DNA-templated Chemical Synthesis of Proteins and Polypeptides

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Proteins are among the important macromolecules in living systems, with diverse biological functions and properties that make them greatly interesting to study in both structure and function. The chemical synthesis of proteins allows researchers to incorporate a wide variety of…

Proteins are among the important macromolecules in living systems, with diverse biological functions and properties that make them greatly interesting to study in both structure and function. The chemical synthesis of proteins allows researchers to incorporate a wide variety of post-translation modifications that can diversify protein functions. It also allows the incorporation of many noncanonical amino acids that enable the study of protein structure and function, as well as the control of their activity in living cells. The work presented in this dissertation focuses on two DNA-templated chemical synthesis approaches for the synthesis of proteins: i) DNA-templated native chemical ligation (NCL), and ii) DNA-templated click chemistry. NCL and its extended version has been used as a powerful tool to obtain proteins; however, it still struggles to make longer proteins due to aggregation and poor yield. To address these issues, a DNA-templated approach is being developed where two peptide fragments are brought into proximity by an oligonucleotide to facilitate the NCL reaction. The sequential ligation of the peptide fragments will result in full-length proteins with increased yield and improved solubility. This research involves synthesis of small molecule auxiliaries, thioester peptides, DNA-peptide conjugates, and ligation of peptides through NCL. This method has the potential to be applied to synthesize large hydrophobic proteins. A DNA-templated click chemistry method was also reported where duplex DNA was utilized as a template for enhancing the copper click reaction between peptide fragments into functional mini-proteins. As a proof of principle, peptide fragments were synthesized with click functional groups and conjugated with distinct DNA handles through a disulfide exchange bioconjugation reaction. The DNA-peptide conjugates were assembled with the template to bring the two peptides into proximity and enhance the effective molarities of the functional groups. The peptides were coupled efficiently using a copper click reaction. The designed DNA-templated method is being implemented to synthesize a designed mini-protein (called LCB1), which can bind tightly to the spike protein of SARS-CoV-2 and inhibit its interaction with the human angiotensin-converting enzyme 2 (ACE2) receptor. This method allows researchers to introduce multiple non-natural amino acids in the protein and has the potential to extend to larger proteins, synthetic polymers, and DNA-peptide biomaterials.
Date Created
2024
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Processes and Properties Refinement of Photovoltaic Hybrid Perovskites and Their Integration into Solar Cells

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Perovskite solar cells are one of the rising stars in the solar cell industry. This thesis explores several approaches to enhance the properties of the perovskite layer and the solar cell devices in which they operate. They include studies of…

Perovskite solar cells are one of the rising stars in the solar cell industry. This thesis explores several approaches to enhance the properties of the perovskite layer and the solar cell devices in which they operate. They include studies of different antisolvent additives during spin coating of triple cation perovskites, the use of surfactants to improve the quality of perovskite film microstructures, the applicability of a new fabrication process, and the value of post-deposition thermal and chemical annealing processes.This thesis experimentally analyzes different antisolvents, viz., ethyl acetate, isopropyl alcohol, toluene, and chlorobenzene. It focuses on the antisolvent-assisted crystallization method to achieve homogenous nucleation of the perovskite film. Of all the antisolvents, ethyl acetate-treated films gave the best-performing device, achieving a power conversion efficiency of 15.5%. This thesis also analyzes the effects of mixed antisolvents on the qualities of triple-cation perovskites. Different solution concentrations of chlorobenzene in ethyl acetate and isopropyl alcohol in ethyl acetate are optimized for optimal supersaturation to achieve enlarged perovskite grains. Evaluations are discussed in the context of solution polarity and boiling point of the antisolvents, where 25% chlorobenzene in ethyl acetate antisolvent mixture shows the best film properties. Another study discusses a new fabrication process called electrical field-assisted direct ink deposition for large-scale printing of perovskite solar cells. This process involves the formation of nanodroplets under an electrical field deposited onto ITO/glass substrates. As a result, smooth Poly (3,4-ethylene dioxythiophene) polystyrene sulfonate layers are ii produced with an average effective electrical resistivity of 4.15104  0.26 -m compared to that of spin-coated films. A successive chapter discusses the studies of the electrical field-assisted direct ink deposition of the photoactive CH3NH3PbI2 (MAPbI3) layer. Its focus is on the post-deposition chemical annealing of the MAPbI3 films in methylamine gas, termed as methylamine gas-assisted healing and growth of perovskite films. This treatment improved the smoothness, reduced porosity, increased density, and generated more uniform grain sizes. Moreover, it improved the inter-grain boundary contacts by eliminating secondary, fine-grained boundary structures. Mechanisms behind the initial liquefaction of the MAPbI3 film's subsequent re-solidification are discussed.
Date Created
2023
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Development of Wet Chemical Synthesis Strategies for the Class of MAX Phases

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MAX phases are an intriguing class of materials with exotic combinations of properties, essentially turning them into metallic ceramics. Despite this unique feature, no commercialization has been accomplished yet. Looking at the state of the art within the MAX phase…

MAX phases are an intriguing class of materials with exotic combinations of properties, essentially turning them into metallic ceramics. Despite this unique feature, no commercialization has been accomplished yet. Looking at the state of the art within the MAX phase community, almost all published studies can be summarized using the term “traditional high temperature synthesis”. Contrasting the scientific interest that has been on the rise especially since the discovery of MXenes, the synthetic spectrum has been largely the same as it has been over the past decades.Herein, the newly-emerging sol-gel chemistry is being explored as an alternative non-conventional synthetic approach. Building on the successful sol-gel synthesis of Cr2GaC, this study focuses around the expansion of sol-gel chemistry for MAX phases. Starting with a thorough mechanistic investigation into the reaction pathway of sol-gel synthesized Cr2GaC, the chemical understanding of this system is drastically deepened. It is shown how the preliminary nano-structured metal-oxide species develop into bulk oxides, before the amorphous and disordered graphite partakes in the reaction and reduces the metals into the MAX phase. Furthermore, the technique is extended to the two Ge- based MAX phases V2GeC and Cr2GeC, a critical step needed to prove the viability and applicability of the newly developed technique. Additionally, by introducing Mn into the Cr-Ga-C system, a Mn-doping was achieved, and for the first time for (Cr1–xMnx)2GaC, a unit cell increase could be recorded. Based on magnetometry measurements, the currently widely accepted assumption of statistically distributed Mn in the M-layer is challenged. The versatility of wet chemistry is explored using the model system Cr2GaC. Firstly, the MAX phase can be obtained in a microwire shape leveraging the branched biopolymer dextran, eliminating the need for any post-synthesis machining. Via halide intercalation, the electrical transport properties could be purposefully engineered. Secondly, leveraging the unique and linear biopolymer chitosan, Cr2GaC was obtained as thick films and dense microspheres, drastically opening potential areas of application for MAX phases. Lastly, hollow microspheres with diameters of tens of μm were synthesized via carboxymethylated dextran. This shape once more opens the door to very specific applications requiring sophisticated structures.
Date Created
2022
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Investigating FAK Phosphorylation and Oncogenic Signaling Pathway Changes in NRAS and BRAF Mutant Melanoma

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The 5-year survival rate for late-stage metastatic melanoma is only ~30%. A major reason for this low survival rate is that one of the most commonly mutated genes in melanoma, NRAS, has no FDA-approved targeted therapies. Because the RAS protein…

The 5-year survival rate for late-stage metastatic melanoma is only ~30%. A major reason for this low survival rate is that one of the most commonly mutated genes in melanoma, NRAS, has no FDA-approved targeted therapies. Because the RAS protein does not have any targeted therapies, patients with RAS mutant tumors have an ongoing need for treatments that indirectly target RAS. This thesis project aims to identify expression and phosphorylation levels of proteins downstream of RAS in melanoma cell lines with the most common driver mutations. By analyzing the protein-level differences between these genetic mutants, we hope to identify additional indirect RAS protein targets for the treatment of NRAS mutant melanoma. RAS has several downstream effector proteins involved in oncogenic signaling pathways including FAK, Paxillin, AKT, and ERK. 5 melanoma cell lines (2 BRAF mutant, 2 NRAS mutant, and 1 designated wildtype) were analyzed using western bloting for FAK, Paxillin, AKT, and ERK phosphorylation and total expression levels. The results of western blot analysis showed that NRAS mutant cell lines had increased expression of phosphorylated Paxillin. Increased Paxillin phosphorylation corresponds to increased Paxillin binding at the FAT domain of FAK. Therefore, cell lines with increased FAK FAT – Paxillin interaction would be more sensitive to FAK FAT domain inhibition. The data presented provide an an explanation for the reduction in cell viability in NRAS mutant cell lines infected with Ad-FRNK. This information also has significant clinical relevance as researchers work to develop synthetic FAK FAT domain inhibitors, such as cyclic peptides. Additionally, cell lines with high levels of phosphorylated AKT showed a significant reduction in the amount of phosphorylated ERK. The identification of this inverse relationship may help to explain why BRAF and NRAS mutations are mutually exclusive. To conclude, NRAS mutant cell lines have increased expression of phosphorylated Paxillin and AKT which may explain why NRAS mutant cell lines are more sensitive to FAK FAT domain inhibition.

Date Created
2022-05
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Molecular Modification of (Iso)quinolines via Earth-Abundant Catalysis

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Chemistry has always played a foundational role in the synthesis of pharmaceuticals. With the rapid growth of the global population, the health and medical needs have also rapidly increased. In order to provide drugs capable of mediating symptoms and curing…

Chemistry has always played a foundational role in the synthesis of pharmaceuticals. With the rapid growth of the global population, the health and medical needs have also rapidly increased. In order to provide drugs capable of mediating symptoms and curing diseases, organic chemistry provides drug derivatives utilizing a limited number of chemical building blocks and privileged structures. Of these limited building blocks, this project explores Late–stage C–H functionalization of (iso)quinolines using abundant metal catalysis in order to achieve site-selective molecular modification.

Date Created
2022-05
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Photophysical Studies to Advance Fluorescence Applications in Biophysics

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Fluorescence spectroscopy has been a vital technique in biophysics due to its high sensitivity and specificity. While the recent development of single-molecule (SM) techniques has furthered the molecular-level understanding of complicated biological systems, the full potential of these techniques hinges…

Fluorescence spectroscopy has been a vital technique in biophysics due to its high sensitivity and specificity. While the recent development of single-molecule (SM) techniques has furthered the molecular-level understanding of complicated biological systems, the full potential of these techniques hinges on the development and selection of fluorescent probes with customized photophysical properties. Red region probes are inherently desirable as background noise from typical biological systems tends to be at its minimum in this spectral region. The first part of this work studies the photophysical properties of red cyanine dyes to access their usefulness for particular SM applications.Protein-induced fluorescence enhancement (PIFE) based approaches are increasingly being used to investigate DNA-protein interactions at the SM level. However, a key limitation remains the absence of good red PIFE probes. This work investigates the photophysical properties of a red hemicyanine dye (Dy-630) as a potential PIFE probe. Results shed light on optimal design principles for ideal probes for PIFE applications, opening new avenues for the technique’s broad applicability in biophysical studies. Further, the photophysical behavior of two novel cyanine fluorophores in the far-red (rigidized pentacyanine) and near-Infrared (IR) (rigidized heptacyanine) region are studied. Both probes are designed to eliminate a photoisomerization caused non-radiative pathway by rigidization of the cyanine backbone. The rigidized pentacyanine was found to have desired photophysical properties and improved quantum yield, vital for application in super-resolution imaging. For rigidized heptacyanine, in contrast to the prior project, it was found that photoisomerization does not contribute significantly to the deactivation pathway. Thus, this work clarifies the role of photoisomerization on heptamethine cyanine scaffold and will enable future efforts to optimize NIR dyes for diverse applications. The second part of this work aims to answer the fundamental question of how the physics of DNA can impact its biology. To this end, interlinkage between the flexibility of local sequence context and the efficiency of uracil removal by Uracil-DNA glycosylase (UDG) protein is investigated using fluorescent base analogue, 2-Aminopurine (2-AP). In summary, this work focuses on photophysical investigations, the understanding of which is vital for the selection and development of fluorescent probes for biophysical studies.
Date Created
2021
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Rational Design of Self-Assembling Crystal Scaffolds and DNA-Peptide Hybrid Materials

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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.…

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.
Date Created
2021
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Functional Studies of Interactions in Proteins

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Interactions between proteins form the basis of almost all biological mechanisms. The majority of proteins perform their functions as a part of an assembled complex, rather than as an isolated species. Understanding the functional pathways of these protein complexes helps…

Interactions between proteins form the basis of almost all biological mechanisms. The majority of proteins perform their functions as a part of an assembled complex, rather than as an isolated species. Understanding the functional pathways of these protein complexes helps in uncovering the molecular mechanisms involved in the interactions. In this thesis, this has been explored in two fundamental ways. First, a biohybrid complex was assembled using the photosystem I (PSI) protein complex to translate the biochemical pathways into a non-cellular environment. This involved incorporating PSI on a porous antimony-doped tin oxide electrode using cytochrome c. Photocurrent was generated upon illumination of the PSI/electrode system alone at microamp/cm2 levels, with reduced oxygen apparently as the primary carrier. When the PSI/electrode system was coupled with ferredoxin, ferredoxin-NADP+ reductase (FNR), and NADP+, the resulting light-powered NADPH production was coupled to a dehydrogenase system for enzymatic carbon reduction. The results demonstrated that light-dependent reduction readily takes place. However, the pathways do not always match the biological pathways of PSI in nature. To create a complex self-assembled system such as the one involving PSI that is structurally well defined, there is a need to develop ways to guide the molecular interactions. In the second part of the thesis, this problem was approached by studying a well-defined system involving monoclonal antibodies (mAbs) binding their cognate epitope sequences to understand the molecular recognition properties associated with protein-protein interactions. This approach used a neural network model to derive a comprehensive and quantitative relationship between an amino acid sequence and its function by using sparse measurements of mAb binding to peptides on a high density peptide microarray. The resulting model can be used to predict the function of any peptide in the possible combinatorial sequence space. The results demonstrated that by training the model on just ~105 peptides out of the total combinatorial space of ~1010, the target sequences of the mAbs (cognate epitopes) can be predicted with high statistical accuracy. Furthermore, the biological relevance of the algorithm’s predictive ability has also been demonstrated.
Date Created
2021
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Metal Organic Interactions at Hydrothermal Conditions: Useful Transformations Through Geomimicry

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Organic compounds are influenced by hydrothermal conditions in both marine and terrestrial environments. Sedimentary organic reservoirs make up the largest share of organic carbon in the carbon cycle, leading to petroleum generation and to chemoautotrophic microbial communities. There have been…

Organic compounds are influenced by hydrothermal conditions in both marine and terrestrial environments. Sedimentary organic reservoirs make up the largest share of organic carbon in the carbon cycle, leading to petroleum generation and to chemoautotrophic microbial communities. There have been numerous studies on the reactivity of organic compounds in water at elevated temperatures, but these studies rarely explore the consequences of inorganic solutes in hydrothermal fluids. The experiments in this thesis explore new reaction pathways of organic compounds mediated by aqueous and solid phase metals, mainly Earth-abundant copper. These experiments show that copper species have the potential to oxidize benzene and toluene, which are typically viewed as unreactive. These pathways add to the growing list of known organic transformations that are possible in natural hydrothermal systems. In addition to the characterization of reactions in natural systems, there has been recent interest in using hydrothermal conditions to facilitate organic transformations that would be useful in an applied, industrial or synthetic setting. This thesis identifies two sets of conditions that may serve as alternatives to commonplace industrial processes. The first process is the oxidation of benzene with copper to form phenol and chlorobenzene. The second is the copper mediated dehalogenation of aryl halides. Both of these processes apply the concepts of geomimicry by carrying out organic reactions under Earth-like conditions. Only water and copper are needed to implement these processes and there is no need for exotic catalysts or toxic reagents.
Date Created
2020
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Impact of Vascular Brachytherapy on Patient Reported Outcomes in Patients with Coronary Artery Disease

Description
Background: Vascular brachytherapy (VBT) is an established treatment for the management of in-stent restenosis (ISR). However, whether VBT is associated with improved patient-reported outcomes is unknown and not been previously studied.

Methods: The authors evaluated 51 consecutive patients, age 18…

Background: Vascular brachytherapy (VBT) is an established treatment for the management of in-stent restenosis (ISR). However, whether VBT is associated with improved patient-reported outcomes is unknown and not been previously studied.

Methods: The authors evaluated 51 consecutive patients, age 18 years and older undergoing VBT in one or more coronary arteries from January 2018 to September 2019. Data on baseline characteristics, procedural outcomes, and occurrence of adverse events were obtained. All patients completed the Seattle Angina Questionnaire – 7 (SAQ-7) form before and after the intervention at 1 month and 6 months.

Results: The mean age was 69 ± 9 years and 29 (57%) of patients were males. Most patients had hypertension (n = 44, 86%) and diabetes (n = 29, 57%). The use of aspirin was 90% while 96% of patients were on P2Y12 inhibitors. 48 (94%) patients were on antianginal therapy. The procedural success was 94.1%. The mean summary SAQ-7 score improved significantly (53.2 ± 21 vs. 83 ± 19, p<0.001) at 30-days. The median Quality of Life (QoL) component of the SAQ-7 score at baseline was 31.3 (Interquartile Range [IQR]: 18.8, 62.5) and improved to 82.5 (IQR: 62.5, 100), p<0.001 at 30 days and 87.5 [IQR: 75, 100), p<0.001 at last follow up. Likewise, the median angina frequency component of the SQL-7 score pre-VBT was 55 (IQR: 45, 80) and improved significantly to 90 (IQR: 60, 100) at 30-days, p<0.001 and 100 [IQR: 68.8, 100], p=0.02 at last follow up. Lastly, the median activity component of the SAQ-7 score improved from 83.3 (IQR: 60 – 100) to 100 (IQR: 83, 100), p = 0.01 at 30-days.

Conclusion: This study demonstrated improvement in patient-reported outcome measures following vascular brachytherapy that are evident as early as 1 month after the intervention and sustained at a median follow up of 17 months.
Date Created
2020-05
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