Separation of Uncharged Oligodeoxynucleotide Analogs by Anion-Exchange Chromatography at High pH

3 Pages·1996·0.14 MB·English

by  Schmidt, Jurgen G.

NOTES & TIPS 239
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Separation of "Uncharged"
6
Oligodeoxynucleotide Analogs by
Anion-Exchange Chromatography
at High pH
JilrgenG. Schmidt,*PeterE.Nielsen,t
I
and LeslieE. Orgel* 20m_
*The Salk Institute for Biological Studies, P.O. Box 85800,
San Diego, California 92186-5800; and tCenter for
10,/ 11
Biornolecular Recognition, IMBG, Biochemistry B,
The Panurn Institute, Blegdamsvej 3c, DK-2100 IV,
Copenhagen, Denmark ¢
Received December 14, 1995
gradient
Ion-exchangechromatography isa well-established
method forthe analysisand purificatioonfphospho-
diester-linkeodligonucleotide(s1).Ifelutioniscarried
outunder alkalineconditionst,hesecondarystructure
ofG- and C-richoligomersisdisrupted.Furthermore,
elutiontimesbecome more sensitivetotheG and T
contentoftheoligomer,becauseG and T aredeproto-
natedatpH 10(2).Inrecentwork onpeptide-nucleic
acids(PNAs)Iwe notedthatmixturesofPNA oligomers
_m
G4,Ge,Gs,and Gm arereadilyseparatedbyelutionat
pH 12on an RPC-5 column (3).Here we show thatthis
FIG. 1. Elution profiles of (a) Mixture I (samples 1-5) separated
separationmethod ismore generallyapplicable. using a linear gradient from A to A/B:80/20 in 40 rain; (b) Mixture
The sequencesoftheoligomersofthetypePNA-Lys- II (samples 6-9) separated using a linear gradient from Ato A/B: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
tAbbreviationused:PNAs, peptide-nucleicacids. 40 rain.
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240 NOTE&STIPS
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 i 1 T_C2TCTC 6 1016
I I 2 T2CT2CT4 8 1018
I I 3 T,CTs 9 1019
I 1 4 Tlo 10 10/10
I 1 5 T,G2TGTG 6 4 10/10
II 2 6 GAGAGGA4 4 10/4
II 2 7 A4G2TGAG 1 4 10/5
II 2 8 AaTG2TOAG 2 4 10/6
II 2 9 TGTACGTCACAACTA 4 2 15/6
III 3 10 GTAGATCACT 3 2 10/5
III 3 ii AGTGATCTAC 3 2 10/5
Note.Each oligomerhas alysineamide groupattachedtoitscarboxylterminus.
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 mMNaOH and 1mM Tris-HC104 in water analogs are oi%en sparingly soluble in neutral aqueous
as the A buffer and 20 m_d NaOH, 1 mM Tris-HCIO4, solution and tend to adopt very stable self-structures.
and 0.1 MNaC104 as the B buffer. Stock solutions of This makes chromatography of PNA and other un-
PNA oligomers containing about 5 × 10-s ODU/pl 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/_l 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 atpH 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 ofT 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
Achnowledgrnents. 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. We thank Sylvia Bailey for manuscript prepa-
(Fig.Ic). ration.
HPLC at alkalinepH isnota usefultechniquefor
thepurificatioonfunmodifiedPNAs. PNA rearranges REFERENCES
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NOTES & TIPS 241
8. Pearson, R. L., Weiss, J. F., and Kelmers, A. D. (1971) Biochim.
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ANALYTICAL BIOCHEMISTRY 235, 241-242 (1996)
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0003-2697/96 $18.00
Copyright © 1996 by Academic Press, Inc,
A]| rights ofreproduction in any form reserved.