G-quadruplex (G4) is involved in many biological processes, such as telomere function, gene expression and DNA replication. The selective isolation of G4 using affinity ligands that bind tightly and selectively is a valuable strategy for discovering new G4 binders for the separation of G4 from duplexes or the discrimination of G4 structures. In this work, one affinity chromatographic support was prepared using a naphthalene amine as a G4 binder. The ligand was immobilized on epoxy-activated Sepharose CL-6B using a long spacer arm and was characterized by HR-MAS spectroscopy. The supercoiled (sc) isoform of pVAX1-LacZ and pVAX1-G4 was isolated from a native sample. Also, the recovery and isolation of the plasmid isoforms from Escherichia coli lysate samples were achieved using an ionic gradient with different concentrations of NaCl in 10 mM Tris-HCl (pH 7.4). The retention times of different DNA/single strand sequences that can form G4, such as, c-MYC, c-kit1, c-kit2, tetrameric, telomeric (23AG), thrombin aptamer (TBA) and 58Sγ3 in this support were evaluated. Our experimental results suggest that the support exhibits selectivity for parallel c-MYC and c-kit1 G4s. In vitro transcription was performed using purified sc pVAX1-G4 and pPH600 to induce G4 formation and circular dichroism (CD) analysis confirmed that both transcripts adopt a parallel G4 topology.

Purification of plasmid DNA targeting therapeutic applications still presents many challenges, namely on supports and specific ligand development. Monolithic supports have emerged as interesting approaches for purifying pDNA due to its excellent mass transfer properties and higher binding capacity values. Moreover, arginine ligands were already described to establish specific and preferential interactions with pDNA. Additionally, some studies revealed the ability of arginine based cationic peptides to condense plasmid DNA, which increased lengthening can result in strongest interactions with higher binding capacities for chromatographic purposes of large molecules such as pDNA. In this work, arginine homopeptides were immobilized in monolithic supports and their performance was evaluated and compared with a single arginine monolithic column regarding supercoiled (sc) plasmid DNA purification. Specific interactions of arginine based peptides with several nucleic acids present in a clarified Escherichia coli lysate sample showed potential for the sc pDNA purification. Effectively, the immobilization of the arginine homopeptides became more functional compared with the single arginine amino acid, showing higher binding capacities, which was also reflected in the intensity of the interactions. The combination of structural versatilities of monoliths with the specificity of arginine peptides raised as a promising strategy for sc pDNA purification.

P53 is implicated in various cellular functions and several studies have shown that transfection of cancer cells with wild-type p53-expressing plasmids could directly drive cells into growth arrest and/or apoptosis. In the present work, the 6.07 kbp pcDNA3-FLAG-p53 plasmid, which encodes the p53 tumor suppressor, was produced and recovered from a recombinant cell culture of Escherichia coli DH5α. Following plasmid biosynthesis, the O-phospho-l-tyrosine chromatographic matrix was explored to purify the supercoiled p53-encoding plasmid. In order to quickly determine the optimal chromatographic performance and to obtain the required purity degree, maximizing the recovery yield of the supercoiled plasmid DNA, the Composite Central Face design was applied. The model revealed to be statistically significant (p-value < 0.05), with coefficient of determination of 0.9434 for the recovery yield and 0.9581 for purity and the central point was successfully validated. After the chromatographic process optimization by using the design of experiments tool, 49.7% of the supercoiled p53-encoding plasmid was recovered with 98.2% of purity, when a decreasing ammonium sulphate gradient was applied. The dynamic binding capacity of the O-phospho-l-tyrosine agarose column was 0.35 ± 0.02 mg pDNA/mL matrix at 50% of the breakthrough. Finally, the purified sample was analysed to assess the content of endotoxins, proteins and genomic DNA, showing that all these impurity levels were below the recommendations of the regulatory agencies.

The present study reports the successful production of human pre-miR-29b both intra- and extracellularly in the marine phototrophic bacterium Rhodovulum sulfidophilum using recombinant RNA technology. In a first stage, the optimal transformation conditions (0.025 μg of plasmid DNA, with a heat-shock of 2 min at 35 °C) were established, in order to transfer the pre-miR-29b encoding plasmid to R. sulfidophilum host. Furthermore, the extracellular recovery of this RNA product from the culture medium was greatly improved, achieving quantities that are compatible with the majority of applications, namely for in vitro or in vivo studies. Using this system, the extracellular human pre-miR-29b concentration was approximately 182 μg/L, after 40 h of bacterial growth, and the total intracellular pre-miR-29b was of about 358 μg/L, at 32 h. At the end of the fermentation, it was verified that almost 87 % of cells were viable, indicating that cell lysis is minimized and that the extracellular medium is not highly contaminated with the host intracellular ribonucleases (RNases) and endotoxins, which is a critical parameter to guarantee the microRNA (miRNA) integrity. These findings demonstrate that pre-miRNAs can be produced by recombinant RNA technology, offering novel clues for the production of natural pre-miRNA agents for functional studies and RNA interference (RNAi)-based therapeutics.

This laboratory experiment describes the production and purification of plasmid DNA for undergraduate biochemistry and biotechnology courses. This experiment performed in a one-week period includes the protocols for plasmid pVAX1-LacZ production in Escherichia coli DH5α cells and subsequent purification of supercoiled pVAX1-LacZ. Firstly, the students use a growth medium that favors the replication of the plasmid resulting in a higher plasmid production during exponential growth. Afterwards, alkaline lysis is done to disrupt the bacterial cells and recover pVAX1-LacZ plasmid. Lastly, they perform the purification of pVAX1-LacZ supercoiled isoform by L-histidine chromatography, followed by agarose gel electrophoresis to characterize the separation of supercoiled isoform from contaminants. The proposed experiment provides an opportunity for students to acquire these skills that are routinely used in biochemistry and biotechnology laboratories. © 2019 International Union of Biochemistry and Molecular Biology, 47(6):638-643, 2019.

Well-defined oligo(ethylene glycol) methyl ether methacrylate (OEOMA) based block copolymers with cationic segments composed by N,N-(dimethylamino) ethyl methacrylate (DMAEMA) and/or 2-(diisopropylamino) ethyl methacrylate (DPA) were developed under biorelevant reaction conditions. These brush-type copolymers were synthesized through supplemental activator and reducing agent (SARA) atom transfer radical polymerization (ATRP) using sodium dithionite as SARA agent. The synthesis was carried out using an eco-friendly solvent mixture, very low copper catalyst concentration, and mild reaction conditions. The structure of the block copolymers was characterized by size exclusion chromatography (SEC) analysis and 1H nuclear magnetic resonance (NMR) spectroscopy. The pH-dependent protonation of these copolymers enables the efficient complexation with plasmid DNA (pDNA), yielding polyplexes with sizes ranging from 200 up to 700 nm, depending on the molecular weight of the copolymers, composition and concentration used. Agarose gel electrophoresis confirmed the successful pDNA encapsulation. No cytotoxicity effect was observed, even for N/P ratios higher than 50, for human fibroblasts and cervical cancer cell lines cells. The in vitro cellular uptake experiments demonstrated that the pDNA-loaded block copolymers were efficiently delivered into nucleus of cervical cancer cells. The polymerization approach, the unique structure of the block copolymers and the efficient DNA encapsulation presented can open new avenues for development of efficient tailor made gene delivery systems under biorelevant conditions.

Mitochondrial gene therapy seems to be a valuable and promising strategy to treat mitochondrial disorders. The use of a therapeutic vector based on mitochondrial DNA, along with its affinity to the site of mitochondria, can be considered a powerful tool in the reestablishment of normal mitochondrial function. In line with this and for the first time, we successfully cloned the mitochondrial gene ND1 that was stably maintained in multicopy pCAG-GFP plasmid, which is used to transform E. coli. This mitochondrial-gene-based plasmid was encapsulated into nanoparticles. Furthermore, the functionalization of nanoparticles with polymers, such as cellulose or gelatin, enhances their overall properties and performance for gene therapy. The fluorescence arising from rhodamine nanoparticles in mitochondria and a fluorescence microscopy study show pCAG-GFP-ND1-based nanoparticles' cell internalization and mitochondria targeting. The quantification of GFP expression strongly supports this finding. This work highlights the viability of gene therapy based on mitochondrial DNA instigating further in vitro research and clinical translation.

In this work magnetic nanoparticles composed of maghemite coated with L-lysine or DL-Histidine (crystallite average diameter between 4.5 to 8.1 nm.) were synthesized via the Fe (II) and Fe (III) coprecipitation method. The synthesized solids were dispersed in water to obtain aqueous suspensions called magnetic fluids. The samples were characterized by X-ray diffraction, infrared spectroscopy, thermogravimetry, UV-Vis spectrophotometry, transmission electron microscopy, electrophoretic and dynamic light scattering. The suspended colloidal nanoparticles showed positive zeta potential at physiological pH, which enables the adsorption of negatively charged molecules of chromosomal and plasmid DNA on their surfaces. Their hydrodynamic radius were smaller than those reported in the literature (100nm) allowing phagocytosis of the nanostructure. A maghemite/L-Lysine sample was selected for DNA adsorption (chromosomal DNA of Humicola grisea fungus and shRNA and GFP encoding plasmids) and subsequent coating with dextran, for protection of its genetic information. Plasmids and dextran adsorption was evidenced by the increasing of the hydrodynamic radius of the samples, and the presence of DNA and dextran related bands in the infrared.

A deficiência na qualidade da água de abastecimento e na rede de coleta de esgoto vem contribuindo para o comprometimento da qualidade ambiental e das espécies que habitam esses ecossistemas. Ambientes aquáticos são considerados uma das principais vias de introdução de genes de resistência, o que os torna fonte de disseminação de organismos resistentes aos antimicrobianos entre populações humanas e animais. O Complexo Lagunar de Jacarepaguá encontra-se sob influência de águas ricas em matéria orgânica, devido principalmente a impactos antropogênicos, o que auxilia na degradação do meio ambiente natural local. No meio ambiente, cepas ambientais, como a família das enterobactérias, possuem a capacidade de adquirir resistência quando em contato com organismos patogênicos. A resistência a betalactâmicos, fluorquinolonas, aminoglicosídeos e carbapenêmicos, indica perfis hospitalares em cepas isoladas em ambientes aquáticos. Atualmente Escherichia coli, Klebisiella pneumoniae e Enterobacter cloacae são as três espécies da família das enterobactérias que mais frequentemente estão associadas a infecções humanas. O objetivo do presente estudo foi analisar cepas isoladas de amostras de água do Complexo Lagunar de Jacarepaguá, procurando comparar dois períodos, no que diz respeito à marcadores de resistência específicos para aminoglicosídeos, fluorquinolonas, beta-lactâmicos e carbapenêmicos. As amostras foram obtidas da Lagoa de Jacarepaguá, Lagoa de Marapendi e Canal de Camorim nos anos de 2005, 2007 e 2013. As cepas foram identificadas ao nível de espécie e submetidas testes de susceptibilidade a antimicrobianos. As cepas que apresentaram resistência a três ou mais grupos de antimicrobianos pré determinados (cafalosporinas de terceira e quarta geração, aminoglicosídeos, quinolonas e carbapenêmicos), foram consideradas multirresistentes e submetidas a ensaios de PCR. As cepas com resistência a três ou mais grupos de antimicrobianos foram submetidas a ensaios de conjugação bacteriana. Todas as cepas selecionadas apresentaram capacidade de transferência de resistência. Das 56 cepas analisadas, foi possível detectar a presença de 48 (85,7%) Escherichia coli, 6 (10,7) Klebsiella pneumoniae e duas (3,5%) Enterobacter cloacae. Das 56 cepas analisadas 14 foram submetidas a ensaios de PCR para detectar a presença de genes de ESBL. Três cepas apresentaram produtos de amplificação para o gene blaTEM, uma (para o gene blaCTX-M, uma para o gene blaTOHO e nenhuma apresentou produto de amplificação para o gene blaSHV. Nenhuma das cepas selecionadas apresentou positividade para os genes analisados para fluorquinolonas, aminoglicosídeos e carbapenêmicos. Quando comparados, os dados encontrados em 2005/2007 e 2013 demonstram certa regularidade na proporção de cepas multirresistentes durante os períodos avaliados, porém observamos um aumento na qualidade da água na lagoa de Marapendi. O uso inadequado de antimicrobianos no ambiente favorece a seleção de clones resistentes, por isso, a vigilância microbiológica constante deve permitir detectar a ocorrência, ou não, da progressão do processo de degradação pela urbanização descontrolada e pela descarga ilegal de esgoto.

A resistência a antibióticos representa uma ameaça crescente à saúde pública. Muitos agentes patogénicos, para além dos genes de virulência, possuem também genes de resistência a antibióticos, o que compromete o tratamento por antibioterapia, incluindo o tratamento de infeções causadas pelos agentes patogénicos mais virulentos. Nesse contexto, destaca-se o grupo de bactérias ESKAPEE (que inclui Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Enterobacter spp., e Escherichia coli) que é particularmente problemático, dada a sua elevada capacidade de adquirir resistências que comprometem a eficácia dos antibióticos disponíveis. Os plasmídeos são elementos genéticos extracromossomais, geralmente circulares e de tamanho variável, replicando-se de forma independente do cromossoma hospedeiro e encontrando-se disseminados em inúmeras espécies de bactérias e arqueias. Caracterizam-se por terem uma elevada plasticidade genética que lhes permite adquirir e disseminar genes que conferem vantagens seletivas aos seus hospedeiros, incluindo resistência a antibióticos, virulência e tolerância a metais pesados ou outros compostos tóxicos. Um aspeto central para compreender a relevância e a ecologia dos plasmídeos é a sua mobilidade. Os plasmídeos conjugativos codificam todos os genes necessários para a sua transferência autónoma entre células. Os plasmídeos mobilizáveis, por sua vez, dependem da maquinaria de plasmídeos conjugativos presentes na mesma célula, enquanto os plasmídeos denominados não-transmissíveis não se conseguem transferir através de conjugação porque lhes faltam todos os genes necessários para a sua transferência. A capacidade de os plasmídeos promoverem transferência horizontal de genes contribui para a adaptação das bactérias a diferentes contextos ambientais, através do aumento da sua diversidade genética, possibilitando a rápida resposta a pressões seletivas. Os plasmídeos são, portanto, fatores-chave para a evolução bacteriana.