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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.
- New insights for therapeutic recombinant human miRNAs heterologous production: Rhodovolum sulfidophilum vs Escherichia coliPublication . Pereira, Patrícia; Pedro, Augusto; Queiroz, João; Figueiras, Ana R.; Sousa, FaniRNA interference-based technologies have emerged as an attractive and effective therapeutic option with potential application in diverse human diseases. These tools rely on the development of efficient strategies to obtain homogeneous non-coding RNA samples with adequate integrity and purity, thus avoiding non-targeted gene-silencing and related side-effects that impair their application onto pre-clinical practice. These RNAs have been preferentially obtained by in vitro transcription using DNA templates or via chemical synthesis. As an alternative to overcome the limitations presented by these methods, in vivo recombinant production of RNA biomolecules has become the focus in RNA synthesis research. Therefore, using pre-miR-29b as a model, here it is evaluated the time-course profile of Escherichia coli and Rhodovolum sulfidophilum microfactories to produce this microRNA. As the presence of major host contaminants arising from the biosynthesis process may have important implications in the subsequent downstream processing, it is also evaluated the production of genomic DNA and host total proteins. Considering the rapidly growing interest on these innovative biopharmaceuticals, novel, more cost-effective, simple and easily scaled-up technologies are highly desirable. As microRNA recombinant expression fulfills those requirements, it may take the leading edge in the methodologies currently available to obtain microRNAs for clinical or structural studies.
- miRNA-29 bioseparation and target delivery strategies for Alzheimer's diseasePublication . Pereira, Patrícia Alexandra Nunes; Sousa, Fani Pereira de; Figueiras, Ana Rita; Correia, Ilídio Joaquim SobreiraThe possibility of selectively alter the expression pattern of a particular gene has been sought by scientists and clinicians for a long time. Nowadays, RNA interference (RNAi)-based technology has become a novel tool for silencing gene expression in cells. In addition, this strategy encloses an enormous therapeutic potential that could change the course of the currently applied treatments in several life threatening pathologies and it is expected that this technology can be translated onto clinical applications in a near future. MicroRNA (miRNA) has become a commonly employed tool for gene silencing, since it prevents protein synthesis by inducing the messenger RNA (mRNA) degradation, with a high specificity degree. Consequently, in the last years, the miRNAs have emerged as biopharmaceuticals to regulate several pathways involved in the insurgence and progression of the Alzheimer’s disease (AD), since they might have key regulatory roles in many neuronal functions, such as differentiation, synaptic plasticity and memory formation, and typically they are down-regulated in disease conditions. In the literature there are some studies describing a causal relationship between miR-29 expression and AD, since a loss of miR-29 cluster can contribute to increased beta-amyloid precursor protein-converting enzyme 1 (BACE1) and Amyloid-β (Aβ) levels in sporadic AD patients. Thus, this evidence supports the possibility to use miR-29 as a potential therapeutic target for AD therapy. In general, miRNA-based therapy relies on the use of synthetic microRNAs. However, these synthesized formulations typically present contaminants that can lead to non-targeted gene silencing, which still restricts the pre-clinical or clinical application of these RNAs. Thus, considering this therapeutic purpose and the global distribution of novel biopharmaceuticals it is necessary to develop efficient processes for their preparation. The development of new strategies for microRNA production with high purity degree and biologically active is extremely required. One of the strategies might be the use of the recombinant production of biomolecules using prokaryotic hosts. Hence, the present work intends to develop and establish an integrative biotechnological platform to biosynthesize and purify a recombinant miRNA precursor (pre-miR-29b) to act in the selective silencing of endogenous pathways directly related with AD, in particular BACE1 and Aβ. In addition, the success of these therapies also depends upon the ability to selectively and efficiently deliver the pre-miR-29b in the cytoplasmic compartment of neuronal cells, the location where their function is exerted; therefore the development of miRNA delivery systems was also envisioned. The expression system Rhodovulum sulfidophilum (R. sulfidophilum) DSM 1374 allowed, for the first time, the production of human pre-miR-29b with a straightforward recuperation of pre-miR-29b in a single step, maintaining its biological active form. The application of this recombinant bacterial microorganism is innovative and is supported by the unusual capacity of secreting the nucleic acids to the extracellular space and the absence of host ribonucleases in the culture medium. Therefore, it is expected that the secreted miRNA will be devoid of main bacterial associated impurities. Regarding the growth conditions, and conversely to what was previously described for this bacterium, our results showed to be possible to develop an original approach for the aerobic growth of the R. sulfidophilum, which results in a cell growth improvement followed by an enhanced production of human pre-miR-29b. The extracellular pre-miR-29b concentration was approximately 182 μg/L, after 40 hours of bacterial growth and the total intracellular pre-miR-29b was of about 358 μg/L, at 32 hours of cell growth. To further develop a potential therapeutic application, the major interest is not only to produce high quantities of RNA but also to obtain and preserve its biological active form, fulfilling the requirements of regulatory agencies. Hence, to assure that this prerequisite is met it was used a novel and effective purification strategy, based on affinity chromatography, to purify the pre-miR-29b. Therefore, in order to achieve the selectivity towards the target pre-miRNA and the maximum resolution between the pre-miR-29b and other host biomolecules (transfer RNAs and proteins) it was used an affinity support that exploits the same biological interactions that are established within the cell, by using immobilized amino acids (L-lysine and L-arginine), as specific ligands. The recognition of the pre-miR-29b achieved with these supports, allowed its selective recovery from a complex mixture with high efficiency and high purity. In parallel, the binding of pre-miRNA to these different amino acids was studied by Surface Plasmon Resonance. This information brings important insights concerning the characterization of the pre-miRNA binding onto chromatographic supports. Moreover, it was possible to determine some particular conditions enabling the improvement of the binding specificity of the amino acid ligands used to purify miRNA, preserving the RNA integrity. Taking into account that the structure of the chromatographic supports has been continuously developed to afford rapid and efficient separations, namely for the purification of nucleic acids, it was also tested a monolithic support to purify the pre-miR-29b. The association of the high capacity of these supports with the specificity conferred by the agmatine ligand (a derivative of L-arginine) represented a novelty and an advantage to obtain highly pure pre-miR-29b (90%) with a high recovery yield (95%). The establishment of an effective application of miRNAs is usually constrained by different phenomena, namely their easy degradation when in contact with the body fluids. To overcome this limitation, delivery systems, such as polymeric systems (polyplexes), were developed and characterized in order to encapsulate and protect the pre-miR-29b biopharmaceuticals from degradation, allowing their sustained and targeted release. The formulations prepared with chitosan and polyethylenimine demonstrated high loading capacity, small sizes and exhibited a strong positive charge on their surface. In addition, considering the application field of this work, the delivery systems should also have the ability to penetrate the Blood-Brain Barrier (BBB), causing an increase of the pre-miRNAs concentration in the brain and, consequently the improvement of the therapeutic effect. Actually, BBB is an intrinsic barrier limiting miRNA therapeutic effect on the central nervous system. Thus, to improve the delivery of pre-miRNA therapeutics in the brain, the polyplexes were functionalized with specific ligands, namely lactoferrin and stearic acid which are recognized by cell surface receptors of BBB. Finally, it was evaluated the biological activity of the recombinant pre-miR-29b by measuring the efficiency on human BACE1 knockdown, using in vitro neuronal cell lines. The effect of recombinant pre-miR-29b administration was verified by both assessing the mRNA and protein human BACE1 levels, by using RT-qPCR, Western blot and Imunocytochemistry. Results suggest that recombinant pre-miR-29b can represent a novel biopharmaceutical product for the therapeutic modulation of human BACE1 levels, because high levels of inhibition were achieved, namely 80% of reduction for BACE1 protein expression and 45% for Aβ42 levels. Globally, the implementation of these cutting-edge technologies can have a great impact on the biopharmaceutical industry, providing the basis for the implementation of novel miRNA-based therapeutics, not only for neurological disorders but also for future therapeutic targets that can be of potential interest.
- Cholinium-Based Good’s Buffers Ionic Liquids as Remarkable Stabilizers and Recyclable Preservation Media for Recombinant Small RNAsPublication . Pedro, Augusto; Pereira, Patrícia; Quental, Maria J.; Carvalho, André P.; Santos, Sérgio M.; Queiroz, João; Sousa, Fani; Freire, Mara G.RNA is a biopolymer of high relevance in the biopharmaceuticals field and in fundamental and applied research; however, the preservation of the RNA stability is still a remarkable challenge. Herein, we demonstrate the enhanced potential of aqueous solutions of self-buffering cholinium-based Good's buffers ionic liquids (GB-ILs), at 20 and 50 % (w/w), as alternative preservation media of recombinant small RNAs. The thermal stability of RNA is highly enhanced by GB-ILs, with an increase of 14 °C in the biopolymer melting temperature - the highest increase observed up to date with ILs. Most GB-ILs investigated improve the stability of RNA at least up to 30-days, both at 25 °C and at 4 °C, without requiring the typical samples freezing. Molecular dynamics simulations were applied to better understand the molecular-level mechanisms responsible for the observed RNA improved stability. The number of IL cations surrounding the RNA chain is similar, yet with differences found for the IL anions, which are responsible for the overall charge of the biopolymer first solvation sphere. No cytotoxicity of the studied solutions containing RNA and ILs at 20 % (w/w) was observed onto two distinct human cell lines, reinforcing their potential to act as preservation media when foreseeing biopharmaceutical applications. Finally, RNA was successfully recovered from the ILs aqueous solutions, without changes in its structural integrity, and the ILs successfully recycled and reused.
- Advances in time course extracellular production of human pre-miR-29b from Rhodovulum sulfidophilumPublication . Pereira, Patrícia; Pedro, Augusto; Tomás, Joana; Baptista, Cláudio; Queiroz, João; Figueiras, Ana; Sousa, FaniThe 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.
- HPV-16 targeted DNA vaccine expression: The role of purificationPublication . Almeida, Ana Margarida; Tomás, Joana; Pereira, Patrícia; Queiroz, João; Sousa, Fani; Sousa, ÂngelaDNA vaccines have come to light in the last decades as an alternative method to prevent many infectious diseases, but they can also be used for the treatment of specific diseases, such as cervical cancer caused by Human Papillomavirus (HPV). This virus produces E6 and E7 oncoproteins, which alter the cell cycle regulation and can interfere with the DNA repairing system. These features can ultimately lead to the progression of cervical cancer, after cell infection by HPV. Thus, the development of a DNA vaccine targeting both proteins arises as an interesting option in the treatment of this pathology. Nonetheless, before evaluating its therapeutic potential, the purity levels of a biopharmaceutical must meet the regulatory agency specifications. Previously, our research group successfully purified the supercoiled isoform of the recombinant HPV-16 E6/E7 DNA vaccine with virtual 100% purity by affinity chromatography. The present work was designed to evaluate the effect that pDNA sample purity levels may exert in the expression of a target protein. Thus, in vitro studies were performed to assess the vaccine ability to produce the target proteins and to compare the expression efficiency between the pDNA sample obtained by affinity chromatography, which only presents the sc isoform and fulfils the regulatory agency recommendations, and the same DNA vaccine retrieved by a commercial purification kit, which contains different pDNA isoforms. Our achievements suggest that the E6/E7 DNA vaccine purified by affinity chromatography promotes higher E6 and E7 mRNA and protein expression levels than the DNA vaccine purified with the commercial kit. Overall, these results underline the importance that a purification strategy may present in the therapeutic outcome of recombinant DNA vaccines, envisaging their further application as biopharmaceuticals. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:546-551, 2018.
- Integrated Extraction-Preservation Strategies for RNA Using Biobased Ionic LiquidsPublication . Quental, Maria V.; Pedro, Augusto; Pereira, Patrícia; Sharma, Mukesh; Queiroz, João; Coutinho, João A.P.; Sousa, Fani; Freire, Mara G.The ubiquitous instability of RNA along with issues associated with its purity degree have been preventing its widespread use as low-cost biotherapeutics. On the basis of the well-known capacity of amino acids to specifically interact with RNA when used as chromatographic ligands, a set of amino-acid-based ionic liquids (AA-ILs) was herein investigated, both to act as preservation media and as phase-forming agents of aqueous biphasic systems (ABS). This set of strategies was combined with the goal of developing integrated extraction-preservation platforms. AA-ILs comprising the cholinium cation and anions derived from l-lysine ([Ch][Lys]), l-arginine ([Ch][Arg]), l-glutamic acid ([Ch][Glu]), and dl-aspartic acid ([Ch][Asp]) were studied. It is shown that the stability of RNA is preserved in aqueous solutions of the studied AA-ILs, even in the presence of ribonucleases (RNases). Furthermore, almost all the investigated AA-ILs display no cytotoxicity onto two distinct human cell lines. After identifying the most promising ILs, ABS formed by AA-ILs and polypropylene glycol with a molecular weight of 400 g mol–1 (PPG 400) were investigated as extraction and purification platforms for RNA. Both with pure RNA and bacterial lysate samples, RNA is successfully extracted to the IL-rich phase without compromising its integrity and stability. On the basis of these results, the integrated extraction-preservation process for RNA is finally demonstrated. RNA is initially extracted from the bacterial lysate sample using ABS, after which the IL-rich phase can be used as the preservation medium of RNA up to its use. RNA can be then recovered from the IL-rich phase by ethanol precipitation, and the ABS phase-forming components recovered and reused. Although improvements in the purity level of RNA are still required, the approach here reported represents a step forward in the development of sustainable processes to overcome the critical demand of high-quality/high-purity RNA to be used as biotherapeutics.
- 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.
- Purification of pre-miR-29 by arginine-affinity chromatographyPublication . Pereira, Patrícia; Sousa, Ângela; Queiroz, João; Correia, Ilídio; Figueiras, Ana; Sousa, FaniRecently, differential expression of microRNAs, in patients with Alzheimer's disease (AD) suggests that they might have key regulatory roles in this neurodegenerative disease. Taking into account this fact, several studies demonstrated that the miR-29 is significantly decreased in AD patients, also displaying abnormally high levels of β-site APP-cleaving enzyme 1. Thus, RNA biochemical or structural studies often require a RNA sample that is chemically pure and biologically active. The present work describes a new affinity chromatography method using an arginine support to specifically purify pre-miR-29 from other Rhodovulum sulfidophilum small RNA species. Nevertheless, in order to achieve higher efficiency and selectivity, it is essential to characterize the behavior of pre-miR-29 binding/elution. Thus, three different strategies based on increased sodium chloride (280–500 mM), arginine (25 mM) or decreased ammonium sulfate (2–0.1 M) stepwise gradients are described to purify pre-miR-29. In this way, it was proved that well-defined binding/elution conditions are crucial to enhance the purification performance. As a matter of fact, by employing elution strategies using sodium chloride or arginine, an improvement in the final pre-miR-29 yields (96.5 and 56.7%, respectively) was obtained. Moreover, the quality control analysis revealed high integrity in pre-miR-29 preparations as well as high purity (90 and 98%, respectively), demonstrated by the scarce detection of proteins. This improved method takes advantage of its simplicity, significant cost reduction, due to the elimination of some complex operations, and speed for large-scale purification of pre-miRNAs suitable for biochemical and structural studies.
- Preparation of well-defined brush-like block copolymers for gene delivery applications under biorelevant reaction conditionsPublication . Góis, Joana R.; Reis, Fábio; Almeida, Ana Margarida; Pereira, Patrícia; Sousa, Fani; Serra, Armenio; Coelho, JorgeWell-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.