DNA self-assembly has been explored as an effective approach for nanoconstruction. And Hermann’s group have reported to build a novel nanostructure incorporating L-shape RNA which guided by DNA strands to self-assemble into a hybrid RNA-DNA branched motifs. AP site, as a common lesion in DNA, has been demonstrated to be a good target position to interact with small molecular ligands like ATMND.

This project introduced ATMND and designed derivatives of it to the hybrid nanostructures built by Hermann’s group and detected different selectivity and affinity to different bases opposite AP site. This work demonstrated the feasibility to combine RNA, DNA and small molecules to construct new self-assembled nanostructures and provided a rational pathway to modify and functionalize nano materials.

Drug discovery in the last century has largely been dominated by therapeutics targeting proteins. RNA targeting has remained largely elusive and high-risk, but some recent therapeutics have seen great utility and success. One area where RNA targeting has strong potential is in tackling viral infections. Many viruses, especially RNA viruses have high rates of mutation that sometimes confer treatment resistance. A method to counter the high mutation rates of viruses is to develop treatments which target conserved regions of the viral genome that are highly structured. For my studies, two structured RNA targets were probed: the Zika virus (ZIKV) stem loop A (SLA) and the SARS-CoV-2 RNA pseudoknot. Replication of mosquito-borne pathogenic flaviviruses like Zika virus depends on a noncoding RNA motif in the 5’ untranslated region of the genome which recruits the viral NS5 replicase. The three-dimensional architecture of the replication promoter RNA is termed the SLA. Sequence alignment as well as conservation and covariation analyses as evidence support a canonical RNA three-way junction (3WJ) architecture for the SLA RNA. The SLA 3WJ also influences viral translation by interaction with the E protein coding region and the conformational flexibility of the SLA 3WJ may be exploited to discover selectively binding ligands that interfere with viral replication. As a proof of concept, antisense DNA ligands hybridized at a domain of the SLA RNA served as surrogate ligands eliciting an extensive conformational change in the 3WJ fold which is readily detected by measuring FRET response. The FRET binding assay provides a useful tool for the discovery of small molecule ligands that target the flavivirus replication promoter. Another FRET binding assay was utilized to discover ligands which selectively bind the SARS-CoV-2 RNA pseudoknot. The discovered ligands consisted of aminoglycosides such as tobramycin and were retested with a phenotypic in vitro translation assay to assess for physiological relevance. While the results were not physiologically relevant, the discovered ligands act as a foundation for future work for discovering pan-SARS-CoV-2 mutant treatments and highlight the potential in targeting structured RNA as a drug target.

Benzothiadiazines were synthesized and tested for their binding affinity with hepatitis C virus internal ribosome entry site subdomain IIa using FRET assay. The acetylated compound was designed to improve the solubility and explore the tautomerization involved in targeting the IIa subdomain. An optimized Cu2O catalyzed Ullmann reaction was developed for future use.

Increasing attention has been paid into RNA as a building block to make nanostructures in the past two decades. The goal of this study was to make a nanoprism which was fully made of double-stranded RNA by an efficient self-assembly method. This was done by extending a nanotriangle which was rationally designed and made from an RNA conformational switch in the internal ribosome entry site (IRES) of Seneca Valley virus (SVV) and then combining two extended triangles with DNA or RNA splints. The successful extension and combination of triangles were investigated by native polyacrylamide gel electrophoresis (PAGE). Details of the structure were further analyzed by Förster resonance energy transfer (FRET) methods, fluorescence quenching methods, and atomic force microscopy (AFM).

Nucleic acid has emerged as a new biomaterial to construct new functional biomaterials through mainly Watson-Crick base pairing between complementary sequences. Different from past efforts that used DNA and RNA largely separately to create nucleic acid nano-assemblies, this dissertation outlines a new research direction that sought to assemble discrete DNA and RNA modules to RNA-DNA hybrid nanoparticles. The new direction that marries the structural diversified RNA motifs as architectural joints and chemical flexible DNA modules as functional components gives rise to nanoparticle entities enriched with structural varieties and functional potentials at the sub-10 nanometer scale. The screening methods and multi-step assembly strategy described in the dissertation laid the foundation for designing and preparing complex RNA-DNA hybrid nanoparticles, as well as composite materials based on RNA-DNA hybrid nanoparticles.

Ligand-responsive RNA mechanical switches represent a new class of simple and small switching modules which regulate viral translation initiation by adopting well-defined ligand-free and bound conformational states without undergoing large secondary structure rearrangements, distinguishing them from metabolite-sensing riboswitches. Initially discovered in the internal ribosome entry site (IRES) of hepatitis C virus (HCV), RNA switch motifs have now been discovered in the genomes of diverse other viruses. Although large variations are seen in sequence and local secondary structure of these switches, their function in viral translation initiation requiring recognition of identical cognate ligands is conserved. These simple ligand-responsive viral RNA switches may be the target for therapeutic intervention and may be incorporated as tunable building blocks for nanotechnology.

Phylogenetic analysis and sequence alignments with other Flaviviruses have led us to identify a putative promoter motif located in a putative three-way junction of the 5’UTR RNA, called Stem Loop A (SLA), which allows for binding of the RNA-dependent RNA polymerase (RdRp), and thus replication of the viral RNA genome. To support this hypothesis through structural and biochemical characterization of this domain, milligrams of the SLA RNA were 

produced by in vitro transcription to then crystallize and determine its structure by X-ray diffraction. Determining the structure of the Zika virus SLA domain would contribute to our understanding of its mechanism of action. Although sufficient amounts of RNA were produced for crystallography, diffracting crystals have not yet been obtained. Additionally, a FRET assay has been developed and successfully used to confirm that the SLA domain of the Zika virus adopts a bent three-way junction conformation. FRET experiments with antisense DNA as surrogate ligands to parts of the SLA RNA suggest that the FRET assay will be useful in the future to discover small molecule ligands that alter and capture the SLA three-way junction in a non-functioning conformation.

Benzodiazepinones and cyclopropylquinazolinamines were designed and synthesized with the goal of creating ligands for the subdomain IIa of the hepatitis C virus (HCV) internal ribosome entry site (IRES) RNA and thereby inhibiting viral translation. The heterocyclic scaffolds were designed using insight into molecular recognition of the RNA target by previously discovered synthetic and natural ligands. The novel benzodiazepinones and cyclopropylquinazolinamines were evaluated for RNA target binding by a previously developed FRET assay.

The American Cancer Society estimates that there will be 97,000 new cases of colon cancer and 51,000 deaths in the United States in 2023Approximately 30% of these diagnoses are in patients under the age of 55. These numbers have been declining slightly in recent years as methods of detection are improving and people are making efforts to live healthier lifestyles. If detected early, the 5 year survival rate is approximately 90%; however, if detected late, the survival rate decreases significantly (14% in stage IV disease). With people being diagnosed at earlier ages, and with only approximately 51% of people being up to date on the colorectal cancer screening, it is imperative that we have as many tools as possible at our disposal for treatment of the disease. While palmyramide A is a promising new drug candidate that may potentially help in the treatment of this disease, availability from natural sources is extremely low (471 g of dry bacteria used to isolate 6.1 mg palmyramide A). To aid in having enough of the compound available, we are proposing the first highly convergent total synthesis that will produce enough material for further testing. Part I of this dissertation describes the first total synthesis of palmyramide A, the biological evaluation of the natural product, and modification of the natural product to use as a probe that can determine its molecular mechanism of action.The World Health organization estimates there are approximately 71 million people worldwide living with hepatitis C virus (HCV), with 15-30% of those developing advanced liver diseases such as cirrhosis or liver cancer. While current treatments can be effective, they are extremely cost prohibitive, limiting access for many people. Another issue is that most commercially available drugs target the same viral protein, non-structural protein 5B (NS5B), an RNA dependent RNA polymerase. Because HCV is a positive-sense RNA virus, its genome is translated and replicated with little proofreading, rendering it prone to mutations, and a mutation in NS5B could render these therapeutics ineffective. However, a small segment of the 5’ non-coding RNA, known as the internal ribosome entry site subdomain IIa, is extremely highly conserved, and is an attractive target for therapeutics. Part II of this dissertation describes the design and synthesis of new classes of therapeutics targeting the HCV IRES.