Journal of Chromatography A, 1216 (2009) 1377–1382
`
`Contents lists available at ScienceDirect
`
`Journal of Chromatography A
`
`j o u r n a l h o m e p a g e : w w w . e l s e v i e r . c o m / l o c a t e / c h r o m a
`
`Studying the mechanism of RNA separations using RNA chromatography and its
`application in the analysis of ribosomal RNA and RNA:RNA interactions
`Sakharam P. Waghmare a, Petros Pousinis a, David P. Hornby b, Mark J. Dickman a,∗
`
`a Biological and Environmental Systems, Department of Chemical and Process Engineering, University of Sheffield, Mappin Street, Sheffield S1 3JD, UK
`b Department of Molecular Biology and Biotechnology, University of Sheffield, Western Bank, Firth Court, Sheffield S10 2TN, UK
`
`a r t i c l e
`
`i n f o
`
`a b s t r a c t
`
`Article history:
`Received 28 October 2008
`Received in revised form 12 December 2008
`Accepted 22 December 2008
`Available online 30 December 2008
`
`Keywords:
`RNA/DNA chromatography
`Ion pair reverse phase liquid
`chromatography
`RNA conformation
`RNA:RNA interactions
`
`DNA/RNA chromatography presents a versatile platform for the analysis of nucleic acids. Although the
`mechanism of separation of double stranded (ds) DNA fragments is largely understood, the mechanism
`by which RNA is separated appears more complicated. To further understand the separation mechanisms
`of RNA using ion pair reverse phase liquid chromatography, we have analysed a number of dsRNA and
`single stranded (ss) RNA fragments. The high-resolution separation of dsRNA was observed, in a similar
`manner to dsDNA under non-denaturing conditions. Moreover, the high-resolution separation of ssRNA
`was observed at high temperatures (75 ◦C) in contrast to ssDNA. It is proposed that the presence of
`duplex regions/secondary structures within the RNA remain at such temperatures, resulting in high-
`resolution RNA separations. The retention time of the nucleic acids reflects the relative hydrophobicity,
`through contributions of the nucleic sequence and the degree of secondary structure present. In addition,
`the analysis of RNA using such approaches was extended to enable the discrimination of bacterial 16S
`rRNA fragments and as an aid to conformational analysis of RNA. RNA:RNA interactions of the human
`telomerase RNA component (hTR) were analysed in conjunction with the incorporation of Mg2+ during
`chromatography. This novel chromatographic procedure permits analysis of the temperature dependent
`formation of dimeric RNA species.
`
`© 2009 Elsevier B.V. All rights reserved.
`
`1. Introduction
`
`The role of RNA recognition is assuming increasing significance
`in biological systems. In addition to its role in biological cataly-
`sis, recently exemplified by the structural studies on the ribosome,
`small RNA molecules are emerging as key regulators of gene expres-
`sion in both prokaryotes [1], higher organisms (reviewed in Ref.
`[2]) and are also associated with mediating antiviral response in
`prokaryotes [3,4]. The synthesis, purification and analysis of RNA
`transcripts are key steps in the investigation of such biological
`events. Currently polyacrylamide gel electrophoresis (PAGE) is the
`principle method for defining the structural homogeneity of RNA
`transcripts and for monitoring the purification of chemically syn-
`thesised RNA for in vitro studies [4–6]. This procedure is time
`consuming and suffers from labour intensity, poor product yields
`and is unsuitable for high throughput approaches. More recently,
`capillary electrophoresis-laser induced fluorescence (CE-LIF) has
`been used to analyse RNA conformation, demonstrating advantages
`in sensitivity and reduced analysis times [7]. However, fluorescent
`labeling of the nucleic acid is required prior to analysis.
`
`∗ Corresponding author. Tel.: +44 114 222 27541; fax: +44 114 222 7566.
`E-mail address: M.Dickman@sheffield.ac.uk (M.J. Dickman).
`
`0021-9673/$ – see front matter © 2009 Elsevier B.V. All rights reserved.
`doi:10.1016/j.chroma.2008.12.077
`
`RNA/DNA chromatography is now established as a versatile
`technique for the analysis of nucleic acids [8]. This form of HPLC
`analysis is largely based upon the unique separation properties of
`a non-porous polystyrene-divinylbenzene polymer bead that has
`been functionalised with C18 alkyl groups. An alkylammonium
`salt is added to the eluent and forms neutral ion pairs when a
`DNA sample is introduced into the HPLC instrument. A gradient of
`acetonitrile solvent separates the nucleic acid fragments with the
`smaller fragments eluting from the column first. It has previously
`been used in the sequence independent sizing of duplex DNA (up
`to 2000 base pairs (bp)) under non-denaturing conditions [9] and
`using denaturing conditions, the analysis of oligonucleotides [10],
`the enrichment, separation and analysis of RNA [11–14]. RNA/DNA
`chromatography also provides a versatile platform for the rapid
`analysis of a wide range of nucleic acid modification reactions
`[15]. Further developments have also demonstrated the ability of
`RNA/DNA chromatography to indirectly study RNA conformation
`and DNA–protein interactions by analysing the products of DNA
`and RNA footprinting reactions [16–18].
`Although the mechanism of separation of double stranded (ds)
`DNA fragments is largely understood, the mechanism by which
`RNA is separated appears more complicated. To further understand
`the separation mechanisms of RNA using ion pair reverse phase
`liquid chromatography, we have analysed a number of dsRNA and
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`single stranded (ss) RNA fragments. In addition, the analysis of RNA
`using such approaches was extended to enable the discrimination
`of bacterial 16S rRNA and the direct analysis of RNA:RNA inter-
`actions exemplified by the RNA component of human telomerase
`(hTR).
`
`2. Materials and methods
`
`2.1. HPLC analysis
`
`All samples were analysed by IP-RP-HPLC on an Agilent 1100
`HPLC (Agilent, Palo Alto, CA, USA) using a DNAsep column
`50 mm × 4.6 mm I. D. (Transgenomic, San Jose, CA, USA). The sta-
`tionary phase of the column consists of a 2-␮m I. D. non-porous,
`alkylated poly(styrene-divinylbenzene) matrix. Chromatograms,
`analysed using UV detection, were recorded at a wavelength of
`260 nm.
`
`2.2. RNA/DNA chromatography
`
`The chromatographic analysis was performed using the follow-
`ing conditions: buffer A 0.1 M triethylammonium acetate (TEAA)
`(Fluka, UK), pH 7.0; buffer B 0.1 M TEAA, pH 7.0 containing 25%
`acetonitrile. The HaeIII digest of pUC18 (250 ng/␮l 5 ␮l injected)
`(Bioline, London, UK) and the dsRNA ladder (300 ng/␮l 5 ␮l
`injected) (New England Biolabs (NEB), Hitchin, Herts, UK) was anal-
`ysed at 50 ◦C using the following linear gradient (1): starting at 10%
`buffer B the gradient was extended to 20% buffer B in 2.5 min, fol-
`lowed by a linear extension to 40% buffer B over 2.5 min followed
`by a linear extension to 70% buffer B over 13 min at a flow rate of
`1.0 ml/min. At 75 ◦C the dsRNA ladder and HaeIII digest of pUC18
`was analysed using gradient (1).
`The total RNA and ssRNA ladder was analysed using gradient
`(2) starting at 20% buffer B the linear gradient was extended to 22%
`buffer B in 2 min, followed by a linear extension to 52% buffer B over
`15 min, followed by a linear extension to 65% buffer B over 2.5 min
`at a flow rate of 1.0 ml/min
`The bacterial rRNA was analysed at 50 ◦C using gradient (3) start-
`ing at 35% buffer B the linear gradient was extended to 50% buffer
`B in 3 min, followed by an extension to 65% buffer B over 15 min at
`a flow rate of 1.0 ml/min. Analysis of the hTR RNA was performed
`over a range of temperatures using the following gradient (4): start-
`ing at 30% buffer B the linear gradient was extended to 60% buffer
`B over 10 min at flow rate of 1.0 ml/min.
`
`2.3. Polyacrylamide gel electrophoresis (PAGE)
`
`5–10% non-denaturing polyacrylamide gels were prepared using
`acrylamide:bisacrylamide 29:1 (Bio-Rad, Hemel Hempstead Herts,
`UK) buffered with 45 mM Tris–borate (pH 8.0), 1 mM EDTA and
`run for 1 h, 100 V at 25 ◦C. PAGE gels containing magnesium were
`buffered with 45 mM Tris–borate (pH 8.0), 1 mM MgCl2 and run
`for 1 h, 100 V at 25 ◦C. The gels were visualised after staining with
`fluorescent dyes SYBR green for dsDNA (Sigma, UK) using a Transil-
`luminator.
`
`2.4. In vitro transcription of hTR
`
`The plasmid pGRN164 (a kind gift from Geron, Menlo Park, CA,
`USA) was linearised using FspI (NEB) prior to in vitro transcription.
`2 ␮g of template DNA was incubated overnight at 37 ◦C and tran-
`scribed using a T7 Megascript in vitro transcription kit (Applied
`Biosystems, Warrington, Cheshire, UK) following the manufactur-
`ers instructions. The RNA was then precipitated in ethanol and
`re-suspended in nuclease free water (Applied Biosystems, Warring-
`
`ton, Cheshire, UK). Analysis was then performed using either 4.5%
`denaturing (8 M urea) PAGE or IP-RP-HPLC.
`
`2.5. Folding of RNA transcripts
`
`Following transcription, RNA samples were purified using dena-
`turing gel polyacrylamide gel electrophoresis in 7 M urea. RNA was
`eluted from the gel by the addition of 2 volumes of 0.5 M ammo-
`nium acetate (Sigma, UK), 0.1% SDS (Sigma, UK) and 2 mM EDTA
`(Sigma, UK). The RNA was subsequently precipitated in ethanol and
`re-suspended in 10 mM Tris–HCl (pH 7.4), 10 mM MgCl2 and 40 mM
`NaCl. To ensure folding of the RNA, samples were heated to 75 ◦C
`for 1 min and cooled slowly to 25 ◦C (2 ◦C/min).
`
`2.6. Bacterial growth and RNA extraction
`
`Escherichia coli K12 (NEB, Hitchin, Herts, UK), Salmonella enter-
`ica (ATCC 700720) and Pseudomonas putida (ATCC 49128) were
`grown to mid-log phase (A600 = 0.4–0.6) in Luria–Bertani (LB) media
`and nutrient broth (DIFCO) respectively, with rapid shaking and
`incubation at 37 ◦C. Cell pellets were obtained using centrifugation
`(5 min 10,000 × g) and washed with 1 ml phosphate buffered saline
`(1× PBS). The total RNA was extracted using Ribopure Bacteria Kit
`(Applied Biosystems, Warrington, Cheshire, UK) following the man-
`ufacturers instructions. RNA was re-suspended in RNase free water
`prior to HPLC analysis.
`
`3. Results and discussion
`
`3.1. Elucidating the mechanism of RNA separations using RNA
`chromatography
`
`The rapid, high-resolution separation of ssRNA using denaturing
`RNA/DNA chromatography has been previously reported [11–13],
`offering significant advantages compared to gel electrophoretic
`analysis of RNA. In this study we have analysed the separation of
`a range of dsRNA transcripts (21–500 bp) using RNA chromatog-
`raphy at 50 ◦C (see Fig. 1A). The results demonstrate the rapid,
`high-resolution separation of dsRNA molecules in a similar manner
`observed to the separation of dsDNA using IP-RP-HPLC. However,
`a number of significant differences between the separation of
`dsDNA and dsRNA are observed. A direct comparison of the IP-
`RP-HPLC analysis of the dsRNA marker (21–500 bp) and the dsDNA
`marker, pUC18 HaeIII digest (80–587 bp) analysed under the same
`chromatographic conditions (see Section 2) is shown in Fig. 1A
`and B respectively. The dsRNA fragments can be seen to elute
`earlier than the corresponding dsDNA species of the same size.
`We have previously observed that dsDNA fragments containing
`uracil instead of thymine elute at slightly earlier retention times,
`demonstrating that sequence specific effects can alter the reten-
`tion time of dsDNA [18]. However, the large differences in retention
`time observed between the dsRNA and dsDNA fragments cannot
`completely be accounted for by the small differences in hydropho-
`bicity of thymine and uracil. dsDNA is known to adopt B-DNA
`conformation, whereas dsRNA adopts an alternative A-DNA con-
`formation. These differences in structure may also be reflected in
`the difference in retention times observed between dsRNA and
`dsDNA fragments under the chromatographic conditions. The A-
`DNA conformation is essentially a shorter squatter version of B-DNA
`and this difference in structure may reflect a decrease in overall
`hydrophobicity compared to dsDNA. These results are consistent
`with the previous analysis of non-canonical B-DNA structures using
`DNA chromatography. Holliday junctions which adopt non-uniform
`tertiary structures were previously analysed using DNA chromatog-
`raphy and showed a decrease in hydrophobicity and therefore
`retention time when compared to B-DNA of the same molecular
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`Fig. 1. IP-RP-HPLC analysis of dsDNA and dsRNA at 50 ◦C. (A) dsRNA marker. (B)
`pUC18 HaeIII digest. The size of the nucleic acid fragments in base pairs are high-
`lighted. The samples were analysed using gradient (1). Buffer A: 0.1 M TEAA, pH
`7.0; buffer B: 0.1 M TEAA, pH 7.0, 25% acetonitrile. 10–20% B in 2.5 min, 20–40% B in
`2.5 min, 40–70% B in 13 min at a flow rate of 1.0 ml/min at 50 ◦C.
`
`weight [18]. In addition, the co-axial stacking of helicies in such
`structures in the presence of magnesium ions also caused a change
`in retention time [18]. Furthermore, RNA is known to adopt more
`stable secondary/tertiary conformations in comparison to ssDNA
`which maybe present under IP-RP-HPLC conditions and contribute
`to the decrease in hydrophobicity compared to B-DNA duplex
`fragments.
`
`3.2. Analysis of ssRNA under different temperatures
`
`The high-resolution separation of ssRNA at high temperature is
`demonstrated in the analysis of a ssRNA size marker (100–1000 nt)
`shown in Fig. 2A. The results demonstrate what appears to be the
`size dependent separation of RNA at high temperature consistent
`with previous observations [11–13]. However, a number of aberrant
`retention times were observed in the analysis of RNA, which has also
`been observed in the analysis of ssDNA molecules (<100 nt) under
`fully denaturing conditions [9]. Such aberrant retention times of
`RNA can be clearly observed in the analysis of total RNA extracted
`from mammalian cells (see Fig. 2B). The chromatogram shows the
`co-elution of the 18S rRNA and 28S rRNA (1869 and 5035 nt respec-
`tively) at 75 ◦C. Collection of the corresponding peak and analysis
`using denaturing PAGE confirmed the presence of both the 18S and
`28S rRNA. The high-resolution separations observed (although not
`
`Fig. 2. IP-RP-HPLC analysis of RNA. Chromatograms show the separation of (A)
`ssRNA marker and (B) total RNA extracted from mammalian cells. The size of the
`nucleic acid fragments of the RNA marker in nucleotides and the total RNA species
`are highlighted. The samples were analysed using gradient (2). Buffer A: 0.1 M TEAA,
`pH 7.0; buffer B: 0.1 M TEAA, pH 7.0, 25% acetonitrile. 20–22% B in 2 min, 22–52% B
`over 15 min, 52–65% B over 2.5 min at a flow rate of 1.0 ml/min at 75 ◦C.
`
`completely size dependent) in the analysis of the high molecular
`weight ssRNA transcripts under denaturing conditions were unex-
`pected. It was anticipated that ssRNA would be similar to ssDNA in
`behaviour, where a decrease in resolution is observed (compared to
`dsDNA) as the molecular weight of the fragments increases. The loss
`of resolution in the analysis of ssDNA fragments is clearly observed
`in the analysis of dsDNA pUC18 HaeIII digest at 75 ◦C (see Fig. 3A).
`The chromatogram shows the loss of resolution of the individual
`DNA fragments. In contrast, the analysis of the dsRNA transcripts at
`75 ◦C is shown in Fig. 3B. The chromatograms show clear differences
`in the resolution of ssRNA fragments compared to ssDNA fragments
`at high temperatures using RNA/DNA chromatography. The results
`demonstrate the loss of resolution of ssDNA at high temperatures
`compared to dsDNA at 50 ◦C (see Fig. 1B vs Fig. 3A). However, the
`analysis of the dsRNA at 75 ◦C reveals that each RNA duplex is sub-
`sequently separated into the ssRNA fragments which are resolved
`under the chromatographic conditions, enabling resolution of each
`separate species within each duplex. For clarity the resolution of
`the 80 bp RNA transcript is not shown on the figure, as the mass of
`this fragment in the marker is twice that of the other fragments.
`These results demonstrate that high-resolution separation of the
`ssRNA is achieved at high temperatures. However, such separa-
`tions are not completely size dependent, as each of the two ssRNA
`fragments generated of the same size from each dsRNA duplex are
`resolved.
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`Fig. 3. IP-RP-HPLC analysis of dsDNA and dsRNA at 75 ◦C. (A) pUC18 HaeIII digest (B)
`dsRNA marker. The size of the nucleic acid fragments in nucleotides are highlighted.
`The samples were analysed using gradient (1). Buffer A: 0.1 M TEAA, pH 7.0; buffer B:
`0.1 M TEAA, pH 7.0, 25% acetonitrile. 10–20% B in 2.5 min, 20–40% in 2.5 min, 40–70%
`B in 13 min at a flow rate of 1.0 ml/min at 75 ◦C.
`
`3.3. Analysis of bacterial rRNA
`
`The rapid and accurate detection of micro-organisms is an
`important tool in a wide range of applications including environ-
`mental monitoring, molecular diagnostics and food monitoring.
`rRNA in bacterial cells is often used as a target for bacterial iden-
`tifications and discriminations due to the relative abundance and
`availability of 16S rRNA sequences [19–21]. Furthermore, due to
`sequence variability, it is often possible to use rRNA probes to clas-
`sify bacterial species (reviewed in Ref. [22]). 16S rRNA sequences
`for E. coli K12, S. enterica and P. putida were obtained from the
`NCBI Entrez Genome Project. Each of the bacterial sequences has
`7 operons including a number of sequence differences between
`the individual operons. The 16S rRNA from E. coli (1534 nt) was
`aligned with P. putida (1518 nt) revealing 85% sequence identity
`between E. coli and P. putida. Alignment of E. coli with S. enterica
`(1542–1546 nt) revealed >97% similarity. Total RNA was extracted
`from E. coli and P. putida (see Section 2) and analysed using RNA
`chromatography. The resulting chromatograms are shown in Fig. 4.
`Each bacterial total RNA was run in quadruplicate and the retention
`times of the 16S rRNA highlighted, including the standard devia-
`tions. The result demonstrates the ability of RNA chromatography
`to readily distinguish between closely related E. coli and P. putida
`
`Fig. 4. IP-RP-HPLC analysis of bacterial ribosomal RNA. Chromatograms show the
`analysis of RNA extracted from (A) P. putida and (B) E. coli. The retention times of
`the 16S rRNA are highlighted from quadruplicate injections including the standard
`deviation. (C) Chromatogram of an overlay of the E. coli and P. putida rRNA. The
`bacterial rRNA was analysed at 50 ◦C using gradient (3). Buffer A: 0.1 M TEAA, pH
`7.0; buffer B: 0.1 M TEAA, pH 7.0, 25% acetonitrile. 35–50% B in 3 min, 50–65% B in
`15 min at a flow rate of 1.0 ml/min.
`
`bacterial 16S rRNA (1534 nt vs 1518 nt), following the rapid extrac-
`tion and analysis of the 16S rRNA, by virtue of differences in the
`retention time. No downstream manipulation, design or synthesis
`of probes was necessary. Such studies could also be extended in the
`analysis of complex microbial communities. The chromatography
`was unable to distinguish E. coli and P. putida 16S rRNA which share
`>97% similarity (data not shown).
`
`3.4. Analysis of human telomerase RNA under non-denaturing
`conditions
`
`Human telomerase is a ribonucleoprotein complex comprising
`two essential components, a catalytic protein subunit (hTERT) and
`an RNA species (hTR) [23–25]. In vivo the hTR gene is transcribed
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`Fig. 5. Electrophoretic and chromatographic analysis of hTR. (A) Non-denaturing
`PAGE analysis of hTR. Electrophoretogram showing the presence of two different hTR
`species when analysed under non-denaturing conditions at 25 ◦C. Lanes 1–4 contain
`in vitro transcribed hTR, the two main RNA species are indicated (a and b). Lane M
`contains a 100-bp duplex DNA ladder. (B) IP-RP-HPLC analysis of hTR in the presence
`of Mg2+ ions. Chromatogram showing the temperature dependent analysis of hTR
`in the presence of 1 mM Mg2+ ions. The samples were analysed using gradient (4).
`Buffer A: 0.1 M TEAA, pH 7.0; buffer B: 0.1 M TEAA, pH 7.0, 25% acetonitrile. 30–60%
`B in 10 min at flow rate of 1.0 ml/min.
`
`by RNA polymerase II and processed at its 3(cid:4)-end to produce a
`transcript of 451 nucleotides (nt) in length [25–27]. The telom-
`erase holoenzyme may act as an independent multimer (a dimer)
`with at least two active sites [28–30]. It has been shown that
`human telomerase forms an active complex containing two homo-
`typic hTR molecules per telomerase complex [31]. More recently,
`it has been demonstrated using native PAGE and agarose gel elec-
`trophoresis that hTR can dimerise in vitro [32,33]. Ren et al. further
`demonstrated using single molecule fluorescence coincidence, the
`multimerisation of hTR in solution and through mutagenesis and
`oligonucleotide blocking experiments, defined the internal J7b/8a
`loop as the site of the RNA:RNA interaction [33]. Recent structural
`studies have provided further insight regarding the structure and
`function of hTR [34,35].
`The analysis of hTR via non-denaturing PAGE is shown in Fig. 5A.
`The electrophoretogram shows the presence of two major RNA
`species, predicted to be the monomer and dimer of the 451 nt
`hTR. These results are consistent with previous results using non-
`denaturing PAGE and agarose gel electrophoresis [32,33]. Analysis
`of the hTR species using RNA chromatography at temperatures
`between 30 and 75 ◦C results in the appearance of a single peak
`(data not shown). This implies that hTR still runs as a single species
`(monomer) under non-denaturing conditions. The folding of RNA
`molecules is predominantly dependent on the presence of diva-
`
`Fig. 6. PAGE analysis of the fractionated hTR species. (A) The hTR species (a) and
`(b) were fractionated using IP-RP-HPLC in the presence of 1 mM Mg2+ at 40 ◦C. (B)
`Electrophoretogram showing the analysis of the fractionated hTR samples. Fraction
`(a) was run in lane 2 and fraction (b) run in lane 1. Lane M contains a 100-bp DNA
`ladder.
`
`lent cations [36] under routine chromatographic conditions no
`metal ions are present in the running buffers (most metal ions are
`in fact detrimental to the columns used here). Moreover, the ion
`pair reagent is associated with the negatively charged phosphate
`backbone of the nucleic acid. To stabilise the dimeric (or mul-
`timeric) hTR molecules as observed under non-denaturing PAGE
`conditions, magnesium ions were included in the chromatography
`buffers (magnesium has no effect on column stability). The hTR
`was folded (see Section 2) and subsequently analysed using RNA
`chromatography in the presence of 1 mM Mg2+ over a range of tem-
`peratures (40–70 ◦C), see Fig. 5B. The chromatogram shows that at
`elevated temperatures (60–70 ◦C), the hTR fragment runs as a sin-
`gle species (monomer). However, as the temperature is decreased
`(40–50 ◦C) two peaks emerge. These results indicate the presence
`of at least two different RNA species consistent with those observed
`under non-denaturing PAGE. Further analysis was performed to
`determine whether the two species separated by chromatography
`in the presence of magnesium are the multimeric species observed
`in PAGE. The two hTR species were recovered following chromatog-
`raphy, precipitated and analysed using non-denaturing PAGE. The
`results are shown in Fig. 6. The early eluting RNA species (a) when
`analysed using PAGE migrates as the monomer and the later peak
`(b) migrates as the multimeric species. These results demonstrate
`that in the presence of Mg2+ ions the dimer (or multimeric) hTR
`RNA species, is stabilised and elutes later than the monomer. Fol-
`lowing fractionation, it can also be seen that in the case of the
`later eluting RNA species (b), when analysed using non-denaturing
`PAGE, a small amount of monomer is observed, suggesting that an
`
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`equilibrium exists in the multimerisation of hTR. These results fur-
`ther support the hypothesis for the mechanism of RNA separation
`using RNA chromatography. Under the chromatographic conditions
`employed at elevated temperatures, the secondary structures of
`RNA transcripts remain, and through the incorporation of diva-
`lent metal ions and analysis at lower temperatures this enables the
`stabilisation of tertiary interactions or RNA:RNA interactions. Such
`interactions would only be possible if secondary structures are sta-
`ble under the chromatographic conditions as previously proposed.
`Moreover, these results demonstrate that RNA chromatography
`can be used to analyse RNA:RNA interactions in a tempera-
`ture dependent fashion as exemplified by the multimerisation
`of hTR.
`
`4. Conclusions
`
`Our current understanding of the influence of molecular struc-
`ture upon the biological functions of RNA is largely derived from
`studies on either tRNAs or more recently from specialised catalytic
`RNAs. This situation has arisen primarily owing to the limitations of
`the available analytical and preparative separation methods for this
`class of macromolecules. Whilst recent improvements in the effi-
`ciency of in vitro transcription methods have recently made possible
`the enzymatic synthesis of mg quantities of single RNA species, in
`order to realise the opportunities for the biophysical and structural
`analysis of RNA, separation and purification methods still present
`a bottleneck. The use of RNA chromatography not only provides
`an efficient means of isolating and analysing RNA species; it can
`also provide valuable structural information as demonstrated here.
`Combined with structural studies such approaches will enable fur-
`ther insight in the analysis of RNA. Studying the separation of a wide
`range of ss and dsRNA fragments revealed that RNA chromatogra-
`phy enables the rapid, high-resolution separation of both dsRNA
`under non-denaturing conditions and ssRNA at high temperatures.
`This is in contrast to the analysis of ssDNA at elevated temper-
`atures. It is proposed that the presence of secondary structures
`remain in the RNA fragments at elevated temperatures, leading to
`the high-resolution separation in a pseudo-size dependent fashion.
`The relative hydrophobicity of the RNA fragments is a reflec-
`tion of both the secondary structure present and the nucleic acid
`sequence. Alternative conformations of RNA molecules present dif-
`ferent hydrophobic surfaces to the stationary phase and therefore
`allow the separation of such molecules in the chromatography. The
`degree to which the alternative conformations vary in hydropho-
`bicity will influence the resolution of such species. Furthermore, by
`facile manipulation of the chromatographic conditions, the nature
`of RNA:RNA interactions can be investigated. By incorporating mag-
`nesium ions and low temperatures the chromatography allows the
`stabilisation of RNA:RNA interactions and such complexes can be
`resolved on the basis of differential hydrophobicity in a tempera-
`ture dependent manner.
`The high-resolution separation that can be achieved using
`RNA chromatography, on both an analytical and preparative scale,
`promises to bring even greater opportunities for the study of the
`
`molecular properties of RNA. The ability to further examine the
`dynamic behaviour of RNA involving RNA:RNA interactions is of
`potential importance in the emerging field of small RNA regulation
`of gene expression.
`
`Acknowledgments
`
`This work was supported by the Engineering and Physical Sci-
`ences Research Council UK [EP/DD033713/1]. The authors would
`also like to thank Doug Gjerde for comments and discussions on
`this manuscript.
`
`References
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