Nucleic Acids Research, 2003, Vol. 31, No. 21 e135
`DOI: 10.1093/nar/gng136
`
`Rapid purification of RNA secondary structures
`
`Stacy L. Gelhaus, William R. LaCourse*, Nathan A. Hagan, Gaya K. Amarasinghe and
`Daniele Fabris
`
`Department of Chemistry and Biochemistry, University of Maryland, Baltimore County, Baltimore, MD 21250, USA
`
`Received August 8, 2003; Revised and Accepted September 18, 2003
`
`ABSTRACT
`
`A new method for rapid purification and structural
`analysis of oligoribonucleotides of 19 and 20 nt is
`applied to RNA hairpins SL3 and SL2, which are
`stable secondary structures present on the y recog-
`nition element of HIV-1. This approach uses
`ion-pairing reversed-phase liquid chromatography
`(IP-RPLC) to achieve the separation of the stem–
`loop from the transcription mix. Evidence is
`presented that IP-RPLC is sensitive to the different
`conformers of
`these secondary structures. The
`purity of each stem–loop was confirmed by mass
`spectrometry and PAGE. IP-RPLC purification was
`found to be superior to PAGE in terms of time,
`safety and, most importantly, purity.
`
`INTRODUCTION
`
`Highly pure oligonucleotides are of critical importance for
`in vitro studies of nucleic
`acid–protein interactions.
`Depending on the desired size and quantity, samples can be
`extracted from natural sources, synthesized according to
`phosphoramidite or similar chemistry, or prepared by in vitro
`transcription of DNA templates carried out by the enzyme, T7
`polymerase (1–3). The fact that the coupling efficiency of
`either the chemical or enzymatic procedure is usually <100%
`ensures the presence of a certain level of sample hetero-
`geneity. Synthetic errors can be caused by decoupling in the
`synthesizer, adding the wrong base or adding an extra base.
`Enzymatic synthesis can lead to misincorporation of bases,
`addition of extra bases at the end of the sequence (most
`commonly cytosine) and the initiation of synthesis with a
`mono- or diphosphate nucleotide (4–6). The incidence of
`errors and the level of heterogeneity are generally increased as
`a function of the sequence length. For this reason, it is usually
`necessary to separate the desired sample from the product
`mixture using a variety of established techniques (7,8).
`Regardless of the RNA source, accurate structural analysis
`necessitates the purification of the RNA. Inherent to this
`process is the repetitive separation of the desired entity from
`n – 1 aborts and n + 1 additions caused by various steps in the
`synthesis process.
`
`The most common approach to purifying RNA from a
`transcription mix is based on denaturing polyacrylamide gel
`electrophoresis (PAGE)
`followed by electro-elution (8).
`Unfortunately, this technique comes at a high price in terms
`of time, labor, cost and toxicity. The initial set-up of the gels is
`labor intensive, requires precautions to avoid introducing
`RNase activity into the samples and may lead to toxic
`exposure to polyacrylamide. The separation itself may take
`hours and the final resolution may not be adequate. The
`separated bands are usually excised by hand, increasing the
`risks of error and contamination. Multiple repetitions of the
`process may become necessary. In the long run, PAGE can
`become an expensive method. This is especially true for
`structural work, which requires large quantities.
`An alternative method for the purification of RNA is
`ion-pairing reversed-phase high-performance liquid chroma-
`tography (IP-RPLC). Despite the fact that IP-RPLC is not
`thought of as one of the standard molecular biology tools, the
`use of IP-RPLC for nucleotide analysis has steadily increased
`over the last several decades. IP-RPLC has previously been
`used for deoxyoligonucleotide purification, analysis of short
`tandem repeats, small nucleotide polymorphisms and RNA
`(9,10). Recently, a paper was published showing that
`footprinting of RNA is also possible by IP-RPLC (11).
`Here we report for the first time the differentiation of
`stem–loop conformations using IP-RPLC. The purification of
`the stem–loops (i.e. SL3 and SL2, see Fig. 1) located in the
`y-recognition element, necessary for the retroviral packaging
`of HIV-1 (12–15), is used to highlight this separation effect.
`For purification, the various stable conformers of the RNA
`secondary structure are collected as a single fraction and are
`separated from all components of the transcription mix and
`abortive sequences. Denaturing IP-RPLC, mass spectrometry
`and PAGE were used to confirm that the ‘packet’ of peaks
`collected in this method all had the same sequence (9).
`
`MATERIALS AND METHODS
`
`Reagents
`
`All reagents and solvents were HPLC grade (Fisher Scientific,
`Springfield, NJ). Triethylammonium acetate (TEAA) was
`obtained as a 203 concentrate (Transgenomic, San Jose, CA)
`and diluted with either water and/or acetonitrile (ACN). Water
`was purified using a reverse osmosis system coupled with a
`
`*To whom correspondence should be addressed. Tel: +1 410 455 2105; Fax: +1 410 455 2608; Email: lacourse@umbc.edu
`Present address:
`Gaya Amarasinghe, Department of Biochemistry, University of Texas, Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75390, USA
`
`The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors
`
`Nucleic Acids Research, Vol. 31 No. 21 ª Oxford University Press 2003; all rights reserved
`
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`e135 Nucleic Acids Research, 2003, Vol. 31, No. 21
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`PAGE 2 OF 6
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`wash the system through with the RNA-grade solvents. Also,
`the samples were pipetted into autosampler tubes after wiping
`down the pipette and tips with RNaseZap from Sigma(cid:226).
`Collection was done at 260 nm on the analytical system, and
`off the maximum absorbance wavelength, at 300 nm, on the
`semi-preparative system. This was done to prevent saturation
`of the detector.
`Separation fractions were collected using a model FCW-
`180 Fraction Collector (Transgenomic). The fraction collector
`was set to collect the main stem–loop peaks by setting a
`specific time window and threshold value. Collection would
`not take place unless the peaks exceeded the threshold value
`within that specified time window. New Falcon(cid:226) tubes were
`used for collecting the RNA product.
`
`Mass spectrometry
`
`Mass spectrometry (MS) analyses were performed on a JEOL
`(Tokyo, Japan) HX110/HX110 four-sector mass spectrometer
`equipped with an Analytica of Brandford (Brandford, CT)
`thermally assisted electrospray ionization (ESI)
`source.
`Lyophilized samples from long-term storage were re-dis-
`solved in 10 mM ammonium acetate (pH adjusted to 7.0) to a
`final concentration of ~10 mM. Each determination was
`carried out by injecting 10 ml aliquots through a loop injector
`at a constant flow rate of 1 ml/min. Negative-ion mode spectra
`were the averaged profile of up to 20 scans with a duty cycle of
`~20 s. Resolution was set to 500 by adjusting the slit width,
`accuracy was determined to be ~410 p.p.m. or better.
`
`PAGE
`
`RNA samples before and after IP-RPLC purification were
`analyzed on an analytical 20% denaturing PAGE. Gels were
`prepared according to the standard protocol (8) and run with
`13 Tris-borate–EDTA buffer at 200 V for 60 min. Samples
`were brought up in 50% glycerol before loading. Staining was
`carried out with Stainzall(cid:226) for 20 min and the gel was
`destained overnight.
`
`RESULTS
`
`Initial separation conditions were selected via the Wavemaker
`software(cid:226). Since the software was originally developed for
`DNA, thymine was replaced by uracil when entering the
`
`Figure 1. Sequence and structure of SL2 (A) and SL3 (B).
`
`Filter/
`(US
`station
`multi-tank/ultraviolet/ultrafiltration
`IONPURE, Lowell, MA). The SL2 and SL3 stem–loops
`were prepared as in Milligan and Uhlenbeck (3) and used
`without further treatment. Samples were stored in a freezer at
`–20(cid:176)C or lyophilized and then frozen for long-term storage.
`
`Liquid chromatography
`
`Analytical separations were carried out on the WAVE Nucleic
`Acid Analysis System (Transgenomic) using Hitachi System
`Manager (HSM) software. The HSM program is interfaced
`with the pump (model L7100), autosampler (model L7200),
`oven (model L7300) and UV detector (model L7400) through
`the D-700 interface module. A semi-preparative system
`consisted of the same elements except it was plumbed to
`accept larger flow rates. SL2 and SL3 were purified on the
`analytical and semi-preparative systems, respectively.
`The column (part no. NUC-99-3860) used was a non-
`porous, C-18 modified polystyrene-divinylbenzene (PS-
`DVB), semi-preparative column (6.0 3 79 mm; 3 mM
`particles) from Transgenomic. The oven temperature was set
`at 30(cid:176)C. A two-solvent gradient was used with a mobile phase
`consisting of an ion-pairing agent and organic modifier.
`Solvent A consisted of 0.1 M TEAA and solvent B consisted
`of 0.1 M TEAA and 25% ACN. The solvents were made using
`RNA-grade water. The flow rates were 0.75 and 1.2 ml·min–1
`for the analytical and semi-preparative systems, respectively.
`The gradient used for the stem–loop purification is shown in
`Table 1. Injection volumes were 80 and 500 ml for the
`analytical and semi-preparative systems, respectively. Several
`water injections were run before injecting the RNA in order to
`
`Table 1. Gradient program for the isolation of SL2 and SL3 in IP-RPLC
`
`SL2 (flow rate: 0.75 ml·min–1)
`Time (min)
`Solvent A (%)
`
`Solvent B (%)
`
`SL3 (flow rate: 1.20 ml·min–1)
`Time (min)
`Solvent A (%)
`
`Solvent B (%)
`
`0.0
`5.0
`8.0
`10.0
`12.0
`15.0
`16.0
`18.0
`20.0
`22.0
`22.1
`24.0
`
`90
`80
`75
`70
`62
`60
`53
`50
`0
`0
`90
`90
`
`10
`20
`25
`30
`38
`40
`47
`50
`100
`100
`10
`10
`
`0.0
`5.0
`8.0
`10.0
`12.0
`14.0
`16.0
`18.0
`18.5
`19.0
`25.0
`
`90
`90
`80
`60
`42
`40
`10
`0
`0
`90
`90
`
`10
`10
`20
`40
`58
`60
`90
`100
`100
`10
`10
`
`2
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`PAGE 3 OF 6
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`Nucleic Acids Research, 2003, Vol. 31, No. 21 e135
`
`sequence information. The effect of this base exchange on the
`initial separation parameters was determined to be minimal
`because the structures of the two bases are sufficiently similar.
`Under these conditions, each of the stem–loops eluted earlier
`than predicted for a linear sequence of the same number of
`bases. The conditions for separation were further optimized
`from Wavemaker(cid:226) to increase resolution as well as
`equilibration time for each injection. Table 1 gives the
`optimized gradient programs used for the SL2 and SL3
`separations. The temperature was set at 30(cid:176)C to more closely
`match the conditions used in the nucleic acid–protein inter-
`action studies and to maximize the efficiency of the separation
`of the stem–loops from aborts and other impurities.
`Figure 2 shows the chromatograms of the crude transcrip-
`tion mix for SL3 (Fig. 2A) and SL2 (Fig. 2B) using the
`conditions outlined above. Typical reaction mixtures include a
`DNA template (single stranded), a primer (17 nt, single
`stranded),
`the triphosphate ribonucleotides
`(NTPs),
`the
`enzyme, T7 polymerase, magnesium, Tris buffer and a
`mixture of RNA products, including the target, a number of
`abortive sequences, and possibly the target plus an additional
`base. In each chromatogram, peak I is the solvent front, which
`is mainly comprised of the unused NTPs and T7 polymerase.
`The group II peaks are comprised of aborts of the desired
`sequence. Some of the aborts, which do not form a secondary
`structure, elute later than the stem–loops even though their
`sequence may be shorter (see Fig. 2B). Both SL3 (III) and SL2
`(III) eluted as packets of four main peaks and three main
`peaks, respectively. Chromatograms of the purified SL3
`(Fig. 3A) and SL2 (Fig. 3B) show only the major RNA
`species of interest. Since the ratio of individual peaks of the
`different conformers is concentration dependent, the purified
`SL2 (Fig. 3B) appears to elute as a single peak due to an over
`10-fold decrease in its injected concentration. When the peaks
`of the major species (injected at higher concentrations) were
`collected and re-injected individually the chromatograms
`showed the same profiles of multiple peaks. The return of one
`peak to the same profile of multiple peaks is an indication of
`the presence of conformer equilibrium. As mentioned previ-
`ously, the retention time for these species was shorter than the
`calculated retention time for a single-stranded species with the
`same number of nucleotides. Previous studies have deter-
`mined that the mechanism of IP-RPLC separation is depend-
`ent upon not only the number of charges, but also on charge
`accessibility (16,17). As a consequence, IP-RPLC is sensitive
`to the shape corresponding to the hairpin conformation. It is
`plausible that the difference in the predicted retention time
`between the stem–loops and their hypothetical linear analogs
`is due to restricted access of the charges of the stem–loops.
`
`DISCUSSION
`
`The presence of multiple peaks, which may represent the
`equilibrium of conformers, can be explained by the stem–
`loops’ ability to maintain a certain degree of secondary
`structure during the separation. In agreement with IP-RPLC
`theory, retention times decreased for the ‘packet’ of peaks,
`which is indicative of stable secondary structure over the
`range of organic modifier concentrations used. If an increase
`in organic modifier had denatured the secondary structure,
`allowing for the increased interaction with the stationary
`
`Figure 2. Chromatograms of the crude transcription mix for: (A) SL3
`showing the solvent front (I), abort sequences (II) and main product peaks
`(III); (B) SL2 showing the solvent front (I), abort sequences (II) and main
`product peaks (III). The vertical dashed line indicates where the cuts were
`taken for purification.
`
`phase, the retention would have increased. Further and more
`definitively, varying the temperature from 35 to 50(cid:176)C in 2(cid:176)C
`increments, starting at 36(cid:176)C under the on-line oven control,
`made the three peaks of SL2 coalesce into a single peak
`(Fig. 4). This melting curve was done under
`initial
`Wavemaker(cid:226) gradient conditions before the gradient was
`further optimized. The fact that the melting temperature of
`SL2 calculated by Wavemaker software(cid:226) is 58(cid:176)C suggests
`that the peaks coalescence may be due to the opening of the
`stem’s double-stranded structure. The presence of multiple
`conformers is also consistent with the observation that the ion-
`pairing reagent is a singly charged monoamine, which may not
`offer the same secondary structure stabilization produced by
`coordination of the divalent cation Mg2+.
`By comparison, the pooled fractions containing multiple
`peaks in the SL2 chromatography provided a single band on a
`20% denaturing PAGE, which showed the same migration
`time as the SL2 marker (Fig. 5). Similarly, the ‘packet’ of
`peaks, designated as the major stem–loop product, purified by
`IP-RPLC and by the established PAGE-base protocol were
`analyzed by ESI-MS (Fig. 6A, PAGE and B, IP-RPLC). In
`both spectra, two charge states were detected for SL2, which
`
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`e135 Nucleic Acids Research, 2003, Vol. 31, No. 21
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`PAGE 4 OF 6
`
`Figure 4. Plot of peak retention verses column temperature for SL2. Each
`of the three main peaks comprising the major species collected for SL2 is
`identified by a different symbol, either a square, triangle or a circle. As the
`temperature is increased, the three main peaks coalesce into one.
`
`Figure 3. Chromatograms of the purified (A) SL3 and (B) SL2. The group-
`ing of peaks (III) indicates the main product that was collected from the
`crude transcription mix of each template synthesis.
`
`provides an experimental mass of 6374 6 2 Da for the PAGE
`sample and 6371 6 2 Da for the IP-RPLC sample (18). Both
`determinations were in close correlation with the molecular
`weight calculated from the sequence (6371.6 Da). Both
`3–, which can be
`spectra indicated the presence of SL2-PO3
`attributed to a small percentage of diphosphate (NDP) in the
`NTPs used in the synthesis mixture that has been incorporated
`by the T7 polymerase (6). The presence of an NDP or an NTP
`at the 5¢ of the construct does not change the overall charging
`of the analyte and thus is inconsequential for the separation. It
`should also be noted that the PAGE-purified sample revealed
`the presence of a minor species, which was absent in the
`IP-RPLC sample, and corresponds to the extra-template
`addition of cytosine (an additional experimental mass of
`304 Da) by the T7 polymerase during the synthesis of the
`oligonucleotide (5). It is clear that the resolution afforded by
`PAGE was insufficient to remove these extended products
`from the reaction mixtures, while IP-RPLC showed clearly
`superior results. Similar results were obtained for the SL3
`construct (data not shown). Hence, analysis by IP-RPLC under
`denaturing conditions, mass spectrometry and PAGE all
`showed that
`the packet of peaks representing individual
`conformers of a particular stem–loop consists of a single
`sequence.
`
`Figure 5. PAGE verification of IP-RPLC-purified SL2. Crude transcription
`mix (lane 1), IP-RPLC-collected solvent front (lane 2), IP-RPLC-purified
`SL2 (lane 3) and an SL2 molecular weight marker (lane 4).
`
`Other considerations (Table 2) should be made concerning
`the advantages of IP-RPLC over the PAGE method. While the
`latter involves long electrophoresis and electro-elution times
`(hours time scale), the IP-RPLC method allows one to obtain
`suitable separation and collection in a much shorter time frame
`(minutes). Preparative-scale electrophoresis is plagued by loss
`
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`PAGE 5 OF 6
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`Nucleic Acids Research, 2003, Vol. 31, No. 21 e135
`
`Figure 6. ESI-MS spectra of (A) PAGE-purified and (B) IP-RPLC-purified SL2. The PAGE purification shows the presence of SL2 + 1 cytosine.
`
`Table 2. Comparison of purification methods
`
`Item
`
`SDS–PAGE
`
`Analytical IP-RPLC
`
`Semi-preparative IP-RPLC
`
`Steps in purification
`Overall time
`Maximum sample size
`Cost per run
`
`4
`2 days
`2 ml
`$$
`
`2
`5.5 h
`2 mg/80 ml
`$
`
`2
`30 min
`2 mg/1.8 ml
`$
`
`of resolution due to the frequent practice of overloading. IP-
`RPLC offers the same resolution obtained in the analytical
`scale for the preparative scale up to 2 mg. In addition, there is
`no manual handling of the RNA samples, as no gel excision is
`required and injections can be automated. This results in a
`much lower probability of RNase contamination. Finally, the
`initial cost of the gel apparatus is cheaper than the HPLC
`system; however, cost per run for the chromatography system
`is less costly than PAGE, making IP-RPLC more cost efficient
`in the long run.
`The IP-RPLC method has proven to be a robust, convenient
`and efficient alternative to PAGE for the purification of RNA
`products synthesized in vitro by T7 polymerase transcription.
`IP-RPLC was shown to be superior to PAGE in several areas.
`By cutting the purification process down from several days to
`several hours, more RNA can be processed and more
`experimentation done with the possibility of simultaneously
`cutting the costs of each synthesis. Single-base resolution can
`be maintained for analytical and semi-preparative scale
`separations. A semi-preparative scale (2 mg/injection) is
`
`necessary to purify the large quantities of products required
`for structural and biochemical investigations.
`This technique does not preclude its application to isolation
`of secondary structures synthesized by various methods. It was
`shown that
`the separation mechanism is sensitive to the
`analyte conformation of the stem–loops chosen. It is this
`characteristic of conformer sensitivity that is exploited by the
`chromatography for isolation of the major species from the
`surrounding matrix. This feature could pave the way for
`intriguing biophysical applications to other systems with
`secondary structure and conformer equilibrium where kinetics
`allows.
`
`ACKNOWLEDGEMENTS
`
`We would like to thank Dr Michael Summers, HHMI, for
`donating the stem–loop, SL2, for the project. We are deeply
`grateful for the financial support from Transgenomic Inc., San
`Jose, CA. The SL3 synthesis and MS analysis was supported
`by NIH Grant R01-GM643208 (D.F.).
`
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