Matching Items (41)
Description
DNA nanotechnology uses the reliability of Watson-Crick base pairing to program and generate two-dimensional and three-dimensional nanostructures using single-stranded DNA as the structural material. DNA nanostructures show great promise for the future of bioengineering, as there are a myriad of potential applications that utilize DNA’s chemical interactivity and ability to bind other macromolecules and metals. DNA origami is a method of constructing nanostructures, which consists of a long “scaffold” strand folded into a shape by shorter “staple” oligonucleotides. Due to the negative charge of DNA molecules, divalent cations, most commonly magnesium, are required for origami to form and maintain structural integrity. The experiments in this paper address the discrepancy between salt concentrations required for origami stability and the salt concentrations present in living systems. The stability of three structures, a two-dimensional triangle, a three-dimensional solid cuboid and a three-dimensional wireframe icosahedron were examined in buffer solutions containing various concentrations of salts. In these experiments, DNA origami structures remained intact in low-magnesium conditions that emulate living cells, supporting their potential for widespread biological application in the future.
ContributorsSeverson, Grant William (Author) / Stephanopoulos, Nicholas (Thesis director) / Mills, Jeremy (Committee member) / School of Molecular Sciences (Contributor) / School of Life Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2020-05
Description
Mass nuclear catastrophe is a serious concern for society at large when considering the rising threat of terrorism and the risks associated with harnessing nuclear energy. In the case of a mass nuclear/radiological event that requires hundreds of thousands of individuals to be assessed for radiation exposure, a rapid biodosimetry triage tool is crucial [1]. The Cytokinesis Block Micronucleus Assay (CBMN) is a promising cytogenetic biodosimetry assay for triage [2]; however, it requires shipping samples to a central laboratory (1-3 days) followed by a lengthy cell culture process (~3 days) before the first dose estimate can be available. The total ~ 1 week response time is too long for effective medical care intervention. A shipping incubator could cut the response time in half (~3 days) by culturing samples in transit; however, possible shipping delays beyond 2 days without the addition of a necessary reagent (Cyto-B) would ruin the integrity of the samples—for accurate CBMN assay endpoint observation, Cyto-B must be added within a 24-44 hour window after sample culture is initiated. Here, we propose a “Smart” Shipping Incubator (SSI) that can add Cyto-B while samples are in transit through a centrifugal system equipped with microfluidic capillary valve caps. The custom centrifugal system was constructed with CNC machined and 3D printed plastic parts, controlled by a custom printed circuit board (PBC) microcontroller, and housed inside a commercial shipping incubator (iQ5 from MicroQ Technologies). Teflon-coated, pre-pulled glass micropipettes (FivePhoton BioChemicals) were used as microfluidic capillary valve caps. Release of Cyto-B was characterized by a desktop centrifugal system at different tip sizes and relative centrifugal forces (RCFs). A theoretical model of Cyto-B release was also deduced to aid the optimization of the process. The CBMN assay was conducted both in the SSI with centrifugal Cyto-B release and in a standard CO2 incubator with manual addition of Cyto-B as the control. The expected mechanical shock during shipment was measured to be less than 25g. Optimal Cyto-B release was found to be at 35g RCF with a Teflon-coated 40 µm tip. Similar CBMN dose curves of micronuclei per binucleated cells (MN/BN) vs. exposed radiation (Gy) were produced for samples assessed conventionally and with the SSI. The similarities between the two methods suggest that centrifugation does not significantly affect the CBMN assay.
ContributorsAkkad, Adam Rifat (Author) / Stephanopoulos, Nicholas (Thesis director) / Mills, Jeremy (Committee member) / Gu, Jian (Committee member) / School of Molecular Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2019-12
Description
Extensive efforts have been made to develop efficient and low-cost methods for diagnostics to identify molecular biomarkers that are linked to a wide array of conditions, including cancer. A highly developed method includes utilizing the gene-editing enzyme CRISPR-Cas12a (Cpf1), which demonstrates double-stranded DNase activity with RuvC catalytic domain with high sensitivity and specificity. This DNase activity is RNA-guided and requires a T-rich PAM site on the target sequence for functional cleavage. There have been recent efforts to utilize this DNase activity of Cas12a by combining it with isothermal amplification and analysis by lateral strip tests. This project examined CRISPR-based early detection of microRNA biomarkers. MicroRNA are short RNA molecules that have large roles in post-transcriptional gene regulation. However, due the short length of microRNA and its single-stranded nature, it is challenging to use Cas12a for microRNA detection using existing methods. Thus, this project investigated the potential of two microRNA detection strategies for recognition by CRISPR-Cas12a. These methods were microRNA-splinted ligation with polymerase chain reaction (PCR) and MicroRNA-specific reverse transcriptase PCR (RT-PCR). Gel imaging demonstrated effective amplification of ligated DNA through microRNA-splinted ligation with PCR/RPA. In addition, lateral strips tests showed effective cleavage of the target sequences by Cas12a. However, RT-PCR method demonstrated low amplification by PCR and inefficient poly(A) elongation. This project paves the way for the detection of an extensive range of microRNA biomarkers that are linked to an array of diseases. Future directions include analysis and modifications of RT-PCR method to improve experimental results, extending these detection methods to a larger range of microRNA sequences, and eventually utilizing them for detection in human samples.
ContributorsStaren, Michael Steven (Author) / Green, Alexander (Thesis director) / Stephanopoulos, Nicholas (Committee member) / Diehnelt, Chris (Committee member) / School of Life Sciences (Contributor) / College of Health Solutions (Contributor) / Barrett, The Honors College (Contributor)
Created2019-05
Description
While DNA and protein nanotechnologies are promising avenues for nanotechnology on their own, merging the two could create more diverse and functional structures. In order to create hybrid structures, the protein will have to undergo site-specific modification, such as the incorporation of an unnatural amino, p-azidophenylalanine (AzF), via Shultz amber codon suppression method, which can then participate in click chemistry with modified DNA. These newly synthesized structures will then be able to self-assemble into higher order structures. Thus far, a surface exposed residue on the aldolase protein has been mutated into an amber stop codon. The next steps are to express the protein with the unnatural amino acid, allow it to participate in click chemistry, and visualize the hybrid structure. If the structure is correct, it will be able to self-assemble.
ContributorsAziz, Ann-Marie (Author) / Stephanopoulos, Nicholas (Thesis director) / Mills, Jeremy (Committee member) / School of Social Transformation (Contributor) / School of Molecular Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2017-05
Description
The main objective of this project is to create a hydrogel based material system to capture and release CCRF-CEM Leukemia cancer cells via chemo-mechanical modulation. This system is composed of an aptamer-functionalized hydrogel thin film at the bottom of a microfluidic channel, which changes its film thickness as the temperature of the fluid in the system changes. The functionalized hydrogel film has been created as the primary steps to creating the microfluidic device that could capture and release leukemia cells by turning the temperature of the fluid and length of exposure. Circulating tumor cells have recently become a highly studied area since they have become associated with the likelihood of patient survival. Further, circulating tumor cells can be used to determine changes in the genome of the cancer leading to targeted treatment. First, the aptamers were attached onto the hydrogel through an EDC/NHS reaction. The aptamers were verified to be attached onto the hydrogel through FTIR spectroscopy. The cell capture experiments were completed by exposing the hydrogel to a solution of leukemia cells for 10 minutes at room temperature. The cell release experiments were completed by exposing the hydrogel to a 40°C solution. Several capture and release experiments were completed to measure how many cells could be captured, how quickly, and how many cells captured were released. The aptamers were chemically attached to the hydrogel. 300 cells per square millimeter could be captured at a time in a 10 minute time period and released in a 5 minute period. Of the cells captured, 96% of them were alive once caught. 99% of cells caught were released once exposed to elevated temperature. The project opens the possibility to quickly and efficiently capture and release tumor cells using only changes in temperature. Further, most of the cells that were captured were alive and nearly all of those were released leading to high survival and capture efficiency.
ContributorsPaxton, Rebecca Joanne (Author) / Stephanopoulos, Nicholas (Thesis director) / He, Ximin (Committee member) / Gould, Ian (Committee member) / Materials Science and Engineering Program (Contributor) / School of Molecular Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2016-12
Description
The synthesis of the bis(2-diphenylphosphinoethyl)amine chelating ligand (1) was a crucial component in the preparation of non-canonical amino acids (NCAAs) throughout the project. Studies in this project indicated the need to isolate the ligand from its hydrochloride salt form seen in (1) which led to the synthesis of the brown oil, (Ph2PCH2CH2)2NH, (2). The ligand features a phosphine-nitrogen-phosphine group that is not observed in existing NCAAs. Phosphine groups are rarely seen in existing NCAAs and avoided by biochemists because they tend to oxidize before metal addition. In this project, (1) was used in a 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate (HATU) mediated method and palladium-catalyzed method to tether an amino acid to the nitrogen atom of the ligand framework. Both methods were monitored through the use of Nuclear Magnetic Resonance (NMR) spectroscopy. While the palladium catalyzed method exhibited little to no coupling, the 31P NMR spectrum obtained for the HATU mediated method did reveal that some coupling had occurred. The unsuccessful attempts to tether an amino acid to (1) led to the hypothesis that the phosphine groups were interfering with the palladium catalyst during the cross-coupling reaction. In an effort to test this hypothesis, (2) was reacted with the dimer, [Rh(nbd)Cl]2, to coordinate the rhodium metal to the free phosphorous arms and the nitrogen atom of the isolated PNP ligand. The PNP-based metal complex was used in the palladium catalyzed method, but cross-coupling was not observed. The new PNP-based metal complex was investigated to demonstrate that it exhibits moisture and air stability.
ContributorsManjarrez, Yvonne (Author) / Trovitch, Ryan (Thesis director) / Stephanopoulos, Nicholas (Committee member) / Herckes, Pierre (Committee member) / School of Molecular Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2018-05
Description
Membrane proteins act as sensors, gatekeepers and information carriers in the cell membranes. Functional engineering of these proteins is important for the development of molecular tools for biosensing, therapeutics and as components of artificial cells. However, using protein engineering to modify existing protein structures is challenging due to the limitations of structural changes and difficulty in folding polypeptides into defined protein structures. Recent studies have shown that nanoscale architectures created by DNA nanotechnology can be used to mimic various protein functions, including some membrane proteins. However, mimicking the highly sophisticated structural dynamics of membrane proteins by DNA nanostructures is still in its infancy, mainly due to lack of transmembrane DNA nanostructures that can mimic the dynamic behavior, ubiquitous to membrane proteins. Here, I demonstrate design of dynamic DNA nanostructures to mimic two important class of membrane proteins. First, I describe a DNA nanostructure that inserts through lipid membrane and dynamically reconfigures upon sensing a membrane-enclosed DNA or RNA target, thereby transducing biomolecular information across the lipid membrane similar to G-protein coupled receptors (GPCR’s). I use the non-destructive sensing property of our GPCR-mimetic nanodevice to sense cancer associated micro-RNA biomarkers inside exosomes without the need of RNA extraction and amplification. Second, I demonstrate a fully reversibly gated DNA nanopore that mimics the ligand mediated gating of ion channel proteins. The 20.4 X 20.4 nm-wide channel of the DNA nanopore allows timed delivery of folded proteins across synthetic and biological membranes. These studies represent early examples of dynamic DNA nanostructures in mimicking membrane protein functions. I envision that they will be used in synthetic biology to create artificial cells containing GPCR-like and ion channel-like receptors, in site-specific drug or vaccine delivery and highly sensitive biosensing applications.
ContributorsDey, Swarup (Author) / Yan, Hao (Thesis advisor) / Hariadi, Rizal F (Thesis advisor) / Liu, Yan (Committee member) / Stephanopoulos, Nicholas (Committee member) / Arizona State University (Publisher)
Created2021
Description
Mycobacterial infections, as represented by leprosy and tuberculosis, have persisted as human pathogens for millennia. Their environmental counterparts, nontuberculous mycobacteria (NTM), are commodious infectious agents endowed with extensive innate and acquired antimicrobial resistance. The current drug development process selects for antibiotics with high specificity for definitive targets within bacterial metabolic and replication pathways. Because these compounds demonstrate limited efficacy against mycobacteria, novel antimycobacterial agents with unconventional mechanisms of action were identified. Two highly resistant NTMs, Mycobacterium abscessus (Mabs) a rapid-growing respiratory, skin, and soft tissue pathogen, and Mycobacterium ulcerans (MU), the causative agent of Buruli ulcer, were selected as targets. Compounds that indicated antimicrobial activity against other highly resistant pathogens were selected for initial screening. Antimicrobial peptides (AMPs) have demonstrated activity against a variety of bacterial pathogens, including mycobacterial species. Designed antimicrobial peptides (dAMPs), rationally-designed and synthetic contingents, combine iterative features of natural AMPs to achieve superior antimicrobial activity in resistant pathogens. Initial screening identified two dAMPs, RP554 and RP557, with bactericidal activity against Mabs. Clay-associated ions have previously demonstrated bactericidal activity against MU. Synthetic and customizable aluminosilicates have also demonstrated adsorption of bacterial cells and toxins. On this basis, two aluminosilicate materials, geopolymers (GP) and ion-exchange nanozeolites (IE-nZeos), were screened for antimicrobial activity against MU and its fast-growing relative, Mycobacterium marinum (Mmar). GPs demonstrated adsorption of MU cells and mycolactone, a secreted, lipophilic toxin, whereas Cu-nZeos and Ag-nZeos demonstrated antibacterial activity against MU and Mmar. Cumulatively, these results indicate that an integrative drug selection process may yield a new generation of antimycobacterial agents.
ContributorsDermody, Roslyn June (Author) / Haydel, Shelley E (Thesis advisor) / Bean, Heather (Committee member) / Nickerson, Cheryl (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
Description
Microfluidics has enabled many biological and biochemical applications such as high-throughput drug testing or point-of-care diagnostics. Dielectrophoresis (DEP) has recently achieved prominence as a powerful microfluidic technique for nanoparticle separation. Novel electric field-assisted insulator-based dielectrophoresis (iDEP) microfluidic devices have been employed to fractionate rod-shaped nanoparticles like Single-walled carbon nanotubes (SWNTs) and manipulate biomolecules like Deoxyribonucleic acid (DNA) and proteins. This dissertation involves the development of traditional as well as 3D-printed iDEP devices for the manipulation of nm-to-µm scale analytes. First, novel iDEP microfluidic constriction-based sorting devices were developed to introduce inhomogeneous electric field gradients to fractionate SWNTs by length. SWNTs possess length-specific optical and electrical properties, expanding their potential applications for future nanoscale devices. Standard synthesis procedures yield SWNTs in large-length polydispersity and chirality. Thus, an iDEP-based fractionation tool for desired lengths of SWNTs may be beneficial. This dissertation presents the first study of DEP characterization and fractionation of SWNTs using an iDEP microfluidic device. Using this iDEP constriction sorter device, two different length distributions of SWNTs were sorted with a sorting efficiency of >90%. This study provides the fundamentals of fractionating SWNTs by length, which can help separate and purify SWNTs for future nanoscale-based applications. Manipulation of nm-scale analytes requires achieving high electric field gradients in an iDEP microfluidic device, posing one of the significant challenges for DEP applications. Introducing nm-sized constrictions in an iDEP device can help generate a higher electric field gradient. However, this requires cumbersome and expensive fabrication methods. In recent years, 3D printing has drawn tremendous attention in microfluidics, alleviating complications associated with complex fabrication methods. A high-resolution 3D-printed iDEP device was developed and fabricated for iDEP-based manipulation of analytes. A completely 3D-printed device with 2 µm post-gaps was realized, and fluorescent polystyrene (PS) beads, λ-DNA, and phycocyanin protein trapping were demonstrated. Furthermore, a nm-resolution 3D-printed iDEP device was successfully printed. In the future, these high-resolution 3D-printed devices may lead to exploring DEP characteristics of nanoscale analytes like single protein molecules and viruses. The electric field-assisted unique fractionation phenomena in microfluidic platforms will become a critical solution for nanoparticle separation and manipulating biomolecules.
ContributorsRabbani, Mohammad Towshif (Author) / Ros, Alexandra (Thesis advisor) / Stephanopoulos, Nicholas (Committee member) / Buttry, Daniel (Committee member) / Arizona State University (Publisher)
Created2022