Doctor of Philosophy / Plant Pathology / John P. Fellers / Harold N. Trick / Wheat streak mosaic virus (WSMV) and Triticum mosaic virus (TriMV) are two viruses affecting wheat in the Great Plains region of the United States. Genetic resistance is severely limited, requiring management methods focusing on the deployment of resistant varieties and various cultural practices. Evaluation of resistance is complicated by the lack of a standard rating scale. The objective of this work was to develop new avenues to mitigate these challenges. A standardized virus symptom rating scale was developed using historical Kansas rating scales, and validated using multiple wheat populations. Two independent RNA interference (RNAi) expression vectors targeting portions of viral coat protein (CP) of WSMV and TriMV were previously transformed into wheat. T₂ plants and beyond were evaluated using PCR, reverse transcription-PCR and bioassays in which plants were challenged with their respective virus. These lines were evaluated for resistance through the T₆ generation. Crosses were made with the susceptible winter wheat cultivars, ‘Overley’ and ‘Karl 92.’ Real-time PCR results show viral titer was up to 20-fold lower in the T₆ transgenic lines, the F₁, and the BC₁F₁ compared to control plants. This provides evidence that this RNAi silencing method is stable in wheat over multiple generations. WSMV and TriMV use host eukaryotic initiation factors (eIF) in order to facilitate replication of their genomes. Previously created RNAi expression vectors were derived from the sequences of the wheat genes eIF(iso)4E-2 and eIF4G. Evaluation of these lines began in the T₁ generation. Resistance has been demonstrated in three lines of eIF(iso)4E-2 and four lines of eIF4G, derived by single seed descent. T₆ progeny co-infected with WSMV and TriMV continue to be resistant. Crosses have been performed with the winter wheat ‘Karl 92’ and three Kansas elite lines, KS030887K-6, KS09H19-2-3, and KS10HW78-1-1. RNAi construct effectiveness was evaluated using real-time PCR. Results show up to 18-fold reduction in viral titer in the transgenic lines, the F₁, and the BC₁F₁ in comparison to control plants. This research provides the first evidence that a single host transgene can provide resistance to multiple viruses and has great potential benefits to both breeders and producers.

Une fraction non négligeable de protéines qui contrôlent la dynamique de la chromatine et la transcription est conservée au cours de l'évolution chez les eucaryotes. Ces protéines se retrouvent dérégulées dans de nombreuses maladies, dont les cancers. Dans cette étude, nous avons exploité la purification de deux protéines associées à la chromatine pour étudier de nouveaux acteurs impliqués dans la réduction au silence (ou silencing) de la transcription au sein de l'hétérochromatine et/ou de l'euchromatine chez la levure Schizosaccharomyces pombe, un modèle de référence pour la biologie de la chromatine.Mmi1 est un facteur de liaison à l'ARN capable de guider la formation d'hétérochromatine facultative sur des gènes méiotiques. Parmi les protéines partenaires de Mmi1, nous nous sommes intéressés à Ccr4-Not, un complexe multifonctionnel, conservé de la levure à l'Homme, important pour la maturation de l'extrémité 3' des ARNs et pour le contrôle de l'expression des gènes. Nos travaux montrent que Ccr4-Not est également nécessaire pour le dépôt de la marque H3K9 méthylée aux gènes cibles de Mmi1, ainsi que pour le silencing de la transcription au sein de l'hétérochromatine constitutive, indépendamment de Mmi1.En parallèle, nous avons étudié deux nouveaux partenaires potentiels de RITS (RNA-Induced Transcriptional Silencing), un complexe nécessaire à la formation de l'hétérochromatine et l'inactivation de gène. Ces partenaires agiraient à l'interface entre la régulation de la chromatine et de la transcription. Le premier partenaire est l'histone chaperonne Spt6. Une caractérisation initiale entreprise sur Spt6 a montré son rôle crucial dans le silencing des gènes à l'hétérochromatine constitutive et facultative. Le second partenaire est Abo1, une histone chaperonne putative et homologue à la protéine humaine ATAD2, une protéine exprimée dans de nombreuses tumeurs et considérée comme une cible prometteuse pour le traitement de certains cancers, bien qu'à ce jour il n'y ait que peu d'information disponible sur sa fonction moléculaire. Nous avons dans un premier temps montré qu'Abo1 est nécessaire pour le silencing de la transcription au sein de l'hétérochromatine constitutive. Cependant, l'analyse du transcriptome des cellules abo1Δ a montré qu'Abo1 est également nécessaire au silencing transcriptionel de nombreux gènes codant et non-codant localisés dans l'euchromatine. Par la suite, nous avons purifié Abo1 et identifié par spectrométrie de masse le réseau des protéines qui lui est associé. Cette approche protéomique a montré qu'Abo1 est connectée à de nombreuses protéines impliquées dans le contrôle de la transcription, comme des histones chaperonnes et des complexes de remodelage ATP-dépendant de la chromatine. Enfin, nous avons montré que le défaut de croissance sévère observé dans les cellules abo1Δ est complètement rétabli par l'expression de ATAD2 humain. Ce dernier résultat indique que la caractérisation fonctionnelle d'Abo1, entreprise dans la levure, a le potentiel de fournir des informations importantes sur la fonction moléculaire non seulement d'Abo1, mais aussi d'ATAD2 et de son lien avec les cancers.En résumé, nos résultats permettent une meilleure compréhension de la fonction de trois acteurs impliqués dans le silencing de la transcription chez la levure fissipare. De plus, la caractérisation plus approfondie d'Abo1 pourrait grandement contribuer à élucider la fonction d'ATAD2 et de son rôle dans les cancers. / A sizeable fraction of proteins controlling chromatin dynamics and transcription are conserved throughout eukaryotes and are deregulated in many diseases, including cancer. In this study, we exploited the purification of two chromatin-associated proteins to characterize new actors in the context of euchromatic and/or heterochromatic gene silencing in Schizosaccharomyces pombe, a reference model for the biology of chromatin.Mmi1 is an RNA binding factor that can guide the formation of facultative heterochromatin assembly at meiotic genes. Among new proteins interacting with Mmi1, we examined the function of Ccr4-Not, which is a conserved multifunctional complex processing 3'ends of RNAs and regulating gene expression. We found that Ccr4-Not is also required for the deposition of H3K9 methylation mark at Mmi1 target genes and for gene silencing in a Mmi1-independent manner at constitutive heterochromatin.In parallel, we studied two new potential partners of RITS (RNA-Induced Transcriptional Silencing), a complex required for heterochromatin formation and gene silencing. Both partners are believed to act at the interface between chromatin and transcription regulation. The first one is the histone chaperone Spt6. An initial functional characterization conducted on this protein showed its implication in gene silencing, both at constitutive and facultative heterochromatin. The second one is Abo1, a putative histone chaperone which is homologue to human ATAD2 protein, a male germ factor ectopically expressed in many tumors and considered as a promising target for cancer therapy, although little is known about its molecular function. We first showed that Abo1 is necessary for proper heterochromatin gene silencing at constitutive heterochromatin. However, transcriptomic analysis of abo1∆ cells further extended Abo1's function in gene silencing to protein-coding and non-coding regions within euchromatin. In addition, we purified Abo1 and identified by mass spectrometry the network of its associated proteins. This proteomic approach showed that Abo1 is connected to several chromatin- and transcription-linked proteins, such as histone chaperones and ATP-dependent chromatin remodeling complexes. Finally, we demonstrated that the severe growth defect observed in abo1Δ is completely rescued by the expression of human ATAD2. This later finding indicates that the functional characterization of Abo1 in yeast has the potential to provide important insights into the molecular function not only of Abo1, but also of the cancer linked ATAD2 protein.Altogether, our results permitted a better understanding of three actors involved in chromatin-based gene silencing in fission yeast. In addition, a further characterization of Abo1 may contribute elucidating the function of ATAD2 and its role in cancer.

Submitted by Daniele Amaral (daniee_ni@hotmail.com) on 2016-10-06T19:17:32Z No. of bitstreams: 1 DissCPP.pdf: 3587784 bytes, checksum: 38e18e70b6c78d140d46b17a57600689 (MD5) / Approved for entry into archive by Marina Freitas (marinapf@ufscar.br) on 2016-10-21T13:32:45Z (GMT) No. of bitstreams: 1 DissCPP.pdf: 3587784 bytes, checksum: 38e18e70b6c78d140d46b17a57600689 (MD5) / Approved for entry into archive by Marina Freitas (marinapf@ufscar.br) on 2016-10-21T13:33:00Z (GMT) No. of bitstreams: 1 DissCPP.pdf: 3587784 bytes, checksum: 38e18e70b6c78d140d46b17a57600689 (MD5) / Made available in DSpace on 2016-10-21T13:33:10Z (GMT). No. of bitstreams: 1 DissCPP.pdf: 3587784 bytes, checksum: 38e18e70b6c78d140d46b17a57600689 (MD5) Previous issue date: 2015-10-14 / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Reactive oxygen species (ROS) are products naturally generated by the cell metabolism and at low levels play an important physiological role in intracellular regulation, whereas in excess can cause damage to cells. To combat this damage, cells present a complex defense mechanism including different enzymes which act as antioxidants. Among these enzymes, and especially in cells such as erythrocytes, which are exposed to high levels of molecular oxygen, the Peroxiredoxins (Prxs), stand out for the abundance and great reactivity with its substrates. In this cell type, when occurs hemolytic diseases such as sickle cell anemia and beta thalassemia, increased production of ROS and consequently oxidative damage are observed, greatly aggravating the clinical picture of patients affected by these diseases. In these diseases, the PRDX2 appears to be a major line of antioxidant defense, as it is the third most present protein in the cytosol of the erythrocyte. Therefore, this study aimed to assess the role of PRDX2 in differentiated K562 cells for the expression of erythroid characteristics, through gene silencing using shRNA_PRDX2. It was possible to obtain a 70% of the PRDX2 expression inhibition, which caused a decrease in the proliferation, cell viability and interaction, showing the importance of PRDX2 in oxidative protection on this cell type. In order to evaluate the modulation of antioxidant system in these cells, we also analyzed the pattern of gene and protein expression of all other PRDXs beyond the gene expression of other antioxidant enzymes during the process of differentiation. We found that inhibition of PRDX2 expression adversely affects the expression of PRDX5 and causes increased expression of their biological reducing agents, which increase the recycling PRDX2, compensating for their lack the cell. These data are not get described in the literature and additional analysis is needed to better understand this interaction beyond the molecular mechanisms involved in the expression of related enzymes in protection against ROS. Understanding these mechanisms seems important to work with a better insight of the pathophysiology of hemolytic diseases by identifying possible targets to assist in the management and can mitigate the effects of the disease in these patients. / Espécies reativas de oxigênio (EROs) são produtos gerados naturalmente pelo metabolismo celular e em baixos níveis fisiológicos desempenham importante papel na regulação intracelular, enquanto que em excesso podem causar diversos danos às células. Para combater esses danos, as células apresentam um complexo mecanismo de defesa incluindo diferentes enzimas que atuam como antioxidantes. Dentre estas enzimas e, principalmente em células como os eritrócitos, que são expostas a altos teores de oxigênio molecular, as Peroxirredoxinas (Prxs), se destacam pela abundância e grande reatividade com os seus substratos. Neste tipo celular, quando ocorrem doenças hemolíticas como a anemia falciforme e a beta talassemia, uma maior produção de EROs e consequentemente de danos oxidativos são observados, agravando sobremaneira o quadro clínico dos pacientes acometidos por estas doenças. Nessas doenças, a PRDX2 aparenta ser uma importante linha de defesa antioxidante, já que é a terceira proteína mais presente no citosol do eritrócito. Diante disso, esse trabalho teve como objetivo a avaliação do papel de PRDX2 em células K562 diferenciadas para a expressão de características eritróides, através do silenciamento gênico de PRDX2 utilizando shRNA. Foi possível a obtenção de uma inibição de 70% da expressão de PRDX2, a qual causou diminuição na proliferação, viabilidade e interação celular, mostrando a importância da PRDX2 na proteção oxidativa nesse tipo celular. Com o objetivo de avaliar a modulação do sistema antioxidante nestas células, analisamos também, o padrão de expressão gênica e proteica de todas as outras PRDXs além da expressão gênica de outras enzimas antioxidantes durante o processo de diferenciação. Verificamos que a inibição da expressão de PRDX2 afeta negativamente a expressão de PRDX5 e causa o aumento da expressão de seus redutores biológicos, o que aumentaria a reciclagem de PRDX2 compensando sua falta na célula. Esse dado é inédito na literatura e análises adicionais são necessárias para melhor compreender essa interação além dos mecanismos moleculares envolvidos na expressão de enzimas relacionadas na proteção contra EROS. A compreensão destes mecanismos parece importante para colaborar com o melhor entendimento da fisiopatologia de doenças hemolíticas, identificando possíveis alvos que auxiliem no manejo e que possam amenizar os efeitos da doença desses pacientes.

O câncer de mama é o tumor mais incidente entre as mulheres no mundo. Assim como em outros tumores, a tumorigênese nas mamas é um processo complexo resultante da combinação de fatores genéticos e ambientais que dirigem a transformação das células normais em células malignas. O gene PAWR, conhecido como PAR-4 (Prostatic apoptosis response-4) foi primeiramente identificado em células de câncer de próstata de rato induzidas a apoptose e codifica uma proteína de 342 aminoácidos que é efetiva na indução de apoptose nas células tumorais e causa regressão dos tumores ativando Fas/FasL e inibindo a atividade de NF-B. O aumento de Par-4 é suficiente para causar apoptose seletiva em células tumorais, mas não em células normais ou imortalizadas. Recentemente, um estudo de nosso grupo demonstrou que a expressão reduzida de Par-4 está associada a um pior prognóstico em câncer de mama e que esta proteína pode ter um papel importante na morfogênese da glândula mamária. O estudo funcional do gene Par-4 em diferentes linhagens de mama é importante para o melhor entendimento do papel deste gene no processo tumorigênico da glândula mamária. Portanto, a proposta do presente estudo foi investigar os efeitos do aumento de expressão ou supressão de Par-4 na proliferação celular e sobrevivência das células normais e tumorais de mama. As células MCF10A e MCF-7 foram transfectadas com os vetores de expressão para Par-4 (pCMV6-PAR-4) ou com oligos siRNA para supressão transiente de Par-4. A caracterização dos clones foi feita por Real Time PCR e Western Blot. Os ensaios de proliferação foram feitos por MTT e os ensaios de apoptose por dupla marcação com Laranja de Acridina/ Hoechst 33342 e por citometria de fluxo. O aumento da expressão de Par-4 diminuiu a proliferação de ambas às células comparadas às células-controle (p<0,01). Por outro lado, a diminuição de expressão de Par-4 por siRNA levou ao aumento da proliferação das células tumorais MCF-7 (p<0,01). A quantificação das células em apoptose mostrou um aumento significativo de apoptose nas células MCF-7 com aumento de Par-4 em relação às células controle MCF-7 pcNEO tratadas com ambas as concentrações de docetaxel (5 nM 13,24% versus 3,89%; p=0.001; 100 nM 24,81% versus 6,07%; p=0,0001) por 24 horas. Embora preliminares, os resultados de citometria também mostraram que as células MCF-7 com aumento de Par-4 apresentam maior porcentagem de células em apoptose na ausência de tratamento e após tratamento com 100 nM de docetaxel por 24 horas. Embora novos estudos sejam necessários, nossos dados sugerem que o aumento de expressão de Par-4 sensibilizou as células MCF-7 ao quimioterápico docetaxel. Pela primeira vez, mostramos o efeito inibitório de Par-4 no crescimento e sobrevivência das células epiteliais mamárias / Breast cancer is the most common tumor among women in the world. As for other malignancies the tumorigenic process of the breast involves genetic alterations that drive the progressive transformation of normal cells into malignant cells with and aggressive phenotype. Alterations in cells that upregulate proliferation or downregulate apoptosis are one of essential mechanisms that dictate tumor growth. The PAWR gene, also known as PAR-4 (Prostatic apoptosis response-4), was first identified in prostate cancer cells undergoing apoptosis and encodes a 332 aminoacid protein that is effective in inducing cancer cell apoptosis and cause regression of tumors by activating Fas/FasL and inhibiting NF-B activity. Interestingly, Par-4 overexpression is sufficient to cause apoptosis in cancer cells, but not in normal or immortalized cells. Recently, we demonstrated that reduced expression of PAR-4 is associated with breast cancer poor prognosis and this protein may have a role in the process of the mammary gland morphogenesis. The functional study of Par-4 gene in different cell lines of breast is important to better understand its role in the tumorigenic process of the breast. Therefore, the purpose of the present study was to investigate the effects of overexpression and suppression of PAR-4 in cell proliferation and survival in mammary epithelial cells. MCF10A and MCF-7 cells were transfected with expression vectors for PAR-4 overexpression (pCMV6-PAR-4) or with small interfering RNAs duplexed oligonucleotides. Clone characterization was performed using real time PCR and western blot. Proliferation assays were carried out using MTT and apoptotic assays were performed by doublefluorescence staining technique (Acridine Orange/Hoechst 33342) or using flow cytometry. PAR-4 overexpression decreased the proliferation rates in both MCF10A and MCF-7 cells compared to the parental or control cells (p<0,01). On the other hand, PAR-4 knockdown leads to increased proliferation in MCF7 cells (p<0,01). We observed a significant increase in apoptosis of MCF-7 cells with increased expression of Par-4 compared to control cells (MCF-7 pcNEO) in both concentrations of docetaxel (5 nM 13,24% versus 3,89%, p = 0,001; 100 nM 24,81% versus 6,07%, p < 0,0001). Cytometry analysis, although preliminary, showed that MCF-7 pcPar-4 have a higher percentage of apoptotic cells in the absence of treatment and after treatment of 100 nM of docetaxel. Although more studies are needed, our data suggest that increased expression of Par-4 sensitizes breast cancer cells to treatment with docetaxel. This is the first report showing that PAR-4 has inhibitory effects on mammary epithelial cells proliferation and survival

L’application de nanotubes de carbone (CNTs) dans le domaine biomédical a été largement explorée grâce à leur propriétés physico-chimiques et à leur biocompatibilité. Par la fonctionnalisation extérieur et/ou intérieur des CNTs c’est possible de préparer des nouveaux conjugués avec différentes propriétés et applications. On a exploré la modification des nanotubes par voie covalente pour leur utilisation comme vecteurs de biomolécules pour achever la thérapie anticancéreuse. Pendant ma thèse, j’ai travaillé sur trois projets: l’application de différentes approches pour la conversion des groupes acides carboxyliques de MWCNTs oxydés en amines, dans le but de préparer des conjugués capables de complexer du siRNA (petits ARN interférents). Dans un second projet, j’ai développé des conjugués à base de nanotubes de carbone couplés avec un fragment d’anticorps thérapeutique via une liaison clivable afin d’en étudier le potentiel antitumoral. Dans le dernier projet, on a achevé la fonctionnalisation de CNTs remplis avec des molécules radioactivables par cycloaddition de nitrene et ensuite conjugué un anticorps de ciblage tumoral. Le but été d’utiliser les nanotubes comme vecteurs pour la délivrance de radioactivité à l'intérieur des cellules tumorales ciblées par l’anticorps. On a aussi conduit des investigations biologique, afin d’évaluer la toxicité et l’efficace de ce conjugué. / The application of carbon nanotubes (CNTs) in the biomedical field has been widely explored thanks to their physico-chemical properties and their biocompatibility. By the external and/or internal functionalization of CNTs it is possible to prepare novel conjugates tailoring different properties and applications. We have investigated the covalent derivatization of CNTs by different chemical strategies to achieve suitable carriers for anticancer therapy. In one project, we have explored the conversion of the carboxylic groups of oxidized CNTs into amino groups, and the ability of these conjugates to complex genetic material, for gene delivery. In another project, CNTs have been functionalized with linkers bearing a cleavable disulfide bond, and further conjugated to a therapeutic nanobody for controlled intracellular drug release. Finally, we have investigated the reactivity of close-ended CNTs filled with radioactivable material toward Bingel and nitrene cycloadditions and the conjugation of a targeting antibody, for the target delivery of radioactivity. By several characterization techniques we have proved that the antibody is covalently grafted to the CNT-carrier and it still possesses its targeting ability. Investigations on the biological profile of these conjugates (cytotoxicity, targeting, uptake, biodistribution) have been also carried out.

Therapeutics based on siRNA represent a promising hope for the treatment of many congenital and acquired disorders. This method is based on posttranscriptional silencing of pathological gene or set of genes (RNAi process), which are responsible for the actual cause of the disease. Access is therefore based on the assumption of treatment options for the disease at the point of origin of the defect intervention at the molecular level, which is different from the conventional, so-called symptomatic therapy, which focuses only on the treatment or suppression of symptoms. Despite rapidly increasing understanding of gene function and cause a number of genetic diseases, the expansion of siRNA therapeutics limited the development of efficient and safe transport systems (vectors). In order to ensure efficient transport of siRNA in vivo conditions, the vectors must sufficiently reduce the size of the siRNA, protect it against degradation during transport, and release in the cytoplasm of the target cell. For this purpose they were developed sophisticated transport systems based on viral and non-viral origin. This diploma thesis is focused on the preparation of new transport systems, siRNA-based synthetic hydrophilic polymers, such as non-viral vectors. For in vitro testing the effectiveness during transport of siRNA...

In Saccharomyces cerevisiae, telomeric and HM silencing requires the histone deacetylase Sir2 and the chromatin binding proteins Sir3 and Sir4, which interact to form the SIR complex. Silent chromatin formation begins with a nucleation step, followed by spreading of Sir proteins along chromatin. Overexpression of Sir3 extends silent chromatin domains, however the role of Sir protein interactions within silent chromatin extensions remains unknown. Here, we generated the Sir3 mutant, Sir3-4A, which cannot interact with Sir4 but is capable of forming silent chromatin extensions when overexpressed. Within extended silent domains, Sir2 and Sir4 enrichments are similar whether Sir3 or Sir3-4A is overexpressed, suggesting that silent chromatin extensions require Sir4 but not the interaction between Sir3 and Sir4. Tethering Sir3-4A at an HMR silencer cannot nucleate silencing in the absence of Sir3, suggesting that in addition to Sir3 recruitment, the Sir3-Sir4 interaction has at least one other function during silent chromatin nucleation.

Knowledge of the processes of RNA interference, the regulation of gene expression by small RNAs (sRNAs), has grown at an unprecedented rate over the last 30 years. Some of the findings were literally revolutionary, as they revealed events that overturned many long-held notions. Many phenomena have been shown to be highly conserved and common to organisms of different species, but others are specific to certain lineages or have not yet been fully explored. There is also a lack of knowledge about the interconnection of numerous pathways - for example between silencing at the transcriptional (TGS, leading to the promoter methylation) and post-transcriptional levels (PTGS, affecting mRNA stability or translation). The present work summarizes the findings of two published and two unpublished works and attempts to describe some of the less known sites of RNA interference using various plant model organisms. Research on Solanum tuberosum transgenic lines has revealed the ability of 5-azacytidine to restore the expression of transcriptionally silenced transgenes at the whole plant level. De novo regeneration from leaves of such plants can lead to re-silencing of reactivated transgenes and thus serves as a selection method to exclude lines prone to spontaneous silencing. The nature of changes in the...

Mobile genetic elements represent a large portion of the genome in many species. Posing a danger to the integrity of genetic information, silencing and structural machinery has evolved to suppress the mobility of foreign and transposable elements within the genome. Condensin proteins – which regulate chromosome structure to promote chromosome segregation – have been demonstrated to function in repetitive gene regulation and transposon silencing in several species. In model system Caenorhabditis elegans, microarray analysis studies have implicated Condensin II subunit HCP-6 in the silencing of multiple loci, including DNA transposon MIRAGE. To address the hypothesis that HCP-6 has a direct function in transcriptional gene silencing of the MIRAGE transposon, we queried MIRAGE expression and chromatin profiles in wild-type and hcp-6 mutant animals. Our evidence confirms that HCP-6 does indeed function during silencing of MIRAGE. However, we found no significant indication that HCP-6 binds to MIRAGE, nor that HCP-6 mediates MIRAGE enrichment of H3K9me3, the repressive heterochromatin mark observed at regions undergoing transcriptional silencing. We suggest that the silencing of MIRAGE, a newly evolved transposon and the only tested mobile element considerably derepressed upon loss of HCP-6, is managed by HCP-6 indirectly.

Small silencing RNAs function in almost every aspect of cellular biology. Argonaute proteins bind small RNA and execute gene silencing. The number of Argonaute paralogs range from 5 in Drosophila melanogaster , 8 in Homo sapiens to an astounding 27 in Caenorhabditis elegans. This begs several questions: Do Argonaute proteins have different small RNA repertoires? Do Argonaute proteins behave differently? And if so, how are they functionally and mechanistically distinct? To address these questions, we examined the thermodynamic, kinetic and functional properties of fly Argonaute1 (dAgo1), fly Argonaute2 (dAgo2) and mouse Argonaute2 (mAGO2). Our studies reveal that in fly, small RNA duplexes sort into Argonaute proteins based on their intrinsic structures: extensively paired siRNA duplex is preferentially sorted into dAgo2 while imperfectly paired miRNA duplex is channeled into dAgo1. The sorting of small RNA is uncoupled from its biogenesis. This is exemplified by mir-277, which is born a miRNA but its extensive duplex structure licenses its entry into dAgo2. In the Argonaute protein, the small RNA guide partitions into functional domains: anchor, seed, central, 3' supplementary and tail. Of these domains, the seed initiates binding to target. Both dAgo2 and mAGO2 (more closely related to and a surrogate for dAgo1 in our studies) bind targets at astonishing diffusion-limited rates (~107–108 M−1s−1). The dissociation kinetics between dAgo2 and mAGO2 from their targets, however, are different. For a fully paired target, dAgo2 dissociates slowly (t½ ~2 hr) but for a seed-matched target, dAgo2 dissociates rapidly (t½ ~20 s). In comparison, mAGO2 does not discriminate between either targets and demonstrates an equivalent dissociation rate (t½ ~20 min). Regardless, both dAgo2 and mAGO2 demonstrate high binding affinity to perfect targets with equilibrium dissociation constants, KD ~4–20 pM. Functionally, we also showed that dAgo1 but not dAgo2 silence a centrally bulged target. By contrast, dAgo2 cleaved and destroyed perfectly paired targets 43-fold faster than dAgo1. In target cleavage, dAgo2 can tolerate mismatches, bulged and internal loop in the target but at the expense of reduced target binding affinities and cleavage rates. Taken together, our studies indicate that small RNAs are actively sorted into different Argonaute proteins with distinct thermodynamic, kinetic and functional behaviors. Our quantitative biochemical analysis also allows us to model how Argonaute proteins find, bind and regulate their targets.