Sensitive fluorescent detection of SARS-CoV-2 RNA using an enzymatic-based method.

Rapid, quantitative, and sensitive detection of viral oligonucleotides can help to diagnose the infection before symptoms occur, monitor disease progression, and identify viral subtypes. A one-pot, simple, rapid hairpin-mediated nicking enzymatic signal amplification (HNESA) method was previously developed for nucleic acids detection. In the present work, this method was applied for the detection of SARS-CoV-2 RNA by designing an assistant probe (AP) that contains the complementary sequence for the target, the sequence of hybridization with the loop region of the molecular beacon (MB), and the recognition site of the nicking endonuclease Nt.

Biophysical characterization of microRNA mixtures based on Molecular Beacons.

Diverse studies have shown a relationship between dysregulated microRNAs (miRNAs), including miRNA-29b and miRNA-9, and several diseases. So, it is hypothesized that miRNAs can be studied as potential agents to be exploited in biomedical applications, due to their ability to take part in gene expression regulation at a post-transcriptional level. Considering the possibility of using miRNAs, it is important to characterize and validate this bioproduct, structurally and functionally.

A new approach for extracellular RNA recovery from Rhodovulum sulfidophilum.

The development of RNA-based drugs is highly pursued due to the possibility of creating viable and effective therapies. However, their translation to clinical practice strongly depends on efficient technologies to produce substantial levels of these biomolecules, with high purity and high quality. RNAs are commonly produced by chemical or enzymatic methods, displaying these limitations.

High-density perfusion cultures of the marine bacterium Rhodovulum sulfidophilum for the biomanufacturing of oligonucleotides.

Therapeutic oligonucleotides (ONs) are typically manufactured via solid-phase synthesis, characterized by limited scalability and huge environmental footprint, limiting their availability. Biomanufactured ONs have the potential to reduce the immunogenic side-effects, and to improve the sustainability of their chemical counterparts. Rhodovulum sulfidophilum was demonstrated a valuable host for the extracellular production of recombinant ONs.

Evaluation of novel chromatographic prototypes for supercoiled plasmid DNA polishing.

Since the world first approved gene therapeutics, nucleic acid-based therapies have gained prominence. Several strategies for DNA-based therapy have been approved, and numerous clinical trials for plasmid DNA (pDNA)-based vaccines are currently in development. Due to the rising interest in pDNA for vaccination and gene therapy, plasmid manufacturing must become more effective.

pH-responsive nanoparticles based on POEOMA-b-PDPA block copolymers for RNA encapsulation, protection and cell delivery.

The use of gene-based products, such as DNA or RNA, is increasingly being explored for various innovative therapies. However, the success of these strategies is highly dependent on the effective delivery of these biomolecules to target cells. Therefore, the development of pH-responsive nanoparticles comprises the creation of intelligent delivery systems with high therapeutic efficiency.

Arginine-Modified 3D-Printed Chromatographic Supports.

The increasing progression of biopharmaceutical-based therapies highlights the demand for efficient chromatographic methods that can be used to purify the desired biomolecules (e.g., nucleic acids, enzymes, or monoclonal antibodies) which are presently under consideration in clinical trials or approved by the Food and Drug Administration.

Arginine-Affinity Chromatography for Nucleic Acid (DNA and RNA) Isolation.

Nucleic acid-based therapy has been emerging as a new strategy with great potential for the treatment of numerous diseases, especially those caused by gene defects. In this context, biotechnology plays a critical role on establishing suitable processes for biopharmaceuticals manufacturing, while the purification step still imposes a major burden. Affinity chromatography using amino acids as specific ligands has been successfully applied for plasmid DNA purification.

Supported Ionic Liquids Used as Chromatographic Matrices in Bioseparation-An Overview.

Liquid chromatography plays a central role in biomanufacturing, and, apart from its use as a preparative purification strategy, either in biopharmaceuticals or in fine chemicals industries, it is also very useful as an analytical tool for monitoring, assessing, and characterizing diverse samples. The present review gives an overview of the progress of the chromatographic supports that have been used in the purification of high-value products (e.g.

Understanding the adsorption of plasmid DNA and RNA molecules onto arginine-agarose chromatographic resin.

Background: The production of nucleic acids (plasmid DNA or mRNA) in response to the development of new advanced vaccine platforms has greatly increased recently, mostly resulting from the pandemic situation. Due to the intended pharmaceutical use, nucleic acids preparations must fulfill all the required specifications in terms of purity and quality. Chromatography is a standard operation used to isolate these molecules from impurities, playing a central role in the manufacturing processes.

mRNA, a Revolution in Biomedicine.

The perspective of using messenger RNA (mRNA) as a therapeutic molecule first faced some uncertainties due to concerns about its instability and the feasibility of large-scale production. Today, given technological advances and deeper biomolecular knowledge, these issues have started to be addressed and some strategies are being exploited to overcome the limitations. Thus, the potential of mRNA has become increasingly recognized for the development of new innovative therapeutics, envisioning its application in immunotherapy, regenerative medicine, vaccination, and gene editing.

Advances Brought by Hydrophilic Ionic Liquids in Fields Involving Pharmaceuticals.

The negligible volatility and high tunable nature of ionic liquids (ILs) have been the main drivers of their investigation in a wide diversity of fields, among which is their application in areas involving pharmaceuticals. Although most literature dealing with ILs is still majorly devoted to hydrophobic ILs, evidence on the potential of hydrophilic ILs have been increasingly provided in the past decade, viz., ILs with improved therapeutic efficiency and bioavailability, ILs with the ability to increase drugs' aqueous solubility, ILs with enhanced extraction performance for pharmaceuticals when employed in biphasic systems and other techniques, and ILs displaying low eco/cyto/toxicity and beneficial biological activities.

Molecular Beacon Assay Development for Severe Acute Respiratory Syndrome Coronavirus 2 Detection.

The 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.

Efficient Isolation of Bacterial RNAs Using Silica-Based Materials Modified with Ionic Liquids.

High quality nucleic acids (with high integrity, purity, and biological activity) have become indispensable products of modern society, both in molecular diagnosis and to be used as biopharmaceuticals. As the current methods available for the extraction and purification of nucleic acids are laborious, time-consuming, and usually rely on the use of hazardous chemicals, there is an unmet need towards the development of more sustainable and cost-effective technologies for nucleic acids purification. Accordingly, this study addresses the preparation and evaluation of silica-based materials chemically modified with chloride-based ionic liquids (supported ionic liquids, SILs) as potential materials to effectively isolate RNAs.

New RNA-Based Breakthroughs in Alzheimer's Disease Diagnosis and Therapeutics.

Dementia is described as the fifth leading cause of death worldwide and Alzheimer's disease (AD) is recognized as the most common, causing a huge impact on health costs and quality of patients' lives. The main hallmarks that are commonly associated with the pathologic process are amyloid deposition, pathologic Tau phosphorylation and neurodegeneration. It is still unclear how these events are linked to the disease progression, due to the complex pathologic mechanisms.

The Performance of Minicircle DNA Versus Parental Plasmid in Gene Delivery Into HPV-18-Infected Cervical Cancer Cells.

Minicircle DNA (mcDNA) has been suggested as a vanguard technology for gene therapy, consisting of a nonviral DNA vector devoid of prokaryotic sequences. Unlike conventional plasmid DNA (pDNA), this small vector is able to sustain high expression rates throughout time. Thus, this work describes the construction, production, and purification of mcDNA- and its precursor parental plasmid (PP)- for a comparative study of both DNA vectors in the growth suppression of human papillomavirus (HPV)-18-infected cervical cancer cells.

Insights on the DNA Stability in Aqueous Solutions of Ionic Liquids.

Deoxyribonucleic acid (DNA) carries the genetic information essential for the growth and functioning of living organisms, playing a significant role in life sciences research. However, the long-term storage and preservation of DNA, while ensuring its bioactivity, are still current challenges to overcome. In this work, aqueous solutions of ionic liquids (ILs) were investigated as potential preservation media for double stranded (dsDNA).

Brain-Targeted Delivery of Pre-miR-29b Using Lactoferrin-Stearic Acid-Modified-Chitosan/Polyethyleneimine Polyplexes.

The 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.

Minicircle DNA Vaccine Purification and E7 Antigen Expression Assessment.

Human papillomavirus (HPV ) has been extensively associated with the development of cervical cancer due to the expression of oncoproteins like E7. This protein can interfere with pRB tumor suppressor activity, enabling the uncontrolled proliferation of abnormal cells. DNA vaccines are known as the third-generation vaccines, providing the ability of targeting viral infections such as HPV in a preventive and therapeutic way.

Enhancement of a biotechnological platform for the purification and delivery of a human papillomavirus supercoiled plasmid DNA vaccine.

New biotechnological strategies are being explored, aimed at rapid and economic manufacture of large quantities of DNA vaccines with the required purity for therapeutic applications, as well as their correct delivery as biopharmaceuticals to target cells. This report describes the purification of supercoiled (sc) HPV-16 E6/E7 plasmid DNA (pDNA) vaccine from a bacterial lysate, using an arginine-based monolith, presenting a spacer arm in its configuration. To enhance the performance of the purification process, monolith modification with the spacer arm can improve accessibility of the arginine ligand.

Minicircle DNA: The Future for DNA-Based Vectors?

Minicircle DNA (mcDNA) is a smaller and safer version of non-viral DNA vectors that results from a cutting-edge in vivo recombination process to excise prokaryotic sequences from plasmid DNA (pDNA). Considering the molecule's potential and increasing interest as a non-viral DNA-based therapeutic, biomanufacturing methodologies need to be improved, especially in downstream processing.

Cervical cancer and HPV infection: ongoing therapeutic research to counteract the action of E6 and E7 oncoproteins.

Cervical cancer is the fourth most common cancer among women worldwide and its development is mainly associated with human papillomavirus infection, a highly sexually transmissible virus. The expression of E6 and E7 viral oncoproteins deregulates cell repairing mechanisms through impairment of tumor suppressor protein functions, such as p53 or retinoblastoma protein. Although the implementation of new preventive vaccines has decreased the infection rate and cervical cancer progression, there are still many women who are affected by this pathology.

Plasmid production and purification: An integrated experiment-based biochemistry and biotechnology laboratory course.

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.

Ligand screening to pre-miRNA 149 G-quadruplex investigated by molecular dynamics.

Using 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.

Cholinium-based Good's buffers ionic liquids as remarkable stabilizers and recyclable preservation media for recombinant small RNAs.

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.