Nucleic Acids Research, 2003, Vol. 31, No. 21 e135
© 2003 Oxford University Press
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
*To whom correspondence should be addressed. Tel: +1 410 455 2105; Fax: +1 410 455 2608; Email: lacourse{at}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
Received August 8, 2003; Revised and Accepted September 18, 2003
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ABSTRACT
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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
recognition 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.
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INTRODUCTION
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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 heterogeneity. 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 chromatography (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 -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).
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MATERIALS AND METHODS
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Reagents
All reagents and solvents were HPLC grade (Fisher Scientific,
Springfield, NJ). Triethylammonium acetate (TEAA) was obtained
as a 20
x 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 multi-tank/ultraviolet/ultrafiltration
station (US Filter/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°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 x 79 mm; 3 µM particles) from Transgenomic. The oven temperature was set at 30°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 µl for the analytical and semi-preparative systems, respectively. Several water injections were run before injecting the RNA in order to 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®. 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® 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-dissolved in 10 mM ammonium acetate (pH adjusted to 7.0) to a final concentration of 
10 µM. Each determination was carried out by injecting 10 µl aliquots through a loop injector at a constant flow rate of 1 µl/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 1x 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® for 20 min and the gel was destained overnight.
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RESULTS
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Initial separation conditions were selected via the Wavemaker
software®. Since the software was originally developed for
DNA, thymine was replaced by uracil when entering the 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®
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°C to more closely match the conditions used in the nucleic
acid–protein interaction 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 transcription 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 previously, 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 determined that the mechanism of IP-RPLC separation is dependent 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.
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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.
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Figure 3. Chromatograms of the purified (A) SL3 and (B) SL2. The grouping of peaks (III) indicates the main product that was collected from the crude transcription mix of each template synthesis.
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DISCUSSION
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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 phase, the retention
would have increased. Further and more definitively, varying
the temperature from 35 to 50°C in 2°C increments, starting
at 36°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® gradient conditions before
the gradient was further optimized. The fact that the melting
temperature of SL2 calculated by Wavemaker software® is
58°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 Mg
2+.
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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.
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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 provides an experimental
mass of 6374 ± 2 Da for the PAGE sample and 6371 ±
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 spectra indicated the presence
of SL2-PO
33–, which can be 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.
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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).
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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.
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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 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.
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ACKNOWLEDGEMENTS
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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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