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. In this sense, recombinant production arises as a promising, cost-effective method, allowing large-scale production of RNA. Rhodovulum sulfidophilum (R. sulfidophilum), a marine purple bacterium, offers the advantage of RNA secretion into the extracellular medium, which contains low levels of RNases and other impurities. Therefore, RNA recovery can be simplified compared to standard extraction protocols involving cell lysis, resulting in a more clarified sample and an improved downstream process. In this work, R. sulfidophilum was transformed with a plasmid DNA encoding pre-miR-29b-1, which is known to be involved in the Alzheimer's disease pathway. After production, the pre-miR-29b-1 was recovered through different extraction methods to verify the most advantageous one. A protocol for extracellular RNA recovery was then established, revealing to be a simpler and less time-consuming method, reducing around 16 h in execution time and the quantity of reagents needed when compared to other established methods. The new strategy brings the additional advantage of eliminating the toxic organic compounds routinely used in intracellular RNA extractions. Overall, the optimized process described here, using isopropanol as the precipitation agent, offers a greener, safer, and faster alternative for recombinant RNA recovery and concentration, with an extracellular RNA recovery of 7 µg/mL and target pre-miRNA-29b-1 recovery of 0.7 µg/L of medium.

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., small molecules, organic compounds, proteins, and nucleic acids). Despite the diversity of currently available chromatographic matrices, the interest in innovative biomolecules emphasizes the need for novel, robust, and more efficient supports and ligands with improved selectivity. Accordingly, ionic liquids (ILs) have been investigated as novel ligands in chromatographic matrices. Given herein is an extensive review regarding the different immobilization strategies of ILs in several types of supports, namely in silica, Sepharose, and polymers. In addition to depicting their synthesis, the main application examples of these supports are also presented. The multiple interactions promoted by ILs are critically discussed concerning the improved selectivity towards target molecules. Overall, the versatility of supported ILs is here considered a critical point to their exploitation as alternatives to the more conventional liquid chromatographic matrices used in bioseparation processes.

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. In this protocol, we describe the process for nucleic acids production and extraction, as well as the chromatographic matrix synthesis for separation between DNA and RNA. This novel arginine-macroporous support presents excellent binding capacity and great robustness for nucleic acids isolation.

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. Nonetheless, to fully potentiate mRNA therapeutic application, its efficient production, stabilization and delivery into the target cells are required. In recent years, intensive research has been carried out in this field in order to bring new and effective solutions towards the stabilization and delivery of mRNA. Presently, the therapeutic potential of mRNA is undoubtedly recognized, which was greatly reinforced by the results achieved in the battle against the COVID-19 pandemic, but there are still some issues that need to be improved, which are critically discussed in this review.