A new affinity approach to isolate Escherichia coli 6S RNA with histidine‐chromatography

Abstract

6S RNA is an abundant non-coding RNA in Escherichia coli (E. coli), but its function has not been discovered until recently. The first advance on 6S RNA function was the demonstration of its ability to bind the s 70 -holoenzyme form of RNA polymerase, inhibiting its activity and consequently the transcription process. The growing interest in the investigation of non-coding small RNAs (sRNA) calls for the development of new methods for isolation and purification of RNA. This work presents an optimized RNA extraction procedure and describes a new affinity chromatography method using a histidine support to specifically purify 6S RNA from other E. coli sRNA species. The RNA extraction procedure was optimized, and a high yield was obtained in the separation of sRNA and ribosomal RNA (rRNA) from total RNA (RNAt). This improved method takes advantage of its simplicity and significant cost reduction, since some complex operations have been eliminated. A purification strategy was also developed to separate 6S RNA from an sRNA mixture. Pure RNA can be advantageously obtained using the histidine-affinity chromatography method, aiming at its application to structural or functional studies.

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

“…B). In a previous work (Martins et al , ), we have successfully exploited RNA stringency properties, which enables rRNA separation from sRNA population through an ammonium sulfate precipitation. Building on these results, the separation between sRNA and rRNA in the crude lysate was accomplished by adjusting the ionic strength of the lysate solution with 1.6 m of (NH 4 ) 2 SO 4, as schematically shown in Fig.…”

“…In fact, RNA purification using amino‐acid based affinity chromatography was recently initiated with an isolation strategy for E . coli 6S RNA using histidine affinity chromatography (Martins et al , ). In that work, the histidine support was able to isolate 6S RNA from a mixture of sRNAs owing to a biorecognition phenomena occurring between the ligands and the RNA bases.…”

“…coli ) cell lysate. The potential of histidine has already been demonstrated in plasmid DNA purification (Sousa et al , ) and recently in RNA purification (Martins et al , ). In this last work, we optimized an RNA extraction procedure and developed a new affinity chromatography method using histidine–agarose support to specifically purify 6S RNA from other E .…”

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.…”

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].…”