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- All Subjects: DNA
- Creators: Lindsay, Stuart
Description
Solution conformations and dynamics of proteins and protein-DNA complexes are often difficult to predict from their crystal structures. The crystal structure only shows a snapshot of the different conformations these biological molecules can have in solution. Multiple different conformations can exist in solution and potentially have more importance in the biological activity. DNA sliding clamps are a family of proteins with known crystal structures. These clamps encircle the DNA and enable other proteins to interact more efficiently with the DNA. Eukaryotic PCNA and prokaryotic β clamp are two of these clamps, some of the most stable homo-oligomers known. However, their solution stability and conformational equilibrium have not been investigated in depth before. Presented here are the studies involving two sliding clamps: yeast PCNA and bacterial β clamp. These studies show that the β clamp has a very different solution stability than PCNA. These conclusions were reached through various different fluorescence-based experiments, including fluorescence correlation spectroscopy (FCS), Förster resonance energy transfer (FRET), single molecule fluorescence, and various time resolved fluorescence techniques. Interpretations of these, and all other, fluorescence-based experiments are often affected by the properties of the fluorophores employed. Often the fluorescence properties of these fluorophores are influenced by their microenvironments. Fluorophores are known to sometimes interact with biological molecules, and this can have pronounced effects on the rotational mobility and photophysical properties of the dye. Misunderstanding the effect of these photophysical and rotational properties can lead to a misinterpretation of the obtained data. In this thesis, photophysical behaviors of various organic dyes were studied in the presence of deoxymononucleotides to examine more closely how interactions between fluorophores and DNA bases can affect fluorescent properties. Furthermore, the properties of cyanine dyes when bound to DNA and the effect of restricted rotation on FRET are presented in this thesis. This thesis involves studying fluorophore photophysics in various microenvironments and then expanding into the solution stability and dynamics of the DNA sliding clamps.
ContributorsRanjit, Suman (Author) / Levitus, Marcia (Thesis advisor) / Lindsay, Stuart (Committee member) / Yan, Hao (Committee member) / Arizona State University (Publisher)
Created2013
Description
This thesis describes several experiments based on carbon nanotube nanofludic devices and field-effect transistors. The first experiment detected ion and molecule translocation through one single-walled carbon nanotube (SWCNT) that spans a barrier between two fluid reservoirs. The electrical ionic current is measured. Translocation of small single stranded DNA oligomers is marked by large transient increases in current through the tube and confirmed by a PCR (polymerase chain reaction) analysis. Carbon nanotubes simplify the construction of nanopores, permit new types of electrical measurement, and open new avenues for control of DNA translocation. The second experiment constructed devices in which the interior of a single-walled carbon nanotube field-effect transistor (CNT-FET) acts as a nanofluidic channel that connects two fluid reservoirs, permitting measurement of the electronic properties of the SWCNT as it is wetted by an analyte. Wetting of the inside of the SWCNT by water turns the transistor on, while wetting of the outside has little effect. This finding may provide a new method to investigate water behavior at nanoscale. This also opens a new avenue for building sensors in which the SWCNT functions as an electronic detector. This thesis also presents some experiments that related to nanofabrication, such as construction of FET with tin sulfide (SnS) quantum ribbon. This work demonstrates the application of solution processed IV-VI semiconductor nanostructures in nanoscale devices.
ContributorsCao, Zhai (Author) / Lindsay, Stuart (Thesis advisor) / Vaiana, Sara (Committee member) / Ros, Robert (Committee member) / Marzke, Robert (Committee member) / Shumway, John (Committee member) / Arizona State University (Publisher)
Created2011
Description
Single molecules in a tunnel junction can now be interrogated reliably using chemically-functionalized electrodes. Monitoring stochastic bonding fluctuations between a ligand bound to one electrode and its target bound to a second electrode ("tethered molecule-pair" configuration) gives insight into the nature of the intermolecular bonding at a single molecule-pair level, and defines the requirements for reproducible tunneling data. Importantly, at large tunnel gaps, there exists a regime for many molecules in which the tunneling is influenced more by the chemical identity of the molecules than by variability in the molecule-metal contact. Functionalizing a pair of electrodes with recognition reagents (the "free analyte" configuration) can generate a distinct tunneling signal when an analyte molecule is trapped in the gap. This opens up a new interface between chemistry and electronics with immediate implications for rapid sequencing of single DNA molecules.
ContributorsChang, Shuai (Author) / Lindsay, Stuart (Thesis advisor) / Ros, Robert (Committee member) / Zhang, Peiming (Committee member) / Tao, Nongjian (Committee member) / Shumway, John (Committee member) / Arizona State University (Publisher)
Created2012
Description
CpG methylation is an essential requirement for the normal development of mammals, but aberrant changes in the methylation can lead to tumor progression and cancer. An in-depth understanding of this phenomenon can provide insights into the mechanism of gene repression. We present a study comparing methylated DNA and normal DNA wrt its persistence length and contour length. Although, previous experiments and studies show no difference between the physical properties of the two, the data collected and interpreted here gives a different picture to the methylation phenomena and its effect on gene silencing. The study was extended to the artificially reconstituted chromatin and its interactions with the methyl CpG binding proteins were also probed.
ContributorsKaur, Parminder (Author) / Lindsay, Stuart (Thesis advisor) / Ros, Robert (Committee member) / Tao, Nongjian (Committee member) / Vaiana, Sara (Committee member) / Beckenstein, Oliver (Committee member) / Arizona State University (Publisher)
Created2012
Description
DNA, RNA and Protein are three pivotal biomolecules in human and other organisms, playing decisive roles in functionality, appearance, diseases development and other physiological phenomena. Hence, sequencing of these biomolecules acquires the prime interest in the scientific community. Single molecular identification of their building blocks can be done by a technique called Recognition Tunneling (RT) based on Scanning Tunneling Microscope (STM). A single layer of specially designed recognition molecule is attached to the STM electrodes, which trap the targeted molecules (DNA nucleoside monophosphates, RNA nucleoside monophosphates or amino acids) inside the STM nanogap. Depending on their different binding interactions with the recognition molecules, the analyte molecules generate stochastic signal trains accommodating their “electronic fingerprints”. Signal features are used to detect the molecules using a machine learning algorithm and different molecules can be identified with significantly high accuracy. This, in turn, paves the way for rapid, economical nanopore sequencing platform, overcoming the drawbacks of Next Generation Sequencing (NGS) techniques.
To read DNA nucleotides with high accuracy in an STM tunnel junction a series of nitrogen-based heterocycles were designed and examined to check their capabilities to interact with naturally occurring DNA nucleotides by hydrogen bonding in the tunnel junction. These recognition molecules are Benzimidazole, Imidazole, Triazole and Pyrrole. Benzimidazole proved to be best among them showing DNA nucleotide classification accuracy close to 99%. Also, Imidazole reader can read an abasic monophosphate (AP), a product from depurination or depyrimidination that occurs 10,000 times per human cell per day.
In another study, I have investigated a new universal reader, 1-(2-mercaptoethyl)pyrene (Pyrene reader) based on stacking interactions, which should be more specific to the canonical DNA nucleosides. In addition, Pyrene reader showed higher DNA base-calling accuracy compare to Imidazole reader, the workhorse in our previous projects. In my other projects, various amino acids and RNA nucleoside monophosphates were also classified with significantly high accuracy using RT. Twenty naturally occurring amino acids and various RNA nucleosides (four canonical and two modified) were successfully identified. Thus, we envision nanopore sequencing biomolecules using Recognition Tunneling (RT) that should provide comprehensive betterment over current technologies in terms of time, chemical and instrumental cost and capability of de novo sequencing.
To read DNA nucleotides with high accuracy in an STM tunnel junction a series of nitrogen-based heterocycles were designed and examined to check their capabilities to interact with naturally occurring DNA nucleotides by hydrogen bonding in the tunnel junction. These recognition molecules are Benzimidazole, Imidazole, Triazole and Pyrrole. Benzimidazole proved to be best among them showing DNA nucleotide classification accuracy close to 99%. Also, Imidazole reader can read an abasic monophosphate (AP), a product from depurination or depyrimidination that occurs 10,000 times per human cell per day.
In another study, I have investigated a new universal reader, 1-(2-mercaptoethyl)pyrene (Pyrene reader) based on stacking interactions, which should be more specific to the canonical DNA nucleosides. In addition, Pyrene reader showed higher DNA base-calling accuracy compare to Imidazole reader, the workhorse in our previous projects. In my other projects, various amino acids and RNA nucleoside monophosphates were also classified with significantly high accuracy using RT. Twenty naturally occurring amino acids and various RNA nucleosides (four canonical and two modified) were successfully identified. Thus, we envision nanopore sequencing biomolecules using Recognition Tunneling (RT) that should provide comprehensive betterment over current technologies in terms of time, chemical and instrumental cost and capability of de novo sequencing.
ContributorsSen, Suman (Author) / Lindsay, Stuart (Thesis advisor) / Zhang, Peiming (Thesis advisor) / Gould, Ian R. (Committee member) / Borges, Chad (Committee member) / Arizona State University (Publisher)
Created2016
Description
For reading DNA bases more accurately, a series of nitrogen-containing aromatic heterocycles have been designed and synthesized as candidates of universal reader to interact with all naturally occurring DNA nucleobases by hydrogen bonding interaction and eventually is used to read DNA by recognition tunneling. These recognition molecules include 6-mercapto-1H-benzo[d]imidazole-2-carboxamide, 5-(2-mercaptoethyl)-1H-imidazole-2-carboxamide, 5-(2-mercaptoethyl)-4H-1,2,4-traizole-3-carboxamide and 1-(2-mercaptoethyl)-1H-pyrrole-3-carboxamide. Their formation of hydrogen bonding complexes with nucleobases was studied and association constants were measured by proton NMR titration experiments in deuterated chloroform at room temperature. To do so, the mercaptoethyl chain or thiol group of these reading molecules was replaced or protected with the more lipophilic group to increase the solubility of these candidates in CDCl3. The 3' and 5' hydroxyl groups of deoxyadenosine (dA), deoxyguanosine (dG), deoxycytidine (dC) and thymidine (dT) were protected with tert-butyldimethylsilyl (TBDMS) to eliminate hydrogen bonding competition from the hydroxyl protons with these candidates as well as to increase the solubility of the nucleosides in CDCl3 for NMR titration experiment. Benzimidazole and imidazole containing readers exhibited the strongest H-bonding affinity towards DNA bases where pyrrole containing reader showed the weakest affinity. In all cases, dG revealed the strongest affinity towards the readers while dA showed the least.
The molecular complex formation in aqueous solution was studied by electrospray ionization mass spectrometry (ESI-MS) and tandem mass spectrometry. The formation of both 1:1 and 2:1 complexes between one or two reading molecules and a DNA nucleotide were observed by ESI mass. A series of amino acids and carbohydrates were also examined by mass spectrometry to show the formation of non-covalent complexes with imidazole reader in aqueous solution. The experimental results were compared by calculating energies of ground state conformers of individual molecules and their complexes using computer modeling study by DFT calculations. These studies give insights into the molecular interactions that happen in a nanogap during recognition tunneling experiments.
The molecular complex formation in aqueous solution was studied by electrospray ionization mass spectrometry (ESI-MS) and tandem mass spectrometry. The formation of both 1:1 and 2:1 complexes between one or two reading molecules and a DNA nucleotide were observed by ESI mass. A series of amino acids and carbohydrates were also examined by mass spectrometry to show the formation of non-covalent complexes with imidazole reader in aqueous solution. The experimental results were compared by calculating energies of ground state conformers of individual molecules and their complexes using computer modeling study by DFT calculations. These studies give insights into the molecular interactions that happen in a nanogap during recognition tunneling experiments.
ContributorsBiswas, Sovan (Author) / Lindsay, Stuart (Thesis advisor) / Zhang, Peiming (Thesis advisor) / Borges, Chad (Committee member) / Gould, Ian (Committee member) / Arizona State University (Publisher)
Created2016
Description
Charge transport in molecular systems, including DNA (Deoxyribonucleic acid), is involved in many basic chemical and biological processes. Studying their charge transport properties can help developing DNA based electronic devices with many tunable functionalities. This thesis investigates the electric properties of double-stranded DNA, DNA G-quadruplex and dsDNA with modified base.
First, double-stranded DNA with alternating GC sequence and stacked GC sequence were measured with respect to length. The resistance of DNA sequences increases linearly with length, indicating a hopping transport mechanism. However, for DNA sequences with stacked GC, a periodic oscillation is superimposed on the linear length dependence, indicating a partial coherent transport. The result is supported by the finding of delocalization of the highest occupied molecular orbitals of Guanines from theoretical simulation and by fitting based on the Büttiker’s theory.
Then, a DNA G4-duplex structures with a G-quadruplex as the core and DNA duplexes as the arms were studied. Similar conductance values were observed by varying the linker positions, thus a charge splitter is developed. The conductance of the DNA G-tetrads structures was found to be sensitive to the π-stacking at the interface between the G-quadruplex and DNA duplexes by observing a higher conductance value when one duplex was removed and a polyethylene glycol (PEG) linker was added into the interface. This was further supported by molecular dynamic simulations.
Finally, a double-stranded DNA with one of the bases replaced by an anthraquinone group was studied via electrochemical STM break junction technique. Anthraquinone can be reversibly switched into the oxidized state or reduced state, to give a low conductance or high conductance respectively. Furthermore, the thermodynamics and kinetics properties of the switching were systematically studied. Theoretical simulation shows that the difference between the two states is due to a difference in the energy alignment with neighboring Guanine bases.
First, double-stranded DNA with alternating GC sequence and stacked GC sequence were measured with respect to length. The resistance of DNA sequences increases linearly with length, indicating a hopping transport mechanism. However, for DNA sequences with stacked GC, a periodic oscillation is superimposed on the linear length dependence, indicating a partial coherent transport. The result is supported by the finding of delocalization of the highest occupied molecular orbitals of Guanines from theoretical simulation and by fitting based on the Büttiker’s theory.
Then, a DNA G4-duplex structures with a G-quadruplex as the core and DNA duplexes as the arms were studied. Similar conductance values were observed by varying the linker positions, thus a charge splitter is developed. The conductance of the DNA G-tetrads structures was found to be sensitive to the π-stacking at the interface between the G-quadruplex and DNA duplexes by observing a higher conductance value when one duplex was removed and a polyethylene glycol (PEG) linker was added into the interface. This was further supported by molecular dynamic simulations.
Finally, a double-stranded DNA with one of the bases replaced by an anthraquinone group was studied via electrochemical STM break junction technique. Anthraquinone can be reversibly switched into the oxidized state or reduced state, to give a low conductance or high conductance respectively. Furthermore, the thermodynamics and kinetics properties of the switching were systematically studied. Theoretical simulation shows that the difference between the two states is due to a difference in the energy alignment with neighboring Guanine bases.
ContributorsXiang, Liming (Author) / Tao, Nongjian (Thesis advisor) / Lindsay, Stuart (Committee member) / Gould, Ian (Committee member) / Arizona State University (Publisher)
Created2016
Description
Emerging pathogens present several challenges to medical diagnostics. Primarily, the exponential spread of a novel pathogen through naïve populations require a rapid and overwhelming diagnostic response at the site of outbreak. While point-of-care (PoC) platforms have been developed for detection of antigens, serologic responses, and pathogenic genomes, only nucleic acid diagnostics currently have the potential to be developed and manufactured within weeks of an outbreak owing to the speed of next-generation sequencing and custom DNA synthesis. Among nucleic acid diagnostics, isothermal amplification strategies are uniquely suited for PoC implementation due to their simple instrumentation and lack of thermocycling requirement. Unfortunately, isothermal strategies are currently prone to spurious nonspecific amplification, hindering their specificity and necessitating extensive empirical design pipelines that are both time and resource intensive. In this work, isothermal amplification strategies are extensively compared for their feasibility of implementation in outbreak response scenarios. One such technology, Loop-mediated Amplification (LAMP), is identified as having high-potential for rapid development and PoC deployment. Various approaches to abrogating nonspecific amplification are described including a novel in silico design tool based on coarse-grained simulation of interactions between thermophilic DNA polymerase and DNA strands in isothermal reaction conditions. Nonspecific amplification is shown to be due to stabilization of primer secondary structures by high concentrations of Bst DNA polymerase and a mechanism of micro-complement-mediated cross-priming is demonstrated as causal via nanopore sequencing of nonspecific reaction products. The resulting computational model predicts primer set background in 64% of 67 test assays and its usefulness is illustrated further by determining problematic primers in a West Nile Virus-specific LAMP primer set and optimizing primer 3’ nucleotides to eliminate micro-complements within the reaction, resulting in inhibition of background accumulation. Finally, the emergence of Orthopox monkeypox (MPXV) as a recurring threat is discussed and SimCycle is utilized to develop a novel technique for clade-specific discrimination of MPXV based on bridging viral genomic rearrangements (Bridging LAMP). Bridging LAMP is implemented in a 4-plex microfluidic format and demonstrates 100% sensitivity in detection of 100 copies of viral lysates and 45 crude MPXV-positive patient samples collected during the 2022 Clade IIb outbreak.
ContributorsKnappenberger, Mark Daniel (Author) / Anderson, Karen S (Thesis advisor) / LaBaer, Joshua (Committee member) / Roberson, Robert (Committee member) / Lindsay, Stuart (Committee member) / Arizona State University (Publisher)
Created2023
Description
My research centers on the design and fabrication of biomolecule-sensing devices that combine top-down and bottom-up fabrication processes and leverage the unique advantages of each approach. This allows for the scalable creation of devices with critical dimensions and surface properties that are tailored to target molecules at the nanoscale.
My first project focuses on a new strategy for preparing solid-state nanopore sensors for DNA sequencing. Challenges for existing nanopore approaches include specificity of detection, controllability of translocation, and scalability of fabrication. In a new solid-state pore architecture, top-down fabrication of an initial electrode gap embedded in a sealed nanochannel is followed by feedback-controlled electrochemical deposition of metal to shrink the gap and define the nanopore size. The resulting structure allows for the use of an electric field to control the motion of DNA through the pore and the direct detection of a tunnel current through a DNA molecule.
My second project focuses on top-down fabrication strategies for a fixed nanogap device to explore the electronic conductance of proteins. Here, a metal-insulator-metal junction can be fabricated with top-down fabrication techniques, and the subsequent electrode surfaces can be chemically modified with molecules that bind strongly to a target protein. When proteins bind to molecules on either side of the dielectric gap, a molecular junction is formed with observed conductances on the order of nanosiemens. These devices can be used in applications such as DNA sequencing or to gain insight into fundamental questions such as the mechanism of electron transport in proteins.
My first project focuses on a new strategy for preparing solid-state nanopore sensors for DNA sequencing. Challenges for existing nanopore approaches include specificity of detection, controllability of translocation, and scalability of fabrication. In a new solid-state pore architecture, top-down fabrication of an initial electrode gap embedded in a sealed nanochannel is followed by feedback-controlled electrochemical deposition of metal to shrink the gap and define the nanopore size. The resulting structure allows for the use of an electric field to control the motion of DNA through the pore and the direct detection of a tunnel current through a DNA molecule.
My second project focuses on top-down fabrication strategies for a fixed nanogap device to explore the electronic conductance of proteins. Here, a metal-insulator-metal junction can be fabricated with top-down fabrication techniques, and the subsequent electrode surfaces can be chemically modified with molecules that bind strongly to a target protein. When proteins bind to molecules on either side of the dielectric gap, a molecular junction is formed with observed conductances on the order of nanosiemens. These devices can be used in applications such as DNA sequencing or to gain insight into fundamental questions such as the mechanism of electron transport in proteins.
ContributorsSadar, Joshua Stephen (Author) / Qing, Quan (Thesis advisor) / Lindsay, Stuart (Committee member) / Vaiana, Sara (Committee member) / Ros, Robert (Committee member) / Arizona State University (Publisher)
Created2019
Description
Driven by the curiosity for the secret of life, the effort on sequencing of DNAs and other large biopolymers has never been respited. Advanced from recent sequencing techniques, nanotube and nanopore based sequencing has been attracting much attention. This thesis focuses on the study of first and crucial compartment of the third generation sequencing technique, the capture and translocation of biopolymers, and discuss the advantages and obstacles of two different nanofluidic pathways, nanotubes and nanopores for single molecule capturing and translocation. Carbon nanotubes with its constrained structure, the frictionless inner wall and strong electroosmotic flow, are promising materials for linearly threading DNA and other biopolymers for sequencing. Solid state nanopore on the other hand, is a robust chemical, thermal and mechanical stable nanofluidic device, which has a high capturing rate and, to some extent, good controllable threading ability for DNA and other biomolecules. These two different but similar nanofluidic pathways both provide a good preparation of analyte molecules for the sequencing purpose. In addition, more and more research interests have move onto peptide chains and protein sensing. For proteome is better and more direct indicators for human health, peptide chains and protein sensing have a much wider range of applications on bio-medicine, disease early diagnoses, and etc. A universal peptide chain nanopore sensing technique with universal chemical modification of peptides is discussed in this thesis as well, which unifies the nanopore capturing process for vast varieties of peptides. Obstacles of these nanofluidic pathways are also discussed. In the end of this thesis, a proposal of integration of solid state nanopore and fixed-gap recognition tunneling sequencing technique for a more accurate DNA and peptide readout is discussed, together with some early study work, which gives a new direction for nanopore based sequencing.
ContributorsSong, Weisi (Author) / Lindsay, Stuart (Thesis advisor) / Ros, Robert (Committee member) / Qing, Quan (Committee member) / Zhang, Peiming (Committee member) / Arizona State University (Publisher)
Created2015