, with similarity to the ion parameterisation, the ϸ values at the negative and positive solvent dipolar atoms correlate with the solvent’s Lewis basicity and acidity respectively. Additionally, these analyses can be related to macroscopic solvent parameters such as the relative permittivity. The data deficiency issue facing the SIE field was more generally addressed in this thesis by the generation of IonSolvR, a repository containing over 3000 distinct QM/MD trajectories of up to 52 ions in 28 bulk solvents on nanosecond-scales. Finally, the key findings of this thesis are summarised and an outlook on the field of SIEs and the broader implications arising from this thesis is presented.