Many viruses use long (thousands of nucleotides) single-stranded (ss)RNAs as their genomic material. Such viruses can be as simple as a single RNA molecule encapsidated inside a shell (capsid) composed of many copies of a single capsid protein (CP). Cowpea chlorotic mottle virus (CCMV) and brome mosaic virus (BMV), the two sibling viruses studied throughout this work, are model ssRNA viruses that are capable of spontaneous self-assembly \textit{in vitro}. This work aims to elucidate how RNA content affects both the structure of the particles formed and their physical properties. In particular, particles containing one or another of a variety of RNA molecules are compared using physical and biological tools. The results of this work further our understanding of the self-assembly of ssRNA viruses, and the physical forces that result in their structure and dynamical properties.

Chapter one is an introductory chapter, introducing the reader to viruses, ssRNAs and their secondary/tertiary structures, CCMV and BMV, and the \textit{in vitro} self-assembly of these viruses. Chapters two and three discuss the production of viral-length polyU, an RNA molecule that lacks secondary structure due to its inability to base-pair or base-stack, and the packaging of this unique, structureless RNA with CCMV CP. Chapter four discusses the cryo-electron microscopy (cryoEM) asymmetric reconstruction of BMV virions, which allows for unprecedented visualization of the RNA genome inside a multipartite virus--a virus with a genome comprised of more than one RNA packaged into several indistinguishable particles. Chapter five summarizes the results of this work, and provides some future experiments related to the work discussed herein.

One of the holy grails in cancer therapy is to simultaneously image and deliver drugs to the tumor site. The first part of the thesis has developed new ideas in cancer theranostics and the second part is about the development of a novel contrast agent for risk-free imaging of the tumor. In the first project of the thesis, I have discussed the development of a liposome-based cargo delivery strategy that can simultaneously monitor alternating magnetic field–induced drug release by observing the change in MRI relaxation parameter R1, and the location and condition of liposomal site (such as tumor) from MRI parameter R2. However, the loading of a contrast agent in liposomes generally results in poor contrast and suffers from various artifacts in in vivo experiments, compared to the use of free contrast agent. Thus, the second project of my work in this part demonstrates the effective filtering of artifacts and contrast enhancement to obtain high quality sensitive imaging of the tumor site in a mouse model using paramagnetic liposomes, a novel pulsing sequence in active-feedback MRI, and robust data analysis. The second part of my work is about using a self-replicating viral-RNA molecule derived from Nodamura, an insect virus, to express and amplify ferritin, leading to increased iron content of the cells in the form of ferrihydrite that acts as a novel contrast agent. In summary, my thesis is about the development of cancer theranostics and a novel contrast agent. In the last part of the thesis, I have reviewed the current development of rapidly growing state of the art magnetic resonance cancer theranostics for commonly used polymer-based nanovehicles.

The first half of this dissertation (Chapters 1-3) deals with the in vitro self-assembly of Cowpea Chlorotic Mottle Virus (CCMV) from its single-stranded (ss) RNA genome and its capsid protein (CP). We have systematically investigated how the assembly depends on three previously unaddressed factors: (1) the length of the RNA; (2) the strength of attraction between the CP and the RNA; and (3) the electrostatic charge of the CP. Our results support a theoretically predicted but previously unverified self-assembly mechanism in which CP initially binds RNA in a disordered manner, and then reorganizes into the final icosahedral capsid structure. Additionally, we find that any length of RNA (from 140 to 12,000 nucleotides) can be efficiently packaged so long as two key requirements are met. First, the reaction mixture must contain a specific excess of CP to provide "charge-matching" between the relevant basic residues of the CP and the phosphate backbone of the RNA. Second, the assembly reaction must be carried out by a two-step, pH- and salt-dependent protocol.

In the second part (Chapters 4 and 5) we explore the potential use of self-assembled CCMV virus-like particles (VLPs), both as fluorescent nano-particles with controllable size (Chapter 4), and as gene delivery vectors (Chapter 5). We find that the fluorescent properties of dye-labeled polymers encapsidated by CCMV CP are robust against quenching by external quencher molecules. Additionally, we find that VLPs consisting of RNA genes packaged in CCMV capsids, once delivered to cytoplasm of mammalian cells, are released from their capsids and efficiently expressed, allowing them to be exploited for gene delivery.

In Chapter 6, we have examined the complicated branching networks of ss-RNA secondary structures by direct visualization with cryo-electron microscopy and confirmed a previously reported secondary structure of the entire genome of satellite tobacco mosaic virus. Finally, we discuss current preliminary data and future work in the Summary section.

Throughout this work we have repeatedly exploited the techniques of native agarose gel electrophoresis, velocity sedimentation, fluorescence spectroscopy (and microscopy), and electron microscopy.

Measurements are described that begin to elucidate the mechanism by which some RNA viruses deliver their genetic information to their hosts. The work focuses on two closely related icosahedral viruses, Cowpea Chlorotic Mottle Virus and Brome Mosaic Virus. Each consists of a 28-nm diameter icosahedral protein shell, the capsid, containing an approximately 3000-nt long single-stranded RNA. We hypothesize that the RNA inside the capsid is delivered to the host’s ribosomes by a thermal fluctuation of an end of the RNA being made available in a process called co-translational delivery. In this process, an end of the RNA fluctuates out of an intact capsid, is captured by the ribosomal machinery and is pulled out of the capsid under force.

We conducted studies to characterize RNA fluctuations that occur through capsid pores by capturing an RNA end. To realize this capture we have utilized ribosomes and purified eukaryotic initiation factor complex eIF4F. This complex is known to initiate the cap-dependent translation process of mRNA by eukaryotic ribosomes. In other studies, the fluctuations of the RNA outside of the capsid were captured by utilizing biotinylated fluorescent RNA which were caught by streptavidin-coated surfaces or nanoparticles.

Additionally we obtained preliminary data using magnetic tweezers to measure the forces required to extract RNA from a capsid with a protruding RNA end. Rather than capture a fluctuation in this force measurement work, we designed a virus like particle construct dubbed a “cherry bomb”. We produced this construct by packaging, in vitro, an RNA where its 5’ terminus had been made rigid by hybridizing to it a length of complementary DNA that is sufficiently long, such that it cannot be accommodated within the virus capsid.

Finally, we investigated how RNA can be condensed at the single-molecule level using polyvalent cations and observe changes in how a condensed RNA can be packaged into virus like particles by viral capsid protein.

Our results hint at the inherent dynamics of the viral RNA genome and its capsid shell, and the complex processes involved in a virus interacting with its host, processes that have been difficult to measure using other structural biological or physical chemical techniques.

Because of the unique simplicity of their life cycles, compared to all other evolving organisms, double-stranded (ds) DNA bacteriophages have served as an extremely valuable model for elucidating the basic physics and molecular biology of gene replication and expression. In this dissertation, we present experimental work on two phages, lambda and P22, to study the general genome delivery (ejection) mechanisms of dsDNA bacteriophage.

Using lambda phage, we systematically investigated the effects on DNA genome ejection of external osmotic pressure controlled by the concentration of osmolyte (PEG 8000) and the presence of polyvalent cations (tetravalent polyamine spermine, Sp4+). We found that the internal pressure of the capsid decreases from 38 to 17 atm as the [Sp4+] is increased from 0 to 1.5 mM. The existence of Sp4+ can also induce incomplete ejection under zero osmotic pressure when its concentration reaches 0.15 mM or higher; for [Sp4+] below this threshold, the ejection is complete. Further, we observed that the self-attraction induced by Sp4+ affects the configurational dynamics of the encapsidated genome, causing it to get stuck in a broad range of non-equilibrated structures.

In order to further study the DNA ejection mechanism from phage capsids, we have systematically determined how DNA transcription in vitro is affected by the presence of different osmolyte and viscogen molecules, so that we can test the transcription-pulling hypothesis of genome delivery in the presence of crowded environments mimicking the cytoplasm of the bacterial cell hosts of phages. We found that at high concentrations of DNA templates, macromolecules can increase the RNA yield due to crowding effects on the initiation step of transcription, while small molecules decrease the yield because of viscosity effects on the elongation step. Experiments carried out at low concentrations show a decrease in yield for large and small molecules, confirming the dominant effect of viscosity effects in the elongation step.

Having established and quantified the nature of the spontaneous driving force for DNA delivery, we then studied the ejection behavior of internal proteins from another phage, P22, and their functions in an in vitro osmotic suppression system controlled by PEG 8000. We found that the Outer Membrane Protein A (OmpA) from Salmonella, the natural bacterial host of P22, can significantly enhance the rate of DNA ejection in the presence of the primary receptor, LPS. While the DNA is ejected in the presence of LPS, no ejection of the internal proteins occurs unless OmpA is also present. We also find that their ejection is largely complete before any of the genome is ejected. This finding helps us understand the possible roles that the internal proteins play during infection.