NASA-CR-204_44 NOTES & TIPS 239 5 2 4 i L 20min 9 7 Separation of "Uncharged" 6 Oligodeoxynucleotide Analogs by Anion-Exchange Chromatography at High pH Jtirgen G. Schmidt,* Peter E. Nielsen,t I and Leslie E. Orgel* 20min *The Salk Institute for Biological Studies, P.O. Box 85800, San Diego, California 92186-5800; and tCenter for 10 11 / Biomolecular Recognition, IMBG, Biochemistry B, \ The Panum Institute, Blegdamsvej 3c, DK-2100 N, Copenhagen, Denmark Received December 14, 1995 gradient Ion-exchange chromatography is a well-established method for the analysis and purification of phospho- diester-linked oligonucleotides (1). If elution is carried out under alkaline conditions, the secondary structure of G- and C-rich oligomers is disrupted. Furthermore, elution times become more sensitive to the G and T content of the oligomer, because G and T are deproto- nated at pH 10 (2). In recent work on peptide-nucleic I acids (PNAs) 1we noted that mixtures of PNA oligomers 2Omin G4, G_, Gs, and Gjo are readily separated by elution at pH 12 on an RPC-5 column (3). Here we show that this FIG. 1. Elution profiles of (a) Mixture I (samples 1-5) separated separation method is more generally applicable. using a linear gradient from A to A/B:80/20 in 40 min; (b) Mixture The sequences of the oligomers of the type PNA-Lys- II (samples 6-9) separated using a linear gradient from Ato AfB:90/ 10 in 50 min; and (c) Mixture III (samples 10 and 11) separated on a degressive gradient, as shown in the figure, from A to A/B:96/4 in 1Abbreviation used: PNAs, peptide-nucleic acids. 40 min. ANALYTICALBIOCHEMISTRY235, 239--241 (1996) ARTICLE NO. 0119 0003-2697/96 $18.00 Copyright © 1996byAcademic Press, Inc. All rights ofreproduction in any form reserved. 240 NOTES & TIPS TABLE 1 List of Sequences, T/G Contents, and Overall Charges of the PNAs Used in This Study Mixture No. Fig. No. PNA No. Sequence T G Length/"charges" I 1 1 T4C_TCTC 6 10/6 I 1 2 T2CT2CT4 8 10/8 I 1 3 T,CT5 9 10/9 I 1 4 T,0 10 10/10 ! 1 5 T4G2TGTG 6 4 10/10 II 2 6 GAGAGGA4 4 10/4 II 2 7 A4G2TGAG 1 4 10/5 II 2 8 A3TGeTGAG 2 4 10/6 II 2 9 TGTACGTCACAACTA 4 2 15/6 III 3 10 GTAGATCACT 3 2 10/5 III 3 11 AGTGATCTAC 3 2 10/5 Note. Each oligomer has a lysine amide group attached to its carboxyl terminus. NH2 (4-6) used in this work are listed in Table 1.Ana- solubility is observed with longer (15-20 bases), pu- lytical anion-exchange chromatography was carried rine-rich PNAs (10), and some PNAs also show a ten- out on an RPC-5 column as previously described (2, 7, dency to aggregate. Other uncharged oligonucleotide 8), using 20 mM NaOH and 1mM Tris-HC104 in water analogs are often sparingly soluble in neutral aqueous as the A buffer and 20 mM NaOH, 1 mM Tris-HC104, solution and tend to adopt very stable self-structures. and 0.1 M NaC104 as the B buffer. Stock solutions of This makes chromatography of PNA and other un- PNA oligomers containing about 5 × 10 3 ODU/#I at charged oligomers less effective than it is for standard 254 nm were prepared. Sample mixtures for analysis DNA oligomers. Elution at a pH of 10 or greater helps contained 1 pl of each relevant stock solution in 1 ml to overcome these difficulties. The method should be total volume. The gradients used are described in the applicable to any DNA analog that is sufficiently stable figure legend. Peaks were assigned on the basis of their at pH 10. retention times and the assignments were confirmed The RPC-5 column that we use routinely could be by co-injection. replaced by a commercially available, alkali-stable The components of mixture I separated principally anion-exchange column such as the Mono-Q column on the basis of charge (the number of G and T residues) from Pharmacia (11), enabling separations on analyti- (Fig. la). However, the replacement of T by G resulted cal or preparative scales. Finally, we believe that cat- in an increased retention time (Fig. la, peaks 4 and 5). ion-exchange chromatography under acidic conditions The elution profile for mixture II, which contains G- could probably be used in an analogous way, since C rich oligomers, shows that the separation method is and A residues are protonated in the pH range 3-4 probably restricted to oligomers containing 4 or more and many oligonucleotide analogs including PNA are ionizable residues (Fig. lb) since the peak correspond- stable against depurination under acidic conditions. ing to PNA 6 has a short retention time and is very Acknowledgments. This work was supported by NSCORT/EXO- broad. A pair ofoligomers with the same base composi- BIOLOGY Grant NAGW-2881 from the National Aeronautics and tion but different seqeunces was resolved successfully Space Administration. Wethank Sylvia Bailey formanuscript prepa- (Fig. lc). ration. HPLC at alkaline pH is not a useful technique for the purification of unmodified PNAs. PNA rearranges REFERENCES slowly at neutral pH and more rapidly under alkaline conditions via the attack of the main-chain terminal 1. Haupt, W., and Pingoud, A. (1983) J. Chromatogr. 260, 419- 426. amino group on the carbonyl function of the adjacent 2. Stribling, R. (1991) J. Chromatogr. 538, 474-479. side chain (9). We were able to resolve PNA G6 from 3. BShler, C., Nielsen, P. E., and Orgel, L. E. (1995) Nature 376, its rearrangement product and hence to determine that 578-581. the half-life for the rearrangement at room tempera- 4. Nielsen, P. E., Egholm, M., Berg, R. H., and Burchhardt, O. ture is somewhat less than 2 h at pH 12 and about 5 (1991) Science 254, 1497-1500. days at pH 10. With longer oligomers, PNA and its 5. Egholm, M.,et al. (1993) Nature 365, 566-568. rearrangement product are not resolved. Modified PNA 6. Wittung, P., Nielsen, P. E., Burchhardt, O., Egholm, M., and suitable for practical applications could be purified by Nord_n, B.(1994) Nature 368, 561-563. HPLC in the pH range 10-12. 7. Larson, J. E., Hardies, S. C., Patient, R. K., and Wells, R. D. PNAs are generally quite water soluble, but reduced (1979) J. Biol. Chem. 254, 5535-5541. NOTES & TIPS 241 8. Pearson, R. L., Weiss, J. F., and Kelmers, A. D. (1971)Biochim. Biophys. Aeta 228, 770-774. 9. Christensen, L., et al. (1995) J. Pept. Sci. 3, 175-183. 10. Noble, S. A., et al. (1995) Drug. Dev. Res. 34, 184-185. 11. Cubellis, M. V., Marino, G., Mayol, L., Picialli, G., and Sannia, G. (1985) J. Chromatogr. 329, 406-414. ANALYTICAL BIOCHEMISTRY 235, 241--242 (1996) ARTICLE NO. 0120 0003-2697/96 $18.00 Copyright © 1996 byAcademic Press, Inc. All rights of reproduction in any form reserved.