ANBCA2
`ISSN 0003-2697
`
`VOLUME 216, NUMBER 1, JANUARY 1994
`
`ANALYTICAL
`IOCHEMISTRY
`
`Methods in the Biological Sciences
`
`Review
`Direct Sequencing of Polymerase Chain
`Reaction-Amplified DNA
`Venigalla B. Rao
`
`ACADEMIC PRESS, INC.
`Harcourt Brace & Company
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`ANALYTICAL BIOCHEMISTRY
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`Volume 216, Number 1, january 1994
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`Copyright © 1994 by Academic Press, Inc.
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`

`

`ANALYTICAL BIOCHEMISTRY 216, 83-88 (1994)
`
`polystyrene Reverse-Phase lon-Pair Chromatography of
`Chi meric Ribozymes
`
`Piotr M . Swiderski, Edouard L. Bertrand, and Bruce E. Kaplan
`Beckman Research Institute of the City of Hope, Duarte, California 91010
`
`Received April 26, 1993
`
`The use of a reverse-phase polystyrene resin for ion(cid:173)
`pair HPLC purification of large amounts of synthetic
`chimeric DNA-RNA oligomers that is faster and more
`reliable t han previously used techniques has been
`developed. The preparation of synthetic oligomers con(cid:173)
`taining R NA requires the use of tetrabutylammonium
`fluoride i n the final step, the cleavage of the tert-butyl(cid:173)
`dimethyl silyl protecting group from the ribonucleo(cid:173)
`tides. Cleavage is accompanied by the serendipitous
`formation of ion pairs between tetrabutylammonium
`cations a nd the oligomer phosphates. The formation of
`these ion pairs retards the elution of the oligomer dur(cid:173)
`ing H PLC, which allows rapid removal of excess tetra(cid:173)
`butylammonium fluoride and the concomitant purifica(cid:173)
`tion of chimeric ribozymes. This technique is based on a
`correlation between the length of ion-paired oligomers
`and their retardation during HPLC. The advantages of
`reverse-phase ion-pair HPLC on polystyrene resin for
`the fast purification of oligoribonucleotides are dis(cid:173)
`cussed a nd illustrated through the examples of synthe(cid:173)
`sized chimeric ribozymes. © 1994 Academic Press, Inc.
`
`raphy (5-9). Reversed-phase chromatography is usually
`accomplished by separating the desired oligomer, which
`is still carrying the lipophilic trityl protecting group,
`from all prematurely terminated sequences (10). The
`trityl group is sufficiently lipophilic to retard the mobil(cid:173)
`ity of an oligonucleotide through a reversed-phase (10)
`HPLC column. The purity of an oligomer obtained after
`removal of the trityl group is satisfactory for most bio(cid:173)
`logical studies. Without employing this trityl-on trityl(cid:173)
`off modality it is not possible, using a reversed-phase
`column and standard
`triethylammonium acetate
`(TEAA) 2 buffers, to completely separate oligomers of
`different length. In this buffer system, ion-pairs be(cid:173)
`tween the triethylammonium cations and the phos(cid:173)
`phates of the oligomer have been reported (12). Al(cid:173)
`though this phenomenon of ion-pairing does "order the
`elution of oligodeoxyribonucleotides according to the
`number of phosphate groups present" (12), the actual
`differential separation is often too small to be useful.
`The synthesis of oligoribonucleotides requires that
`the 2'-position of the ribose moiety be protected, both
`during synthesis and during the base and phosphate de-
`
`Synthetic oligoribonucleotides are becoming central
`to studies of ribozymes, molecules possessing catalytic
`activity (1- 3), and their potential action on RNA vi(cid:173)
`ruses. W hile the synthesis of fully protected oligoribonu(cid:173)
`cleotides is now possible for most laboratories equipped
`with an oligonucleotide synthesizer using commercially
`available reagents, their deprotection and purification
`still remains challenging and time consuming. The puri(cid:173)
`ficati on of DNA oligomers by HPLC was reported over
`15 years ago ( 4) and most oligomers are still purified in a
`manner quite similar to these original methods. The
`Primary modes used for purification of oligonucleotides
`are reversed-phase 1 (5,6) and ion-exchange chromatog-
`
`1 We use the term reversed-phase to refer to silica stationary
`Phases that are covalently linked to a lipophilic orga nic moiety, such
`
`as C-4, C-8, or C-18 alkylsilyl ethers, and in which the eluting solvent
`is run from higher concentrations of the more polar solvent (i.e.,
`water) to higher concentrations of the less polar solvent (i.e., acetoni(cid:173)
`trile). The term reverse phase (11) refers to a material that operates
`in a manner similar to a reversed- phase re sin but has not had its
`surface chemistry modified (i.e., reve rsed), so as to make it more lipo(cid:173)
`philic, but where t his property is inherent to the surface chemistry of
`the material (i.e. , polystyrene).
`2 Abbreviations used: ACN, acetonitrile; C-4, butylsilyl bonded
`phase; C-18, octadecyl bonded phase; CPG, controlled pore glass;
`DEPC, diet hyl pyrocarbonate; DMT, 4,4'-dimethoxytrityl; LCAA(cid:173)
`CPG, long-chain alkyl amine controlled pore glass; ODS, octadecylsi(cid:173)
`lyl bonded phase; ODU, optical density unit; PAGE, polyacrylamide
`gel electrophoresis; PRP-1, 12- to 20-11m spherical chromatographic
`resin; PRP-IPC, polystyrene reverse-phase ion-pair chromatogra(cid:173)
`phy; TBDMS, tert-butyldimet hylsily l; TBAA, tetra-n-butylammo(cid:173)
`nium acetate; TBAF, tetra-n-butylammonium flu oride; TBAP, tetra(cid:173)
`n.-butylammonium phosphate; TEAA, triethylammonium acetate;
`THF, tetrahydrofurane.
`
`0003-2697 /94 $5.00
`Copyri ght © 1994 by Academic Press, Inc.
`All rights of reproduct ion in any form reserved.
`
`83
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`3
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`

`84
`
`SWIDERSKI, BERTRAND, AND KAPLAN
`
`protection steps. The protecting group commonly used
`is the tert-butyldimethylsilyl (TBDMS) moiety. The
`final deprotection step in the synthesis of oligoribonu(cid:173)
`cleotides requires the cleavage of this TBDMS group by
`treatment with a large excess of tetrabutylammonium
`(TBA) fluoride. The oligoribonucleotide, under these
`conditions, forms very lipophilic ion-pairs between
`TBA cations and oligonucleotide phosphates. This phe(cid:173)
`nomenon of ion pairing causes a dramatic increase in
`the retention of oligomers on polystyrene reverse-phase
`HPLC columns and therefore complicates the use of a
`purification scheme identical to that used for the purifi(cid:173)
`cation of synthetic DNA oligomers. The differential in(cid:173)
`crease in lipophilicity, conferred by the trityl group, is
`no longer sufficient to separate a series of oligomers
`which differ only in presence or absence of the trityl
`group. Although it might appear that ion-exchange col(cid:173)
`umns would be the ideal method for the purification of
`all oligonucleotides, these columns can be relatively ex(cid:173)
`pensive and may exhibit exceedingly short life times.
`Whereas a typical polystyrene reverse -phase column
`might last for several hundred purifications, an ion ex(cid:173)
`change column can become unreliable after many fewer
`purifications. It is this inherent unreliability, plus the
`need to subsequently "desalt" the product, that has
`kept ion exchange chromatography from becoming the
`method of choice for oligomer purification.
`We have developed a technique which combines the
`reliability of polystyrene reverse phase HPLC with a
`degree of resolution that is associated with ion-ex(cid:173)
`change columns. This technique is referred to as poly(cid:173)
`styrene reverse-phase ion-pair chromatography (PRP(cid:173)
`IPC) to differentiate from reversed-phase ion-pair
`chromatography (RP-IPC) (13). In both RP-IPC and
`PRP-IPC, a bulky lipophilic ion is introduced into the
`mobile phase in relatively high concentration in order to
`significantly increase the retention of ions of opposite
`charge (13- 16) . Several mechanisms have been postu(cid:173)
`lated (16,17) to explain the phenomenon of increased
`retention of an ionic species by the introduction of a
`lipophilic counter ion. According to the "ion-pair"
`model (16), the lipophilic counterions pair with ions
`that are separated in the mobile phase. The ion-pair
`then partitions between the nonpolar stationary phase
`and the mobile phase. Another mechanism is postulated
`by the "dynamic ion exchange" model (16,18). In this
`model, the lipophilic counter ion is thought to adsorb
`onto the nonpolar stationary phase. The stationary
`phase then has associated with it an adsorbed cationic
`species, which causes it to behave as an ion exchanger.
`
`MATERIALS AND METHODS
`The assembly of the chimeric ribozymes was per(cid:173)
`formed on an automated synthesizer (Applied Biosys(cid:173)
`tems Model 394 DNA/RNA Synthesizer, Foster City,
`
`CA). D eoxyribophosphoramidites (Millipore, Milford
`MA) and 2'-0-TBDMS ribophosphoramidites (Chem~
`Genes Corp., Waltham, MA) were used for the synthe(cid:173)
`sis. Two terminal phosphate units of the chimeric oligo(cid:173)
`mers were thiolated (19) in order to reduce degradation
`by nucleases (20). Syntheses were performed in t rityl(cid:173)
`off mode. Recommended coupling time for ribophos(cid:173)
`phoramidites was empirically determined to be optimal
`at 800 s. The average coupling yield, determined by
`quantitation of the released trityl cation, was 97.5-98.5%.
`Two chimeric ribozymes were synthesized (sequences
`shown below), which were used to demonstrate the use(cid:173)
`fulness of this novel technique,
`
`A. (32-mer) 5'-TGT CTC Acu gau gag cTT
`
`TTg ega aaG CTA*G*G-3'
`
`B . (38-mer) 5'-G*G*C TTT Tgc uga uga
`
`gTC CGT GAG GAc gaa acT CTG*C*T-3'
`
`where A, G, C, Tare deoxyribonucleotides; a, g, c, u are
`ribonucleotides; and asterics mark the phosphor(cid:173)
`othioate. The assembled chimeric ribozymes were de(cid:173)
`protected as follows:
`(i) The phosphate deprotection and deacylation were
`accomplished by treatment with saturated (0°C) , anhy(cid:173)
`drous ethanolic ammonia for 8 h at 55°C (21,22). We
`found deprotection with anhydrous ethanolic am m onia
`more reliable than treatment with 35 % aq ammonia(cid:173)
`ethanol 3:1 (v/v) (23).
`(ii) The tube containing ethanolic ammonia was then
`cooled to - 20°C before it was opened; the resin was t hen
`filtered off and washed with 5 ml of dry ethanol. Most of
`the remaining partially protected oligonucleotides were
`separated from the CPG by a final wash with warm
`water (70°C). Combined washes were concentrated to
`dryness in a SpeedVac (Model SVC 200H, Savant Corp.,
`Farmingdale, NY).
`(iii) The product of deacylation was then treated with
`1 M TBAF solution in THF (0.8 ml) (Aldrich) for 18 h
`at rt.
`(iv) The reaction mixture was rapidly dried down for 1
`min under reduced pressure while being vortexed and
`then dissolved in 1.5 ml of water, centrifuged for 1 min,
`and immediately injected into the PRP-1 HPLC col(cid:173)
`umn. The reaction mixture was then simultaneously de(cid:173)
`salted and purified in a nonlinear gradient of 5- 80%
`buffer B for 75 min (A: 10 mM tetrabutylammonium
`acetate, 1 mM
`tetrabutylammonium phosphate
`in
`water, pH 7.5; B: 10 mM tetrabutylammonium acetate, 1
`mM tetrabutylammonium phosphate in acetonitrile:
`water, 8:2 (v /v), pH 7.5) (Fig. 1) . The convex gradient
`profile (curve 3 on the Waters Automated Gradient
`controller) is described by an equation (25),
`
`4
`
`4
`
`

`

`A
`
`B
`
`POLYSTYRENE REVERSE -PHASE ION-PAIR CHROMATOGRAPHY
`
`85
`
`is strongly recommended as an
`latex gloves
`ile,
`additional precaution.
`(v) Results were visualized by analytical PAGE (Fig.
`2) . Fractions containing product were combined, con(cid:173)
`centrated under reduced pressure in a SpeedVac, and
`precipitated from ethanol.
`Oligomers used as HPLC markers (Fig. 3, dT 10 , dT 15 ,
`dT20 , dT25 , dT30 , dT 45 , dT60 , dT90 ) were synthesized in
`DMT -off mode, and were purified by preparative PAGE
`in accordance with established methods.
`
`RESULTS AND DISCUSSION
`Molecular biologists are making ever increasing de(cid:173)
`mands on synthesis laboratories for large amounts of
`
`E
`c
`
`f2 N
`b
`"'
`u c
`0 -e g
`
`.c
`<t
`
`60
`
`75
`
`FIG . 1. A typical PRP-IPC chromatograms of large scale sy nth eses
`(2 11mol) of chimeric ribozymes on a PRP-1 column. Samples were
`loaded onto a PRP-1 (300-7 mm) column and eluted with a co nvex
`gradie nt of5- 80 % buffer B (A: 10 mM TBAA, 1 mM TBAP, pH 7.5; 8 :
`10 mM TBAA, 1 mM TBAP, pH 7.5, acetonitrile:wate r, 8: 2 (v/v). The
`flow rate was 4 ml/m in , 75 min. The shadowed area was collected, and
`fraction s we re analyzed by PAGE (Fig. 2) . (A) 32-mer, (B) 38-mer.
`
`where %A is the percentage flow rate of pump A, tis the
`elapsed time from injection, t0 = 0 is the elapsed time of
`the beginning of segment, t 1 = 75 min is the elapsed time
`of the end of the segment, %A end = 20% is the end point
`value of pump A at the end of the segment and %Astart =
`95% is t he starting point value of pump A at the begin(cid:173)
`ning of t he segment.
`HPLC purification of the chimeric DNA-RNA ribo(cid:173)
`zymes was performed on a Waters Model 510 instru(cid:173)
`ment with an automated gradient controller (Millipore
`Corp., Waters Chromatography Division, Milford, MA).
`All H PLC buffers were treated with 0.1 % DEPC (Al(cid:173)
`drich) and 0.1 % EDT A, followed by autoclaving at
`120°C for 2 h; 0.001 % NaN 3 (w/v) was added to prevent
`bacterial growth. All HPLC columns used for purifica(cid:173)
`tion of R NA were treated with 0.2 % DEPC in water
`overnight and washed with 1% EDT A prior to use. Only
`disposable, sterile plasticware was used during final pu (cid:173)
`rification. After the final deprotection step strictly ster(cid:173)
`ile conditions were maintained. Use of disposable, ster-
`
`A
`
`B
`
`--51-mer
`
`--35-mer
`
`--22-mer
`
`--51-mer
`
`--35-mer
`
`--22-mer
`
`R M
`FIG. 2. Gel assay of chimeric ribozymes after purification by PRP(cid:173)
`IPC on PRP-1 column. Oligomers in collected fractions were 5' end(cid:173)
`labeled with 32P and then resolved on a 20%, 8 M urea polyacrylamide
`gel. Results were visualized by autoradiography. (A) 32-mer, (B) 38-
`mer; R, ribozyme sy nthesis reaction mixture after final deprotection;
`M, a mixture of DNA markers of indicated length.
`
`5
`
`5
`
`

`

`86
`
`SWIDERSKI, BERTRAND, AND KAPLAN
`
`A
`
`B
`
`(1-8)
`
`/
`
`.~~
`~ w 00
`00 ~0 ~ w 00
`Time (min)
`
`00 ~
`
`FIG. 3. A PRP-IPC chromatogram of the mixture of DNA markers
`(1) dT 10 , (2) dT 15 , (3) dT20 , (4) dT25 , (5) dT30 , (6) dT45 , (7) dT60 , (8)
`dT90 • Samples were loaded onto a PRP -1 (250- 2 mm) column and
`eluted with linear gradient of: (A) 0-100% of bufl"er B (A: 10 mM
`TEAA in water, pH 7.5; B: 10 mM TEAA, pH 7.5, acetonitrile:wate r,
`8:2 at flow rate of 1 ml/min, 100 min); (B) 0- 100% of buffer B (A: 10
`mM TBAA, 1 mM TBAP, in water, pH 7.5; B : 10 mM TBAA, 1 mM
`TBAP, pH 7.5, acetonitrile:water, 8:2 at flow rate of 1 ml/min , 100
`min.
`
`synthetic RNAs and chimeric DNA-RNA oligomers. Al(cid:173)
`though the syntheses of such molecules is straightfor(cid:173)
`ward, their deprotection and purification have not yet
`achieved the simplicity of methods utilized for the de(cid:173)
`protection and purification of DNA oligomers. The base
`and phosphate protecting groups are easily removed by
`treatment of the fully protected oligoribonucleotide
`with ethanolic ammonia (21,22). The final reaction in(cid:173)
`volves the removal of the TBDMS group from the 2'-po(cid:173)
`sition of the ribose moiety. This deprotection is accom(cid:173)
`plished by treatment of the partially deprotected
`oligoribonucleotide with tetrabutylammonium fluoride
`(TBAF) in tetrahydrofuran (THF). When cleavage has
`been completed a problem arises in separating the fully
`deprotected oligoribonucleotide from the TBAF before
`final purification by PAGE. High concentration of salts
`in the sample makes direct purification by PAGE diffi(cid:173)
`cult. The separation of excess TBAF from the oligori(cid:173)
`bonucleotide has been accomplished by several meth(cid:173)
`ods, including gel filtration (23,26,27), ion exchange
`protocols (21,22), and reversed-phase silica chromatog(cid:173)
`raphy (28) . Each of these techniques contains inherent
`problems, which are briefly discussed below.
`Oligonucleotides containing ribonucleotides are very
`susceptible to nucleases (29) and cleavage of the inter(cid:173)
`nucleotide bonds in the presence of traces metals (30).
`Thus RNA as well as chimeric RNA- DNA ribozymes is
`
`extremely labile in solution. The techniques of dialysis
`and gel filtration are so time consuming that the RNAs
`are often cleaved in the course of the purification, and
`thus yields are substantially reduced. Anoth er tech(cid:173)
`nique, which involves the use of various ion exchange
`resins, is effective in the removal of TBAF and some(cid:173)
`what effective in the purification of RNA, but is unde(cid:173)
`sirable because it requires an additional desalting step
`prior to PAGE.
`Alternately, reversed-phase resins such as octadecyl(cid:173)
`silyl (C-18, ODS) and butylsilyl (C-4) bonded phases,
`although shown to be effective for TBAF removal and
`purification of synthetic RNA (28), have proven to be
`unreliable. The chemical linkage between the silica and
`alkylsilyl (31) moieties is chemically similar t o t hat of
`the linkage between ribose and its 2'-0- protecting
`group. All of these linkages are silyl ethers, whic h are
`susceptible to cleavage with TBAF. When this re agent
`is passed through an alkylsilyl bonded phase, a signifi(cid:173)
`cant amount of cleavage occurs, causing immediate and
`permanent damage to the column, loss of sign ifi cant
`amounts of sample, and contamination of the R NA with
`products of resin cleavage.
`Polystyrene reverse-phase resin (PRP-1 from H amil(cid:173)
`ton), unlike alkylsilyl bonded phases, is stable under
`most conditions used for chromatography and tolerates
`extremes of pH and fluoride anions without hydro lysis.
`The long lifetime of the column and very good recover(cid:173)
`ies of the samples (95 %) are additional advantages of
`PRP resins. HPLC on PRP-1 is an efficient method for
`the fast removal of TBAF. While developi ng this
`method for the removal of fluoride, we noted that the
`presence of the TBA cation dramatically affected reten(cid:173)
`tion of oligomers during chromatographic separation.
`This phenomenon of chromatographic retardation is
`known (14,32) to be caused by ion pairing, affecting the
`mobility of the oligomer through the polystyrene re(cid:173)
`verse-phase column . Ion-pair HPLC was previously
`suggested as a technique for the purification of DNA
`oligomers (5,13).
`We have developed a new method for the purification
`of oligoribonucleotides, which we refer to as polystyrene
`reverse -phase ion-pair chromatography. We have com(cid:173)
`bined the important and time-consuming step of re(cid:173)
`moval of tetrabutylammonium fluoride, after fi na l de(cid:173)
`protection, with ion-pair-based HPLC purification,
`creating a rapid technique for the purification of syn(cid:173)
`thetic chimeric ribozymes and oligoribonucleotides.
`Using PRP-IPC, the longer the oligomer, the greater
`its retention time (5,13) . The ion-pairing retardatio n ef(cid:173)
`fect has also been observed with the DNA-TEA ion-pair
`(12) but this retardation is not strong enough to be use(cid:173)
`ful (Fig. 3A). The resolution that can be achieved by
`analytical PRP-IPC is presented on an HPLC chro(cid:173)
`matogram of synthesized model DNA oligomers (Fig.
`3B). Thus, it can be seen that the ion-pair effect has
`
`6
`
`6
`
`

`

`POLYSTYRENE REVERSE-PHASE ION-PAIR CHROMATOGRAPHY
`
`87
`
`made possible the efficient separation of oligonucleo(cid:173)
`tides based on their length (5,13). Such ion-pair effects
`are a co mmon feature of liquid chromatography (33)
`and have been very useful for peptide purification (34),
`separatio n of polyanionic nucleotides (35,36), and sepa(cid:173)
`ration of lipophilic acids and sulfonates (32). In order to
`maint ain the maximal possible conversion of oligomers
`into their tetrabutylammonium ion-pairs, TEA buffers
`were u sed during chromatography. Use of TEA buffers
`is necessary to maintain the RNA- TEA ion-pair during
`HPLC. W hen using other buffers, such as TEA, we have
`observed partial replacement of the cation (TEA) in the
`RNA-TE A ion-pairs by the TEA cation derived frorri
`the buffe r. When this replacement occurs during an
`HPLC, t he continuous readjustment of the equilibrium
`between the RNA-TEA and the RNA-TEA causes re(cid:173)
`tention t imes to decrease and the concomitant resolu(cid:173)
`tion to be dramatically decreased. In order to achieve
`reproducible resolution and retention during PRP-IPC
`it is n ecessary to maintain the concentration of the TEA
`cation at a value sufficient to maintain the RNA- TEA
`ion-pair while keeping this concentration as low as pos(cid:173)
`sible, so as to make possible an analytical PAGE with(cid:173)
`out any additional desalting steps.
`We demonstrate the usefulness of preparative PRP(cid:173)
`IPC wit h t wo examples of novel chimeric ribozymes (32-
`and 38-mer). After the final deprotection step the crude
`oligomer (synthesized on a 2.0-~-tmol scale) mixture was
`injected onto a PRP-1 column (300-7 mm) and eluted
`with a gradie nt of 5-80% buffer E (A: 10 mM TEAA, 1
`mM T EAP, pH 7.5; E: 10 mM TEAA, 1 mM TEAP, aceto(cid:173)
`nitrile:water, 8:2 (v/v) at flow rate of 4 ml/min, 75 min
`(Fig. 1) . Portions of collected fractions were 5' end-la(cid:173)
`beled wit h 32P, resolved on a 20%, 8 M urea polyacryl(cid:173)
`amide, and visualized by autoradiography (Fig. 2). The
`PAGE shown in Fig. 2E had a visibly strong edge effect
`which created a "smiling effect" which made it appear
`that the mobilities of the 38-mer and 35-mer were simi(cid:173)
`~ar. Figure 2 shows that PRP-IPC is capable ofseparat(cid:173)
`mg the desired oligomer from oligomers shorter than (n
`- 1) but is not capable of offering unit resolution in a
`prepar ative mode. In a "real life" synthesis, in which the
`1) oligomer is present in small amounts, this
`(n -
`method is capable of yielding oligomers of estimated
`95% purity at a length of 38 nucleotide units and of even
`greater p urity for shorter oligomers. Res'.llts of PRP(cid:173)
`IPC pu rification are presented in Table 1. Injecting 240
`ODU of crude oligomer and pooling marked () fractions
`we obtained 120- 135 ODU of 95 % pure chimeric ribo(cid:173)
`zymes. T his level of purification is comparable to that
`wh~ch might be achieved by preparative gel electropho(cid:173)
`resis, but PRP-IPC is able to purify much larger sam(cid:173)
`ples more rapidly and in principle can be scaled up to
`any level desired.
`~h e use of RNA, chimeric RNA- DNA ribozymes, and
`antisense oligomers for large-scale in vitro or in vivo
`
`TABLE
`
`[ODU] p e r fraction, collected amount
`
`Fraction
`
`Ribozyme A a
`
`Ribozyme Bb
`
`1
`2
`3
`4
`5
`6
`7
`8
`9
`10
`11
`12
`13
`14
`15
`16
`17
`18
`19
`
`5.4
`6.2
`8.0
`9.4
`13.4
`15.3
`15.8
`(16.7)
`(17.2)
`(17.9)
`(16.4)
`(13.8)
`(11.7)
`(9.7)
`(7.7)
`(6.1)
`(4.6)
`
`4.1
`6.5
`7.1
`7.5
`9.0
`11.0
`12.0
`14.1
`(15.2)
`(16.2)
`(17.1)
`(17.2)
`(16.6)
`(13 .5)
`(12 .5)
`(10.1)
`(8.1)
`(6.2)
`(4.1)
`
`a Ribozyme A, 32- mer, sy nthesized with overall yield of 41 %. In (cid:173)
`jected sample was 240 ODUs.
`b Ribozyme B, 38- mer, sy nth esized with overall yield of 56%. In (cid:173)
`jected sa mple was 240 ODUs.
`
`studies will soon require gram amounts of such mate(cid:173)
`rials. These large amounts may be refractory to PAGE
`purification and traditional reversed phase or ion-ex(cid:173)
`change chromatographic techniques. The use of PRP-1
`HPLC columns in conjunction with TEA buffers has
`allowed us to prepare relatively large amounts of high
`purity, chimeric ribozymes (24,37) with elimination of
`purification by PAGE. There are no obvious reasons
`why this technique cannot be scaled up and utilized for
`purification of large quantities of ribozymes and anti(cid:173)
`sense oligomers in order to satisfy the demands of large(cid:173)
`scale biological studies which require the use of purified
`oligoribonucleotides. If it is necessary to replace the
`TEA cation, for biological or for structural studies (i.e.,
`NMR) , this can be easily accomplished by passing a so(cid:173)
`lution of the TEA-oligomer through a Dowex 50-Na+
`form which then yields the sodium form of the oligomer.
`This can also be easily accomplished by precipitation of
`the oligomer in the presence of sodium acetate.
`
`ACKNOWLEDGMENTS
`
`We t h a nk Dr. Dan Peter Lee from Hamilton Co. (Reno, NV) for t he
`supply of PRP-1, 12- to 20-pm spherical chromatog raphic resin. W e
`thank Dr. John Termini for his carefu l reading of the manuscript.
`
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