Expanding the natural functions of biomolecules: polymerase redesign &amp; new advances in RNA- and lipid-based biosensors
Creators: name: Aggarwal, Tushar
Publisher: name: No Publisher Supplied
Year: 2023

description: This dissertation delineates systematic research initiatives centered on the strategic remodeling and deliberate repurposing of biomolecules, with the aim of integrating these innovative tools as advanced biosensors within the framework of life sciences. This mission constituted a methodical effort to develop novel approaches and to address some of the current challenges in chemical biology. In this thesis document, I shall describe my contributions to three specific areas; (1) Reengineering of a DNA polymerase for enzymatic synthesis of a novel phosphoramidate bond, (2) Development of nucleic acid aptamers and small molecules for sensing biomarkers and redox species, and (3) Utilization of a designer lipid as a peroxynitrite sensor in vivo and ex vivo.In chapter one, I have outlined the narrative and broader purpose behind each research undertaking. This introductory chapter conveys to the reader the philosophical exploration behind each chapter of the thesis.Chapter two delves into the potential of engineering a DNA polymerase to enzymatically form internucleotidyl N–P linkages (phosphoramidate bonds) using 3′-NH2-ddNTPs to form a 3′-nitrogen-5′-phosphorus-linked DNA (NP-DNA), where each 3′-O atom is replaced by a 3′- NH. These unconventional nucleic acid backbones show promise in therapeutic realms, with their nuclease resistance potentially ensuring prolonged and enhanced treatments. Wild type (WT) DNA polymerases naturally catalyze the formation of genetic polymers. An archaeal B-family DNA polymerase, 9°N, can utilize non-standard deoxynucleoside triphosphate monomers for primer extensions but does not create internucleotidyl N−P linkages. Through a strategic, structure-based design approach, we substituted a key aspartic acid in the active-site of 9°N, resulting in its ability to incrementally extend 3′-amino terminated primers using 3′-NH2-ddNTP monomers. Further mutations in distal finger subdomain yielded a quadruple mutant 9°N-NRQS, that can extend a 3′-amino-G primer on a DNA template at rates similar to nonenzymatic primer extensions with activated nucleotide building blocks. These copolymers, which include DNA and NP-DNA linkages, could offer special structural and functional traits. For instance, DNA/NP-DNA chimeras might assume intricate structures resembling DNA/RNA while being more resistant to nucleolytic degradation. This work has the potential to profoundly influence synthetic biology, biotechnology, and molecular medicine by pushing the boundaries of designer biological systems and crafting new nucleic acid structures with unique properties and applications.Chapter three presents our recent ventures into the promising territory of fluorescent light-up aptamer (FLAP) systems. Historically, the innate challenge has been the inability of these systems to detect small inorganic molecules, especially short-lived redox species effectively. New aptamer sequences are typically identified using systematic evolution of ligands by exponential enrichment (SELEX), a method that allows for the rapid identification of aptamers with desired target specificities. Here, we have designed a pre-ligand library using small molecule synthesis which, when integrated into our current system of genetically encoded RNA aptamer, enable the detection of inorganic targets—namely hydrogen sulfide (H2S) and hydrogen peroxide (H2O2). These structures, when interacting with H2S or H2O2, get transformed into native ligands, facilitating real-time detection. An exciting breakthrough came when we tested this in a live-cell environment. Using E. coli. cells, we were able to generate a detectable fluorescence signal in response to these inorganic molecules. What's particularly exciting for us, and the broader field, is the efficiency this method offers. Instead of venturing down the time-consuming and resource-intensive path of creating new aptamer sequences, we have effectively reworked our existing toolkit. This approach not only streamlines our processes but also significantly expands the capabilities of FLAP systems.In Chapter Four, I outline the development of a split DNA-based aptamer biosensor designed for microRNA detection. microRNA (miRNA) has a crucial function in normal and abnormal biological processes through its mediation of intercellular signaling, leading to increasing interest in using miRNAs as diagnostic and prognostic biomarkers. Despite the growing list of clinically relevant miRNA biomarkers, adapting existing chemical tools for miRNA research in the native environments of cells and tissues remains challenging. This study outlines a fast approach to create aptamer-based fluorescent biosensors that are designed to recognize miRNAs under biologically relevant conditions (10–30% v/v), including media from HeLa cell cultures, human serum, and plasma. The strategy is based on a semi-rational design of hybridization between DNA oligonucleotides and the target miRNA, resulting in a range of potential aptamers. Following a screening process, we identified aptamers with optimal signal-to-background ratios and strict target specificity. The DNA oligonucleotides used in this process are readily available and versatile, requiring no chemical modification, making them adaptable to a wide array of both established and emerging miRNA targets. Using this approach, we developed sensors capable of detecting specific oncogenic miRNAs (miR-19b, miR-21, and miR-92a) at concentrations as low as 5 nM without the need for amplification, demonstrating strong discrimination against single-nucleotide variants. This work provides a simplified framework for developing aptamer-based biosensors for miRNA detection.The concluding chapter elucidates our endeavors directed towards the development of novel detection methodologies for inorganic redox species within biological systems, culminating in the creation of a meticulously designed, lipid-based membrane-localizing biosensor. Peroxynitrite (ONOO–) is a reactive agent produced during nitrative stress, and its activity in lipid-rich areas is a concern due to the lack of effective detection tools. Addressing this gap, our study unveils DPPC-TC-ONOO–, a pioneering phospholipid probe tailored to detect ONOO– in varied lipid environments like biomimetic vesicles, the endoplasmic reticulum, and the lung lining. When this novel probe is combined with another lipid molecule, it can form large vesicles. These vesicles can detect ONOO– in a fluorogenic manner, meaning they emit fluorescence upon contact. This confirmed the effectiveness of probe in a laboratory setting. For real-world application and clinically relevant settings, we employed lipid nanoparticles to transport our probe into live mammalian cells. This was transformative; post introducing nitrative stress, we observed a heightened fluorescence in the endoplasmic reticulum. Even more impressive, our approach allowed us to detect ONOO– activity in lung tissues during acute lung injury. Specific observations revealed ONOO– activity in specific lung cells in standard mice post-injury, but this activity was noticeably absent in genetically modified variants.In conclusion, this thesis work signifies a substantial advancement in the field of biosensing. Through the judicious amalgamation of principles derived from synthetic organic chemistry, molecular &amp; cell biology, we are charting a course toward the realization of more sophisticated and versatile cell-based sensors in synthetic biology for the future.