Matching Items (94)
Eradication of multidrug-resistant bacteria using biomolecule-encapsulated two-dimensional materials
Description
The increasing pervasiveness of infections caused by multidrug-resistant bacteria (MDR) is a major global health issue that has been further exacerbated by the dearth of antibiotics developed over the past 40 years. Drug-resistant bacteria have led to significant morbidity and mortality, and ever-increasing antibiotic resistance threatens to reverse many of the medical advances enabled by antibiotics over the last 40 years. The traditional strategy for combating these superbugs involves the development of new antibiotics. Yet, only two new classes of antibiotics have been introduced to the clinic over the past two decades, and both failed to combat broad spectrum gram-negative bacteria. This situation demands alternative strategies to combat drug-resistant superbugs. Herein, these dissertation reports the development of potent antibacterials based on biomolecule-encapsulated two-dimensional inorganic materials, which combat multidrug-resistant bacteria using alternative mechanisms of strong physical interactions with bacterial cell membrane. These systems successfully eliminate all members of the ‘Superbugs’ set of pathogenic bacteria, which are known for developing antibiotic resistance, providing an alternative to the limited ‘one bug-one drug’ approach that is conventionally used. Furthermore, these systems demonstrate a multimodal antibacterial killing mechanism that induces outer membrane destabilization, unregulated ion movement across the membranes, induction of oxidative stress, and finally apoptotic-like cell death. In addition, a peptide-encapsulation of the two-dimensional material successfully eliminated biofilms and persisters at micromolar concentrations. Overall, these novel systems have great potential as next-generation antimicrobial agents for eradication of broad spectrum multidrug-resistant bacteria.
ContributorsDebnath, Abhishek (Author) / Green, Alexander A (Thesis advisor) / Liu, Yan (Committee member) / Stephanopoulos, Nicholas (Committee member) / Arizona State University (Publisher)
Created2019
Description
The fundamental building blocks for constructing complex synthetic gene networks are effective biological parts with wide dynamic range, low crosstalk, and modularity. RNA-based components are promising sources of such parts since they can provide regulation at the level of transcription and translation and their predictable base pairing properties enable large libraries to be generated through in silico design. This dissertation studies two different approaches for initiating interactions between RNA molecules to implement RNA-based components that achieve translational regulation. First, single-stranded domains known as toeholds were employed for detection of the highly prevalent foodborne pathogen norovirus. Toehold switch riboregulators activated by trigger RNAs from the norovirus RNA genome are designed, validated, and coupled with paper-based cell-free transcription-translation systems. Integration of paper-based reactions with synbody enrichment and isothermal RNA amplification enables as few as 160 copies/mL of norovirus from clinical samples to be detected in reactions that do not require sophisticated equipment and can be read directly by eye. Second, a new type of riboregulator that initiates RNA-RNA interactions through the loop portions of RNA stem-loop structures was developed. These loop-initiated RNA activators (LIRAs) provide multiple advantages compared to toehold-based riboregulators, exhibiting ultralow signal leakage in vivo, lacking any trigger RNA sequence constraints, and appending no additional residues to the output protein. Harnessing LIRAs as modular parts, logic gates that exploit loop-mediated control of mRNA folding state to implement AND and OR operations with up to three sequence-independent input RNAs were constructed. LIRA circuits can also be ported to paper-based cell-free reactions to implement portable systems with molecular computing and sensing capabilities. LIRAs can detect RNAs from a variety of different pathogens, such as HIV, Zika, dengue, yellow fever, and norovirus, and after coupling to isothermal amplification reactions, provide visible test results down to concentrations of 20 aM (12 RNA copies/µL). And the logic functionality of LIRA circuits can be used to specifically identify different HIV strains and influenza A subtypes. These findings demonstrate that toehold- and loop-mediated RNA-RNA interactions are both powerful strategies for implementing RNA-based computing systems for intracellular and diagnostic applications.
ContributorsMA, DUO (Author) / Green, Alexander (Thesis advisor) / Mangone, Marco (Committee member) / Liu, Yan (Committee member) / Arizona State University (Publisher)
Created2019
Description
Nature is a master at organizing biomolecules in all intracellular processes, and researchers have conducted extensive research to understand the way enzymes interact with each other through spatial and orientation positioning, substrate channeling, compartmentalization, and more.
DNA nanostructures of high programmability and complexity provide excellent scaffolds to arrange multiple molecular/macromolecular components at nanometer scale to construct interactive biomolecular complexes and networks. Due to the sequence specificity at different positions of the DNA origami nanostructures, spatially addressable molecular pegboard with a resolution of several nm (less than 10 nm) can be achieved. So far, DNA nanostructures can be used to build nanodevices ranging from in vitro small molecule biosensing to sophisticated in vivo therapeutic drug delivery systems and multi-enzyme networks.
This thesis focuses on how to use DNA nanostructures as programmable biomolecular scaffolds to arranges enzymatic systems. Presented here are a series of studies toward this goal. First, we survey approaches used to generate protein-DNA conjugates and the use of structural DNA nanotechnology to engineer rationally designed nanostructures. Second, novel strategies for positioning enzymes on DNA nanoscaffolds has been developed and optimized, including site-specific/ non site-specific protein-DNA conjugation, purification and characterization. Third, an artificial swinging arm enzyme-DNA complex has been developed to mimic substrate channeling process. Finally, we extended to build a artificial 2D multi-enzyme network.
DNA nanostructures of high programmability and complexity provide excellent scaffolds to arrange multiple molecular/macromolecular components at nanometer scale to construct interactive biomolecular complexes and networks. Due to the sequence specificity at different positions of the DNA origami nanostructures, spatially addressable molecular pegboard with a resolution of several nm (less than 10 nm) can be achieved. So far, DNA nanostructures can be used to build nanodevices ranging from in vitro small molecule biosensing to sophisticated in vivo therapeutic drug delivery systems and multi-enzyme networks.
This thesis focuses on how to use DNA nanostructures as programmable biomolecular scaffolds to arranges enzymatic systems. Presented here are a series of studies toward this goal. First, we survey approaches used to generate protein-DNA conjugates and the use of structural DNA nanotechnology to engineer rationally designed nanostructures. Second, novel strategies for positioning enzymes on DNA nanoscaffolds has been developed and optimized, including site-specific/ non site-specific protein-DNA conjugation, purification and characterization. Third, an artificial swinging arm enzyme-DNA complex has been developed to mimic substrate channeling process. Finally, we extended to build a artificial 2D multi-enzyme network.
ContributorsYang, Yuhe Renee (Author) / Yan, Hao (Thesis advisor) / Liu, Yan (Thesis advisor) / Chen, Julian (Committee member) / Hayes, Mark (Committee member) / Arizona State University (Publisher)
Created2016
Description
DNA and DNA nanoassemblies such as DNA origamis have large potential in biosensing, drug delivery, nanoelectronic circuits, and biological computing requiring suitable methods for migration and precise positioning. Insulator-based dielectrophoresis (iDEP) provides an efficient and matrix-free approach for manipulation of micro-and nanometer-sized objects. In order to exploit iDEP for naturally formed DNA and DNA nanoassemblies, a detailed understanding of the underlying polarization and dielectrophoretic migration is essential. The shape and the counterion distribution are considered two essential factors in the polarization mechanism. Here, the dielectrophoretic behavior of 6-helix bundle (6HxB) and triangle DNA origamis with identical sequences but substantial topological differences was explored. The polarizability models were discussed for the two species according to their structural difference. The experimental observations reveal distinct iDEP trapping behavior in low frequency AC electric fields in addition to numerical simulations showing a considerable contribution of the electrophoretic transport of the DNA origami species in the DEP trapping regions. Furthermore, the polarizabilities of the two species were determined by measuring the migration times through a potential landscape exhibiting dielectrophoretic barriers. The resulting migration times correlate to the depth of the dielectrophoretic potential barrier and the escape characteristics of the DNA origamis according to an adapted Kramer’s rate model. The orientations of both species in the escape process were studied. Finally, to study the counterion distribution around the DNA molecules, both λ-DNA and 6HxB DNA were used in a phosphate buffer containing magnesium, revealing distinctive negative dielectrophoretic trapping behavior as opposed to positive trapping in a sodium/potassium phosphate buffer system.
ContributorsGan, Lin (Author) / Ros, Alexandra (Thesis advisor) / Buttry, Daniel (Committee member) / Liu, Yan (Committee member) / Arizona State University (Publisher)
Created2015
Description
Humanity’s demand for energy is increasing exponentially and the dependence on fossil fuels is both unsustainable and detrimental to the environment. To provide a solution to the impending energy crisis, it is reasonable to look toward utilizing solar energy, which is abundant and renewable. One approach to harvesting solar irradiation for fuel purposes is through mimicking the processes of natural photosynthesis in an artificial design to use sunlight and water to store energy in chemical bonds for later use. Thus, in order to design an efficient energy conversion device, the underlying processes of the natural system must be understood. An artificial photosynthetic device has many components and each can be optimized separately. This work deals with the design, construction and study of some of those components. The first chapter provides an introduction to this work. The second chapter shows a proof of concept for a water splitting dye sensitized photoelectrochemical cell followed by the presentation of a new p-type semiconductor, the design of a modular cluster binding protein that can be used for incorporating catalysts, and a new anchoring group for semiconducting oxides with high electron injection efficiency. The third chapter investigates the role of electronic coupling and thermodynamics for photoprotection in artificial systems by triplet-triplet energy transfer from tetrapyrroles to carotenoids. The fourth chapter describes a mimic of the proton-coupled electron transfer in photosystem II and confirms that in the artificial system a concerted mechanism operates. In the fifth chapter, a microbial system is designed to work in tandem with a photovoltaic device to produce high energy fuels. A variety of quinone redox mediators have been synthesized to shuttle electrons from an electron donor to the microbial system. Lastly, the synthesis of a variety of photosensitizers is detailed for possible future use in artificial systems. The results of this work helps with the understanding of the processes of natural photosynthesis and suggests ways to design artificial photosynthetic devices that can contribute to solving the renewable energy challenge.
ContributorsBrown, Chelsea L (Author) / Moore, Ana L (Thesis advisor) / Gust, Devens (Committee member) / Woodbury, Neal (Committee member) / Arizona State University (Publisher)
Created2015
Description
The manipulation of biological targets using synthetic compounds has been the focal point of medicinal chemistry. The work described herein centers on the synthesis of organic small molecules that act either as probes for studying protein conformational changes or DNA–protein interaction, or as multifunctional radical quenchers.
Fluorescent labeling is of paramount importance to biological studies of proteins. For the development of new extrinsic small fluorophores, a series of tryptophan analogues has been designed and synthesized. Their pdCpA derivatives have been synthesized for tRNA activation and in vitro protein synthesis. The photophysical properties of the tryptophan (Trp) analogues have been examined, some of which can be selectively monitored even in the presence of multiple native tryptophan residues. Further, some of the Trp analogues form efficient FRET pairs with acceptors such as acridon-2-ylalanine (Acd) or L-(7-hydroxycoumarin-4-yl)ethylglycine (HCO) for the selective study of conformational changes in proteins.
Molecules which can bind with high sequence selectivity to a chosen target in a gene sequence are of interest for the development of gene therapy, diagnostic devices for genetic analysis, and as molecular tools for nucleic acid manipulations. Stereoselective synthesis of different alanyl nucleobase amino acids is described. Their pdCpA derivatives have been synthesized for tRNA activation and site-specific incorporation into the DNA-binding protein RRM1 of hnRNP LL. It is proposed that the nucleobase moieties in the protein may specifically recognize base sequence in the i-motif DNA through H-bonding and base-stacking interactions.
The mitochondrial respiratory chain accumulates more oxidative damage than any other organelle within the cell. Dysfunction of this organelle is believed to drive the progression of many diseases, thus mitochondria are an important potential drug target. Reactive oxygen species (ROS) are generated when electrons from the respiratory chain escape and interact with oxygen. ROS can react with proteins, lipids or DNA causing cell death. For the development of effective neuroprotective drugs, a series of N-hydroxy-4-pyridones have been designed and synthesized as CoQ10 analogues. All the analogues synthesized were evaluated for their ability to quench lipid peroxidation and reactive oxygen species (ROS).
Fluorescent labeling is of paramount importance to biological studies of proteins. For the development of new extrinsic small fluorophores, a series of tryptophan analogues has been designed and synthesized. Their pdCpA derivatives have been synthesized for tRNA activation and in vitro protein synthesis. The photophysical properties of the tryptophan (Trp) analogues have been examined, some of which can be selectively monitored even in the presence of multiple native tryptophan residues. Further, some of the Trp analogues form efficient FRET pairs with acceptors such as acridon-2-ylalanine (Acd) or L-(7-hydroxycoumarin-4-yl)ethylglycine (HCO) for the selective study of conformational changes in proteins.
Molecules which can bind with high sequence selectivity to a chosen target in a gene sequence are of interest for the development of gene therapy, diagnostic devices for genetic analysis, and as molecular tools for nucleic acid manipulations. Stereoselective synthesis of different alanyl nucleobase amino acids is described. Their pdCpA derivatives have been synthesized for tRNA activation and site-specific incorporation into the DNA-binding protein RRM1 of hnRNP LL. It is proposed that the nucleobase moieties in the protein may specifically recognize base sequence in the i-motif DNA through H-bonding and base-stacking interactions.
The mitochondrial respiratory chain accumulates more oxidative damage than any other organelle within the cell. Dysfunction of this organelle is believed to drive the progression of many diseases, thus mitochondria are an important potential drug target. Reactive oxygen species (ROS) are generated when electrons from the respiratory chain escape and interact with oxygen. ROS can react with proteins, lipids or DNA causing cell death. For the development of effective neuroprotective drugs, a series of N-hydroxy-4-pyridones have been designed and synthesized as CoQ10 analogues. All the analogues synthesized were evaluated for their ability to quench lipid peroxidation and reactive oxygen species (ROS).
ContributorsTalukder, Poulami (Author) / Hecht, Sidney M. (Thesis advisor) / Woodbury, Neal (Committee member) / Gould, Ian (Committee member) / Arizona State University (Publisher)
Created2016
Description
Deoxyribonucleic acid (DNA) has emerged as an attractive building material for creating complex architectures at the nanometer scale that simultaneously affords versatility and modularity. Particularly, the programmability of DNA enables the assembly of basic building units into increasingly complex, arbitrary shapes or patterns. With the expanding complexity and functionality of DNA toolboxes, a quantitative understanding of DNA self-assembly in terms of thermodynamics and kinetics, will provide researchers with more subtle design guidelines that facilitate more precise spatial and temporal control. This dissertation focuses on studying the physicochemical properties of DNA tile-based self-assembly process by recapitulating representative scenarios and intermediate states with unique assembly pathways.
First, DNA double-helical tiles with increasing flexibility were designed to investigate the dimerization kinetics. The higher dimerization rates of more rigid tiles result from the opposing effects of higher activation energies and higher pre-exponential factors from the Arrhenius equation, where the pre-exponential factor dominates. Next, the thermodynamics and kinetics of single tile attachment to preformed “multitile” arrays were investigated to test the fundamental assumptions of tile assembly models. The results offer experimental evidences that double crossover tile attachment is determined by the electrostatic environment and the steric hindrance at the binding site. Finally, the assembly of double crossover tiles within a rhombic DNA origami frame was employed as the model system to investigate the competition between unseeded, facet and seeded nucleation. The results revealed that preference of nucleation types can be tuned by controlling the rate-limiting nucleation step.
The works presented in this dissertation will be helpful for refining the DNA tile assembly model for future designs and simulations. Moreover, The works presented here could also be helpful in understanding how individual molecules interact and more complex cooperative bindings in chemistry and biology. The future direction will focus on the characterization of tile assembly at single molecule level and the development of error-free tile assembly systems.
First, DNA double-helical tiles with increasing flexibility were designed to investigate the dimerization kinetics. The higher dimerization rates of more rigid tiles result from the opposing effects of higher activation energies and higher pre-exponential factors from the Arrhenius equation, where the pre-exponential factor dominates. Next, the thermodynamics and kinetics of single tile attachment to preformed “multitile” arrays were investigated to test the fundamental assumptions of tile assembly models. The results offer experimental evidences that double crossover tile attachment is determined by the electrostatic environment and the steric hindrance at the binding site. Finally, the assembly of double crossover tiles within a rhombic DNA origami frame was employed as the model system to investigate the competition between unseeded, facet and seeded nucleation. The results revealed that preference of nucleation types can be tuned by controlling the rate-limiting nucleation step.
The works presented in this dissertation will be helpful for refining the DNA tile assembly model for future designs and simulations. Moreover, The works presented here could also be helpful in understanding how individual molecules interact and more complex cooperative bindings in chemistry and biology. The future direction will focus on the characterization of tile assembly at single molecule level and the development of error-free tile assembly systems.
ContributorsJiang, Shuoxing (Author) / Yan, Hao (Thesis advisor) / Liu, Yan (Thesis advisor) / Hayes, Mark (Committee member) / Wang, Xu (Committee member) / Arizona State University (Publisher)
Created2016
Description
Sunlight, the most abundant source of energy available, is diffuse and intermittent; therefore it needs to be stored in chemicals bonds in order to be used any time. Photosynthesis converts sunlight into useful chemical energy that organisms can use for their functions. Artificial photosynthesis aims to use the essential chemistry of natural photosynthesis to harvest solar energy and convert it into fuels such as hydrogen gas. By splitting water, tandem photoelectrochemical solar cells (PESC) can produce hydrogen gas, which can be stored and used as fuel. Understanding the mechanisms of photosynthesis, such as photoinduced electron transfer, proton-coupled electron transfer (PCET) and energy transfer (singlet-singlet and triplet-triplet) can provide a detailed knowledge of those processes which can later be applied to the design of artificial photosynthetic systems. This dissertation has three main research projects. The first part focuses on design, synthesis and characterization of suitable photosensitizers for tandem cells. Different factors that can influence the performance of the photosensitizers in PESC and the attachment and use of a biomimetic electron relay to a water oxidation catalyst are explored. The second part studies PCET, using Nuclear Magnetic Resonance and computational chemistry to elucidate the structure and stability of tautomers that comprise biomimetic electron relays, focusing on the formation of intramolecular hydrogen bonds. The third part of this dissertation uses computational calculations to understand triplet-triplet energy transfer and the mechanism of quenching of the excited singlet state of phthalocyanines in antenna models by covalently attached carotenoids.
ContributorsTejeda Ferrari, Marely (Author) / Moore, Ana (Thesis advisor) / Mujica, Vladimiro (Thesis advisor) / Gust, John (Committee member) / Woodbury, Neal (Committee member) / Arizona State University (Publisher)
Created2016
Description
Colloidal quantum dots (QDs) or semiconductor nanocrystals are often used to describe 2 to 20 nm solution processed nanoparticles of various semiconductor materials that display quantum confinement effects. Compared to traditional fluorescent organic dyes, QDs provide many advantages. For biological applications it is necessary to develop reliable methods to functionalize QDs with hydrophilic biomolecules so that they may maintain their stability and functionality in physiological conditions. DNA, a molecule that encodes genetic information, is arguably the smartest molecule that nature has ever produced and one of the most explored bio-macromolecules. DNA directed self-assembly can potentially organize QDs that are functionalized with DNA with nanometer precision, and the resulting arrangements may facilitate the display of novel optical properties. The goal of this dissertation was to achieve a robust reliable yet simple strategy to link DNA to QDs so that they can be used for DNA directed self assembly by which we can engineer their optical properties. Presented here is a series of studies to achieve this goal. First we demonstrate the aqueous synthesis of colloidal nanocrystal heterostructures consisting of the CdTe core encapsulated by CdS/ZnS or CdSe/ZnS shells using glutathione (GSH), a tripeptide, as the capping ligand. We next employed this shell synthesis strategy to conjugate PS-PO chimeric DNA to QDs at the time of shell synthesis. We synthesized a library of DNA linked QDs emitting from UV to near IR that are very stable in high salt concentrations. These DNA functionalized QDs were further site-specifically organized on DNA origami in desired patterns directed by DNA self-assembly. We further extended our capability to functionalize DNA to real IR emitting CdxPb1-xTe alloyed QDs, and demonstrated their stability by self-assembling them on DNA origami. The photo-physical properties of the QDs were further engineered by attaching a QD and a gold nanoparticle in controlled distances on the same DNA origami, which revealed a much longer range quenching effect than usual Forster Resonance Energy Transfer. We are currently engaged in enhancing photoluminescence intensity of the QDs by bringing them in the plasmonic hot spots generated by cluster of larger plasmonic nanoparticles.
ContributorsSamanta, Anirban (Author) / Yan, Hao (Thesis advisor) / Liu, Yan (Thesis advisor) / Buttry, Daniel (Committee member) / Gould, Ian (Committee member) / Arizona State University (Publisher)
Created2014
Description
DNA is a unique, highly programmable and addressable biomolecule. Due to its reliable and predictable base recognition behavior, uniform structural properties, and extraordinary stability, DNA molecules are desirable substrates for biological computation and nanotechnology. The field of DNA computation has gained considerable attention due to the possibility of exploiting the massive parallelism that is inherent in natural systems to solve computational problems. This dissertation focuses on building novel types of computational DNA systems based on both DNA reaction networks and DNA nanotechnology. A series of related research projects are presented here. First, a novel, three-input majority logic gate based on DNA strand displacement reactions was constructed. Here, the three inputs in the majority gate have equal priority, and the output will be true if any two of the inputs are true. We subsequently designed and realized a complex, 5-input majority logic gate. By controlling two of the five inputs, the complex gate is capable of realizing every combination of OR and AND gates of the other 3 inputs. Next, we constructed a half adder, which is a basic arithmetic unit, from DNA strand operated XOR and AND gates. The aim of these two projects was to develop novel types of DNA logic gates to enrich the DNA computation toolbox, and to examine plausible ways to implement large scale DNA logic circuits. The third project utilized a two dimensional DNA origami frame shaped structure with a hollow interior where DNA hybridization seeds were selectively positioned to control the assembly of small DNA tile building blocks. The small DNA tiles were directed to fill the hollow interior of the DNA origami frame, guided through sticky end interactions at prescribed positions. This research shed light on the fundamental behavior of DNA based self-assembling systems, and provided the information necessary to build programmed nanodisplays based on the self-assembly of DNA.
ContributorsLi, Wei (Author) / Yan, Hao (Thesis advisor) / Liu, Yan (Thesis advisor) / Chen, Julian (Committee member) / Gould, Ian (Committee member) / Arizona State University (Publisher)
Created2014