abstract: The highly predictable structural and thermodynamic behavior of deoxynucleic acid (DNA) and ribonucleic acid (RNA) have made them versatile tools for creating artificial nanostructures over broad range. Moreover, DNA and RNA are able to interact with biological ligand as either synthetic aptamers or natural components, conferring direct biological functions to the nucleic acid devices. The applications of nucleic acids greatly relies on the bio-reactivity and specificity when applied to highly complexed biological systems. This dissertation aims to 1) develop new strategy to identify high affinity nucleic acid aptamers against biological ligand; and 2) explore highly orthogonal RNA riboregulators in vivo for constructing multi-input gene circuits with NOT logic. With the aid of a DNA nanoscaffold, pairs of hetero-bivalent aptamers for human alpha thrombin were identified with ultra-high binding affinity in femtomolar range with displaying potent biological modulations for the enzyme activity. The newly identified bivalent aptamers enriched the aptamer tool box for future therapeutic applications in hemostasis, and also the strategy can be potentially developed for other target molecules. Secondly, by employing a three-way junction structure in the riboregulator structure through de-novo design, we identified a family of high-performance RNA-sensing translational repressors that down-regulates gene translation in response to cognate RNAs with remarkable dynamic range and orthogonality. Harnessing the 3WJ repressors as modular parts, we integrate them into biological circuits that execute universal NAND and NOR logic with up to four independent RNA inputs in Escherichia coli. / Dissertation/Thesis / Doctoral Dissertation Biochemistry 2019

Diagnostics are critical tools to assist in the identification of pathogens, the assessment of medical conditions, and helping to inform therapeutic decisions. Nevertheless, commonly used molecular diagnostics often require sophisticated instruments and skilled technicians, and therefore can only be done in centralized, well-equipped laboratories, which leads to long turnaround times, increased costs, and limited accessibility. These limitations have motivated the development of rapid, low-cost, decentralized diagnostics that are more widely accessible, affordable, and suitable for point-of-care applications. Synthetic biology, by creating rationally designed biological components that can sense disease markers, provides innovative and promising diagnostic solutions to achieve highly sensitive and specific detection for targets of interest, while at the same time being time- and cost-efficient, field-deployable, and shelf-stable. This dissertation focuses on the development of novel biosensing elements and their diagnostic applications. First, I introduce the methods for the computational design of riboregulators using automated algorithms. Followed by that, I describe the development, optimization, and applications of toehold-switch-based platforms for the detection of coccidioides, noroviruses, and severe acute respiratory syndrome coronavirus 2 (SARS‑CoV‑2). Next, I introduce the development of an ultra-specific riboregulator system termed single-nucleotide specific programmable riboregulators (SNIPRs) and their use for detecting different variants of concern of SARS-CoV-2. It is shown that riboregulators can be ideal solutions for various pathogen diagnostics with comparable accuracy and reduced cost. Lastly, I describe the use of peptide reporters derived from split protein systems to detect gene mutations. By incorporating peptide reporters into amplification primers, detection can be achieved by a quick isothermal amplification step and cell-free gene expression. Together, this research brings advancements in diagnostics based on riboregulators and cell-free systems that will increase the accessibility of these essential healthcare tools.

abstract: 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. / Dissertation/Thesis / Doctoral Dissertation Biochemistry 2019