Nucleases are enzymes that can specifically recognize nucleic acids and hydrolyze their phosphodiester bonds effectively. As is the case with many hydrolases, nucleases often carry one or more metal centers. Cooperation between such metal centers and other interactions involving general acid-base activities are believed to be essential in multifunctional catalyses. Combination of such interactions in model compounds often resulted in larger than additive effects. This work is aimed at synthesizing nuclease models that combine the ability to recognize phosphate groups and/or nitrogen bases of DNA together with the ability to catalyze phosphodiester hydrolysis. These models were designed to achieve optimum interaction between the recognition and the catalytic functionalities. Towards this goal, we chose phenonthiazonium ions (methylene blue analogues) and anthracene as spacers.

DNA in cells is constantly damaged by both endogenous and exogenous genotoxic agents. DNA repair is a cellular machinery that counters DNA damage and thus preserve genome integrity. Nucleotide excision repair (NER) in Escherichia coli (E. coli) is one of the DNA repair systems that recognizes and removes a variety of DNA damage such as pyrimidine dimers, bulky chemical adducts, DNA intrastrand cross-links, etc. The genes responsible for E. coli NER incisions are UvrA, UvrB, and UvrC. As the first step of E. coli NER, DNA damage recognition is achieved through the UvrA2B complex. Purification of UvrA, UvrB, and UvrC is essential for research to understand the molecular mechanisms of NER and carcinogenesis. Although UvrA, a 115 kDa protein, has been successfully purified in our lab in the past, the experimental procedures were very time-consuming and technically challenging. In this study we employed IMPACT (Intein Mediated Purification with an Affinity Chitin-binding Tag) system to subclone the cDNA of UvrA and express and purify the recombinant UvrA protein by a single-column step using the cloned expression construct. Furthermore, the purified protein was found to be fully functional in the UvrABC incision assay in which the DNA adduct of FABP [N-(20-deoxyguanosin-8-yl)-4-fluoro-4-aminobiphenyl] was efficiently cleaved in a time course-dependent manner.

Hepatitis B virus chronically infects 350-400 million people worldwide. It often leads to hepatocellular carcinoma, which causes >1 million deaths yearly. Current therapies prevent new viral genome formation but do not target pre-existing viral genomic DNA, thus curing only ~1/2 of patients. We targeted hepatitis B virus DNA for cleavage using zinc finger nucleases, which cleave as dimers. Co-transfection of our zinc finger nuclease pair with a target plasmid containing the hepatitis B virus genome resulted in specific cleavage. After three days in culture, 26% of the target remained linear, while ~10% was cleaved and mis-joined tail-to-tail. A portion of cleaved plasmids are repaired in cells, often with deletions and insertions. To track misrepair, we introduced an XbaI restriction site in the spacer between the zinc finger nuclease sites. Targeted cleavage and misrepair destroys the XbaI site. After three days in culture, ~6% of plasmids were XbaI resistant. 13 of 16 clones sequenced contained frameshift mutations that would lead to dramatic truncations of the viral core protein. These results demonstrate for the first time the feasibility of targeting episomal viral DNA genomes in cells using zinc finger nucleases. This strategy is broadly applicable toward inactivating other DNA viruses within cells. A major concern for the therapeutic use of zinc finger nucleases is off-target cleavage. To measure specificity, we employed in vitro assays and developed a bioinformatics method to find off-target cleavage sites in cultured cells. These sites can then be PCR amplified and tested using a mutation detection assay that we developed.

No description available.

Herpes Simplex Virus Type 1 (HSV-1) establishes latent infections in neurons in vivo and lytic infections in epithelial cells and fibroblasts. During latent infections, HSV-1 transcription is restricted and the genomes are not replicated. Latent HSV-1 genomes are chromatinized, such that digestion with micrococcal nuclease (MCN) releases DNA fragments with sizes characteristic of nucleosomal DNA. During lytic infections, in contrast, all HSV-1 genes are expressed, the genomes are replicated, and their digestion produces primarily heterogeneously sized fragments. However, as evaluated by ChIP assays, HSV-1 DNA interacts with histones during lytic infections, although in most cases only a small percentage of HSV-1 DNA co-immunoprecipitates with histones (or is cleaved to nucleosome sizes following MCN digestion). Therefore, although current models propose that chromatin regulates HSV-1 transcription, it remains unclear how the association of histones with only a small percentage of HSV-1 DNA can globally regulate viral transcription. Moreover, the physical properties of the complexes containing histones and HSV-1 DNA are unknown. My objective was therefore to evaluate the biophysical properties of the HSV-1 DNA-containing complexes during lytic infection. Differing from pervious studies, however, I used classical chromatin purification techniques. I show that most HSV-1 DNA is in unstable nucleoprotein complexes and, consequently, more accessible to MCN than DNA in cellular chromatin. This HSV-1 DNA is protected from MCN redigestion only after crosslinking, similar to unstable cellular nucleosomes. HSV-1 DNA is in such complexes throughout lytic infection. Using unrelated small-molecule inhibitors, I further show that inhibition of HSV-1 transcription is associated with a decrease in MCN accessibility of HSV-1 DNA. Roscovitine, a cyclin-dependent kinase inhibitor, prevents activation but not elongation of IE, E, and L HSV-1 transcription. Consistent with a functional association between accessibility and transcription, roscovitine only decreases the accessibility of DNA templates of which it also inhibits transcription, independent of specific promoter sequences. In summary, I show that most HSV-1 DNA is in unstable nucleosome-like complexes during lytic infection and that accessibility to HSV-1 DNA likely plays a key role in regulating HSV-1 transcription.

A short oligonucleotide sequence as in a single-stranded antisense oligo nucleotides (AON) or in double-stranded small interfering RNAs (siRNA) can modulate the gene expression by targeting against the cellular mRNA, which can be potentially exploited for therapeutic purposes in the treatment of different diseases. In order to improve the efficacy of oligonucleotide-based drugs, the problem of target affinity, nuclease stability and delivery needs to be addressed. Chemical modifications of oligonucleotides have been proved to be an effective strategy to counter some of these problems. In this thesis, chemical synthesis of conformationally constrained nucleosides such as 7′-Me-carba-LNA-A, -G, -MeC and -T as well as 6′, 7′-substituted α-L-carba-LNA-T (Papers I-III) was achieved through a key free-radical cyclization. 1D and 2D NMR techniques were employed to prove the formation of bicyclic ring system by free-radical ring closure as well as to identify the specific constrained conformations in sugar moieties. These sugar-locked nucleosides were transformed to the corresponding phosphoramidites and incorporated into antisense oligonucleotides in different sequences, to evaluate their physicochemical and biochemical properties for potential antisense-based therapeutic application. AONs modified with 7′-Me-carba-LNA analogues exhibited higher RNA affinities (plus 1-4°C/modification) (Papers I & III), but AONs containing α-L-carba-LNA analogues showed decreased affinities (minus 2-3°C/ modification) (Paper II) towards complementary RNA compared to the native counterpart.  It has been demonstrated in Papers I-III that 7′-methyl substitution in α-L-carba-LNA caused the Tm drop due to a steric clash of the R-configured methyl group in the major groove of the duplex, whereas 7′-methyl group of carba-LNA locating in the minor groove of the duplex exerted no obviously negative effect on Tms, regardless of its orientation. Moreover, AONs containing 7′-Me-carba-LNA and α-L-carba-LNA derivatives were found to be nucleolytically more stable than native AONs, LNA modified AONs as well as α-L-LNA modified ones (Papers I-III). We also found in Paper II & III that the orientations of OH group in C6′ of α-L-carba-LNAs and methyl group in C7′ of 7′-Me-carba-LNAs can significantly influence the nuclease stabilities of modified AONs. It was proved that the methyl substitution in cLNAs which points towards the vicinal 3′-phosphate were more resistant to nuclease degradation than that caused by the methyl group pointing away from 3′-phosphate. Additionally, AONs modified with 7′-Me-carba-LNAs and α-L-carba-LNAs were found to elicit the RNase H mediated RNA degradation with comparable or higher rates (from 2-fold to 8-fold higher dependent upon the modification sites) as compared to the native counterpart. We also found that the cleavage patterns and rates by E. coli RNase H1 were highly dependent upon the modification sites in the AON sequences, regardless of the structural features of modifications (Papers II & III). Furthermore, we have shown that the modulations of Tms of AON/RNA duplexes are directly correlated with the aqueous solvation (Paper III).

Traditional methods of pest control are threatened by the development of insecticide resistance, both to traditional insecticides and Bt toxins. Discovery of RNA interference (RNAi) has created opportunities to develop new insect control mechanisms. However, RNAi responses appear to be robust in coleopteran pests, but other orders, e.g. Lepidoptera and Hemiptera, present varied or ineffective RNAi responses. Current delivery strategies for double-stranded RNA (dsRNA) include microinjection, ingestion, and soaking. These approaches have benefits and problems. This study investigates the potential for porous silica nanoparticles (pSNPs) to improve the delivery of dsRNA and induce an RNAi response in Spodoptera frugiperda cells. Initially, the binding conditions of RNA onto porous and nonporous silica nanoparticles was examined, and the movement of RNA on and within pSNPs was observed. That information was then applied to in vitro studies for examining the capacity of silica nanoparticles to protect dsRNA from degradation by nucleases. This work culminated in an in vivo assay for measuring apoptosis when dsRNA is delivered to insect cells by pSNPs. Results of these studies show that silica nanoparticles bind nucleic acids and that dsRNA is mobile, pSNPs protect dsRNA from nuclease degradation, and pSNP/dsRNA complexes can induce apoptosis in lepidopteran insect cells.

A crucial step of the viral life cycle of Kaposi’s Sarcoma Herpesvirus (KSHV) lytic infection is the triggering of a massive RNA decay event termed “Host Shutoff”. Host Shutoff is driven by the viral endonuclease SOX which leads to the destruction of over 70% of the total transcriptome. This process cripples cellular gene expression and allows for viral reprograming of the cell for the purpose of viral replication. Co-evolution has led to the host developing a multitude of antiviral defenses aimed at preserving certain cellular RNAs linked to antiviral responses. One such defense are RNA secondary structures located within the 3’UTR of select host transcripts that protect them from SOX degradation. This structure, known as the SOX Resistant Element or SRE, has previously been isolated to a 200-nucleotide region found within the 3’UTR of the host transcript Interleukin-6. In this thesis, I sought to further define the structure of the IL-6 and other SREs using SHAPE-MaP to generate chemically-probed RNA structural models. Through this work, I demonstrated that the IL-6 SRE confers a form of active resistance to SOX cleavage, and based on structural analyses, likely acts as a scaffold for the recruitment of a protective ribonucleoprotein complex. This research highlights the importance of RNA secondary structures in influencing mRNA fate during viral infection and establishes the groundwork for understanding how these structural features can facilitate escape of cellular transcripts from viral endonucleases.

Les translocations chromosomiques sont des événements cellulaires rares signatures de nombreux cancers, pouvant mener à l’expression de nouveaux gènes de fusion oncogènes ou à la dérégulation d’un oncogène existant. Cependant, le lien direct entre la formation de translocations et la tumorigenèse n’est pas toujours bien établi. Jusqu’à présent, la modélisation de translocations se limitait principalement à la surexpression du gène de fusion créé. Pour mieux comprendre leur contribution à l’oncogenèse, nous avons développé une nouvelle méthode pour induire des translocations oncogéniques de novo, afin de recréer plus fidèlement les premières étapes de la transformation cellulaire.Pour cela, nous nous appuyons sur la technologie des nucléases artificielles telles que les nucléases à doigt de zinc, les TALEN (TALE Nucleases) et le système CRISPR/Cas9 (Clustered Regularly Interspaced Palindromic Repeats) pour générer des cassures ciblées de l’ADN et induire la formation de remaniements chromosomiques. Nous nous sommes particulièrement concentrés sur l’induction de la translocation modèle t(2;5)(p23;q32) et du gène de fusion NPM-ALK, associés au Lymphome Anaplasique à Grandes Cellules (ALCL), dans divers modèles cellulaires. Nous avons ainsi mis en évidence des propriétés oncogéniques du gène de fusion NPM-ALK exprimé sous son promoteur endogène suite à la formation du réarrangement chromosomique. Cependant, l’induction de la translocation dans des lymphocytes T primaires suggère que cet événement ne suffit pas à lui seul à initier l’oncogenèse, et nécessite probablement un contexte génétique ou épigénétique favorable. / Chromosomal translocations are signatures of numerous cancers and lead to expression of fusion genes that act as oncogenes. However, the wealth of genomic aberrations found in cancer makes it challenging to assign a specific phenotypic change to a specific aberration. We set out to use genome editing with Zinc Finger Nucleases (ZFN), Tale Effector Nucleases (TALEN), and the CRISPR/Cas9 (Clustered Regularly Interspaced Palindromic Repeats) to induce de novo specific chromosomal translocations in human cells, thus generating new models to interrogate the contribution of tumor-related translocations in first steps of oncogenesis. We specially focused on Anaplastic Large Cell Lymphoma (ALCL) t(2;5) translocation and NPM-ALK consequent fusion gene. For the first time, we highlighted oncogenic properties for NPM-ALK fusion expressed under endogenous promoter. However, translocation induction in primary T cells suggests that t(2;5) is not sufficient to initiate ALCL oncogenesis, and likely requires favourable genetic or epigenetic or context.

Designer nucleases have greatly simplified small genome modifications in many genomes. They can precisely target a specific DNA sequence within a genome and make a double stranded break (DSB). DNA repair mechanisms of the DSB lead to gene mutations or gene modification by homologous directed repair (HDR) if a repair template is exogenously supplied. Thus, small, site directed mutations are easily and quickly achieved. However, strategies that utilize designer nucleases for more complex tasks are emerging and require optimization. To optimize CRISPR/Cas9 assisted targeting, an HPRT rescue assay was utilized to measure the relationship between targeting frequency and homology arm length in targeting constructs in mouse embryonic stem cells. The results show that different gene engineering exercises had different homology requirements.