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Authors
Abstract(s)
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.
Description
Keywords
Ácido ribonucleico (RNA) - Purificação Cromatografia de afinidade Cromatografia de afinidade - Aminoácidos Arginina Listidina