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- All Subjects: Chemistry
- Creators: Stephanopoulos, Nicholas
- Resource Type: Text
Description
Multivalency is an important phenomenon that guides numerous biological interactions. It has been utilized in design of therapeutics and drug candidates. Hence, this study attempts to develop analytical tools to study multivalent interactions and design multivalent ligands for drug delivery and therapeutic applications.
Atomic Force Microscopy (AFM) has been envisioned as a means of nanodiagnostics due to its single molecule sensitivity. However, the AFM based recognition imaging lacks a multiplex capacity to detect multiple analytes in a single test. Also there is no user friendly wet chemistry to functionalize AFM tips. Hence, an uncatalyzed Click Chemistry protocol was developed to functionalize AFM tips. For multiplexed recognition imaging, recognition heads based on a C3 symmetrical three arm linker with azide functionalities at its ends were synthesized and the chemistry to attach them to AFM tips was developed, and these recognition heads were used in detecting multiple proteins simultaneously using AFM.
A bis-Angiopeptide-2 conjugate with this three-arm linker was synthesized and this was conjugated with anti-West Nile virus antibody E16 site specifically to target advanced West Nile virus infection in the Central Nervous System. The bis-Angiopeptide-2 conjugate of the antibody shows higher efficacy compared to a linear linker-Angiopeptide-2 conjugate of the antibody in in vitro studies and currently the efficacy of this antibody conjugate in studied in mice. Surface Plasmon Resonance imaging (SPRi) results indicate that the conjugation does not affect the antigen binding activity of the antibody very significantly.
A Y-shaped bisbiotin ligand was also prepared as a small sized antibody mimic. Compared to a monovalent biotin ligand, the y-Bisbiotin can cooperatively form a significantly more stable complex with streptavidin through intramolecular bivalent interactions, which were demonstrated by gel electrophoresis, SPR and AFM. Continuing on these lines, a four-arm linker was synthesized containing three single chain variable fragments (scFv) linked to the scaffold to form a tripod base, which would allow them to concomitantly interact with a trimeric Glycoprotein (GP) spike that has a “chalice” configuration. Meanwhile, a human IgG1 Fc is to be installed on the top of the tetrahedron, exerting effector functions of a monoclonal antibody.
Atomic Force Microscopy (AFM) has been envisioned as a means of nanodiagnostics due to its single molecule sensitivity. However, the AFM based recognition imaging lacks a multiplex capacity to detect multiple analytes in a single test. Also there is no user friendly wet chemistry to functionalize AFM tips. Hence, an uncatalyzed Click Chemistry protocol was developed to functionalize AFM tips. For multiplexed recognition imaging, recognition heads based on a C3 symmetrical three arm linker with azide functionalities at its ends were synthesized and the chemistry to attach them to AFM tips was developed, and these recognition heads were used in detecting multiple proteins simultaneously using AFM.
A bis-Angiopeptide-2 conjugate with this three-arm linker was synthesized and this was conjugated with anti-West Nile virus antibody E16 site specifically to target advanced West Nile virus infection in the Central Nervous System. The bis-Angiopeptide-2 conjugate of the antibody shows higher efficacy compared to a linear linker-Angiopeptide-2 conjugate of the antibody in in vitro studies and currently the efficacy of this antibody conjugate in studied in mice. Surface Plasmon Resonance imaging (SPRi) results indicate that the conjugation does not affect the antigen binding activity of the antibody very significantly.
A Y-shaped bisbiotin ligand was also prepared as a small sized antibody mimic. Compared to a monovalent biotin ligand, the y-Bisbiotin can cooperatively form a significantly more stable complex with streptavidin through intramolecular bivalent interactions, which were demonstrated by gel electrophoresis, SPR and AFM. Continuing on these lines, a four-arm linker was synthesized containing three single chain variable fragments (scFv) linked to the scaffold to form a tripod base, which would allow them to concomitantly interact with a trimeric Glycoprotein (GP) spike that has a “chalice” configuration. Meanwhile, a human IgG1 Fc is to be installed on the top of the tetrahedron, exerting effector functions of a monoclonal antibody.
ContributorsManna, Saikat (Author) / Lindsay, Stuart (Thesis advisor) / Zhang, Peiming (Thesis advisor) / Gould, Ian (Committee member) / Stephanopoulos, Nicholas (Committee member) / Arizona State University (Publisher)
Created2016
Description
The two chapters of this thesis focus on different aspects of DNA and the properties of nucleic acids as the whole. Chapter 1 focuses on the structure of DNA and its relationship to enzymatic efficiency. Chapter 2 centers itself on threose nucleic acid and optimization of a step in the path to its synthesis. While Chapter 1 discusses DNA and Uracil-DNA Glycosylase with regards to the base excision repair pathway, Chapter 2 focuses on chemical synthesis of an intermediate in the pathway to the synthesis of TNA, an analogous structure with a different saccharide in the sugar-phosphate backbone.
Chapter 1 covers the research under Dr. Levitus. Four oligonucleotides were reacted for zero, five, and thirty minutes with uracil-DNA glycosylase and subsequent addition of piperidine. These oligonucleotides were chosen based on their torsional rigidities as predicted by past research and predictions. The objective was to better understand the relationship between the sequence of DNA surrounding the incorrect base and the enzyme’s ability to remove said base in order to prepare the DNA for the next step of the base excision repair pathway. The first pair of oligonucleotides showed no statistically significant difference in enzymatic efficiency with p values of 0.24 and 0.42, while the second pair had a p value of 0.01 at the five-minute reaction. The second pair is currently being researched at different reaction times to determine at what point the enzyme seems to equilibrate and react semi-equally with all sequences of DNA.
Chapter 2 covers the research conducted under Dr. Chaput. Along the TNA synthesis pathway, the nitrogenous base must be added to the threofuranose sugar. The objective was to optimize the original protocol of Vorbrüggen glycosylation and determine if there were better conditions for the synthesis of the preferred regioisomer. This research showed that toluene and ortho-xylene were more preferable as solvents than the original anhydrous acetonitrile, as the amount of preferred isomer product far outweighed the amount of side product formed, as well as improving total yield overall. The anhydrous acetonitrile reaction had a final yield of 60.61% while the ortho-xylene system had a final yield of 94.66%, an increase of approximately 32%. The crude ratio of preferred isomer to side product was also improved, as it went from 18% undesired in anhydrous acetonitrile to 4% undesired in ortho-xylene, both values normalized to the preferred regioisomer.
Chapter 1 covers the research under Dr. Levitus. Four oligonucleotides were reacted for zero, five, and thirty minutes with uracil-DNA glycosylase and subsequent addition of piperidine. These oligonucleotides were chosen based on their torsional rigidities as predicted by past research and predictions. The objective was to better understand the relationship between the sequence of DNA surrounding the incorrect base and the enzyme’s ability to remove said base in order to prepare the DNA for the next step of the base excision repair pathway. The first pair of oligonucleotides showed no statistically significant difference in enzymatic efficiency with p values of 0.24 and 0.42, while the second pair had a p value of 0.01 at the five-minute reaction. The second pair is currently being researched at different reaction times to determine at what point the enzyme seems to equilibrate and react semi-equally with all sequences of DNA.
Chapter 2 covers the research conducted under Dr. Chaput. Along the TNA synthesis pathway, the nitrogenous base must be added to the threofuranose sugar. The objective was to optimize the original protocol of Vorbrüggen glycosylation and determine if there were better conditions for the synthesis of the preferred regioisomer. This research showed that toluene and ortho-xylene were more preferable as solvents than the original anhydrous acetonitrile, as the amount of preferred isomer product far outweighed the amount of side product formed, as well as improving total yield overall. The anhydrous acetonitrile reaction had a final yield of 60.61% while the ortho-xylene system had a final yield of 94.66%, an increase of approximately 32%. The crude ratio of preferred isomer to side product was also improved, as it went from 18% undesired in anhydrous acetonitrile to 4% undesired in ortho-xylene, both values normalized to the preferred regioisomer.
ContributorsTamirisa, Ritika Sai (Author) / Levitus, Marcia (Thesis director) / Stephanopoulos, Nicholas (Committee member) / Windman, Todd (Committee member) / School of Molecular Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2016-05
Description
Cyclodextrins are known for their pharmaceutical applications in a range of pathologies. Beta(ꞵ)-cyclodextrins have been suggested to be effective scaffolds that can ligate to peptides when chemically modified, which has the potential to be cost-effective in comparison to other available treatments for antiviral therapeutics. It is hypothesized that a ꞵ-cyclodextrin platform can be modified through a few-step reaction process to develop a ꞵ-cyclodextrin-DBCO-GFP nanobody. The findings of this few-step reaction support the general approach of conjugating the ꞵ-cyclodextrin derivative to GPF nanobody for developing a cyclodextrin antiviral scaffold.
ContributorsTaniguchi, Tohma (Author) / Hariadi, Rizal (Thesis director) / Stephanopoulos, Nicholas (Committee member) / Sasmal, Ranjan (Committee member) / Barrett, The Honors College (Contributor) / School of Molecular Sciences (Contributor)
Created2023-05
Description
Molecular tessellation research aims to elucidate the underlying principles that govern intricate patterns in nature and to leverage these principles to create precise and ordered structures across multiple scales, thereby facilitating the emergence of novel functionalities. DNA origami technology enables the fabrication of nearly arbitrary DNA architectures with nanoscale precision, which can serve as excellent building blocks for the construction of tessellation patterns. However, the size and complexity of DNA origami tessellation systems are currently limited by several unexplored factors relevant to the accuracy of essential design parameters, the applicability of design strategies, and the compatibility between different tiles. Here, a general design and assembly method are described for creating DNA origami tiles that grow into tessellation patterns with micrometer-scale order and nanometer-scale precision. A critical design parameter, interhelical distance (D), was identified, which determined the conformation of monomer tiles and the outcome of tessellation. Finely tuned D facilitated the accurate geometric design of monomer tiles with minimized curvature and improved tessellation capability. To demonstrate the generality of the design method, 9 tile geometries and 15 unique tile designs were generated. The designed tiles were assembled into single-crystalline lattices ranging from tens to hundreds of square micrometers with micrometer-scale, nearly defect-free areas readily visualized by atomic force microscopy. Two strategies were applied to further increase the complexity of DNA origami tessellation, including reducing the symmetry of monomer tiles and co-assembling tiles of various geometries. The designed 6 complex tilings that includes 5 Archimedean tilings and a 12-fold quasicrystal tiling yielded various tiling patterns that great in size and quality, indicating the robustness of the optimized tessellation system. The described design and assembly approach can also be employed to create square DNA origami units for algorithmic self-assembly. As the square units assembled and expanded, they executed the binary function XOR, which generated the Sierpinski triangular pattern according to the predetermined instructions. This study will promote DNA-templated, programmable molecular and material patterning and open up new opportunities for applications in metamaterial engineering, nanoelectronics, and nanolithography.
ContributorsTang, Yue (Author) / Yan, Hao (Thesis advisor) / Guo, Jia (Committee member) / Stephanopoulos, Nicholas (Committee member) / Arizona State University (Publisher)
Created2023
Description
Without a doubt, protein is the most crucial biomolecule performing life and biological functions of any living cell. Profiling various protein expression in individual cells has raised a great interest for scientist and researchers over decades in attempts to reveal cell-to-cell variation, which used to be masked in many previous population average measurement methods. Immunofluorescence (IF) has been a well-established single cell protein analysis technique as for its fast and high-resolution detection and localization, simple and adaptable workflows, and affordable instrumentation. However, inadequate detection sensitivity and multiplexing capability are the two limitation of this platform that remain incompletely addressed in many decades. In this work, several improvements have been proposed and demonstrated to improve existing drawbacks of conventional immunofluorescence. An azide-based linker featured in the novel fluorescent probes synthesis has enable iterative protein staining on the same tissue sample, which subsequently increase the multiplex capacity of IF. Additionally, the multiple fluorophore introduction to the proteins target via either layer by layer biotin-cleavable fluorescent streptavidin or tyramide signal amplification (TSA) have significantly increase the detection sensitivity of the platform. With these advances, IF has the potential to detect, image and quantify up to 100 protein targets in single cell in the tissue sample. In addition of desirable features of IF, these improvements have further turned the technique into a powerful proteomic study platform for not only research setting but also clinical study setting. It is anticipated this highly sensitive and multiplexed, renovated IF method will soon be translated into biomedical studies.
ContributorsPham, Thai Huy (Author) / Guo, Jia (Thesis advisor) / Stephanopoulos, Nicholas (Committee member) / Chiu, Po-Lin (Committee member) / Arizona State University (Publisher)
Created2023
Description
Molecular recognition forms the basis of all protein interactions, and therefore is crucial for maintaining biological functions and pathways. It can be governed by many factors, but in case of proteins and peptides, the amino acids sequences of the interacting entities play a huge role. It is molecular recognition that helps a protein identify the correct sequences residues necessary for an interaction, among the vast number of possibilities from the combinatorial sequence space. Therefore, it is fundamental to study how the interacting amino acid sequences define the molecular interactions of proteins. In this work, sparsely sampled peptide sequences from the combinatorial sequence space were used to study the molecular recognition observed in proteins, especially monoclonal antibodies. A machine learning based approach was used to study the molecular recognition characteristics of 11 monoclonal antibodies, where a neural network (NN) was trained on data from protein binding experiments performed on high-throughput random-sequence peptide microarrays. The use of random-sequence microarrays allowed for the peptides to be sparsely sampled from sequence space. Post-training, a sequence vs. binding relationship was deduced by the NN, for each antibody. This in silico relationship was then extended to larger libraries of random peptides, as well as to the biologically relevant sequences (target antigens, and proteomes). The NN models performed well in predicting the pertinent interactions for 6 out of the 11 monoclonal antibodies, in all aspects. The interactions of the other five monoclonal antibodies could not be predicted well by the models, due to their poor recognition of the residues that were omitted from the array. Furthermore, NN predicted sequence vs. binding relationships for 3 other proteins were experimentally probed using surface plasmon resonance (SPR). This was done to explore the relationship between the observed and predicted binding to the arrays and the observed binding on different assay platforms. It was noted that there was a general motif dependent correlation between predicted and SPR-measured binding. This study also indicated that a combined reiterative approach using in silico and in vitro techniques is a powerful tool for optimizing the selectivity of the protein-binding peptides.
ContributorsBisarad, Pritha (Author) / Woodbury, Neal W (Thesis advisor) / Green, Alexander A (Committee member) / Stephanopoulos, Nicholas (Committee member) / Arizona State University (Publisher)
Created2022
Description
Protein-nucleic acid interactions are ubiquitous in biological systems playing a pivotal role in fundamental processes such as replication, transcription and translation. These interactions have been extensively used to develop biosensors, imaging techniques and diagnostic tools.This dissertation focuses on design of a small molecule responsive biosensor that employs transcription factor/deoxyribonucleic acid (DNA) interactions to detect 10 different analytes including antibiotics such as tetracyclines and erythromycin. The biosensor harnesses the multi-turnover collateral cleavage activity of Cas12a to provide signal amplification in less than an hour that can be monitored using fluorescence as well as on paper based diagnostic devices. In addition, the functionality of this assay was preserved when testing tap water and wastewater spiked with doxycycline. Overall, this biosensor has potential to expand the range of small molecule detection and can be used to identify environmental contaminants.
In second part of the dissertation, interactions between nonribosomal peptide synthetases (NRPS) and ribonucleic acid (RNA) were utilized for programming the synthesis of nonribosomal peptides. RNA scaffolds harboring peptide binding aptamers and interconnected using kissing loops to guide the assembly of NRPS modules modified with corresponding aptamer-binding peptides were built. A successful chimeric assembly of Ent synthetase modules was shown that was characterized by the production of Enterobactin siderophore. It was found that the programmed RNA/NRPS assembly could achieve up to 60% of the yield of wild-type biosynthetic pathway of the iron-chelator enterobactin.
Finally, a cas12a-based detection method for discriminating short tandem repeats where a toehold exchange mechanism was designed to distinguish different numbers of repeats found in Huntington’s disease, Spinocerebellar ataxia type 10 and type 36. It was observed that the system discriminates well when lesser number of repeats are present and provides weaker resolution as the size of DNA strands increases. Additionally, the system can identify Kelch13 mutations such as P553L, N458Y and F446I from the wildtype sequence for Artemisinin resistance detection.
This dissertation demonstrates the great utility of harnessing protein-nucleic acid interactions to construct biomolecular devices for detecting clinically relevant nucleic acid mutations, a variety of small molecule analyte and programming the production of useful molecules.
ContributorsChaudhary, Soma (Author) / Green, Alexander (Thesis advisor) / Stephanopoulos, Nicholas (Committee member) / Mangone, Marco (Committee member) / Arizona State University (Publisher)
Created2022
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
Since the inception of DNA nanotechnology, DNA has found itself poised as one of the most robust self-assembling building blocks due to its well understood double helix structure formed by two anti-parallel strands of DNA held together by hydrogen bond from nucleobases which also provides the material programmability due to the well-understood Watson Crick base pairing rules. These capabilities have led to the exponential increase in publications showing off intricate and remarkable designs alongside ever-expanding applications. However, as the field expands there is an apparent lack of chemical diversity and functionality. To combat this my research focused on creating hybrid peptide oligonucleotide conjugates (POC) where the conjugated peptide could add chemical and structural diversity using the 20 canonical amino acids and various peptide secondary structures. In this work, I conjugate DNA to the self-assembling peptide building block the coiled coil. The coiled coil motif is formed from the self-assembly of two or more α-helical peptides and, like DNA, the coiled coil has well understood programmability. Together as a conjugate, the DNA and coiled coil, create a new self-assembling building block capable of two orthogonal self-assembling modes that can work in tandem. In this work, I used DNA coiled coil conjugates to show the capability to create first of their kind hybrid DNA/coiled coil one-dimensional fibers (chapter 2), integrate proteins (chapter 3), and to create hybrid cage structures (chapter 4). Finally, a POC hydrogel is created using the polypeptide gelatin with DNA crosslinks to create a reversible stiffening gel using toe-hold mediated strand displacement (chapter 5).
ContributorsBuchberger, Alex Richard (Author) / Stephanopoulos, Nicholas (Thesis advisor) / Mills, Jeremy (Committee member) / Van Horn, Wade (Committee member) / Arizona State University (Publisher)
Created2021
Description
Since the conception of DNA nanotechnology, the field has evolved towards the development of complex, dynamic 3D structures. The predictability of Watson-Crick base pairing makes DNA an unparalleled building block, and enables exceptional programmability in nanostructure shape and size. The work presented in this dissertation focuses on expanding two facets of the field: (1) introducing functionality through the incorporation of peptides to create DNA-peptide hybrid materials, and (2) the development of self-assembling DNA crystal lattices for scaffolding biomolecules. DNA nanostructures have long been proposed as drug delivery vehicles; however, they are not biocompatible because of their low stability in low salt environments and entrapment within the endosome. To address these issues, a functionalized peptide coating was designed to act as a counterion to a six-helix bundle, while simultaneously displaying numerous copies of an endosomal escape peptide to enable cytosolic delivery. This functionalized peptide coating creates a DNA-peptide hybrid material, but does not allow specific positioning or orientation of the peptides. The ability to control those aspects required the synthesis of DNA-peptide or DNA-peptide-DNA conjugates that can be incorporated into the nanostructure. The approach was utilized to produce a synbody where three peptides that bind transferrin with micromolar affinity, which were presented for multivalent binding to optimize affinity. Additionally, two DNA handle was attached to an enzymatically cleavable peptide to link two unique nanostructures. The second DNA handle was also used to constrain the peptide in a cyclic fashion to mimic the cell-adhesive conformations of RGD and PHSRN in fibronectin.
The original goal of DNA nanotechnology was to use a crystalline lattice made of DNA to host proteins for their structural determination using X-ray crystallography. The work presented here takes significant steps towards achieving this goal, including elucidating design rules to control cavity size within the scaffold for accommodating guest molecules of unique sizes, approaches to improve the atomic detail of the scaffold, and strategies to modulate the symmetry of each unique lattice. Finally, this work surveys methodologies towards the incorporation of several guest molecules, with promising preliminary results that constitute a significant advancement towards the ultimate goal of the field.
ContributorsMacCulloch, Tara Lynn (Author) / Stephanopoulos, Nicholas (Thesis advisor) / Borges, Chad (Committee member) / Gould, Ian (Committee member) / Arizona State University (Publisher)
Created2021