Utilizing logic-gated DNA strand displacement to induce cancer prodrug activation

Highly selective cancer therapeutics with minimal off target effects that preserve patient quality of life are in high demand. Towards creating more targeted therapies, advances in bioinformatics and systems biology reinforce that cancer is an extremely complex disease, and multiple biomarkers must be considered to exclusively identify cancer cells. Furthermore, unique multi-input cancer signatures can vary between cancer subtypes, individual patients, and even over time within an individual. To address this, we have developed a flexible platform based on synthetic nucleic acid computation for a “smart drug” that can match the complex and dynamic nature of cancer. Harnessing the powerful properties of toehold-mediated strand displacement, our technology has the ability to control protein assembly through input-triggered nucleic acid circuits. By conjugating DNA to fluorescent proteins or a split version of yeast cytosine deaminase, a cancer prodrug activating enzyme, we can use nucleic acid computation to control protein behavior for sensing or therapeutic applications, respectively. We first created multi-input logic-gated circuits controlled by various cancer-specific miRNA inputs. Realizing the limitations that low input concentrations inside cells could place on our protein-DNA devices, we utilized catalytic hairpin assembly (CHA) for signal amplification. Next, we integrated both Boolean-logic and amplification architectures into a programmable and streamlined design. Moving beyond an in vitro demonstration, we executed our protein-DNA computation device in various cancer cells lines that endogenously express cancer-specific RNA. To our knowledge, this is the first time protein assembly/activity controlled by strand displacement computation has been executed inside live mammalian cells, a significant step in realizing the power of nucleic acid nanotechnology for future applications

Doctor of Philosophy

dissertationSpontaneous preterm birth (SPTB) is defined as birth before 37 completed weeks gestation that is not secondary to iatrogenic intervention. SPTB is both a pressing personal and public health issue that is not well understood. Previous studies have shown genetics as an important factor to SPTB. Here I present my PhD work on genetic analyses of SPTB. I dissected the problem with: (1) candidate gene, (2) quantitative genetics, and (3) genome-wide approaches. In Chapter 1, I explore why published candidate gene studies are inconsistent with each other. Allele frequency difference across populations provides one reason. I conducted a meta-analysis on the single nucleotide polymorphism (SNP) rs1800795, located in the promoter region of interleukin-6. With population stratification, I unmasked the signal showing that the CC genotype is protective against PTB in women of European descent. This also highlights how positive genetic signals can become obscured when the population structure is not controlled for. In Chapter 2, I use quantitative genetic approaches to decompose the etiologies of SPTB with massive data from the Utah Population Database. A generation effect, which confounds the heritability estimate, was discovered. I then utilized a sibling analysis and partitioned components contributing to the phenotypic variance of SPTB: iv heritability (13.33%), dominance genetic (11.12%), maternal effect (15.23%), and individual environment (60.33%). These findings shed light on the architecture of SPTB pathogenesis and quantify the maternal and fetal contribution to SPTB. In Chapter 3, I perform an unbiased genome-wide association study for SPTB on 22 autosomal chromosomes. The data, which contain cases and controls collected by the Danish National Birth Cohort, were acquired from the National Center for Biotechnology Information Genotypes and Phenotypes Database. No SNP reached genome-wide significance. Although negative, the result provides proof of principle regarding the relatively low heritability of SPTB. Finally, I address insights learned from these analyses that may help guide future SPTB research and may be applicable to other human genetic studies. With these lessons, progress can be made in understanding the genetics of SPTB

Conditional protein rescue (CPR) by binding-induced protective shielding

The rational regulation of protein concentration remains an elusive problem in synthetic biology. Several strategies have been developed at the transcriptional level, but these suffer from suboptimal response kinetics due to slow turnover of any protein already synthesized. The direct fusion of degradation tags to a protein of interest (POI) results in rapid fluctuations in protein concentration, and several degradation tags have been developed that function conditionally on the presence of a small molecule. However, no solutions currently exist that allow the intracellular concentration of a POI to be regulated by the cellular environment. Please click Additional Files below to see the full abstract

Programmable control of CRISPR-Cas9 systems by engineering sgRNA as toehold- switchable riboregulators

Robust control over gene expression is necessary for diverse applications in molecular biology, synthetic biology, and biotechnology. One of the most promising strategies to exert these types of control is the recently developed CRISPR interference (CRISPRi) and activation (CRISPRa) approach, which provides simple and highly effective RNA-based methods for targeted silencing and upregulation of transcription in bacterial and eukaryotic cells. While these current methods are capable of sequence-specific targeting Please click Additional Files below to see the full abstract

Controlled EGFR ligand display for tunable targeted intracellular delivery of cancer suicide enzymes

Advances in molecular engineering have led to customizable proteins for a multitude of clinical applications, leading to the rapid growth of the protein pharmaceutical market. Proteins can be advantageous compared to other treatment methods because of their functional complexities and high specificity that cannot be mimicked by small molecule drugs. Although there has been great interest and investment in advanced protein therapeutics, a majority of marketed proteins continue to have extracellular targets, despite the therapeutic relevance for intracellular protein therapies. Engineering efforts to improve cytosolic protein delivery often rely on modifying proteins through direct conjugation of polymers and peptides using reactive residues on amino acids. While this method has shown some success, the inability to modify a specific site within a protein can significantly hinder pharmacological action.Additionally, such approaches do not offer control over design variables that can be important determinants of targeting efficacy, such as ligand clustering. Previous work has demonstrated the ability to site-specifically insert biorthogonal reactive residues into proteins through unnatural amino acid incorporation, enabling direct protein conjugation with simple ‘click’ chemistries. Such an approach could be applied to protein therapeutics to explore the effect arrangement of delivery molecules has on protein bioactivity and intracellular delivery capabilities. In our work, we have demonstrated application of this approach for conjugation of epidermal growth factor receptor (EGFR) targeting peptides in fluorescent proteins. By varying EGFR peptide arrangements we have demonstrated the ability to tune cellular internalization in inflammatory breast cancer (IBC) cells. Furthermore, this system has been adapted for delivery of a cancer suicide enzyme to enable IBC-targeted cell death through prodrug activation. Through this approach, we have identified the importance of ligand display for targeted protein delivery and applied these finding to enhance enzyme delivery to IBC cells. Future efforts will refine the efficacy of this method though incorporation of endosomal escaping peptides and hydrophilic polymers to address additional challenges associated with in vivo intracellular protein delivery

Engineering a blue light inducible SpyCatcher system (BLISS) as a tool for protein photopatterning and optogenetics

The SpyTag-SpyCatcher protein conjugation system has recently exploded in popularity due to its fast kinetics and high yield under biologically favorable conditions in both in vitro and intracellular settings. We imagine we can further expand the utility of this system by introducing the ability to spatially and temporally control the conjugation event. Taking inspiration from photoreceptor proteins in nature, we designed a method to integrate light dependency into the protein conjugation reaction. The light-oxygen-voltage 2 domain of Avena sativa (AsLOV2) undergoes a dramatic conformational change in response to blue light. We have thus genetically fused the SpyTag into the AsLOV2 domain to create a Blue Light Inducible SpyCatcher System (BLISS). In this design (Figure 1), the SpyTag is blocked from reacting with the SpyCatcher in the dark, but upon irradiation with blue light, the Jα-helix of the AsLOV2 undocks to expose the SpyTag. We screened several likely insertion points in the Jα-helix, and found a variant with desirable light switching behavior where after one hour of irradiation, the reaction is 80% complete, while only 10% of the AsLOV2-SpyTag protein reacted in the dark. This reaction can be quenched within minutes by returning the reaction to the dark. We demonstrated the spatial aspect of this light control mechanism through photopatterning proteins onto Ni-NTA coated slides. As our system is made solely from protein components, which can be genetically encoded, we can extend the same spatiotemporal control of proteins inside cells. We anticipate BLISS will be a strong tool for fabricating protein microassays, crafting biomaterial composition, as well as optically controlling enzyme activity and protein localization in cells. Please click Additional Files below to see the full abstract

1/ f Noise and Machine Intelligence in a Nonlinear Dopant Atom Network

Noise exists in nearly all physical systems ranging from simple electronic devices such as transistors to complex systems such as neural networks. To understand a system's behavior, it is vital to know the origin of the noise and its characteristics. Recently, it was shown that the nonlinear electronic properties of a disordered dopant atom network in silicon can be exploited for efficiently executing classification tasks through “material learning.” Here, we study the dopant network's intrinsic 1/f noise arising from Coulomb interactions, and its impact on the features that determine its computational abilities, viz., the nonlinearity and the signal‐to‐noise ratio (SNR), is investigated. The findings on optimal SNR and nonlinear transformation of data by this nonlinear network provide a guideline for the scaling of physical learning machines and shed light on neuroscience from a new perspective

Versatile microbial surface-display for environmental remediation and biofuels production

Surface display is a powerful technique that utilizes natural microbial functional components to express proteins or peptides on the cell exterior. Since the reporting of the first surface-display system in the mid-1980s, a variety of new systems have been reported for yeast, Gram-positive and Gram-negative bacteria. Non-conventional display methods are emerging, eliminating the generation of genetically modified microorganisms. Cells with surface display are used as biocatalysts, biosorbents and biostimulants. Microbial cell-surface display has proven to be extremely important for numerous applications ranging from combinatorial library screening and protein engineering to bioremediation and biofuels production

International Full-Scale Test Facility for Structural Control

Planning is underway to establish an international full-scale test facility for structural control on the campus of the Hong Kong University of Science and Technology. The purpose of the test facility is to facilitate the development and application of structural response control and health monitoring technologies to improve the safety, serviceability and economy of structures required to resist wind loads, as well as the development of wind engineering information under various wind conditions. The test facility will be centered around a 30-meter high (ten-story) structure with state-of-the-art equipment for the experimental study of structural response control and real-time health monitoring, and it will be available for use by the international structural control community. Current plans are described for the full-scale test structure and associated test facilities, and for a proposed research program, international cooperation and industrial participation