abstract: RNA aptamers adopt tertiary structures that enable them to bind to specific ligands. This capability has enabled aptamers to be used for a variety of diagnostic, therapeutic, and regulatory applications. This dissertation focuses on the use RNA aptamers in two biological applications: (1) nucleic acid diagnostic assays and (2) scaffolding of enzymatic pathways. First, sensors for detecting arbitrary target RNAs based the fluorogenic RNA aptamer Broccoli are designed and validated. Studies of three different sensor designs reveal that toehold-initiated Broccoli-based aptasensors provide the lowest signal leakage and highest signal intensity in absence and in presence of the target RNA, respectively. This toehold-initiated design is used for developing aptasensors targeting pathogens. Diagnostic assays for detecting pathogen nucleic acids are implemented by integrating Broccoli-based aptasensors with isothermal amplification methods. When coupling with recombinase polymerase amplification (RPA), aptasensors enable detection of synthetic valley fever DNA down to concentrations of 2 fM. Integration of Broccoli-based aptasensors with nucleic acid sequence-based amplification (NASBA) enables as few as 120 copies/mL of synthetic dengue RNA to be detected in reactions taking less than three hours. Moreover, the aptasensor-NASBA assay successfully detects dengue RNA in clinical samples. Second, RNA scaffolds containing peptide-binding RNA aptamers are employed for programming the synthesis of nonribosomal peptides (NRPs). Using the NRP enterobactin pathway as a model, RNA scaffolds are developed to direct the assembly of the enzymes entE, entB, and entF from E. coli, along with the aryl-carrier protein dhbB from B. subtilis. These scaffolds employ X-shaped RNA motifs from bacteriophage packaging motors, kissing loop interactions from HIV, and peptide-binding RNA aptamers to position peptide-modified NRP enzymes. The resulting RNA scaffolds functionalized with different aptamers are designed and evaluated for in vitro production of enterobactin. The best RNA scaffold provides a 418% increase in enterobactin production compared with the system in absence of the RNA scaffold. Moreover, the chimeric scaffold, with E. coli and B. subtilis enzymes, reaches approximately 56% of the activity of the wild-type enzyme assembly. The studies presented in this dissertation will be helpful for future development of nucleic acid-based assays and for controlling protein interaction for NRPs biosynthesis. / Dissertation/Thesis / Doctoral Dissertation Biochemistry 2020

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The emerging field of RNA nanotechnology necessitates creation of functional RNA nanoparticles, but has been limited by particle instability. Previously, it was found the three-way junction (3WJ) of the Phi29 DNA packaging motor pRNA was found to be ultra-stable and assemble in solution without the presence of metal ions. The three-way junction is composed of three short oligo RNA strands and proven to be thermodynamically stable. Here the assembly mechanism, thermodynamic and enzymatic stabilities, and kinetics are examined in order to understand the stability behind this unique motif. Thermodynamic and kinetics studies found that the pRNA 3WJ formed out of three components at a rapid rate creating a single-step three component collision with a lack of dimer intermediate formation while being governed by entropy, instead of the commonly seen enthalpy. Furthermore, the pRNA 3WJ proved to be stable at temperatures above 50 °C, concentrations below 100 pM, and produced a free energy of formation well below other studied RNA structures and motifs. With the high stability and folding efficiency of the pRNA 3WJ, it serves as an ideal platform for multi-branched RNA nanoparticles constructed through bottom-up techniques. RNA nanoparticles were constructed for the specific targeting of prostate cancer cells expressing Prostate Specific Membrane Antigen (PSMA) by receptor mediated endocytosis through the addition of an RNA aptamer; and the delivery of anti-miRNA sequences for gene regulation. The resulting nanoparticles remained stable while showing highly specific binding and entry in PSMA positive cells through cell surface receptor endocytosis. Furthermore, the entry of the nanoparticles allowed for the knockdown of against onco-miRNAs. Nanoparticles harboring antimiRNAs led to the upregulation of tumor suppressor genes, and signaling of apoptotic pathways. These findings display RNA nanotechnology can result in the production of stable nanoparticles and result in the specific treatment of cancers, specifically prostate cancer.

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