This article is published in Journal of Molecular Recognition as a special issue on Affinity 2009 edited by Gideon Fleminger, Tel-Aviv University, Tel-Aviv, Israel and George Ehrlich, Hoffmann-La Roche, Nutley, NJ, USA.
Small RNAs (sRNA) are a family of regulatory non-coding RNAs, which are targeted in several research studies on all organisms. Their activities have been associated to RNA processing, mRNA stability, translation, protein stability and secretion in bacteria, acting mostly by base pairing and, to a lesser extent, by protein binding (Wassarman et al., 1999). Escherichia coli (E. coli) 6S RNA was one of the first sRNA to be discovered in the late 1960s, its sequence and secondary structure having been promptly proposed (Willkomm and Hartmann, 2005). Although 6S RNA was soon recognized, its function remained unknown for several years. This RNA species has always been found at high abundance in total cellular RNA (RNAt). However, studies of 6S RNA depletion and over-expression have not demonstrated any evident change in cell metabolism behaviour (Willkomm and Hartmann, 2005). In 2000, the first breakthrough on 6S RNA function was the demonstration of its ability to bind the σ70-holoenzyme form of RNA polymerase, inhibiting its activity in the stationary phase of cell growth. The high structural similarity observed between 6S RNA and a DNA open promoter in RNA transcription process, as well as the identification of 6S RNA in a specific complex linked to RNA polymerase, has suggested that this RNA type functions by mimicking the transcription promoter. Hence, 6S RNA seems to directly compete with DNA promoters for the σ70-RNA polymerase active site to inhibit the transcription process (Wassarman and Storz, 2000; Wassarman, 2007).
Over the last years, new insights triggered a better understanding of the molecular mechanism of 6S RNA (Gildehaus et al., 2007; Karen, 2007), while the techniques employed in the isolation of this RNA species were still plasmid design and enzymatic purification. These techniques involve time-consuming and expensive procedures. Therefore, the development of new tools for 6S RNA purification would be of great importance to simplify the current genetic-based approaches.
The use of amino acid-based affinity chromatography has been described as a promising approach for nucleic acids purification. This is due to the biological selectivity, which occurs between the amino acid ligand and the nucleic acid molecule under study. In general, the selectivity found in affinity chromatography can be explained by some biological recognition or individual chemical structure, which favours the interaction (Sousa et al., 2008). In the present study, the isolated E. coli sRNA population was tested in an affinity chromatographic support with immobilized histidine, combining the reliable and economical chromatographic operation with the high selectivity of histidine, which has been shown to have great applicability in the purification of nucleic acids. Histidine–agarose chromatographic support has demonstrated to efficiently separate supercoiled (SC) and open circular (OC) plasmid DNA (pDNA) isoforms, via a bio-recognition phenomenon with the nucleic acid bases, involving also hydrophobic interactions between the support and the pDNA molecules (Sousa et al., 2005). Furthermore, in these previous studies, the RNA showed to be strongly retained on the column, because of the higher base exposure, and a decrease in salt concentration was deemed necessary for its elution (Sousa et al., 2006). Building on the interesting results obtained for RNA separation, this report explores the possibility of using histidine–agarose chromatography to purify 6S RNA from other sRNA.
MATERIALS AND METHODS
L-histidine–agarose gel, oligo(dT) cellulose and all the organic compounds used in the RNAt extractions were obtained from Sigma (St. Louis, MO, USA). Glycogen was from Roche (Mannheim, Germany) and the RNA/DNA midi kit was purchased from Qiagen (Hilden, Germany). The PD-10 desalting columns were from GE Healthcare (Uppsala, Sweden) and the RNA molecular weight marker was obtained from Invitrogen (Carlsbad, CA, USA). Specific primers for E. coli 6S RNA cDNA were purchased from Stab Vida (Lisbon, Portugal). All salts used were of analytical grade.
Bacterial growth conditions
The RNA used in this study was obtained from a cell culture of E. coli DH5α. Growth was carried out in shake flasks at 37°C and 250 rpm with 250 mL of Terrific Broth medium (12 g/L tryptone, 24 g/L yeast extract, 4 mL/L glycerol, 0.017 M KH2PO4, 0.072 M K2HPO4). It was suspended in the late log phase (OD600 ≈ 9). Cells were recovered by centrifugation and were stored at −20°C.
Lysis and RNAt isolation
Cells were lysed and RNAt was extracted using the acid guanidinium thiocyanate–phenol–chloroform method described by Chomczynski and Sacchi (2006) with some modifications. The bacterial pellets were resuspended in 5 mL of Solution D (4 M guanidinium thiocyanate; 25 mM sodium citrate, pH 4.0; 0.5% (w/m) N-laurosylsarcosine (sarcosyl) and 0.1 M β-mercaptoethanol) to perform lysis. After incubating on ice for 10 min, cellular debris, genomic DNA and proteins were precipitated by gently adding and mixing 10 mL of water-saturated phenol and 1 mL of 2 M sodium acetate (pH 4.0). The RNAt isolation was achieved by adding 2 mL of chloroform/isoamyl alcohol (49:1), and by mixing vigorously until two immiscible phases were obtained. The upper aqueous phase, which contained mostly RNA, was recovered and concentrated by the addition of 11 µL of 20 µL/mL glycogen and 10 mL of ice-cold isopropanol. After incubating for 10 min at −20°C, RNAt was recovered by centrifugation at 15 000 g for 20 min at 4°C. The RNAt pellet was resuspended in 3 mL of Solution D. It was concentrated again with glycogen and 3 mL of ice-cold isopropanol. After centrifuging for 10 min at 15 000 g (4°C), the RNAt pellet was washed with 7.5 mL of 75% ethanol and incubated at room temperature for 10 min, followed by a 5 min centrifugation at 15 000 g (4°C). The air-dried RNAt pellet was solubilized in 400 µL of 0.05% DEPC-treated water, and its optical density was determined in order to assess RNAt quantity and purity.
Isolation of sRNA population from RNAt extract
The isolation of the sRNA population was first performed using the method previously described by Stellrecht and Gandhi (2002). However, the complexity of this protocol and its frequent use in eucaryotic cells led us to develop a new methodology for sRNA extraction from procaryotic cells.
In the optimized protocol, RNAt extract was precipitated with 2 M (NH4)2SO4 and incubated at 4°C for several hours. The sRNA recovery was accomplished by centrifugation at 15 000 g for 20 min at 4°C, as the sRNA population was in the supernatant, while the ribosomal RNA (rRNA) was in the pellet. Next, the supernatant was applied to a PD-10 desalting column (GE Healthcare, Uppsala, Sweden), following the manufacturer's instructions. The sRNA concentration was achieved with 2 volumes of ice-cold isopropanol and 11 µL of 20 µL/mL glycogen, followed by centrifugation at 15 000 g for 20 min at 4°C. Finally, the sRNA pellet was solubilized with 200 µL of 0.05% DEPC-treated water. The quantification and purity of sRNA was assessed as previously described for RNAt.
Chromatography was performed in an ÄKTA purifier system with UNICORN software (GE Healthcare, Sweden). A 10 mm diameter × 20 mm long (∼2 mL) column was packed with the commercial L-histidine–agarose gel. The manufacturer characterizes this support as a cross-linked 4% beaded agarose matrix with a 12-atom spacer and an extent of labelling between 1 and 2 µmol/mL. The column was equilibrated with 2.2 M (NH4)2SO4 in 10 mM Tris–HCl (pH 7.0) buffer at a flow rate of 1 mL/min. The isolated sRNA samples were injected onto the column using a 100 µL loop at the same flow rate. The absorbance of the eluent was continuously monitored at 260 nm. After elution of unbound species with 2.2 M (NH4)2SO4 in 10 mM Tris–HCl (pH 7.0) buffer, the ionic strength of the buffer was decreased stepwise to 1.6 M (NH4)2SO4 in 10 mM Tris–HCl (pH 7.0) buffer. Finally, tightly bound RNA species were removed by changing to ammonium sulphate-free 10 mM Tris–HCl (pH 7.0) buffer. Fractions were pooled according to the chromatograms obtained. Following concentration and desalting with Vivaspin concentrators (Vivascience), the pools were kept for further analysis as described below.
Pooled fractions were analysed by vertical electrophoresis using Amersham Biosciences system (GE Healthcare, Sweden) with 7.5% polyacrylamide gel and were then stained with ethidium bromide (0.5 µg/mL). Electrophoresis was carried out at 120 V for 90 min with TBE buffer (0.84 M Tris base, 0.89 M boric acid and 0.01 M EDTA, pH 8.3). The pooled fractions were previously denatured with 97.5% formamide and the denatured conditions were kept in the gel due to the presence of 8 M urea. sRNA in the gel was visualized using a Vilber Lourmt system (ILC Lda).
Reverse-transcription PCR analysis
6S RNA identification was assessed using reverse-transcriptase polymerase chain reaction (PCR) in a MyCycler™Thermal Cycle (Biorad). Samples collected after the chromatographic purification process with histidine–agarose column were pre-treated with DNase I (Sigma Chemical Co., St. Louis, MO) according to the manufacturer's instructions. cDNA was synthesized from 150 or 500 ng of 6S RNA which was denatured for 5 min at 65°C with 500 µM of deoxynucleotide triphosphates (Amersham, Uppsala, Sweden) and 250 ng of random primers (Invitrogen, Karlsruhe LMA, Germany). Reverse transcription was carried out at 37°C for 60 min in a 20 µL reaction containing reverse transcriptase buffer (50 mM Tris–HCl, 75 mM KCl and 3 mM MgCl2), 0.1 M DTT, 60 U of RNaseOUT (Invitrogen) and 200 U of M-MLV RT (Invitrogen). The reaction was stopped at 75°C for 15 min. PCR reactions were carried out using 1 µL of synthesized cDNAs in a 25 µL reaction containing 1× Taq DNA polymerase buffer (20 mM Tris–HCl and 50 mM KCl), 500 µM deoxynucleotide triphosphates (Amersham), 3 mM of MgCl2 (Promega, Madison), 300 nM of each primer and 0.125 U of Taq DNA polymerase (Promega). Specific primers for E. coli 6S RNA cDNA (sense: 5′-GCT CCG CGG TTG GTG AGC AT-3′; antisense: 5′-GAT GCC GCC GCA GGC TGT AA-3′) whose design was achieved on RNA database were used to amplify a fragment of 95 bp. After an initial denaturation at 95°C for 5 min, the cycling conditions were used as follows: 25 cycles consisting of denaturation at 95°C for 30 s, annealing at 60°C for 30 s and extension at 72°C for 10 s. PCR products were analysed using 1.5% agarose-gel electrophoresis.
RESULTS AND DISCUSSION
The available protocols to obtain sRNA molecules are complex, time-consuming and require the use of highly toxic compounds. Therefore, they may not be adequate to recover a product for biological application, which justifies the need for the development of an efficient, tolerable and scalable process to purify non-coding RNA. This study was developed to evaluate the applicability of amino acid-based affinity chromatography, namely the potential of histidine ligand, in the purification of the different sRNA molecules.
Optimization of the RNA extraction methods
The methods commonly used to isolate the different types of RNA require a variety of organic compounds and saline solutions, as well as the application of commercial kits. In fact, this may be considered a drawback to their broader application. However, the developed protocol allowed the efficient isolation of RNA while significantly reducing the number of operation steps.
Figure 1 shows the global extraction procedure used in our experiments to obtain sRNA molecules while Figure 2 highlights the differences between the described multi-step method (Stellrecht and Gandhi, 2002) and the optimized protocol that we have developed to isolate the sRNA population from RNAt.
The herein proposed protocol was based on the stringent properties of RNA, consisting of the manipulation of several variables, such as the ionic strength, which influence the nucleic acid structure (Farrell, 2005). The effect of high salt concentrations on stringency allows single-stranded nucleic acid molecules to form stable hydrogen bonds between their complementary bases. The monovalent cations present in the salts minimize the tendency for natural electrostatic repulsion between two negatively charged phosphodiester backbones and, as a result, the nucleic acid structure is stabilized and compacted. In particular, salts with NH cations stabilize the tertiary structure of rRNA due to the formation of specific bonds involving hydrogen bonds with a tetrahedral rearrangement of the rRNA carbonyl bases (Wang et al., 1993). Thus, the separation of sRNA molecules was achieved via the precipitation of rRNA in the RNAt extract with 2 M (NH4)2SO4 at 4°C for several hours. This procedure revealed to be practical, economical and effective in the isolation of the sRNA population for chromatographic application.
Chromatographic separation of sRNA 6S
The ability of the histidine–agarose support to separate the SC and OC pDNA isoforms present in a ‘native’ (SC + OC) pDNA sample prepared with a commercial purification kit or from a E. coli lysate has been recently described (Sousa et al., 2005; Sousa et al., 2006). The previous works demonstrated that RNA interacts with the histidine–agarose matrix in the presence of higher salt concentration and elutes with the decrease of (NH4)2SO4 concentration in the elution buffer to 1.5 M. Following these results, the general aim of the present study was to explore the retention pattern of the different sRNA in the histidine–agarose support in order to investigate the interactions that might arise from RNA retention.
The 6S RNA purification process started with the extraction procedure for RNAt followed by the isolation of the sRNA species, which were required to perform the chromatographic studies in the histidine–agarose matrix.
The chromatographic assays performed initially were intended to test the ionic strength effects on the sRNA retention (data not shown). In the first set of experiments, a two-step elution with 2.2 M (NH4)2SO4 in 10 mM Tris–HCl (pH 7.0) buffer and ammonium sulphate-free Tris buffer was carried out. The higher salt concentration promoted total sRNA retention. Its elution was achieved with ammonium sulphate-free Tris buffer. In another set of experiments, we used 1.6 M (NH4)2SO4 in 10 mM Tris–HCl (pH 7.0) buffer in the first step and the Tris buffer in the last step. It was observed that sRNA molecules eluted immediately with 1.6 M (NH4)2SO4. These results were important to study the behaviour of the sRNA mixture in different salt concentrations and to develop the best purification strategy for 6S RNA. In fact, high salt concentration plays a key role on the 6S RNA binding to histidine ligand.
Figure 3a shows the chromatographic profile obtained after injection of the sRNA sample (≈100 µg). The presence of different peaks in the chromatogram indicates that the RNAs present in the sRNA population interact differently with the histidine–agarose support. A denaturing polyacrylamide electrophoresis was used to detect and identify the different species eluting in each peak (Figure 3b). The sRNA sample injected on the histidine–agarose matrix (Figure 3b, lane S) was also run on the gel for comparative purposes. This sample was qualitatively characterized by three distinct bands, which corresponded to 6S RNA, 5S RNA and other sRNA, including tRNAs. The electrophoresis analysis indicates that the first peak corresponds to the elution of a small part of 5S RNAs and other sRNA (peak 1 and lane 1 in Figure 3). However, the complete elution of these RNAs was achieved in the second peak, following the reduction of the ionic strength of the elution buffer (peak 2 and lane 2). Finally, with the Tris buffer, 6S RNA was eluted, as it is observed in the third peak of the chromatogram (peak 3 and lane 3).
The main explanation for the specific interactions occurring between the sRNA population and the histidine–agarose matrix is the single-stranded nature of RNA, which is normally involved in RNA recognition, due to the high base exposure and availability for interactions. Furthermore, atomic studies performed on protein-RNA complex structures have shown that histidine has a strong tendency to interact with nucleotides (Jeong et al., 2003). As for the exact type of interactions, these may include (i) hydrogen-bonding between H-donor (NτH) and H-acceptor (Nπ) atoms in the non-protonated histidine with base edges; (ii) ring stacking/hydrophobic interactions and (iii) water-mediated hydrogen bonds (Hoffman et al., 2004; Morozova et al., 2006). Thus, and considering the fact that at the working pH (7.0), histidine (pKa = 6.5) is not significantly protonated (Özkara et al., 2002; Pitiot and Vijayalakshmi, 2002) the elution of sRNA when salt concentration is decreased suggests that ring stacking/hydrophobic interactions and histidine–RNA direct hydrogen-bonding are the dominant effects.
Additionally, 6S RNA structural features seem to be relevant on its distinct behaviour with the histidine–agarose matrix. 6S RNA presents a DNA promoter-like secondary structure consisting of two long irregular double-stranded stem regions, which are interrupted by small bulge loops and a largely single-stranded internal loop in the central region. Along the central bulge and through the continuous stem sequences there are mostly adenines (A) and guanines (G) (Barrick et al., 2005), which were described to interact preferably with histidine (Hoffman et al., 2004). Interestingly, in newly described aspects on the function of 6S RNA, A and G were also identified as specific nucleotides involved in close contact with RNA polymerase (Gildehaus et al., 2007).
Identification of the purified sRNA species
A more accurate identification of 6S RNA species purified by the histidine–agarose matrix was performed. Hence, 6S RNA levels were determined during different stages of E. coli growth, taking into account the fact that this RNA is highly expressed in the stationary phase which is already known (Wassarman and Storz, 2000). After the purification process, the fractions of 6S RNA were further identified by reverse-transcription PCR.
The 6S RNA accumulation in the late growing phase of cells is clearly attested in the electrophoretic analysis of the sRNA samples (Figure 4) because there was a significant increase in 6S RNA concentration from 3 to 24 h of cell growth (Figure 4, lanes S3 and S24). The 6S RNAs from the sRNA populations of the different cell extracts were successfully purified using the above-described chromatographic technique with a histidine–agarose support. For the PCR analysis, the purified 6S RNA pools were treated with DNase I because contaminated DNA could be amplified giving rise to false-positive results. As it can be observed from the electrophoresis analysis of the PCR products (Figure 5), by using specific primers for 6S RNA cDNA, the PCR reaction allowed the amplification of 6S RNA cDNA fragments within 3 h (lane 1) and 24 h (lane 2) of cell growth. No further amplification was seen in the controls (lanes 3–5). The negative control was made of PCR reaction solutions without cDNA, while the positive controls consisted of 6S RNA samples treated with the DNase I, which was used to initially synthesize cDNAs. Therefore, we verified the identity of the sRNA isolated from the original population as the 6S RNA.
The isolation of biologically competent and chemically stable RNA continues to be a central procedure in molecular biology, which has even greater relevance today due to the progress of new disciplines, such as functional genomics and proteomics. The development of new RNA isolation methodologies, such as the purification strategy for 6S RNA isolation that is presented here, is an asset to the research now regularly exploring the transcriptome, the proteome, the metabolome and the genome.
In conclusion, this study provided an optimization of the protocol for the separation of sRNA and rRNA from RNAt by reducing the complexity of the general procedure. Moreover, we have developed a purification strategy to separate 6S RNA from E. coli sRNA mixture by affinity chromatography with immobilized amino acids, using histidine as a specific ligand. The underlying mechanism involves not only hydrophobic interaction, but also a bio-recognition of nucleic acid bases with histidine. The successful results obtained with this support reveal an efficient technique to obtain a reproducible and appropriate RNA quality with potential applicability for RNA structural and functional studies and gene therapy.