Journal of Chromatography, 536 (1991) 85-93
`Elsevier Science Publishers B.V., Amsterdam
`
`CHROMSYMP. 2028
`
`Large-scale purification of haptenated oligonucleotides using
`high-performance liquid chromatography
`
`RONALD L. MORGAN and JOSEPH E. CELEBUSKI*
`Research and Development, Abbott Laboratories, Abbott Park, JL 60064 (U.S.A.)
`
`ABSTRACT
`
`We report methodology for the succesful separation of unreactcd oligonucleotide from end labeled
`material (fluorescein or biotin) on an milligram scale using high-performance liquid chromatography
`(HPLC). The oligonucleotides (19-24-mers) were synthesized on an automated instrument using cya(cid:173)
`noethylphosphoramidite chemistry. These oligonucleotides possessed a primary amino group at either the
`5' -end or the 3' -end. After trityl-on HPLC purification and detritylation, the amine-terminated oligonucle(cid:173)
`otides were treated with either fluorescein isothiocyanate or biotin-(aminocaproyl) 2-N-hydroxysuccini(cid:173)
`mide active ester to give the haptenated materials. After removal of excess labeling reagent, the labeled
`oligonucleotides were purified by reversed-phase HPLC using a polystyrene-based column, with C 18
`groups on the phenyl part of the polystyrene backbone. The terminally labeled oligonucleotides hybridized
`to their complementary sequences, as observed by size-exclusion chromatography.
`
`INTRODUCTION
`
`In connection with a program designed to commercialize non-radioactive DNA
`probe technology [1], we investigated the covalent labeling of oligonucleotides with
`non-radioactive reporter groups. Several technologies exist to accomplish this ob(cid:173)
`jective. Keller and coworkers [2,3] have introduced haptens onto bulk DNA using
`both nucleophilic attack and photochemistry. Another technique for introduction of
`haptens onto DNA is to synthesize terminally [4] and internally [5] aminated oligonu(cid:173)
`cleotides which can then be specifically labeled with an appropriate hapten. Tous et
`a!. [6] and Smith et al. [7] have obtained ftuoresceinated, terminally labeled oligonu(cid:173)
`cleotides in this fashion. Telser eta!. [8] have prepared oligonucleotides which were
`internally labeled with biotin, fluorescein and pyrene, and studied their thermody(cid:173)
`namic characteristics. Others have chemically labeled oligonucleotides with biotin
`[9-11 ]. While the synthetic chemistry of labeling DNA with small molecules has been
`dealt with in detail, the chromatographic techniques for adequate separation of start(cid:173)
`ing oligonucleotide from product, especially on a milligram scale, are less well in(cid:173)
`vestigated. The aforementioned references use polyacrylamide gel electrophoresis
`(PAGE) or reversed-phase high-performance liquid chromatography (HPLC) col(cid:173)
`umns for their oligonucleotide separations. However, PAGE suffers from a low load(cid:173)
`ing capacity, while reversed-phase HPLC will give coelution of fluorescein isothiocya(cid:173)
`nate (FITC) labeled oligonucleotide with non-labeled oligonucleotide. Biotinylated
`
`0021-9673/91 j$03.50
`
`![:1
`
`1991 Elsevier Science Publishers B.V.
`
`1
`
`MTX1016
`
`

`

`86
`
`R. L. MORGAN, J. E. CELEBUSKI
`
`oligonucleotides show no separation at all on reversed-phase HPLC. In this paper we
`describe the results of a comparative study of several separation conditions for hapte(cid:173)
`nated oligonucleotides.
`
`EXPERIMENTAL
`
`Materials
`Fluorescein isothiocyanate was purchased from Eastman Kodak (Rochester,
`NY, U.S.A.). Biotin-(aminocaproyl)2-N-hydroxysuccinimide ester and Aminomod(cid:173)
`ifier II were obtained from Clontech (Palo Alto, CA, U.S.A.). Triethylamine was
`supplied by Aldrich (Milwaukee, WI, U.S.A.); glacial acetic acid by J. T. Baker
`(Phillipsburg, NJ, U.S.A.); ethanol by Aaper Alcohol and Chemical (Shelbyville, KY,
`U.S.A.); concentrated ammonium hydroxide by Mallinckrodt (Paris, KY, U.S.A.);
`HPLC-grade water and acetonitrile by Fisher (Fair Lawn, NJ, U.S.A.), acrylamide(cid:173)
`N,N' -methylenebisacrylamide (Bis), tetramethylethylenediamine (TEMED), ammo(cid:173)
`nium persulfate, xylene cyanol and Bromophenol Blue by Bio-Rad (Richmond, CA,
`U.S.A.); urea by Boehringer Mannheim (Indianapolis, IN, U.S.A.). NAP-5 Sephadex
`columns were obtained from Pharmacia (Piscataway, NJ, U.S.A.). Amino-controlled
`pore glass was purchased from Glen Research (Herndon, VA, U.S.A.).
`
`Equipment
`A Waters Assoc. (Milford, MA, U.S.A.) HPLC system was used for all HPLC
`separations. This consisted of a 600E system controller, U6K injector, 745B data
`module or NEC Powermate 386/20 and a 484 detector or a 991 photodiode array
`detector. The columns used were Waters J1Bondapak C 18 , 30 x 0.39 em, Hamilton
`(Reno, NV, U.S.A.) PRP-1, 25 x 0.41 em, and EM Science (Cherry Hill, NJ, U.S.A.)
`Polyspher RP-18, 15 x 0.46 em. Oligonucleotides were synthesized on Applied Bio(cid:173)
`systems (Foster City, CA, U.S.A.) 380A or 380B DNA Synthesizers using fJ-cya(cid:173)
`noethylphosphoramidite chemistry with the SYN 11 program, for both the 5'-am(cid:173)
`inated (Clontech) and 3'-aminated (Glen Research) oligonucleotides. Deprotection of
`the oligonucleotides was performed at 5YC for 6 h in concentrated ammonium hy(cid:173)
`droxide. Gel electrophoresis was performed on a Bio-Rad Sequi-Gen apparatus, with
`an E-C apparatus (St. Petersburg, FL, U.S.A.) EC 650 power supply. All HPLC
`separations were performed at ambient temperature. A Beckman (San Ramon, CA,
`U.S.A.) DU-70 spectrophotometer was used for measuring ultraviolet (UV) absorp(cid:173)
`tion spectra. A Savant SpeedVac (Farmingdale, NY, U.S.A.) was used for concentra(cid:173)
`tion of samples and a Jouan (Saint Herblain, France) centrifuge was used for centrifu(cid:173)
`gation.
`
`HPLC purification of trityl-on oligonucleotides
`The aqueous mobile phase (A) for the trityl-on HPLC purification was 0.1 M
`triethylammonium acetate (pH 7.0). The organic mobile phase (B) was acetonitrile.
`The following linear gradient was used: 10 to 40% B in 15 min, hold at 40% B for 10
`min, then linear to 10% Bin 5 min. A Waters J1Bondapak C 18 at 1.5 ml(min was used
`for the trityl-on chromatography. The purified product is collected at approximately
`14 min and concentrated to a residue on a SoeedVac.
`
`2
`
`

`

`HPLC PURIFICATION OF HAPTENATED OLIGONUCLEOTIDES
`
`87
`
`Manual detritylation of oligonucleotides
`The trityl-on purified oligonucleotides were taken up into 1.0 ml of acetic acid(cid:173)
`water (80:20, vfv) and were left to stand at room temperature for 1 h. The solvent was
`removed on a SpeedVac and the residue was taken up into 100 J.ll of 0.3 M sodium
`acetate. Precipitation of the oligonucleotide was initiated by addition of 1.0 ml of
`- 20T ethanol and was effected by immersion of the suspension in a -7SOC (dry
`ice-isopropanol) bath. Centrifugation of the suspension (-lOT, I2 560 g, 15 min)
`gave a pellet which was used for the haptenation reactions.
`
`Synthesis of haptenated oligonucleotides
`Fluoresceinated oligonucleotides. To a solution of 1 J.lmol of detritylated, termi(cid:173)
`nally aminated 24-mer oligonucleotide (determined by UV absorption) in 250 J.ll
`sodium borate buffer (pH 9.0) 8 mg (20 J.lmol) fluorescein isothiocyanate dissolved in
`250 J.ll of dimethylformamide (DMF) was added. The reaction was left to proceed in
`the dark at room temperature for I6 h. The reaction mixture was loaded onto a
`NAP-5 column which had been equilibrated with 10 ml water. The column was eluted
`with 1.0 ml water and the eluate was collected and concentrated on the SpeedVac.
`Biotinylated oligonucleotides. To a solution of 1 J.lmol detritylated, terminally
`aminated 24-mer oligonucleotide in 250 J.ll sodium phosphate buffer (pH 7 .2) II mg
`(20 J.lmol) of biotin-(aminocaproyl)l-N-hydroxysuccinimide ester in 250 t-tl of DMF
`was added. The reaction was left to stand in the dark at room temperature for I7 h.
`The reaction was worked up as in the fluorescein case to give the concentrate, ready
`for HPLC or gel separation.
`
`Polyacrylamide gel electrophoresis of haptenated oligonucleotides
`A 150 ml solution of 12% acrylamide-Bis/8 M urea/89 mM Tris/89 mM sodi(cid:173)
`um borate/2 mM EDT A, pH 8.0 was left to polymerize for 4 h in a 2I x 40 x 0.2 em
`Bio-Rad Sequi-Gen apparatus, after addition of ammonium persulfate and TEMED.
`After pre-electrophoresis of the cell (1 h, 20 W), the oligonecleotides were loaded onto
`the gel. Generally, no more than l t-tmol of oligonucleotide in I 50 t-tl of formamide
`could be applied at once. After 4--5 hat 40 W, the bromophenol blue dye had eluted
`off the gel, signaling the end of the run. At this point, the gel was overlaid on a silica
`gel plate and the DNA bands were visualized by UV shadowing. The product (slowest
`moving major) bands were excised and extracted with I.O M triethylammonium bi(cid:173)
`carbonate overnight. The extracts were concentrated, taken up into 0.5 ml of water
`and desalted on a NAP-5 column. The eluate was checked for DNA concentration by
`UV absorbance.
`
`Reversed-phase HP LC purification of haptenated oligonucleotides (Hamilton P RP-1
`procedure)
`The aqueous mobile phase (A) for the purification of haptenated oligonucleo(cid:173)
`tides was 0.1 M sodium phosphate (pH 8.9). The organic mobile phase (B) was 50%
`aqueous acetonitrile. The following linear gradient was used: 10 to 30% Bin 23 min,
`then 30 to IO% Bin IO min. The flow-rate was I ml/min and the injection volume was
`150 f.ll. The detector was adjusted to 290 nm and 2.0 a.u.f.s.
`
`3
`
`

`

`88
`
`R. L. MORGAN, J. E. CELEBUSKI
`
`Reversed-phase HPLC purification of haptenated oligonucleotides ( Polyspher RP-18,
`EM Science procedure)
`The aqueous mobile phase (A) for the purification of haptenated oligonucleo(cid:173)
`tides was 0.1 M sodium phosphate (pH 8.8). The organic mobile phase (B) was 50%
`aqueous acetonitrile. The following linear gradient was used: 20 to 50% B in 36 min,
`then 50 to 20% Bin 14 min. The flow-rate was 0.6 ml/min and the injection volume
`was 150 J,tL The detector was adjusted to 290 nm and 2.0 a.u.f.s.
`
`Hybridization determination by size-exclusion HPLC
`The ability of haptenated complementary single stranded oligonucleotides to
`hybridize was demonstrated by size-exclusion HPLC using a Bio-Rad Bio-Sil
`SEC-125 as follows: the mobile phase was 0.1 M sodium phosphate (pH 7 .0), the
`flow-rate was 1 ml/min at 280 p.s.i. and the detector was adjusted to 260 nm and 0.5
`a.u.f.s. An aqueous solution of each oligonucleotide, concentration approximately 1
`J,tg per 40 J,tl, was injected individually and each retention time determined for the
`single stranded oligonucleotides. Equivalent amounts of complementary single
`stranded oligonucleotides were mixed at room temperature and injected under the
`previous conditions. Typical retention times for single stranded 24-mers were 7.7-7.9
`min and changed to 7 min when hybridized.
`
`RESULTS AND DISCUSSION
`
`We have found that a 2-mm thick, 21 x 40 em polyacrylamide gel will separate
`labeled from non-labeled material quite well on a 0.5--1.0 J,tmol scale upon electropho(cid:173)
`resis. In all cases observed, labeled material migrates more slowly than non-labeled.
`However, efficiency of separation falls off drastically at higher oligonucleotide load(cid:173)
`ings. Our investigations of separation technology for large-scale work then shifted
`from gel electrophoresis to suitable HPLC columns and conditions. We first exam(cid:173)
`ined the separation afforded by a Waters J,tBondapak c18 HPLC column (30 X 0.39
`em). On an analytical scale (0.1 Jlmol), a C 18 ODS column did not separate biotiny(cid:173)
`lated oligonucleotide from the starting oligonucleotide. We then attempted a sep(cid:173)
`aration of FITC-labeled oligonucleotide from starting, amine-terminated oligonucle(cid:173)
`otide. Due to fluorescein's lipophilicity, one would expect good separation of labeled
`from non-labeled material for fluoresceinated oligonucleotide. In fact, separation was
`seen between fluoresceinated and non-fluoresceinated oligonucleotide (Fig. 1 ). How(cid:173)
`ever, upon further investigation of the fractions by denaturing gel electrophoresis, it
`was determined that peak 1, from Fig. I, was starting oligonucleotide, while peak 2
`was a mixture of starting material and product. Under the non-denaturing chroma(cid:173)
`tographic conditions, it is possible that intercalation of the fluorescein label into a
`neighboring oligonucleotide is occurring. This supposition prompted us to investigate
`the HPLC of oligonucleotides under denaturing conditions.
`One chromatographic support which is well suited for the large-scale separation
`of oligonucleotides under denaturing conditions is polystyrene reversed-phase (PRP)
`material, with divinylbenzene crosslinks. Several studies [12-14] have shown that
`PRP columns possess outstanding loading capacities and will function under ex(cid:173)
`tremes of pH (2-13), as well as at high salt concentration. As shown in Fig. 2, the
`Hamilton PRP-1 column (25 x 0.41 em) will fractionate oligonucleotides. Unfortu-
`
`4
`
`

`

`HPLC PURIFICATION OF HAPTENATED OLIGONUCLEOTIDES
`
`89
`
`1.0
`
`0.8
`
`Absorbance
`A290 run
`
`peak 1 -
`
`-
`
`peak2
`
`0.0
`
`10
`
`20
`
`30
`
`Time
`(minutes)
`
`Fig. 1. Injection of 2.4 pg of a 23-mer, 5'-end labeled with FITC. Peak I, eluting at 16.3 min, is starting
`23-mer. Peak 2, eluting at 17.98 min, is a mixture of starting and fluoresceinated oligonucleotide. Column is
`Waters pBondapak C 18 , flow-rate I ml/min. Gradient table: initial, 5% B, 95% A; 5 min, 20% B, 80% A;
`35 min, 50% B 50% A. Solvent A is 0.1 M triethylammonium acetate, solvent B is acetonitrile.
`
`nately, "ghosting" frequently occurs. Ghosting is a phenomenon in which some in(cid:173)
`jected oligonucleotide is retained on the column during a sample run. The retained
`oligonucleotide is eluted in a subsequent run, when the gradient conditions that elut(cid:173)
`ed the major part of the initial charge are attained. Ghosting was detected when, after
`thoroughly washing the HPLC injector port and syringe, the ghost peak still eluted in
`a blank injection. This even occurs after repeated column washes. Ghosting is seen in
`trityl-on as well as trityl-off HPLC and so is not limited to the case of haptenated
`oligonucleotides. The phenomenon is largely restricted to injections of 1 ,umol and
`larger. This phenomenon is not a problem if the column is used for only one oligonu(cid:173)
`cleotide. If the intended use for the column is for multiple oligonucleotide separa(cid:173)
`tions, the probability of contamination is unacceptably high. From the above experi(cid:173)
`ences, the criteria for a suitable HPLC support were determined to be high loading
`capacity, good resolution, stability to extremes of pH and salt concentration and no
`ghosting. As the PRP-1 column fulfilled all of the criteria save the last one, it seemed a
`
`5
`
`

`

`90
`
`R. L. MORGAN, J. E. CELEBUSKI
`
`peak 1 -
`
`-
`
`peak 2
`
`16.19
`
`16.40
`
`Absorbance
`A29onm
`
`0
`
`5
`
`10
`
`15
`
`20
`
`25
`
`Time
`(minutes)
`
`Fig. 2. Injection of 200 flg of a 25-mer, 5'-end labeled with FITC. Column is Hamilton PRP-1. Peak I,
`eluting at 16.19 min, is unreacted starting material, while peak 2, eluting at 16.4 min, is haptenated
`oligonucleotide.
`
`worthwhile exercise to examine other polystyrene-based supports which were cova(cid:173)
`lently modified. The EM Science Polyspher RP-18 column (15 x 0.46 em) is a 3-C 18
`styrene/3-C 18-1,4-divinylbenzene cross-linked polymeric support which is generally
`used in situations requiring ODS column resolution under unusual salt or pH condi(cid:173)
`tions. We have found that this support is admirably suited to the task of resolving
`haptenated from non-haptenated oligonucleotide, as well as removal of excess hapte(cid:173)
`nation reagent. The separation time between starting material and product ranges
`between 9 and II min, with a flow-rate of0.6 mljmin on the analytical column. Fig. 3
`shows the resolution seen for a typical preparation of a fluoresceinated, end-labeled
`oligonucleotide, while Fig. 4 depicts the separation between terminally biotinylated
`oligonucleotide from terminally aminated starting oligonucleotide. We have obtained
`1.5 mg of purified 25-mer from the analytical EM column in a single crude injection of
`2.2 mg, with recovery of unreacted starting material. Importantly, no ghosting is
`observed when blanks are injected onto an RP-18 column after a sample run. Best
`separation results are obtained on the RP-18 when the pH of the load matches of the
`pH of the eluent.
`
`6
`
`

`

`HPLC PURIFICATION OF HAPTENATED OLIGONUCLEOTIDES
`
`91
`
`1.5
`
`s
`
`1.0
`
`Absorbance
`A290 nm
`
`0.5
`
`0
`
`10
`
`20
`
`30
`
`Time
`(minutes)
`
`Fig. 3. HPLC trace of 249 J.tg loading of a 24-mer, 5'-end labeled with FITC. Column is EM Science
`Polyspher RP-18. Peaks 3 and4, eluting at 9.9 and 12.3 min, are starting oligonucleotide. Peak 5, eluting at
`21.3 min, is FITC-labeled material.
`
`Finally, hybridization of the haptenated oligonucleotides to their complemen(cid:173)
`tary sequences has been examined qualitatively by hyperchromicity measurements
`and quantitatively by size-exclusion chromatography on a high-performance liquid
`chromatograph (Fig. 5). In all cases, the terminally haptenated oligonucleotides were
`able to hybridize to their complementary strands.
`
`CONCLUSION
`
`We have found that C 18 column HPLC will not adequately separate fluorescei(cid:173)
`nated from non-fluoresceinated oligonucleotide, as checked by polyacrylamide gel
`electrophoresis. PAGE itself has the problem of low loading capacity. Unmodified
`polystyrene reversed-phase columns have high loading capacity and afford some sep(cid:173)
`aration of haptenated from non-haptenated oligonucleotide, but have "ghosting" as
`a setback. Taking all of these limitations into account, we now employ the RP-18
`HPLC column from EM Science to separate haptenated from non-haptenated oligo(cid:173)
`nucleotide. It has the characteristics of high loading capacity, good resolution, and no
`"ghosting" in its favor.
`
`7
`
`

`

`92
`
`R. L. MORGAN, J. E. CELEBUSKI
`
`1.5
`
`Absorbance
`A290DD1
`
`1. 0
`
`0.5
`
`Time
`(minutes)
`
`25
`
`Fig. 4, Injection of 345 pg of a 25-mer, 3' -end labeled with biotin-(aminocaproyi)2-N-hydroxysnccinimide
`ester. Column is EM Science Polyspher RP-18. Peaks 3 and 4, eluting at 10.4 and 13 min, are starting
`oligonucleotide, while peak 5, eluting at 21 min, is biotinylated material.
`
`A
`
`B
`
`7.07
`
`7.77
`
`Absorbance
`A260 nm
`
`0
`
`5
`
`10
`
`0
`
`5
`
`10
`
`Time
`(minutes)
`
`Fig. 5. (A) Injection of 24-mer, 5' -end labeled with FITC and hybridized to complementary strand, 7.07
`min peak; (B) 24-mer by itself, 7. 77 min retention time. Note the residual peak at 7. 74 min from unhybrid(cid:173)
`ized material in (A). This disappears upon further admixture with complementary DNA. Column is Bio-Sil
`SEC-125.
`
`8
`
`

`

`HPLC PURIFICATION OF HAPTENATED OLIGONUCLEOTIDES
`
`93
`
`ACKNOWLEDGEMENTS
`
`We thank Cliff Chan for the oligonucleotide synthesis work and Julie Johanson
`for some of the purifications. We thank Abbott Laboratories for allowing the publi(cid:173)
`cation of this manuscript. Finally, we thank a reviewer for calling ref. 7 to our atten(cid:173)
`tion.
`
`REFERENCES
`
`I A.M. Maxam and W. Gilbert, Proc. Nat!. Acad. Sci. U.S.A., 74 (1977) 560--564.
`2 G. H. Keller, C. U. Cumming, C.-P. Huang, M. M. Manak and R. Ting, Anal. Biochem., 170 (1988)
`441-450.
`3 G. H. Keller, D.-P. Huang and M. M. Manak, Anal. Biochem., 177 (1989) 392-395.
`4 P. S. Nelson, R. Sherman-Gold and R. Leon, Nucleic Acids Res., 17 (1989) 7179-7186.
`5 G. B. Dreyer and P. B. Dervan, Proc. Nat/. Acad. Sci. U.S.A., 82 (1985) 968-972.
`6 G. Tous, J. Fausnaugh, P. Vieira and S. Stein, J. Chromatogr., 444 (1988) 67-77.
`7 L. M. Smith, S. Fung, M. W. Hunkapiller, T. J. Hunkapiller and L. E. Hood Nucleic Acids Res., 13
`(1985) 2399-2412.
`8 J. Telser, K. A. Cruickshank, L. E. Morrison and T. L. Netzel, J. Am. Chern. Soc., 111 (1989) 6966-
`6976.
`9 A. C. Forster, J. L. Mcinnis, D. C. Skingle and R. H. Symons, Nucleic Acids Res., 13 (1985) 745-761.
`10 R. P. Viscidi, C. J. Connelly and R. H. Yolken, J. Clin. Microbial., 23 (1986) 311-317.
`II A.Reisfeld, J. M. Rothenberg, E. A. Bayer and M. Wilchek, Biochem. Biophys. Res. Commun., 142
`(1987) 519-526.
`12 M. W. Germann, R. T. Pon and J. H. van de Sande, Anal. Biochem., 165 (1987) 399-405.
`13 S. Ikuta, R. Chattopadhyaya and R. E. Dickerson, Anal. Chern., 56 (1984) 2253-2257.
`14 User Bulletin. No. 50, Applied Biosystems, Foster City, CA, 1988.
`
`9
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`