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- Naphthalene amine support for G-quadruplex isolationPublication . Ferreira, João; Santos, Tiago; Pereira, Patrícia; Corvo, Marta C.; Queiroz, João; Sousa, Fani; Cruz, CarlaG-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.
- Plasmid production and purification: An integrated experiment‐based biochemistry and biotechnology laboratory coursePublication . Santos, Tiago; Pereira, Patrícia; Queiroz, João; Cruz, Carla; Sousa, FaniThis 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.
- Brain-Targeted Delivery of Pre-miR-29b Using Lactoferrin-Stearic Acid-Modified-Chitosan/Polyethyleneimine PolyplexesPublication . Pereira, Patrícia; Barreira, Maria; Cruz, Carla; Tomás, Joana; Luís, Ângelo; Pedro, Augusto; Queiroz, João; Sousa, FaniThe efficacy of brain therapeutics is largely hampered by the presence of the blood-brain barrier (BBB), mainly due to the failure of most (bio) pharmaceuticals to cross it. Accordingly, this study aims to develop nanocarriers for targeted delivery of recombinant precursor microRNA (pre-miR-29b), foreseeing a decrease in the expression of the BACE1 protein, with potential implications in Alzheimer's disease (AD) treatment. Stearic acid (SA) and lactoferrin (Lf) were successfully exploited as brain-targeting ligands to modify cationic polymers (chitosan (CS) or polyethyleneimine (PEI)), and its BBB penetration behavior was evaluated. The intracellular uptake of the dual-targeting drug delivery systems by neuronal cell models, as well as the gene silencing efficiency of recombinant pre-miR-29b, was analyzed in vitro. Labeled pre-miR-29b-CS/PEI-SA-Lf systems showed very strong fluorescence in the cytoplasm and nucleus of RBE4 cells, being verified the delivery of pre-miR-29b to neuronal cells after 1 h transfection. The experiment of transport across the BBB showed that CS-SA-Lf delivered 65% of recombinant pre-miR-29b in a period of 4 h, a significantly higher transport ratio than the 42% found for PEI-SA-Lf in the same time frame. Overall, a novel procedure for the dual targeting of DDS is disclosed, opening new perspectives in nanomedicines delivery, whereby a novel drug delivery system harvests the merits and properties of the different immobilized ligands.
- Ligand screening to pre-miRNA 149 G-quadruplex investigated by molecular dynamicsPublication . Carvalho, Josué; Santos, Tiago; Carrilho, Rui; Sousa, Fani; Salgado, Gilmar; Queiroz, João; Cruz, CarlaUsing a molecular dynamics approach, the study of the interaction between six different known ligands and a predicted pre-miRNA 149 RNA G-quadruplex (rG4) structure is reported. The stabilization of rG4 structures formed within the pre-miRNA stem-loop regions using small ligands is an attractive anticancer strategy. Particularly, miRNA-149 is upregulated in a variety of cancers such as prostate cancer and is therefore a potential target for drug development. The results show that ligands C8 and PhenDC3 interact with the rG4 structure via stacking interactions with the end G-quartets. Ligands [16]phenN2, [32]phen2N4 and pyridostatin on the other hand bind the loops/groove interface of the rG4 being H-bonding and electrostatic interactions the driving force of the interaction. The C8 precursor, C8-NH2, emphasizes the structural nuances of the rG4 short loops as the lack of a large terminal aromatic moiety produced a mixed stacking-groove binding mode. Overall, this study may help the design of specific ligands for pre-miRNA rG4 towards anticancer therapeutics development. Communicated by Ramaswamy H. Sarma.
- Molecular Beacon Assay Development for Severe Acute Respiratory Syndrome Coronavirus 2 DetectionPublication . Carvalho, Josué; Nunes, J. Lopes; Figueiredo, Joana; Santos, Tiago; Miranda, André; Riscado, Micaela; Sousa, Fani; Duarte, A. P.; Socorro, Sílvia; Tomaz, Cândida; Felgueiras, Mafalda; Teixeira, Rui; Faria, Conceição; Cruz, CarlaThe fast spread of SARS-CoV-2 has led to a global pandemic, calling for fast and accurate assays to allow infection diagnosis and prevention of transmission. We aimed to develop a molecular beacon (MB)-based detection assay for SARS-CoV-2, designed to detect the ORF1ab and S genes, proposing a two-stage COVID-19 testing strategy. The novelty of this work lies in the design and optimization of two MBs for detection of SARS-CoV-2, namely, concentration, fluorescence plateaus of hybridization, reaction temperature and real-time results. We also identify putative G-quadruplex (G4) regions in the genome of SARS-CoV-2. A total of 458 nasopharyngeal and throat swab samples (426 positive and 32 negative) were tested with the MB assay and the fluorescence levels compared with the cycle threshold (Ct) values obtained from a commercial RT-PCR test in terms of test duration, sensitivity, and specificity. Our results show that the samples with higher fluorescence levels correspond to those with low Ct values, suggesting a correlation between viral load and increased MB fluorescence. The proposed assay represents a fast (total duration of 2 h 20 min including amplification and fluorescence reading stages) and simple way of detecting SARS-CoV-2 in clinical samples from the upper respiratory tract.