RESUMO
RNA-protein complexes (RNPs) are essential components in a variety of cellular processes, and oftentimes exhibit complex structures and show mechanisms that are highly dynamic in conformation and structure. However, biochemical and structural biology approaches are mostly not able to fully elucidate the structurally and especially conformationally dynamic and heterogeneous nature of these RNPs, to which end single molecule Förster resonance energy transfer (smFRET) spectroscopy can be harnessed to fill this gap. Here we summarize the advantages of strategic smFRET studies to investigate RNP dynamics, complemented by structural and biochemical data. Focusing on recent smFRET studies of three essential biological systems, we demonstrate that investigation of RNPs on a single molecule level can answer important functional questions that remained elusive with structural or biochemical approaches alone: The complex structural rearrangements throughout the splicing cycle, unwinding dynamics of the G-quadruplex (G4) helicase RHAU, and aspects in telomere maintenance regulation and synthesis.
Assuntos
Transferência Ressonante de Energia de Fluorescência , Quadruplex G , RNA/química , Imagem Individual de Molécula , Animais , Bovinos , Análise por Conglomerados , Cristalografia por Raios X , Humanos , Cadeias de Markov , Conformação de Ácido Nucleico , Ligação Proteica , Desnaturação Proteica , Dobramento de Proteína , Estrutura Secundária de Proteína , Splicing de RNA , Ribonucleoproteínas , Spliceossomos/química , Telomerase/química , Telômero/química , Telômero/ultraestruturaRESUMO
Due to the high toxicity of currently used chemotherapeutics, novel methods of cancer treatment are needed. Gold nanoparticles (AuNPs) seem to be an interesting alternative due to penetration through biological membranes and systemic barriers. AuNPs as carriers of chemotherapeutics allow for reduced concentrations whilst maintaining the expected effect, and thus reducing the costs of therapy and adverse effects. We synthesized AuNPs stabilized with reduced glutathione (GSH) and conjugated with doxorubicin (DOX), gemcitabine (GEM) or cytarabine (CTA). This is the first study in which cytarabine-AuNPs were synthesized and characterized. Transmission electron microscopy (TEM), thermogravimetric analysis (TGA), nuclear magnetic resonance spectroscopy (NMR) and high-performance liquid chromatography (HPLC) were used to chemically characterize obtained nanoparticles. Antitumor activity and safety of application were assessed by MTT assay in in vitro model (human osteosarcoma cells -143B, human osteoblast- hFOB1.19, breast cancer cells - MCF7, breast epithelial cells - MCF10A, pancreatic cancer cells - PANC-1, and pancreatic cells - hTERT-HPNE cells). We have shown that cellular response varies according to the type and concentration of AuNPs. At some concentrations, we were able to show selective cytotoxicity of our AuNPs conjugates only to cancer cell lines. Synthesized nanoparticles were more cytotoxic to tumor cell lines than chemotherapeutics alone.
Assuntos
Glutationa/farmacologia , Ouro/química , Nanopartículas Metálicas/química , Neoplasias/tratamento farmacológico , Cromatografia Líquida de Alta Pressão , Citarabina/química , Citarabina/farmacologia , Desoxicitidina/análogos & derivados , Desoxicitidina/química , Desoxicitidina/farmacologia , Doxorrubicina/química , Doxorrubicina/farmacologia , Glutationa/química , Ouro/efeitos adversos , Humanos , Células MCF-7 , Nanopartículas Metálicas/efeitos adversos , Microscopia Eletrônica de Transmissão , Neoplasias/genética , Neoplasias/patologia , Osteoblastos/efeitos dos fármacos , Telomerase/química , GencitabinaRESUMO
Maintenance of telomeres by telomerase permits continuous proliferation of rapidly dividing cells, including the majority of human cancers. Despite its direct biomedical significance, the architecture of the human telomerase complex remains unknown. Generating homogeneous telomerase samples has presented a significant barrier to developing improved structural models. Here we pair single-molecule Förster resonance energy transfer (smFRET) measurements with Rosetta modeling to map the conformations of the essential telomerase RNA core domain within the active ribonucleoprotein. FRET-guided modeling places the essential pseudoknot fold distal to the active site on a protein surface comprising the C-terminal element, a domain that shares structural homology with canonical polymerase thumb domains. An independently solved medium-resolution structure of Tetrahymena telomerase provides a blind test of our modeling methodology and sheds light on the structural homology of this domain across diverse organisms. Our smFRET-Rosetta models reveal nanometer-scale rearrangements within the RNA core domain during catalysis. Taken together, our FRET data and pseudoatomic molecular models permit us to propose a possible mechanism for how RNA core domain rearrangement is coupled to template hybrid elongation.
