Biorecognition by amino acid-based affinity chromatography for RNA purification

Abstract

Following the decoding of the human genome, a new era was opened for developing new gene therapy strategies employing nucleic acids. Recently, RNA was renowned a central molecule in cellular processes with implications in many diseases as well as in understanding of evolution, becoming one of the most exciting research areas of molecular biology. From basic to applied research, many procedures employ pure and intact RNA molecules. On one hand, RNA purification is a first critical step of a number of molecular biology procedures and its quality is crucial to ensure reproducibility and biological relevance of an experiment. On the other hand, the promising and revolutionary RNA-based therapies of RNA vaccination, gene therapy or recombinant biopharmaceuticals involves RNA formulations which should fulfill rigorous quality criteria recommended by international regulatory agencies. However, the isolation and purification of RNA are critical steps because of the easy degradability of RNA, which can impair chemical stability and biological functionality essential for analysis. Many techniques have been development to overcome the challenges of purifying RNA molecules; nonetheless they still have several limitations in regard to time demanding and the requirement of toxic solvents and denaturing conditions. Therefore, there is a growing demand for the evaluation and improvement of the methodologies currently used for RNA isolation and purification. Chromatography is undoubtedly one of the most diverse and potent methods in biotechnology, both at analytical, preparative and industrial level due to its simplicity, robustness, versatility and high reproducibility. Affinity chromatography is recognized as a powerful technique with great applicability in the purification of many biomolecules, including plasmid DNA and proteins because it exploits the principle of biomolecular recognition. The work that we have been developing considers new chromatographic strategies for RNA purification, using amino acids as affinity ligands. These studies are based on the fact that many different interactions exist between proteins and nucleic acids in biological systems, involving in particular basic amino acids such as histidine or arginine. New methodologies were accomplished that allowed obtaining RNA preparations from different sources with high recovery yields, purity and integrity. A new analytical method for RNA quantification was also developed in this work. The applicability of histidine-based affinity chromatography in the purification of RNA molecules was first demonstrated in the separation of 6S RNA, a regulatory non-coding RNA of the prokaryotic Escherichia coli (E.coli). A specific recognition between the histidine support and 6S RNA allowed its selective purification from a complex mixture of other small RNAs (sRNA). In another strategy, the simultaneous isolation of sRNA and ribosomal RNA from E.coli cell lysates, eliminating host DNA and proteins, was also attained by a histidine chromatographic-based method. Furthermore, arginine matrix was employed in RNA purification from eukaryotic cells demonstrating an exceptional ability to interact with all functional classes of RNA, despite their structural diversity and different folding states, enabling their isolation from impurities of eukaryotic crude cell extracts. Moreover, an analytical technique based on arginine affinity support for quantification and quality assessment of total RNA from different eukaryotic cells and synthetic RNA samples was also developed and validated, according to international and European legislation for bioanalytical methods. More efforts into RNA purification were developed with amino acid-based matrices, in particular with arginine-agarose matrix, in order to approach this technique to therapeutic application of RNA. The new goal was to exploit its applicability in purifying messenger RNA (mRNA) molecules not from cells, but from synthetic crudes of in vitro transcription reactions, pursuing mRNA vaccination for cervical cancer. In this work, arginine-based chromatography also showed its singular capability to improve purification processes, showing the advantages of eliminating additional steps and improving global economics of the production process. The development of these new methodologies revealed several interesting characteristics of RNA molecules, including their chromatographic behavior and natural interactions that can occur between amino acids-based supports and RNA molecules. Accordingly, these methods demonstrated a potential multipurpose applicability by aiding in molecular biology RNA-based analysis and RNA therapeutics, which support the interest in applying amino acid-based affinity chromatography for the future development of new RNA isolation, purification and quantification processes.