Stabilizing DNA Nanostructures via Oligolysine-Based Polymers

Description

DNA nanotechnology, the self-assembly of DNA into 2D and 3D nanoscale structures facilitated via Watson and Crick base pairing, provides alternative solutions for biomedical challenges, especially for therapeutic cargo delivery, because it is easily fabricated, exhibits low cytotoxicity, and high…

DNA nanotechnology, the self-assembly of DNA into 2D and 3D nanoscale structures facilitated via Watson and Crick base pairing, provides alternative solutions for biomedical challenges, especially for therapeutic cargo delivery, because it is easily fabricated, exhibits low cytotoxicity, and high biocompatibility. However, the stability of these DNA nanostructures (DN) under cellular environment presents an issue due to their requirements for high salt conditions and susceptibility to nuclease degradation. Furthermore, DNs are typically trapped in endolysosomal compartments rather than the cytosol, where most of their cargo must be delivered. Many attempts to mitigate the stability issue have been made in recent years. Previously, our lab designed an endosomal escape peptide, Aurein 1.2 (denoted “EE, for endosomal escape)”, combined with a decalysine sequence (K10) proven to electrostatically adhere to and protect DNs under cell culture conditions. Unfortunately, this molecule, termed K10-EE, only resulted in endosomal escape in absence of serum due to formation of a protein corona on the surface of the coated DN.6 Therefore, we now propose to electrostatically coat the DN with a polymer composed of decalysine (K10), polyethylene glycol (PEG, which demonstrates antibiofouling properties), and peptide EE: K10- PEG1k-EE. Described herein are the attempted synthetic schemes of K10-PEG1k-EE, the successful synthesis of alternative products, K10-(EK)5 and K10-(PEG12)2-EE, and their resulting impacts on DN stability under biological conditions. Coating of the K10-(EK)5 with a DNA barrel origami demonstrated inefficient stabilizing capability in serum. Future studies include testing K10- (PEG12)2-EE protection for a variety of nucleic acid-based structures.

Date Created
2023-05
Agent

Molecular Structure in Amorphous Pharmaceuticals and Biopolymers

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Description
Molecular structures and dynamics in amorphous materials present unique experimental challenges compared with the characterization of crystalline solids. Liquids and glassy solids have many applications in industry such as ionic liquids for fuel cells or biomolecule stabilizing agents, enhancing pharmaceuticals…

Molecular structures and dynamics in amorphous materials present unique experimental challenges compared with the characterization of crystalline solids. Liquids and glassy solids have many applications in industry such as ionic liquids for fuel cells or biomolecule stabilizing agents, enhancing pharmaceuticals dissolution rates, and modified high performance biopolymers like silk for textile, biomedical, drug delivery, among many others. Amorphous materials are metastable, with kinetic profiles of phase transitions depending on relaxation dynamics, thermal history, plus factors such as temperature, pressure, and humidity. Understanding molecular structure and phase transitions of amorphous states of small molecules and biopolymers is broadly important for realizing their applications. The structure of liquid and glassy states of the drugs carbamazepine (CBZ) and indomethacin (IMC) were studied with solid-state nuclear magnetic resonance (ssNMR) spectroscopy, high energy X-ray diffraction, Fourier Infrared Transform Spectroscopy (FTIR), differential scanning calorimetry (DSC), and Empirical Potential Structure Refinement (EPSR). Both drugs have multiple crystalline polymorphs with slow dissolution kinetics, necessitating stable glassy or polymer dispersed formulations. More hydrogen bonds per CBZ molecule and a larger distribution of oligomeric states in the glass versus the liquid than expected. The chlorobenzyl ring of crystalline and glassy IMC measured with ssNMR were surprisingly found to have similar mobility. Crucially, humidity strongly affects glass structure, highlighting the importance of combining modeling techniques like EPSR with careful sample preparation for proper interpretation. Highly basic protic ionic liquids with low ∆pKa were synthesized with metathesis rather than proton transfer and characterized using NMR and dielectric spectroscopy. Finally, the protein secondary structure of spider egg sac silk was studied using ssNMR, FTIR, and scanning electron microscopy. Tubuliform silk found in spider egg sacs has extensive β-sheet domains which form nanocrystallites within an amorphous matrix. Structural predictions and spectroscopic measurements of tubuliform silk solution are mostly α-helical, with the mechanism of structural rearrangement to the β-sheet rich fiber unknown. The movement of spiders during egg silk spinning make in situ experiments difficult practically. This work is the first observation that tubuliform silk of Argiope aurantia after liquid crystalline spinning exits the spinneret as a predominantly (~70%) β-sheet fiber.
Date Created
2022
Agent

Membrane Protein Mimetic Dynamic DNA Nanostructures for Biosensing Applications

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…

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.
Date Created
2021
Agent

Photophysical Studies to Advance Fluorescence Applications in Biophysics

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Description
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
Agent

Functional Studies of Interactions in Proteins

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Description
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
Agent

Nanomedical Treatments for Cancer: Breakthroughs and Challenges

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Description

The purpose of this project is to analyze the current state of cancer nanomedicine and its challenges. Cancer is the second most deadly illness in the United States after heart disease. Nanomedicine, the use of materials between 1 and 100…

The purpose of this project is to analyze the current state of cancer nanomedicine and its challenges. Cancer is the second most deadly illness in the United States after heart disease. Nanomedicine, the use of materials between 1 and 100 nm to for the purpose of addressing healthcare-related problems, is particularly suited for treating it since nanoparticles have properties such as high surface area-to-volume ratios and favorable drug release profiles that make them more suitable for tasks such as consistent drug delivery to tumor tissue. The questions posed are: What are the current nanomedical treatments for cancer? What are the technical, social, and legal challenges related to nanomedical treatments and how can they be overcome? To answer the questions mentioned above, information from several scientific papers on nanomedical treatments for cancer as well as from social science journals was synthesized. Based on the findings, nanomedicine has a wide range of applications for cancer drug delivery, detection, and immunotherapy. The main technical challenge related to nanomedical treatments is navigating through biological barriers such as the mononuclear phagocyte system, the kidney, the blood-brain barrier, and the tumor microenvironment. Current approaches to meeting this challenge include altering the size, shape, and charge of nanoparticles for easier passage. The main social and legal challenge related to nanomedical treatments is the difficulty of regulating them due to factors such as the near impossibility of detecting nanowaste. Current approaches to meeting this challenge include the use of techniques such as scanning tunneling microscopy and atomic force microscopy to help distinguish nanowaste from the surroundings. More research will have to be done in these and other areas to enhance a major cancer-fighting tool.

Date Created
2021-05
Agent

Investigating the Effect of Salts and Small Molecule on Dissociation and Association Kinetics of the DNA Processivity Clamps using Fluorescence Techniques

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Description
In this study, the stability of two protein homo-oligomers, the β clamp (homodimer) from E. coli and the Proliferation Cell Nuclear Antigen (PCNA) from the yeast cell, were characterized. These clamps open through one interface by another protein called clam…

In this study, the stability of two protein homo-oligomers, the β clamp (homodimer) from E. coli and the Proliferation Cell Nuclear Antigen (PCNA) from the yeast cell, were characterized. These clamps open through one interface by another protein called clamp loader, which helps it to encircle the DNA template strand. The β clamp protein binds with DNA polymerase and helps it to slide through the template strand and prevents its dissociation from the template strand. The questions need to be to answered in this research are, whether subunit stoichiometry contributes to the stability of the clamp proteins and how does the clamp loader open up the clamp, does it have to exert force on the clamp or does it take advantage of the dynamic behavior of the interface?

The x-ray crystallography structure of the β clamp suggests that there are oppositely charged amino acid pairs present at the interface of the dimer. They can form strong electrostatic interactions between them. However, for Proliferation Cell Nuclear Antigen (PCNA), there are no such charged amino acids present at its interface. High sodium chloride (NaCl) concentrations were used to disrupt the electrostatic interactions at the interface. The role of charged pairs in the clamp interface was characterized by measuring the apparent diffusion times (\tau_{app}) with fluorescence correlation spectroscopy (FCS). However, the dissociation of the Proliferation Cell Nuclear Antigen (PCNA) trimer does not depend on sodium chloride (NaCl) concentration.

In the next part of my thesis, potassium glutamate (KGlu) and glycine betaine (GB) were used to investigate their effect on the stability of both clamp proteins. FCS experiments with labeled β clamp and Proliferation Cell Nuclear Antigen (PCNA) were performed containing different concentrations of potassium glutamate and glycine betaine in the solution, showed that the apparent diffusion time\ {(\tau}_{app}) increases with potassium glutamate and glycine betaine concentrations, which indicate clamps are forming higher-order oligomers. Solute molecules get excluded from the protein surface when the binding affinity of the protein surface for water molecules is more than solutes (potassium glutamate, and glycine betaine), which has a net stabilizing effect on the protein structure.
Date Created
2020
Agent

RNA-Based Computing Devices for Intracellular and Diagnostic Applications

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Description
The fundamental building blocks for constructing complex synthetic gene networks are effective biological parts with wide dynamic range, low crosstalk, and modularity. RNA-based components are promising sources of such parts since they can provide regulation at the level of transcription…

The fundamental building blocks for constructing complex synthetic gene networks are effective biological parts with wide dynamic range, low crosstalk, and modularity. RNA-based components are promising sources of such parts since they can provide regulation at the level of transcription and translation and their predictable base pairing properties enable large libraries to be generated through in silico design. This dissertation studies two different approaches for initiating interactions between RNA molecules to implement RNA-based components that achieve translational regulation. First, single-stranded domains known as toeholds were employed for detection of the highly prevalent foodborne pathogen norovirus. Toehold switch riboregulators activated by trigger RNAs from the norovirus RNA genome are designed, validated, and coupled with paper-based cell-free transcription-translation systems. Integration of paper-based reactions with synbody enrichment and isothermal RNA amplification enables as few as 160 copies/mL of norovirus from clinical samples to be detected in reactions that do not require sophisticated equipment and can be read directly by eye. Second, a new type of riboregulator that initiates RNA-RNA interactions through the loop portions of RNA stem-loop structures was developed. These loop-initiated RNA activators (LIRAs) provide multiple advantages compared to toehold-based riboregulators, exhibiting ultralow signal leakage in vivo, lacking any trigger RNA sequence constraints, and appending no additional residues to the output protein. Harnessing LIRAs as modular parts, logic gates that exploit loop-mediated control of mRNA folding state to implement AND and OR operations with up to three sequence-independent input RNAs were constructed. LIRA circuits can also be ported to paper-based cell-free reactions to implement portable systems with molecular computing and sensing capabilities. LIRAs can detect RNAs from a variety of different pathogens, such as HIV, Zika, dengue, yellow fever, and norovirus, and after coupling to isothermal amplification reactions, provide visible test results down to concentrations of 20 aM (12 RNA copies/µL). And the logic functionality of LIRA circuits can be used to specifically identify different HIV strains and influenza A subtypes. These findings demonstrate that toehold- and loop-mediated RNA-RNA interactions are both powerful strategies for implementing RNA-based computing systems for intracellular and diagnostic applications.
Date Created
2019
Agent

Eradication of multidrug-resistant bacteria using biomolecule-encapsulated two-dimensional materials

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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…

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.
Date Created
2019
Agent

Computational design and study of structural and dynamic nucleic acid systems

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Description
DNA and RNA are generally regarded as one of the central molecules in molecular biology. Recent advancements in the field of DNA/RNA nanotechnology witnessed the success of usage of DNA/RNA as programmable molecules to construct nano-objects with predefined shapes and…

DNA and RNA are generally regarded as one of the central molecules in molecular biology. Recent advancements in the field of DNA/RNA nanotechnology witnessed the success of usage of DNA/RNA as programmable molecules to construct nano-objects with predefined shapes and dynamic molecular machines for various functions. From the perspective of structural design with nucleic acid, there are basically two types of assembly method, DNA tile based assembly and DNA origami based assembly, used to construct infinite-sized crystal structures and finite-sized molecular structures. The assembled structure can be used for arrangement of other molecules or nanoparticles with the resolution of nanometers to create new type of materials. The dynamic nucleic acid machine is based on the DNA strand displacement, which allows two nucleic acid strands to hybridize with each other to displace one or more prehybridized strands in the process. Strand displacement reaction has been implemented to construct a variety of dynamic molecular systems, such as molecular computer, oscillators, in vivo devices for gene expression control.

This thesis will focus on the computational design of structural and dynamic nucleic acid systems, particularly for new type of DNA structure design and high precision control of gene expression in vivo. Firstly, a new type of fundamental DNA structural motif, the layered-crossover motif, will be introduced. The layered-crossover allow non-parallel alignment of DNA helices with precisely controlled angle. By using the layered-crossover motif, the scaffold can go through the 3D framework DNA origami structures. The properties of precise angle control of the layered-crossover tiles can also be used to assemble 2D and 3D crystals. One the dynamic control part, a de-novo-designed riboregulator is developed that can recognize single nucleotide variation. The riboregulators can also be used to develop paper-based diagnostic devices.
Date Created
2019
Agent
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