The NTD Nanoscope: potential applications and implementations

<p>Abstract</p> <p>Background</p> <p>Nanopore transduction detection (NTD) offers prospects for a number of highly sensitive and discriminative applications, including: (i) single nucleotide polymorphism (SNP) detection; (ii) targeted DNA re-sequencing; (iii) protein isoform assaying; and (iv) biosensing via antibody or aptamer coupled molecules. Nanopore event transduction involves single-molecule biophysics, engineered information flows, and nanopore cheminformatics. The NTD Nanoscope has seen limited use in the scientific community, however, due to lack of information about potential applications, and lack of availability for the device itself. Meta Logos Inc. is developing both pre-packaged device platforms and component-level (unassembled) kit platforms (the latter described here). In both cases a lipid bi-layer workstation is first established, then augmentations and operational protocols are provided to have a nanopore transduction detector. In this paper we provide an overview of the NTD Nanoscope applications and implementations. The NTD Nanoscope Kit, in particular, is a component-level reproduction of the standard NTD device used in previous research papers.</p> <p>Results</p> <p>The NTD Nanoscope method is shown to functionalize a single nanopore with a channel current modulator that is designed to transduce events, such as binding to a specific target. To expedite set-up in new lab settings, the calibration and troubleshooting for the NTD Nanoscope kit components and signal processing software, the NTD Nanoscope Kit, is designed to include a set of test buffers and control molecules based on experiments described in previous NTD papers (the model systems briefly described in what follows). The description of the Server-interfacing for advanced signal processing support is also briefly mentioned.</p> <p>Conclusions</p> <p>SNP assaying, SNP discovery, DNA sequencing and RNA-seq methods are typically limited by the accuracy of the error rate of the enzymes involved, such as methods involving the polymerase chain reaction (PCR) enzyme. The NTD Nanoscope offers a means to obtain higher accuracy as it is a single-molecule method that does not inherently involve use of enzymes, using a functionalized nanopore instead.</p

Nanopore Sensing Of Peptides And Proteins

In recent years the application of single-molecule techniques to probe biomolecules and intermolecular interactions at single-molecule resolution has expanded rapidly. Here, I investigate a series of peptides and proteins in an attempt to gain a better understanding of nanopore sensing as a single-molecule technique. The analysis of retro, inversed, and retro-inversed isomers of glucagon and α-helical Fmoc-D2A10K2 peptide showed that nanopore sensing utilizing a wild-type α-hemolysin pore can distinguish between all four isomers while circular dichroism can only distinguish between chiral isomers, but not between directional isomers. The investigation of a series of proteins of different chemical and physical properties revealed important information about nanopore analysis of proteins. Contrary to some reports in the literature, all proteins analysed here induced large blockade events. The frequency of total events and the proportion of large blockade events were significantly reduced in tris(hydroxymethyl)aminomethane or 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid buffers and were only restored by the addition of ethylenediaminetetraacetic acid or the use of phosphate buffer, both of which can sequester metal ions. Furthermore, the results obtained with the proteins in the presence of ligands demonstrated that transient or partial unfolding of proteins can be detected by nanopore analysis confirming the usefulness of this technique for conformational studies or for protein/ligand interactions. Interestingly, while the blockade current histograms were different for each protein there was no obvious correlation between the properties of the proteins and the blockade current histograms. In an attempt to identify whether the large blockade events were translocation or intercalation, both an indirect and a direct approach were taken. The indirect approach which relies on the effect of voltage on the interaction of the molecule with the pore provided no conclusive answer to the question of protein translocation through the α-hemolysin pore. In contrast, the direct approach in which ribonuclease A is added to the cis side of the pore and then the trans side is tested for enzyme activity showed that ribonuclease A doesn't translocate through the α-hemolysin pore

Applications of bacterial enterotoxins, ribosome-inactivating proteins and viral cytotoxins

A toxin can be described as a foreign substance that inflicts damages to living organisms. Naturally occurring proteinaceous toxins can derive from bacteria, fungi, plants, animal venoms and even viruses. Identifying the toxins’ underlying mechanisms of action has been a major research interest in order to develop inhibitors against their effects. Nonetheless, various findings have sparked the use of toxic moieties for the medical benefit resulting in treatment options as for example for cancers. To gain novel insights into the structure and function of a toxin, the toxin itself has to be synthesized. In vivo production can involve high laboratory safety standards as well as a low total protein amount since the toxin might harm the overexpressing cell. An alternative to circumvent these drawbacks is cell-free protein synthesis (CFPS). Within this doctoral thesis CFPS was established as a platform technology for the production and application of proteinaceous toxins in diagnostic and medical fields. As a first step, various bacterial toxins were analyzed. The mechanisms of action of the tripartite pore-forming toxins (PFT) Hbl and Nhe were studied by hemolytic activity assays, cell-based toxicity assessments and electrophysiological recordings. Next, the PFT CytK was analyzed to identify its potential as a biological nanopore that can be used as a diagnostic tool. This thesis identified the CytK1 variant as a candidate for a nanopore development. Further, two AB5 toxins, namely the cholera toxin and the heat-labile enterotoxin, were modified. These modified toxins could be fluorescently labeled and tested for their functional activity. These data are a proof-of-concept for using CPFS for intracellular trafficking of toxins and coupling of payloads for drug delivery. In a second step, a targeted toxin combining the plant-derived toxin Dianthin and the epidermal growth factor (EGF) was assessed for its potency as a potential cancer therapeutic. The medical benefit of this Dianthin-EGF targeted toxin was demonstrated on human squamous cell carcinoma samples. 0.1 nM Dianthin-EGF in combination with an endosomal escape enhancer suppressed the growth of carcinoma colonies by almost 50%. As a third and last step, CFPS was assessed for its potential as a rapid response system against novel viral pathogens using SARS-CoV2 viral proteins. All SARS-CoV2 proteins could be synthesized and analyzed. The cytotoxic behaviors of the nsp1 and envelope protein were determined. The nucleocapsid protein was quantitatively detected by specific antibodies thereby facilitating cell-free systems for the validation of available antibodies. All in all, this thesis successfully developed a platform technology for the cell-free synthesis, functional characterization and application of toxic proteins in clinical and diagnostic fields

Carbon nanotube based potentiometric aptasensors for protein detection

El diagnóstico rápido de la mayoría de las enfermedades tiene una importancia vital para proporcionar el remedio adecuado y, por lo tanto, el control de problemas de salud. La detección rápida y selectiva de biomoléculas grandes, específicamente proteínas, es uno de los objetivos importantes en este campo. Las técnicas basadas en inmunoensayos son las más comúnmente utilizadas, aunque requieren un marcaje específico. Por lo general, estos métodos también requieren personal altamente capacitado y equipos complejos que se traduce en una metodología relativamente cara y lenta. En la presente tesis aportamos por primera vez un nuevo tipo de aptasensores potenciométricos de estado sólido basados en nanotubos de carbono que pueden detectar analitos grandes, como por ejemplo proteínas, de manera rápida (casi instantánea), selectiva, y sensible sin necesidad de marcaje químico. Los sensores desarrollados responden correctamente a la proteína analito (α-trombina humana) dentro de los niveles fisiológicos en el suero humano.Rapid diagnosis of most illnesses has a vital importance for providing the appropriate cure and hence controlling public health concerns. Fast and accurate detection of large biomolecules, specifically proteins, is one of the major steps regarding the subject. Over recent years, several detection methodologies have been developed. However, almost all of the developed methods either required very complex techniques to be applied, or a long time to obtain the results. The most commonly used techniques were specific label requiring immunoassays. They generally require highly trained staff and complex equipment which results in an expensive and relatively slow methodology. With the present thesis we report a new type of label-free potentiometric solid state carbon nanotube based aptasensors that can detect large analytes, as case example proteins, in a rapid (almost instantaneous), selective and sensitive way, for the first time. The developed sensors successfully responded to analyte protein (human α-thrombin) within physiological human serum levels

Fluorescence-based nanofluidic biosensor platform for real-time measurement of protein binding kinetics

L'analyse cinétique d'interactions de protéines offre une multitude d'informations sur les fonctions physiologiques de ces molécules au sein de l'activité cellulaire, et peut donc contribuer à l'amélioration des diagnostics médicaux ainsi qu'à la découverte de nouveaux traitements thérapeutiques. La résonance plasmonique de surface (SPR) est la technique de biodétection optique de référence pour les études cinétiques d'interaction de molécules biologiques. Si la SPR offre une détection en temps réel et sans marquage, elle nécessite en revanche des équipements coûteux et sophistiqués ainsi que du personnel qualifié, limitant ainsi son utilisation au sein de laboratoires de recherche académiques. Dans ces travaux de thèse, nous avons développé une plateforme de biodétection basée sur l'utilisation de nanofentes biofonctionnalisées combinées avec une détection par microscopie à fluorescence. Ce système permet l'observation en temps réel d'interactions protéines-protéines et la détermination des constantes cinétiques associées, avec des temps de réponse optimisés et une excellente efficacité de capture. La fonctionnalité du système a été démontrée par l'étude des cinétiques d'interaction de deux couples modèles de différentes affinités : le couple streptavidine/biotine et le couple IgG de souris/anti-IgG de souris. Une très bonne cohérence entre les constantes cinétiques extraites, celles obtenues par des expériences similaires réalisées en SPR et les valeurs rapportées dans la littérature montre que notre approche pourrait être facilement applicable pour l'étude cinétique d'interactions de protéines avec une sensibilité allant jusqu'au pM, sur une large gamme de constantes de dissociation. De plus, nous avons intégré un générateur de gradient de concentrations microfluidique en amont de nos nanofentes, permettant ainsi des mesures simultanées de cinétiques d'interactions à différentes concentrations d'analyte en une seule expérience. Ce système intégré offre de nombreux avantages, tels qu'une réduction de la consommation des réactifs et des temps d'analyse par rapport aux approches séquentielles classiques. Cette technologie innovante pourrait ainsi être un outil précieux non seulement pour les domaines du biomédical et de la médecine personnalisée mais aussi pour la recherche fondamentale en chimie et biologie.Kinetic monitoring of protein-protein interactions offers fundamental insights of their cellular functions and is a vital key for the improvement of diagnostic tests as well as the discovery of novel therapeutic drugs. Surface plasmon resonance (SPR) is an established biosensor technology routinely used for kinetic studies of biomolecular interactions. While SPR offers the benefits of real-time and label-free detection, it requires expensive and sophisticated optical apparatus and highly trained personnel, thus limiting the accessibility of standard laboratories. In this PhD project, we have developed an alternative and cost-effective biosensor platform exploiting biofunctionalized nanofluidic slits, or nanoslits, combined with a bench-top fluorescence microscope. Our approach enables the visualization of protein interactions in real-time with the possibility to determine associated kinetic parameters along with optimized response times and enhanced binding efficiency. We have demonstrated the effectiveness of our devices through kinetic studies of two representative protein-receptor pairs with different binding affinities: streptavidin-biotin and mouse IgG/anti-mouse IgG interactions. Good agreement of extracted kinetic parameters between our device, SPR measurements and literature values indicated that this approach could be readily applicable to study kinetics of protein interactions with sensitivity down to 1 pM on a large scale of dissociation constants. In addition, we have incorporated a microfluidic gradient generator to our validated nanoslit device, which has allowed one-shot parallel kinetic measurements to be realized in a single-experiment. This integrated system provides advantages of diminished material consumption and analysis time over the conventional kinetic assays. We believe that this innovative technology will drive future advancements not only in the discipline of biomedical and personalized medicine, but also in basic chemical/biological research

RNA, the Epicenter of Genetic Information

The origin story and emergence of molecular biology is muddled. The early triumphs in bacterial genetics and the complexity of animal and plant genomes complicate an intricate history. This book documents the many advances, as well as the prejudices and founder fallacies. It highlights the premature relegation of RNA to simply an intermediate between gene and protein, the underestimation of the amount of information required to program the development of multicellular organisms, and the dawning realization that RNA is the cornerstone of cell biology, development, brain function and probably evolution itself. Key personalities, their hubris as well as prescient predictions are richly illustrated with quotes, archival material, photographs, diagrams and references to bring the people, ideas and discoveries to life, from the conceptual cradles of molecular biology to the current revolution in the understanding of genetic information. Key Features Documents the confused early history of DNA, RNA and proteins - a transformative history of molecular biology like no other. Integrates the influences of biochemistry and genetics on the landscape of molecular biology. Chronicles the important discoveries, preconceptions and misconceptions that retarded or misdirected progress. Highlights major pioneers and contributors to molecular biology, with a focus on RNA and noncoding DNA. Summarizes the mounting evidence for the central roles of non-protein-coding RNA in cell and developmental biology. Provides a thought-provoking retrospective and forward-looking perspective for advanced students and professional researchers