- Email: [email protected]
Use of a volatile buffer system in ion-exchange high-performance liquid chromatography of oligonucleotides
178,320-323
ANALYTICALBIOCHEMISTRY
(1989)
Use of a Volatile Buffer System in Ion-Exchange HighPerformance Liquid Chromatography of Oligonucleotides Janet M. Munholland, Karen A. Bright, and Ross N. Nazar Department of Molecular Biology and Genetics,University of Guelph, Guelph, Ontario, CanadaNlG 2 Wl
Received
September
16,1988
A volatile buffer has been adapted for use with ionexchange high-performance liquid chromatography in the analysis and preparation of oligonucleotides. The system employs a commercial weakly basic anion-exchange column containing a DEAE-derivatized silica gel and eluted with a volatile buffer gradient of triethylamine acetate and acetonitrile. Nucleic acid digests and oligonucleotides synthesized by chemical or enzymatic methods can be analyzed or purified with nearly quantitative recovery following solvent volatilization. 0 1989 Academic Press, Inc.
A large number of methods are now available for the synthesis of oligonucleotides. While oligodeoxyribonucleotides are almost entirely synthesized by chemical methods (see Ref. (1)) and numerous automated instruments (“gene machines”) are commercially available, enzymatic approaches are normally used for the synthesis of RNA fragments. The two most common involve the transcription of RNA from DNA templates by a number of different RNA polymerases (2,3) or the use of T, RNA ligase in the synthesis of short RNA fragments (45). T4 RNA ligase has been successfully used for the synthesis of oligoribonucleotides for a number of years but the numerous purification steps which are required with intermediate substrates and the products have been very time consuming. The original methods made use of open column chromatography (e.g., Refs. (4,6)) for this purpose and more recent approaches have been based on the use of polyacrylamide gels (7,8), high-performance liquid chromatography (9,10), or a combination of all the methods. While polyacrylamide gels offer a high resolution and are somewhat less time consuming than the open columns, they require eluting and desalting/concentrating steps which also are quite labor intensive. The HPLC methods which have been reported are much less labor intensive but still pose disadvantages. The use 320
of an ion-exchange column normally requires a nonvolatile salt buffer which necessitates that samples be desalted after each separation. On the other hand, reversephase chromatography avoids the need for desalting but does not necessarily result in separations according to nucleotide chain length and can lead to confusion regarding the elution positions of the substrates and products. In the present paper we report HPLC methods based on a DEAE-derivatized silica ion-exchange column and a volatile buffer system, thereby avoiding the need for desalting and retaining separation based on the number of phosphate residues. The approach is especially useful for the enzymatic synthesis of oligonucleotides but also can be conveniently used for the purification of chemically synthesized fragments or the analysis of nucleic acid digests. MATERIALS
AND
METHODS
High-Pressure Liquid Chromatography of Oligonucleotides All separations were carried out using a Waters’ HPLC system consisting of two Model 6000 pumps, data module, system controller, and a Model 441 absorbance detector at 254 nm. The column (250 X 4.6 mm) containing DEAE-Si 100 Polyol (0.005 mm) was from Serva Feinbiochemica (Heidelberg, FRG) together with a guard column packed with Si 100 Polyol (0.01 nm). Unless otherwise stated, the column was equilibrated with 25 mM triethylamine (TEA)l--acetate, pH 7.2-7.5, containing 20% acetonitrile and eluted using a linear gradient from 25 mM to 1 M TEA-acetate, pH 7.2-7.5, 20% acetonitrile (0.8 ml per min). The triethylamine and acetic acid were reagent grade and the acetonitrile was HPLC grade; all solutions were filtered through a 0.451 Abbreviations used: 2,2-bis(p-chlorophenyl)ethane; zineethanesulfonic acid.
TEA,
triethylamine; DDT, l,l,l-trichloroHepes, 4-(2-hydroxyethyl)-l-pipera-
0003-2697/89
$3.00
Copyright 0 1989 by Academic Press, Inc. All rights of reproduction in any form reserved.
CHROMATOGRAPHIC
SYSTEM
FOR
Ill I
OLIGONUCLEOTIDES
pm nylon filter. All samples were spun in a microfuge for l-2 min prior to injection in order to remove particulate contaminants. Preparation
of RNA and Oligonucleotide Precursors
IV
Whole cell RNA was prepared from yeast or rat liver cells by SDS/phenol extraction and fractionated by gel electrophoresis as previously described (11-13). Oligonucleotides for HPLC analysis or RNA synthesis were prepared by digesting purified RNA dissolved in 10 mM Tris, pH 8.0, with pancreatic ribonuclease (1:20 enzyme: substrate) for 30 min at 37°C (14). Digests were directly analyzed by HPLC or, when oligomers were used as substrates for RNA synthesis, the RNA fragments were fractionated by chromatography on DEAE-Sephadex A25 (11) or the present HPLC methods. Oligonucleotide Synthesis with RNA Ligase Oligonucleotides were synthesized with T, RNA ligase essentially as described by England and Uhlenbeck (4) except the present HPLC methods were used to fractionate the intermediates and products. The 3’-phosphorylated ribooligonucleotides AAUp and GUp were prepared from whole cell RNA as described above and dephosphorylated or 5’-phosphorylated, respectively, with bacterial alkaline phosphatase or polynucleotide kinase (14). For dephosphorylation, AUUp or the synthetic intermediate AAUGUp (2-3 Azm nm units per 50 ~1) were incubated in 10 mM Tris-HCl, pH 9.0, containing 200 U/ml alkaline phosphatase for 4 h at 37°C. The mixture was extracted with buffer-saturated phenol and twice with ether before approximately 1 AzeOnm unit or 20 ~1 was injected. The solvent was removed under an air stream or in uacuo by a Speedvac (Savant Instr., Hicksville, NY) and dried again from 10 to 50 ~1 distilled water. For 5’-phosphorylation, GUp (l-2 Azso nm units per 50 ~1) was incubated in 50 mM Tris-HCl, pH 7.6,lO mM MgC&, 5 mM DDT, 0.1 mM spermidine, 0.1 mM EDTA with 300 nmol ATP and 5-10 units T, polynucleotide kinase at 37°C for 60 min. Reaction mixtures were loaded directly on the HPLC after a short spin to remove particulates; the last peak was collected and dried as above. For RNA synthesis, the phosphorylated donor and dephosphorylated acceptor (approximately 1 Azsonm unit of each) were incubated with 70 nmol ATP in 40 ~1 of 50 mM Hepes-NaOH, pH 8.3,20 mM MgC&, 3 mM DDT, 20 pg/ml BSA, and 9 units of T, RNA ligase (Pharmacia, Canada) overnight at 17°C. Reactions were stopped by heating to 90°C for 90 s and the samples were either stored at -20°C or injected for HPLC analysis. RESULTS
AND
DISCUSSION
In the course of synthesizing oligonucleotide fragments either by chemical or by enzymatic methods, we
321
8 2
v VI
II
w
5
FIG. digest (1:20, linear indicate peak.
10
I 15ml
1. Fractionation of a yeast 5S rRNA pancreatic ribonuclease by ion-exchange HPLC. The RNA was digested at 37°C for 6 h enzyme to substrate) and 12 ng was injected and eluted using a 0.05-l M TEA-acetate, pH 7.5, gradient. The roman numerals the major chain length of fragments constituting each major
previously experienced a variety of technical problems including a need for concentrating, separation distortions due to excess contaminants, and a confusion of fragment chain lengths when the composition of intermediates and products was significantly different. Ionexchange chromatography appeared to offer the best overall solution with two limitations, the need for desalting and potential base composition or hydrophobic interaction effects. In an effort to overcome these limitations three options were examined: the use of a volatile buffer gradient, the use of apolar solvents to suppress hydrophobic interactions, and the use of alternate column supports. Because TEA-carbonate (triethylamine) has been so successfully used for the elution of oligonucleotides from DEAE paper (see Ref. (14)) and a variety of DEAE-substituted HPLC columns are now available, we examined the use of triethylamine gradients to separate oligonucleotides on such columns. Since ribonuclease digests of whole cell RNA extracts or specific RNAs are useful sources of short oligonucleotides for enzymatic synthesis, we first examined the resolution of the oligoribonucleotides mixture from a pancreatic ribonuclease digest of the yeast 5S rRNA. To avoid bubble formation and to allow for a more accurate pH, the column was eluted with a TEA-acetate (pH 7.27.5) buffer gradient instead of TEA-carbonate. While, as predicted from the nucleotide sequence, there were seven major peaks (Fig. l), many minor peaks were clearly visible. Furthermore, when peaks were collected, dried, labeled with [c~-~~P]ATP and further characterized by separation on a 20% polyacrylamide gel (results
322
MUNHOLLAND,
BRIGHT,
AND
NAZAR
AUU
1
JVb A.f i
2. 5
10
15m
5
10
15ml
5
GUP
10
I
15ml
FIG. 2. Effect of organic solvents on the separation of oligonucleotides by ion-exchange HPLC. RNA was digested and analyzed as described in Fig. 1 except the elution buffer contained 20% (v/v) methanol (left), ethanol (center), or acetontrile (right).
not shown), a very significant overlap between the fragments in the peaks and a trail of fragments in the background was evident. These results suggested very substantial hydrophobic interactions with the stationary phase. Other commercially available DEAE-substituted columns were also examined but the profiles were very similar or even more diffuse. The rise in background which was observed in Fig. 1 resulted from the intrinsic absorbancy of triethylamine at 254 nm as the concentration increased. This was reduced slightly when a very high purity solvent was used or when the triethylamine was redistilled, but an elevating baseline remained a characteristic feature of the TEA-buffer system. Nevertheless, because the absorbancy of oligonucleotides is very high, very small amounts still could be analyzed with the detector at high sensitivity levels (0.05-0.2 scale settings). Previous reports (15) have indicated that hydrophobic interactions with the stationary phase could be suppressed by the addition of an apolar solvent. As indicated in the examples shown in Fig. 2, when methanol, ethanol, or acetonitrile was added to a 20% (v/v) final concentration, the profiles were substantially improved. Small peaks or split peaks were completely or largely eliminated particularly with ethanol or acetronitrile. Furthermore, especially with acetonitrile, larger quantities of oligonucleotides were eluted in each peak, approximately twice that observed with TEA-buffer alone. When these peaks were further analyzed by gel electrophoresis, the peaks were found essentially to contain only anticipated oligonucleotides and buffer that was collected after the final peak had eluted contained virtually no oligonucleotides (results not shown). Again, results with other commercially available DEAE-substituted columns ranged from very similar separations to more diffuse profiles. Figures 3 and 4 illustrate the usefulness of the optimized solvent system in the enzymatic synthesis of oligonucleotides. A heptaribonucleotide AAUGUGU was prepared by a two-step synthesis from two small oligo-
L
20ml
*
20ml
10
FIG. 3. Purification of phosphorylated or dephosphorylated substrates for enzymatic synthesis of oligonucleotides. GUp or AAUp was phosphorylated (right) or dephosphorylated (left) with alkaline phosphatase or polynucleotide kinase and purified by HPLC as described under Materials and Methods.
nucleotides (AAUp and GUp) which were purified from a digest of mammalian whole cell RNA. The substrates were phosphorylated or dephosphorylated with polynucleotide kinase or alkaline phosphatase, respectively, as required, and rapidly purified by HPLC using the TEA-acetate (pH 7.2-7.5)-20% acetonitrile buffer gradient (Fig. 3). As shown in Fig. 4 (left), the product of the first ligation step was a pentanucleotide, AUUGUp. After further dephosphorylation this intermediate was used as an acceptor to which pGUp was again ligated to produce the final product (right). Because the products were separated according to their charge, each component could be readily identified in the elution profile and the products were available after drying without further desalting. Furthermore, preparations as TEA-salts were not inhibitory to further enzyme reactions.
AAUGU
rAU
lOUp
AAlJQUGlb
G ,TP
ADP
20ml
J-K 10
2oml
FIG. 4. Preparative analysis of oligonucleotides synthesized with T4 RNA ligase. The first ligation step between pGUp and AAU is shown on the left, the second step between pGUp and AAUGU is shown on the right.
CHROMATOGRAPHIC
SYSTEM
10
FIG.
20
30ml
323
OLIGONUCLEOTIDES
ion-exchange HPLC methods for the separation of oligonucleotides provides a flexible, efficient, and convenient method for use with both chemically and enzymatically synthesized oligomers.
17mer
E 4*3
FOR
I,, 10
ACKNOWLEDGMENT This Council
study was supported of Canada.
by a grant
from
the Medical
Research
Note added in proof. Recent experiments indicate that ammonium acetate can be effectively substituted for triethylamine acetate when a lower baseline is desirable and ammonium ions will not interfere with subsequent procedures.
20ml
5. Purification of chemically synthesized deoxyribonucleotides. The oligonucleotides were synthesized on a Biosearch 8600 automated synthesizer and deprotected and purified by HPLC as described under Materials and Methods. The sequences CATAGTCATCGTCCTCC (left) and CCACCACCACCACCA (right) were separated with a linear gradient, as described under Materials and Methods.
REFERENCES 1. Carruthers, 2. Kraimer,
M. H. (1985) Science 230,281-285. A., Maniatis, T., Ruskin, N. A., and Green,
M. R. (1984)
Cell36,993-1005. 3. Meltan, 4.
D. A., Kreig, P., and Green, M. R. (1984) Nucleic Res. 12,7035-7056. England, T. E., and Uhlenbeck, 0. C. (1978) Biochemistry
Acids
17,
2069-2076.
TEA-acetate separations are also very useful in the chemical synthesis of oligonucleotides. As illustrated in Fig. 5, chemically synthesized deoxyribooligonucleotides can be rapidly purified using the same system. Even when failed reaction products (right) are present in large amounts, the oligonucleotide of interest is clearly resolved as the slowest peak. More important, the large amounts of chemical contaminants which are present after chemical synthesis and deprotection could be loaded directly onto the column without a great reduction in column capacity or column life. The failed reaction products also illustrate the very high resolution which the present system is capable of; the complex profile of shorter failed products which is resolved is clearly equal to the resolution which has been reported with other nonvolatile systems (e.g., Ref. (10)). In summary, therefore, the use of a volatile solvent in
5. Romaniuk, P. J., and Uhlenbeck, 0. C. (1983) in Methods in Enzymology (Wu, R., Grossman, L., and Moldave, K., Eds.), Vol. 100, Academic Press, San Diego, CA. 6. Ohtsuka, E., Nishikawa, S., Fukumoto, R., Vemura, H., Tanaka, T., Nakagawa, E., Miyake, T., and Ikehara, M. (1980) Eur. J. Biothem. 105,481-487. 7. Krug, M., de Haseth, P. L., and Uhlenbeck, 0. C. (1982) Biochemistry 21,4713. 8. Nazar, R. N. (1985) Canad. J. Biochem. Cell Biol. 63,313318. 9. Romaniuk, E., McLaughlin, L. W., Neilson, T., and Romaniuk, P. J. (1982) Eur. J. Biochem. 126,639. 10. Vandenberghe, A., Nelles, L., and DeWachter, R. (1980) Anal. Biochem. 107,369-376. 11. Nazar, R. N., and Busch, H. (1974) J. Biol. Chem. 249,919-929. 12. Lo, A. C., and Nazar, R. N. (1982) J. Biol. Chem. 257,3516-3524. 13. McDougall, J., and Nazar, R. N. (1985) J. Biol. Chem. 258,5256-
5259. 14. Brownlee,
G. G. (1972) in Laboratory Techniques in Biochemistry and Molecular Biology (Work, T. S., and Work, E., Eds.), Vol. pp. l-265, North-Holland, Amsterdam. 15. Van Haastert, P. J. M. (1981) J. Chromatogr. 210,241-254.
3,
ANALYTICALBIOCHEMISTRY
(1989)
Use of a Volatile Buffer System in Ion-Exchange HighPerformance Liquid Chromatography of Oligonucleotides Janet M. Munholland, Karen A. Bright, and Ross N. Nazar Department of Molecular Biology and Genetics,University of Guelph, Guelph, Ontario, CanadaNlG 2 Wl
Received
September
16,1988
A volatile buffer has been adapted for use with ionexchange high-performance liquid chromatography in the analysis and preparation of oligonucleotides. The system employs a commercial weakly basic anion-exchange column containing a DEAE-derivatized silica gel and eluted with a volatile buffer gradient of triethylamine acetate and acetonitrile. Nucleic acid digests and oligonucleotides synthesized by chemical or enzymatic methods can be analyzed or purified with nearly quantitative recovery following solvent volatilization. 0 1989 Academic Press, Inc.
A large number of methods are now available for the synthesis of oligonucleotides. While oligodeoxyribonucleotides are almost entirely synthesized by chemical methods (see Ref. (1)) and numerous automated instruments (“gene machines”) are commercially available, enzymatic approaches are normally used for the synthesis of RNA fragments. The two most common involve the transcription of RNA from DNA templates by a number of different RNA polymerases (2,3) or the use of T, RNA ligase in the synthesis of short RNA fragments (45). T4 RNA ligase has been successfully used for the synthesis of oligoribonucleotides for a number of years but the numerous purification steps which are required with intermediate substrates and the products have been very time consuming. The original methods made use of open column chromatography (e.g., Refs. (4,6)) for this purpose and more recent approaches have been based on the use of polyacrylamide gels (7,8), high-performance liquid chromatography (9,10), or a combination of all the methods. While polyacrylamide gels offer a high resolution and are somewhat less time consuming than the open columns, they require eluting and desalting/concentrating steps which also are quite labor intensive. The HPLC methods which have been reported are much less labor intensive but still pose disadvantages. The use 320
of an ion-exchange column normally requires a nonvolatile salt buffer which necessitates that samples be desalted after each separation. On the other hand, reversephase chromatography avoids the need for desalting but does not necessarily result in separations according to nucleotide chain length and can lead to confusion regarding the elution positions of the substrates and products. In the present paper we report HPLC methods based on a DEAE-derivatized silica ion-exchange column and a volatile buffer system, thereby avoiding the need for desalting and retaining separation based on the number of phosphate residues. The approach is especially useful for the enzymatic synthesis of oligonucleotides but also can be conveniently used for the purification of chemically synthesized fragments or the analysis of nucleic acid digests. MATERIALS
AND
METHODS
High-Pressure Liquid Chromatography of Oligonucleotides All separations were carried out using a Waters’ HPLC system consisting of two Model 6000 pumps, data module, system controller, and a Model 441 absorbance detector at 254 nm. The column (250 X 4.6 mm) containing DEAE-Si 100 Polyol (0.005 mm) was from Serva Feinbiochemica (Heidelberg, FRG) together with a guard column packed with Si 100 Polyol (0.01 nm). Unless otherwise stated, the column was equilibrated with 25 mM triethylamine (TEA)l--acetate, pH 7.2-7.5, containing 20% acetonitrile and eluted using a linear gradient from 25 mM to 1 M TEA-acetate, pH 7.2-7.5, 20% acetonitrile (0.8 ml per min). The triethylamine and acetic acid were reagent grade and the acetonitrile was HPLC grade; all solutions were filtered through a 0.451 Abbreviations used: 2,2-bis(p-chlorophenyl)ethane; zineethanesulfonic acid.
TEA,
triethylamine; DDT, l,l,l-trichloroHepes, 4-(2-hydroxyethyl)-l-pipera-
0003-2697/89
$3.00
Copyright 0 1989 by Academic Press, Inc. All rights of reproduction in any form reserved.
CHROMATOGRAPHIC
SYSTEM
FOR
Ill I
OLIGONUCLEOTIDES
pm nylon filter. All samples were spun in a microfuge for l-2 min prior to injection in order to remove particulate contaminants. Preparation
of RNA and Oligonucleotide Precursors
IV
Whole cell RNA was prepared from yeast or rat liver cells by SDS/phenol extraction and fractionated by gel electrophoresis as previously described (11-13). Oligonucleotides for HPLC analysis or RNA synthesis were prepared by digesting purified RNA dissolved in 10 mM Tris, pH 8.0, with pancreatic ribonuclease (1:20 enzyme: substrate) for 30 min at 37°C (14). Digests were directly analyzed by HPLC or, when oligomers were used as substrates for RNA synthesis, the RNA fragments were fractionated by chromatography on DEAE-Sephadex A25 (11) or the present HPLC methods. Oligonucleotide Synthesis with RNA Ligase Oligonucleotides were synthesized with T, RNA ligase essentially as described by England and Uhlenbeck (4) except the present HPLC methods were used to fractionate the intermediates and products. The 3’-phosphorylated ribooligonucleotides AAUp and GUp were prepared from whole cell RNA as described above and dephosphorylated or 5’-phosphorylated, respectively, with bacterial alkaline phosphatase or polynucleotide kinase (14). For dephosphorylation, AUUp or the synthetic intermediate AAUGUp (2-3 Azm nm units per 50 ~1) were incubated in 10 mM Tris-HCl, pH 9.0, containing 200 U/ml alkaline phosphatase for 4 h at 37°C. The mixture was extracted with buffer-saturated phenol and twice with ether before approximately 1 AzeOnm unit or 20 ~1 was injected. The solvent was removed under an air stream or in uacuo by a Speedvac (Savant Instr., Hicksville, NY) and dried again from 10 to 50 ~1 distilled water. For 5’-phosphorylation, GUp (l-2 Azso nm units per 50 ~1) was incubated in 50 mM Tris-HCl, pH 7.6,lO mM MgC&, 5 mM DDT, 0.1 mM spermidine, 0.1 mM EDTA with 300 nmol ATP and 5-10 units T, polynucleotide kinase at 37°C for 60 min. Reaction mixtures were loaded directly on the HPLC after a short spin to remove particulates; the last peak was collected and dried as above. For RNA synthesis, the phosphorylated donor and dephosphorylated acceptor (approximately 1 Azsonm unit of each) were incubated with 70 nmol ATP in 40 ~1 of 50 mM Hepes-NaOH, pH 8.3,20 mM MgC&, 3 mM DDT, 20 pg/ml BSA, and 9 units of T, RNA ligase (Pharmacia, Canada) overnight at 17°C. Reactions were stopped by heating to 90°C for 90 s and the samples were either stored at -20°C or injected for HPLC analysis. RESULTS
AND
DISCUSSION
In the course of synthesizing oligonucleotide fragments either by chemical or by enzymatic methods, we
321
8 2
v VI
II
w
5
FIG. digest (1:20, linear indicate peak.
10
I 15ml
1. Fractionation of a yeast 5S rRNA pancreatic ribonuclease by ion-exchange HPLC. The RNA was digested at 37°C for 6 h enzyme to substrate) and 12 ng was injected and eluted using a 0.05-l M TEA-acetate, pH 7.5, gradient. The roman numerals the major chain length of fragments constituting each major
previously experienced a variety of technical problems including a need for concentrating, separation distortions due to excess contaminants, and a confusion of fragment chain lengths when the composition of intermediates and products was significantly different. Ionexchange chromatography appeared to offer the best overall solution with two limitations, the need for desalting and potential base composition or hydrophobic interaction effects. In an effort to overcome these limitations three options were examined: the use of a volatile buffer gradient, the use of apolar solvents to suppress hydrophobic interactions, and the use of alternate column supports. Because TEA-carbonate (triethylamine) has been so successfully used for the elution of oligonucleotides from DEAE paper (see Ref. (14)) and a variety of DEAE-substituted HPLC columns are now available, we examined the use of triethylamine gradients to separate oligonucleotides on such columns. Since ribonuclease digests of whole cell RNA extracts or specific RNAs are useful sources of short oligonucleotides for enzymatic synthesis, we first examined the resolution of the oligoribonucleotides mixture from a pancreatic ribonuclease digest of the yeast 5S rRNA. To avoid bubble formation and to allow for a more accurate pH, the column was eluted with a TEA-acetate (pH 7.27.5) buffer gradient instead of TEA-carbonate. While, as predicted from the nucleotide sequence, there were seven major peaks (Fig. l), many minor peaks were clearly visible. Furthermore, when peaks were collected, dried, labeled with [c~-~~P]ATP and further characterized by separation on a 20% polyacrylamide gel (results
322
MUNHOLLAND,
BRIGHT,
AND
NAZAR
AUU
1
JVb A.f i
2. 5
10
15m
5
10
15ml
5
GUP
10
I
15ml
FIG. 2. Effect of organic solvents on the separation of oligonucleotides by ion-exchange HPLC. RNA was digested and analyzed as described in Fig. 1 except the elution buffer contained 20% (v/v) methanol (left), ethanol (center), or acetontrile (right).
not shown), a very significant overlap between the fragments in the peaks and a trail of fragments in the background was evident. These results suggested very substantial hydrophobic interactions with the stationary phase. Other commercially available DEAE-substituted columns were also examined but the profiles were very similar or even more diffuse. The rise in background which was observed in Fig. 1 resulted from the intrinsic absorbancy of triethylamine at 254 nm as the concentration increased. This was reduced slightly when a very high purity solvent was used or when the triethylamine was redistilled, but an elevating baseline remained a characteristic feature of the TEA-buffer system. Nevertheless, because the absorbancy of oligonucleotides is very high, very small amounts still could be analyzed with the detector at high sensitivity levels (0.05-0.2 scale settings). Previous reports (15) have indicated that hydrophobic interactions with the stationary phase could be suppressed by the addition of an apolar solvent. As indicated in the examples shown in Fig. 2, when methanol, ethanol, or acetonitrile was added to a 20% (v/v) final concentration, the profiles were substantially improved. Small peaks or split peaks were completely or largely eliminated particularly with ethanol or acetronitrile. Furthermore, especially with acetonitrile, larger quantities of oligonucleotides were eluted in each peak, approximately twice that observed with TEA-buffer alone. When these peaks were further analyzed by gel electrophoresis, the peaks were found essentially to contain only anticipated oligonucleotides and buffer that was collected after the final peak had eluted contained virtually no oligonucleotides (results not shown). Again, results with other commercially available DEAE-substituted columns ranged from very similar separations to more diffuse profiles. Figures 3 and 4 illustrate the usefulness of the optimized solvent system in the enzymatic synthesis of oligonucleotides. A heptaribonucleotide AAUGUGU was prepared by a two-step synthesis from two small oligo-
L
20ml
*
20ml
10
FIG. 3. Purification of phosphorylated or dephosphorylated substrates for enzymatic synthesis of oligonucleotides. GUp or AAUp was phosphorylated (right) or dephosphorylated (left) with alkaline phosphatase or polynucleotide kinase and purified by HPLC as described under Materials and Methods.
nucleotides (AAUp and GUp) which were purified from a digest of mammalian whole cell RNA. The substrates were phosphorylated or dephosphorylated with polynucleotide kinase or alkaline phosphatase, respectively, as required, and rapidly purified by HPLC using the TEA-acetate (pH 7.2-7.5)-20% acetonitrile buffer gradient (Fig. 3). As shown in Fig. 4 (left), the product of the first ligation step was a pentanucleotide, AUUGUp. After further dephosphorylation this intermediate was used as an acceptor to which pGUp was again ligated to produce the final product (right). Because the products were separated according to their charge, each component could be readily identified in the elution profile and the products were available after drying without further desalting. Furthermore, preparations as TEA-salts were not inhibitory to further enzyme reactions.
AAUGU
rAU
lOUp
AAlJQUGlb
G ,TP
ADP
20ml
J-K 10
2oml
FIG. 4. Preparative analysis of oligonucleotides synthesized with T4 RNA ligase. The first ligation step between pGUp and AAU is shown on the left, the second step between pGUp and AAUGU is shown on the right.
CHROMATOGRAPHIC
SYSTEM
10
FIG.
20
30ml
323
OLIGONUCLEOTIDES
ion-exchange HPLC methods for the separation of oligonucleotides provides a flexible, efficient, and convenient method for use with both chemically and enzymatically synthesized oligomers.
17mer
E 4*3
FOR
I,, 10
ACKNOWLEDGMENT This Council
study was supported of Canada.
by a grant
from
the Medical
Research
Note added in proof. Recent experiments indicate that ammonium acetate can be effectively substituted for triethylamine acetate when a lower baseline is desirable and ammonium ions will not interfere with subsequent procedures.
20ml
5. Purification of chemically synthesized deoxyribonucleotides. The oligonucleotides were synthesized on a Biosearch 8600 automated synthesizer and deprotected and purified by HPLC as described under Materials and Methods. The sequences CATAGTCATCGTCCTCC (left) and CCACCACCACCACCA (right) were separated with a linear gradient, as described under Materials and Methods.
REFERENCES 1. Carruthers, 2. Kraimer,
M. H. (1985) Science 230,281-285. A., Maniatis, T., Ruskin, N. A., and Green,
M. R. (1984)
Cell36,993-1005. 3. Meltan, 4.
D. A., Kreig, P., and Green, M. R. (1984) Nucleic Res. 12,7035-7056. England, T. E., and Uhlenbeck, 0. C. (1978) Biochemistry
Acids
17,
2069-2076.
TEA-acetate separations are also very useful in the chemical synthesis of oligonucleotides. As illustrated in Fig. 5, chemically synthesized deoxyribooligonucleotides can be rapidly purified using the same system. Even when failed reaction products (right) are present in large amounts, the oligonucleotide of interest is clearly resolved as the slowest peak. More important, the large amounts of chemical contaminants which are present after chemical synthesis and deprotection could be loaded directly onto the column without a great reduction in column capacity or column life. The failed reaction products also illustrate the very high resolution which the present system is capable of; the complex profile of shorter failed products which is resolved is clearly equal to the resolution which has been reported with other nonvolatile systems (e.g., Ref. (10)). In summary, therefore, the use of a volatile solvent in
5. Romaniuk, P. J., and Uhlenbeck, 0. C. (1983) in Methods in Enzymology (Wu, R., Grossman, L., and Moldave, K., Eds.), Vol. 100, Academic Press, San Diego, CA. 6. Ohtsuka, E., Nishikawa, S., Fukumoto, R., Vemura, H., Tanaka, T., Nakagawa, E., Miyake, T., and Ikehara, M. (1980) Eur. J. Biothem. 105,481-487. 7. Krug, M., de Haseth, P. L., and Uhlenbeck, 0. C. (1982) Biochemistry 21,4713. 8. Nazar, R. N. (1985) Canad. J. Biochem. Cell Biol. 63,313318. 9. Romaniuk, E., McLaughlin, L. W., Neilson, T., and Romaniuk, P. J. (1982) Eur. J. Biochem. 126,639. 10. Vandenberghe, A., Nelles, L., and DeWachter, R. (1980) Anal. Biochem. 107,369-376. 11. Nazar, R. N., and Busch, H. (1974) J. Biol. Chem. 249,919-929. 12. Lo, A. C., and Nazar, R. N. (1982) J. Biol. Chem. 257,3516-3524. 13. McDougall, J., and Nazar, R. N. (1985) J. Biol. Chem. 258,5256-
5259. 14. Brownlee,
G. G. (1972) in Laboratory Techniques in Biochemistry and Molecular Biology (Work, T. S., and Work, E., Eds.), Vol. pp. l-265, North-Holland, Amsterdam. 15. Van Haastert, P. J. M. (1981) J. Chromatogr. 210,241-254.
3,