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- Plasmid purification by using a new naphthalene tripodal supportPublication . Santos, Tiago; Proença, Z.; Queiroz, João; Tomaz, C. T.; Cruz, CarlaThe aim of this work was to employ a new naphthalene tripodal support for the isolation of supercoiled (sc) isoform of plasmid (pDNA) from a native sample. This support is for the first time synthesized and used in pDNA purification. The naphthalene tripodal ligand was synthesized and characterized to assess its purity and subsequently immobilized onto an epoxy-activated Sepharose CL-6B, using mild conditions and resulting in a ligand density of 0.32 mmol naphthalene tripodal/g derivatized Sepharose CL-6B. The complete characterization of naphthalene tripodal Sepharose CL-6B support was performed by High Resolution Magic Angle Spinning (HR-MAS) NMR spectroscopy, scanning electron microscopy (SEM) and elemental analysis. The affinity was measured by SPR biosensor between naphthalene tripodal ligand immobilized on the surface and sc pVAX1-LacZ and the KD was 8.65 10 8 ± 1.0 10 8 M in 10 mM Tris-HCl pH 8.0, at T = 25 C, indicating a high affinity. For comparison reasons, the affinity ligand 3,8-diamino-6-phenylphe nanthridine (DAPP) was also immobilized on the chip surface and the KD for sc pVAX1-LacZ is lower than with naphthalene tripodal. Saturation transfer difference-nuclear magnetic resonance spectroscopy (STD-NMR) experiments showed that the interactions between the naphthalene tripodal–Sepharose CL-6B and DAPP-Sepharose supports and the 50-mononucleotides are mainly hydrophobic and p-p stacking. The isolation of sc pDNA isoform was achieved with low salt concentrations, using 95 mM NaCl in binding step and 550 mM NaCl in elution step at T = 4 C and pH 8, thus reducing the economic and environmental impact.
- 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.
- Influenza DNA vaccine purification using pHEMA cryogel supportPublication . Santos, Tiago; Brito, Andreia; Boto, Renato; Sousa, Pedro; Almeida, Paulo; Cruz, Carla; Tomaz, C. T.Influenza virus is a huge financial and social burden for health care systems over the world. Currently, traditionalapproaches are not effective in the fight of the epidemy and new alternatives like DNA vaccines have been developed. However, the downstream process of DNA vaccines is a constant challenge in the biotechnology industry. Cryogels has several advantages over traditional supports and have been tested as stationary phase in chromatographic separations. In this work, a method based on poly(2-hydroxyethyl methacrylate) cryogel was used to purify the plasmid NTC7482-41H-VA2 HA, which express the Influenza hemagglutinin gene. For this purpose, the cryogel was synthesized by cryo-polymerization of 2-hydroxyethyl methacrylate and characterized by scanning electron microscopy. The purification of supercoiled isoform of the plasmid NTC7482-41H-VA2 HA from a clarified lysate sample was achieved in a two-step experiment using NaCl and the dynamic binding capacity of pHEMA cryogel was determined. The assessment of DNA vaccine allowed to conclude that the level of contaminants such as proteins, genomic DNA, RNA and endotoxins are in accordance with FDA agency.
- 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.
- Recognition of G-quadruplexes in microRNA precursors by nucleolin and its implications in cancerPublication . Santos, Tiago André Afonso dos; Cruz, Carla Patrícia Alves Freire Madeira; Cabrita, Eurico José da SilvaCancer is among the most frequent pathologies, and it is responsible for high mortality rates, which tend to increase year by year. In order to restore the well-being of citizens, numerous efforts have been performed to develop new diagnostic and therapeutic approaches. However, ensuring the effectiveness of these new approaches is a challenging task. Indeed, it is necessary to study the mechanisms and molecular interactions during cancer development. Several molecular mechanisms related to cancer development have been studied in the past decades. Their study encouraged the identification of new nucleic acid structures, which can adopt alternative secondary conformations. Among these structures are G-quadruplexes (G4), three-dimensional structures formed in the genome or transcriptome of regions rich in guanines. G4 DNAs are located in key regions of the genome, including the ends of telomeres and oncogenic promoters, and have received considerable attention from the scientific community in the past two decades. On the other hand, the G4 RNAs found in non-coding regions have recently received this interest due to their importance in controlling multiple biological processes. In this way, the intermediates of microRNA biogenesis (miRNAs) that can adopt a G4 structure have been more intensely studied, focusing on microRNA precursors (pre-miRNAs). The development and/or study of small molecules (G4 ligands, molecular weight < 500 Da) with the ability to bind and stabilize/destabilize G4 structures could strongly contribute to the modulation of miRNA biogenesis. G4 ligands have shown high potential to be applied to the diagnosis and therapy of several pathologies such as neurodegenerative diseases, cancer, and viral infections. Acridine orange derivatives have been demonstrated to bind and stabilize different DNA and RNA G4 structures. Besides small molecules that bind to G4 structures, there are other approaches to control the biogenesis of miRNAs, which are also attractive. Indeed, the process is essentially modulated by proteins, which reveals its potential to be modulated by interfering with those molecules. In this way, the most widely used approach aims to influence Dicer activity, a protein with enzymatic activity that cleaves pre-miRNAs into miRNAs. However, other equally relevant approaches could be explored. Nucleolin is a protein highly expressed in cancer cells. The protein is involved in several cellular processes, including tumorigenesis, angiogenesis, and extracellular signaling pathways. Nucleolin is mainly located in the nucleolus but can also be found in the nucleoplasm, cytoplasm, and cell surface. Recently, it has been described that nucleolin is involved in miRNA biogenesis through its interaction with the microprocessor complex. However, since has been described as a protein with a high affinity for parallel G4 structures, and in cancer cells performs the transport of several molecules between the cytoplasm and the nucleus, its action at this stage of biogenesis is a robust hypothesis. In this way, this thesis aims to deal with the interaction of the G4s adopted by pre-miRNA let 7e, 92b, and 149, with ligands and nucleolin. Indeed, the expression levels of these pre-miRNAs and miRNAs have been found dysregulated in several types of cancer. In addition, the potential biological applications of the recognition of these sequences by ligands and nucleolin were addressed. The formation of G4s in the sequences of the pre-miRNA let 7e, 92b, and 149 were evaluated under different experimental conditions (concentration, ionic strength, and temperature). In addition, the stabilizing/destabilizing effect of the ligands on the G4s was evaluated and they are dependent on the experimental conditions. For example, the ligand C8, depicts a modest impact in stabilizing the G4 of pre-miRNA let 7e, but stabilized the G4s of pre-miRNAs 92b and 149. Once we established the potential of G4 ligands to stabilize the G4s present in miRNAs, we evaluated the G4/nucleolin molecular interactions in the presence and absence of ligands. In the case of the G4 in pre-miRNA let 7e, the formation of the ternary complex G4/ligand/nucleolin was observed, except in the presence of the ligands PhenDC3 and TMPyP4, which seem to destabilize the G4 structure. Thus, these two ligands have the potential to control the miRNA biogenesis by increasing miRNA let 7e expression levels. The formation of the ternary complex was not affected by the presence of C8. In the studies performed with the G4 structure of pre-miRNA 92b, the acridine derivatives showed high potential to stabilize the structure, namely C8. Also, this ligand does not significantly affect the recognition of nucleolin by G4. Therefore, the G4/C8 complex was tested in a microfluidic device to sense nucleolin in plasma samples from prostate cancer patients. Finally, even known for its enormous relevance in several cancer-associated processes and mechanisms, the G4 sequence of pre-miRNA 149 has been less studied than those previously described. Furthermore, the G4 sequence overlaps almost entirely with the miRNA 149-3p, which presents an opportunity to act at different stages of miRNA 149 biogenesis. Considering this evidence, we aim to study in-depth the G4 sequence of pre-miRNA 149 in terms of its interaction with ligands and nucleolin. Furthermore, due to its high potential, the G4 structure was tested as a potential strategy to recognize and detect nucleolin 0n the surface of cancer cells. First, the formation of the G4 structure was confirmed by several biophysical techniques and revealed a G4 structure of high structural complexity. Then, the binding mode and interaction of different G4 ligands with the G4 structure were analyzed, and the results showed a high potential of the ligand C8 to stabilize the G4 structure. We also analyzed the complete sequence of pre-miRNA 149 and demonstrated its ability to detect nucleolin using a microfluidic system manufactured by INESC-MN. Finally, the interaction of G4 and the G4/C8 complex with nucleolin was also investigated and revealed a binding pocket in the 3D structure of nucleolin domains 1 and 2. Overall, these results revealed the biological potential of the G4 sequences in pre-miRNA let 7e, 92b, and 149. The structural regulation of G4 sequences present in pre-miRNAs can enable the development of applications for cancer diagnosis and therapy. Therefore, we anticipated that structural studies of the G4/nucleolin interaction would be available in the future, leading to the emergence of new ligands with inhibitory or enhancing effects on the interaction.
- Production and Purification of DNA G-quadruplex using pPH600 plasmidPublication . Santos, Tiago André Afonso dos; Cruz, Carla Patrícia Alves Freire Madeira; Queiroz, João António de Sampaio RodriguesWhen we think in structure of DNA, the image that comes immediately to mind is the iconic structure in double helix discovered by Watson and Crick in 1953. However, in addition to this structure DNA can assume other secondary structures which are relevant in the biological context. Some important regions of human genome have unusual potential to form this structures upon transcription. The plasmid pPH600 have a sequence of S?3 immunoglobulin switch region of murine that are able to form G-quadruplex upon transcription. The G-loops predominance is more evidenced on supercoiled (sc) topology than on relaxed (oc) or linearized (ln) plasmid. The present work describe the biosynthesis of plasmid pPH600 in E. coli DH5a and the strategies for sc pPH600 purification, directly from native sample (oc + sc) and clarified E. coli lysate. The purification strategies are based on amino acid affinity chromatography taking advantage of biological recognition to pDNA. For this propose two supports were prepared, L-tryptophan Sepharose and L-tyrosine Sepharose, by covalent immobilisation using 1,4-butanediol diglycidyl ether spacer arm. The commercial support L-arginine Sepharose 4B was also used in the strategy for purifying sc pPH600 since it has already been efficiently applied to separate sc isoforms of different plasmids using mild binding and elution conditions. Therefore, an initial screening with pPH600 native sample (oc + sc) was performed to evaluate the behavior of three supports. The better support in isoform separation was selected to purify sc pPH600 directly from clarified lysate. L-tyrosine support shows the prominent result in separation of two isoforms, allowing the recovery of sc pPH600, through a decreasing stepwise gradient from 2.25 to 0 M (NH4)2SO4 in 100 mM HEPES acid (pH 7.4) with temperature at 10ºC. Thereafter, the clarified E. coli lysate sample was injected directly onto L-tyrosine support to separate sc pPH600 from internal impurities of E. coli (gDNA, RNA, proteins, endotoxins and other conformations of pPH600). The total separation of sc pPH600 was totally achieved using a stepwise gradient from 2.25, 1.95 and 0 M (NH4)2SO4 in 100 mM HEPES acid (pH 7.4) with temperature at 10ºC. The underlying mechanism is thought to involve not only hydrophobic but also other non-covalent interactions such as, hydrogen bonds, p-p stacking and van der Waals interactions. Plasmid assessment tests indicated that the sc pPH600 resultant from the purification step presented a purity degree of 98.2%, with an extremely reduced level of impurities. Finally, the sc pPH600 resultant from purification was transcribed to induce the formation of G-quadruplex and it is confirmed by circular dichroism.
- 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.