Over the past years, small RNAs (smRNAs) have been identified as important molecular regulators of gene expression and specifically eukaryotic messenger RNAs (mRNAs). Small RNAs including small-interfering RNAs (siRNAs) and microRNAs (miRNAs) take part in the RNA silencing pathway and regulate various pathways in the cell including transcription, genome integrity, chromatin structure, mRNA stability, and translation. siRNAs are usually from exogenously derived molecules, while miRNAs are expressed endogenously by the genome. The RNA silencing pathway is highly conserved between organisms and plays a critical part in maintaining homeostasis, host-pathogen interaction, and disease progression. Thus, a better understanding of the RNA silencing pathway and probing of the molecules involved in the process is instrumental in developing tools that can better regulate the expression of specific genes. The viral suppressor of RNA silencing (VSRS) p19, is a 19 kDa protein that is expressed by tombusviruses and exhibits the highest reported affinity to small RNAs, including siRNA and miRNA. Further engineering of this protein acts as an interesting means to control the RNA silencing pathway and provides a platform to design novel tools to further modulate the activity of smRNAs in living systems. The ability to incorporate new and useful chemical functionality into proteins within living organisms has been greatly enhanced by technologies that expand the genetic code. These usually involve bioorthogonal transfer RNA (tRNA) /aminoacyl-tRNA synthetase (aaRS) pairs that can selectively incorporate an unnatural amino acid (UAA) site specifically into ribosomally synthesized proteins. Site-specificity is coded for by using a rare codon such as the amber stop codon. In Chapter 2, we demonstrate the engineering of p19 for the development of a Förster resonance energy transfer (FRET) reporter system for the visualization of RNA delivery and release in cells using UAAs and bioorthogonal click chemistry, which was done by incorporating azidophenylalanine (AzF). In Chapter 3, by incorporating UAAs into p19’s binding pocket, we were able to enhance its smRNA suppressing activity by covalently trapping the bound substrates. We have demonstrated the engineering of a molecular switch that contains photo-crosslinking groups that covalently trap smRNAs. In Chapter 4, incorporating a metal-ion chelating UAA (2,2′-bipyridin-5-yl) alanine (BpyAla) into p19’s binding pocket has successfully led to site-specific cleavage of small RNAs including siRNAs and endogenous miRNAs. The genetic introduction of BpyAla provides a unique method of introducing catalytic activity into proteins of interest. The developed unnatural enzyme provides a new tool for catalytic suppression of the RNA silencing pathway. These results demonstrate the power of adding new chemistries to proteins using UAAs to achieve possible, diverse applications in therapy and biotechnology.

Chez les plantes, le RNA silencing (RNAi) est le principal mécanisme de défense antivirale. Il est opéré par de petites molécules d’ARN (siRNA), de 21-22nt de long, générées à partir de l’ARN viral par DCL4 et DCL2, respectivement. Ces siRNA confèrent la séquence-spécificité des réactions de défense intracellulaire et peuvent se déplacer à longue distance pour immuniser les cellules saines. En conséquence, les virus ont développé des protéines (VSRs) capables de supprimer ces deux aspects du RNAi. Au cours de cette thèse, j’ai pu démontrer in vivo que la protéine P15 du Peanut clump virus (PCV) est capable de séquestrer les siRNA de 21 et 22nt et qu’elle bloque le mouvement de ces derniers plus efficacement que ceux de 21nt. Pour compenser cette faiblesse, au cours de l’infection par le PCV, P15 est transportée à l’intérieur des peroxisomes en association avec les siRNA qu’elle séquestre. Le confinement des siRNA mobiles de 21nt à l’intérieur de ces organelles conduit à une inhibition du RNAi systémique et stimule fortement la propagation du PCV à travers la plante. Ces travaux définissent une nouvelle stratégie de pathogénèse virale au cours de laquelle une organelle est utilisé pour neutraliser des molécules de défense produites par l’hôte. / In plants, RNA interference (RNAi) is the main antiviral defense mechanism. It is initiated through the processing of viral RNA into 21-22nt long siRNA by DCL4 and DCL2, respectively. These siRNA can mediate sequence-specific local defense reactions (cell-autonomous RNAi) or move to distant tissues to prime defenses in naive cells (systemic RNAi). Consequently, viruses have evolved proteins (VSRs) to suppress both aspects of RNAi. In this in vivo study, I show that P15, the VSR of Peanut clump virus (PCV), binds and sequesters both 21nt and 22nt siRNA. Importantly, it stops the movement of 22nt siRNA more efficiently than 21nt siRNA. During infection, P15 is shuttled into peroxisomes, and is able to « piggyback » siRNA into these organelles. By confining mobile DCL4-dependent antiviral 21nt siRNA within peroxisomes, P15 is able to shut down systemic RNAi and strongly promote PCV movement. This work describes a novel pathogenic strategy in which an organelle is used to neutralize host defensive molecules.

Molecular modeling is a term referring to the study of proteins, nucleic acids, lipids, and other bio- or macro- or small molecules at the atomistic level using a combination of computational methods, physico-chemical principles, and mathematical functions. It can be generally sub-divided into two areas: molecular mechanics, which is the treatment of atoms and bonds as Newtonian particles and springs, and quantum mechanics, which models electronic behaviors using the Schrödinger equation and wavefunctions. Each technique is a powerful tool that, when used alone or in combination with wet lab experiments, can yield useful results, the products of which have broad applications in studying human disease models, oxidative damage, and other biomolecular processes that are otherwise not easily observed by experiment alone. Within this document, we study seven different such systems. This includes the mode of inhibitor binding to the enzyme monoamine oxidase B, the active site mechanism of that same enzyme, the dynamics of the unstructured p53 C-terminal domain in complex with globular, structured proteins, the process of the viral protein B2 unbinding from double-stranded RNA, and a focus on the dynamics of a variable loop in the antigenic peanut protein Ara h 2. In addition to those conventional molecular modeling studies, several of which were done in tandem with wet lab experiment, we also discuss the validation of charges and charge group parameters for small molecules used in molecular mechanics, and the development of software for the analysis of lipid bilayer systems in molecular mechanics simulations. As computational resources continue to evolve, and as more structural information becomes available, these methods are becoming an integral part of the study of biomolecules in the context of disease. / Ph. D.