Histidine affinity chromatography‐based methodology for the simultaneous isolation ofEscherichia colismall and ribosomal RNA

Abstract

Research on RNA has led to many important biological discoveries and the improvement of therapeutic technologies. In particular, there is a great focus on small RNA and ribosomal RNA owing to their key functions in the cell, which make them excellent therapeutic targets. Although the study of these RNA classes is progressing, some limitations have been found regarding the use of suitable techniques that are able to produce and isolate biologically competent and chemically stable RNA. To address this, we have developed a novel histidine affinity chromatography-based isolation methodology for small and ribosomal RNA molecules. The new procedure involves three main steps: (1) cell lysis with guanidinium buffer, (2) RNA primary isolation with ammonium sulfate precipitation and (3) histidine affinity chromatography to specifically purify small RNA and ribosomal RNA from other Escherichia coli impurities (genomic DNA and proteins). The RNA quality assessment revealed that both RNA species were obtained with a high recovery, integrity and purity. The potential of this method to achieve a reproducible RNA isolation with appropriate quality has been demonstrated and it should have broad application in the structural, biophysical and biomedical investigation of systems involving RNA components.

PolyGuanine methacrylate cryogels for ribonucleic acid purification

“…Recently, chromatographic techniques such as RP (toxic), LC, ion‐pairing RP (toxic), gel filtration and anion‐exchange , and affinity techniques such as base‐pairing , affinity tags and amino acids‐RNA have been used to isolate, separate, and purify RNA and DNA. However, these techniques have some drawbacks such as the need for toxic chemicals, scale‐up problems, time consuming, denaturation, misfolding impurity, instability, or the need for high salt concentration.…”

Dye affinity cryogels for plasmid DNA purification

“…These aromatic rings promote hydrophobic interactions between Cibacron Blue F3GA and pDNA. The intermolecular electrostatic forces and hydrogen bond formation contribute to the driving force for pDNA adsorption on cryogel discs [29][30][31][32][33].…”

Pharmaceutical-grade pre-miR-29 purification using an agmatine monolithic support

“…In the last years, our research group developed a new affinity chromatography approach, named amino acid-affinity chromatography to efficiently purify different RNA species (total RNA, rRNA, sRNA and 6S RNA) [15][16][17]. This powerful technique is based on the application of amino acids as specific ligands to purify RNA on the basis of their biological function or individual chemical structure [18].…”