ANALYTICAL
BIOCHEMISTRY
9,
213-216 (1979)
Fractionation of dG Homooligonucleotides Using RPC-5 Column Chromatography at High pH ERIK SELSING,’ Department
of Biochemistry,
JACQUELYNN College
E. LARSON,
AND ROBERT
of Agricultural and Life Madison, Wisconsin 53706
Sciences.
D. WELLS
University
of Wisconsin,
Received April 13, 1979 dG oligomers, prepared by partial acid hydrolysis of dG,, were fractionated in large quantity according to chain length by RPC-5 column chromatography at high pH (0.1 M NaOH). Resolution of oligonucleotides up to approximately the 30-mer was achieved. The oligomers were characterized by partial chemical digestion and analysis on 20% polyacrylamide-urea gels.
RPC-5 column chromatography has been a valuable tool for the fractionation of DNA and RNA oligonucleotides as well as DNA restriction fragments (l-3). Large-scale isolation of specific oligonucleotides from oligomer mixtures generated by partial hydrolysis of homopolynucleotides has been particularly useful in the preparative synthesis of model nucleic acids for biochemical and biophysical studies (3-9). Using RPC-5 at neutral pH, it has been possible to fractionate oligomers of dA, rA, dC, rC, dT, rU, d1, and r1 having chain lengths as high as 60 (1). Attempts to separate dG oligomers using these same conditions have been unsuccessful. This may be due to the self-association found for deoxyguanosine-rich polymers (10,ll). However, based on the ability of RPC-5 to separate complementary strands of restriction fragments under denaturing conditions (1,12), we have investigated the effect of high pH on dG oligomer separations. At 0.1 M NaOH, fractionation and recovery of dG oligomers ‘Present address: Department of Microbiology and Immunology, X-42, University of Washington, Seattle, Wash. 98195.
is similar to that found for other oligonucleotides at neutral pH. MATERIALS
AND METHODS
Nucleic acids. dG, .dC, was prepared using Micrococcus luteus DNA polymerase as previously described (10) and separated into single-stranded dG, and dC, by alkaline cesium chloride density gradient centrifugation (13). Partial digestion ofdG,. Partial depurination of dG, was achieved by incubation at 37°C in 50% acetic acid. After depurination, the reaction was mixed with 3 vol of 5 M KOH and incubated at 37°C for 16 h in order to cleave the sugar-phosphate backbone at sites of depurination. The resulting mixture of dG oligomers was concentrated and dialyzed into 0.01 M Tris-HCl (pH 8.0), 0.1 mM EDTA. Small-scale depurination reactions (5.0 pmol dG,) were incubated for various times and subsequently analyzed on 20% polyacrylamide gels. While the absorbance patterns of gel scans in these trial experiments were complex, presumably due to the self-aggregation of the dG oligomers, resolution of lower molecular weight oligomers was achieved and the 213
0003-2697/79/150213-04$02.00/O Copyright 0 1979 by Academic Press. Inc. All rights of reproduction in any form reserved.
214
SELSING,
LARSON.
scans seemed to provide a general idea of the particular size distribution of oligomers resulting from various depurination reaction times (data not shown). A large-scale preparation of dG oligomers having an average chain length of lo-20 nucleotide residues, as judged from 20% polyacrylamide gels, was used for RPC-5 chromatography. The large-scale depurination reaction was incubated at 37°C for 24 h. Alkaline ofigomers.
RPC-5
chromatography
of dG
The general procedure used for RPC-5 column chromatography has been described elsewhere (1). RPC-5 was equilibrated with 0.1 M KCl, 0.1 M NaOH and used to prepare a 20 x l.O-cm column. The column was equilibrated with 0.1 M KCl, 0.1 M NaOH under pressure supplied from a peristaltic pump at a flow rate of 0.5 mYmin. Two hundred Azsz units (in a 20-ml volume) of dG oligomers was applied to the column and 100 ml of 0.1 M KCl, 0.1 M NaOH used to wash the column. A 2-liter linear gradient of 0.1 M NaOH containing 0.1-1.0 M KC1 was applied to the column and fractions (6.4 ml) were collected. Absorbances at 252 nm of the eluted fractions were recorded and each fraction brought to neutral pH. Fractions containing peaks of absorbance were pooled, concentrated, and dialyzed into
AND
0.01
M
‘Iris-HCl
Characterization
200 FRACTION
(pH S.O), 0.1 mM EDTA. of dG oligomers.
Sev-
eral pooled fractions of dG oligomers were labeled using polynucleotide kinase and [y-32P]ATP as described by Maxam and Gilbert (14). Approximately 1 nmol of oligonucleotide was labeled in each reaction. The reactions were terminated by phenol extraction and were then extracted with ether and extensively dialyzed. The labeled oligomers were subjected to partial degradation according to the “G-specific” chemical reaction protocol described by Maxam and Gilbert (14) except the incubation with dimethylsulfate was allowed to proceed for 2 h. Both partially degraded and undegraded dG oligomers were electrophoresed on 20% polyacrylamide sequencing gels (14) which had been preelectrophoresed for 10 h. The marker dye, bromophenol blue, present in the samples was allowed to migrate 10 cm into the 40-cm gel and the gel was then subjected to autoradiography. Other materials and methods. Analytical polyacrylamide tube gels (20%) were as described previously (15). Polynucleotide kinase was purchased from P-L. Biochemicals and bacterial alkaline phosphatase was purchased from Sigma (Type III-S).
100 FIG. 1. Fractionation of dG oligomers described under Materials and Methods. oligomer sizes were assigned according gels (see text).
WELLS
300
NUMBER
using RPC-5 column chromatography. The column was run as The linear KCI gradient applied to the column is indicated. dG to subsequent analysis on 20% polyacrylamide sequencing
FRACTIONATION
215
OF dG OLIGOMERS
RESULTS RPC-5 Chromatography of dG Oligomers
A mixture of dG oligomers was prepared as described under Materials and Methods. Figure 1 shows the elution profile of these dG oligonucleotides from a RPC-5 column run under strongly denaturing conditions (0.1 M NaOH). The resolution between peaks in this profile is similar to that seen when A, C, or I (both deoxyribose and ribose derivatives) and dT and rU oligomers are separated using RPC-5 chromatography at neutral pH. Baseline separation of oligomers up to the 15mer (dG oligomer sizing is discussed below) is seen; this range could likely be extended by use of a shallower or convex salt gradient. Each fraction from the column was neutralized within 6 h after elution; long-term storage of the oligomers under alkaline conditions resulted in some degradation. The recovery from this column was approximately 65%. In preliminary studies, we attempted to fractionate dG oligomers under less alkaline conditions (12 mM NaOH) than those reported here. Recovery from these columns was poor (approximately 20%). Characterization
of dG Oligomers
The purities and sizes of oligomers in the pooled peaks from the RPC-5 column shown in Fig. 1 were ascertained by partial digestion and analysis on 20% sequencing gels. Figure 2 shows the results of such an analysis. In lane 6 of this autoradiogram, partial digestion products of the oligomer eluting in fraction 162 from the column are electrophoresed. Counting the number of bands in the pattern indicates that the dG oligomer present in these fractions is 12 residues in length. The purity of the dG,, oligomer eluted from the column is high, as can be seen in lane 5 of the autoradiogram. Comparison of the oligomer bands in lanes l-5 with each other and with the partial digestion products in lane 6 indicates that
-(PG),
PGP
-(PG),
PGP
-(PG),
PGP
- (PG), PGP + - PGPGP
- PGP
2
3
4
5
6
7
FIG. 2. Analysis of dG oligomers eluted from RPC-5. In lanes 1-5, dG oligomer pools labeled using polynucleotide kinase and [-Y-~~P]ATP (Materials and Methods) are electrophoresed. Each pool is denoted by an appropriate column fraction, i.e., the pool comprising fractions 160-164, in Fig. 1 is indicated as fraction 162. In lane 6, partial degradation products of fraction 162, generated as described in the text, are electrophoresed. Lane 7 shows orthophosphate run as a marker. Assignments of partial degradation products are indicated in the right margin. The arrow indicates a contaminant present in all the oligomer preparations remaining from the kinase reaction, presumably residual ATP. In comparing relative oligomer migrations, note that in lanes l-5, oligomers bear no 3’phosphate moieties while, in lane 6, 3’-phosphate groups are present on all the partial degradation products. In this analysis, the oligomer pools containing dG, and dG,, were not included in order to extend the range of the assay.
each peak in the RPC-5 column profile is an individual oligomer and that neighboring peaks differ in length by one nucleotide
216
SELSING,
LARSON,
residue. The gel characterizations permit assignment of the oligomer sizes indicated on the column profile in Fig. 1. The low level contaminant bands seen in some lanes of Fig. 2 are likely due to a small amount of degradation during the labeling of the oligomers rather than coelution from the RPCJ column. Supporting this contention, rechromatography of slightly degraded 32P-labeled oligomers on a small scale alkaline RPC-5 column resulted in several peaks of radioactivity, each of which gave only one band upon subsequent gel analysis (data not shown). DISCUSSION
This study shows that dG oligomers can be successfully purified by RPC-5 chromatography under alkaline conditions. RPC-5 can, therefore, now be used for fractionation of all DNA and RNA homooligonucleotides except those in the rG series. From the results of this study, it may be possible that rG oligomers can also be separated, by use of less alkaline conditions and low temperature during RPC-5 chromatography. The availability of large amounts of these oligomers will allow further studies on the effects of base sequence on DNA properties similar to those reported previously (3-9). ACKNOWLEDGMENTS
AND WELLS
postdoctoral fellowship from the National Institutes of Health (GM 05468).
REFERENCES 1. Wells, R. D., Hardies, S. C., Horn, G. T., Klein, B., Larson, .I. E., Neuendorf, S. K., Panayotatos, N., Patient, R. K., and Selsing, E. (1979) in Methods in Enzymology, Academic Press, New York, in press. 2. Larson, J. E., Hardies, S. C., Patient, R. K., and Wells, R. D. (1979) J. Biol. Chem., 254, 55355541. 3. Selsing, E., and Wells, R. D. (1979)5. Biol. Chem., 254, 5410-5416. 4. Wells, R. D., Blakesley, R. W., Burd, J. F., Chan, H. W., Dodgson, .I. B., Hardies, S. C., Horn, G. T., Jensen, K. F., Larson, J. E., Nes, I. F., Selsing, E., and Wartell, R. M. (1977) Crit.
Chem.
This work was supported by grants from the National Science Foundation (KM 77-15033) and the National Institutes of Health (CA 20279). Erik Selsing was supported during a portion of these studies by a
Rev.
Biochem.
4, 305-340.
5. Selsing, E., Wells, R. D., Early, T. A., and Kearns, D. R. (1978) Nature (London) 275, 249-250. 6. Selsing, E., Wells, R. D., Alden, C. J., and Amott, S. (1979) J. Biol. Chem., 254,5417-5422. 7. Walker, G. C., Uhlenbeck, 0. C., Bedows, E., and Gumport, R. I. (1975) Proc. Nut. Acad. Sci. USA 72, 122-126. 8. Shum, B. W., and Crothers, D. M. (1978) Nucleic Acids Res. 5, 2297-2311. 9. Trip, E. M., and Smith, M. (1978) Nucleic Acids Res. 5, 1529-1538. 10. Wells, R. D., Larson, J. E., Grant, R. C., Shortle, B. E., and Cantor, C. R. (1970) J. Mol. Biol. 54, 465-497. 11. Gray, D. M., and Bollum, F. J. (1974)Biopofymers 13, 2087-2102. 12. Eshaghpour, H., and Crothers D. M. (1978) Nucleic Acids Res. 5, 13-21. 13. Wells, R. D., and Larson, J. E. (1972) J. Biol. 247, 3405-3409.
14. Maxam, A. M., and Gilbert, W. (1977) Proc. Acad.
Sci.
USA
Nur.
74,5&L564.
15. Burd, J. F., and Wells, R. D. (1974)J. Biol. Chem. 249,7094-7101.
BIOCHEMISTRY
9,
213-216 (1979)
Fractionation of dG Homooligonucleotides Using RPC-5 Column Chromatography at High pH ERIK SELSING,’ Department
of Biochemistry,
JACQUELYNN College
E. LARSON,
AND ROBERT
of Agricultural and Life Madison, Wisconsin 53706
Sciences.
D. WELLS
University
of Wisconsin,
Received April 13, 1979 dG oligomers, prepared by partial acid hydrolysis of dG,, were fractionated in large quantity according to chain length by RPC-5 column chromatography at high pH (0.1 M NaOH). Resolution of oligonucleotides up to approximately the 30-mer was achieved. The oligomers were characterized by partial chemical digestion and analysis on 20% polyacrylamide-urea gels.
RPC-5 column chromatography has been a valuable tool for the fractionation of DNA and RNA oligonucleotides as well as DNA restriction fragments (l-3). Large-scale isolation of specific oligonucleotides from oligomer mixtures generated by partial hydrolysis of homopolynucleotides has been particularly useful in the preparative synthesis of model nucleic acids for biochemical and biophysical studies (3-9). Using RPC-5 at neutral pH, it has been possible to fractionate oligomers of dA, rA, dC, rC, dT, rU, d1, and r1 having chain lengths as high as 60 (1). Attempts to separate dG oligomers using these same conditions have been unsuccessful. This may be due to the self-association found for deoxyguanosine-rich polymers (10,ll). However, based on the ability of RPC-5 to separate complementary strands of restriction fragments under denaturing conditions (1,12), we have investigated the effect of high pH on dG oligomer separations. At 0.1 M NaOH, fractionation and recovery of dG oligomers ‘Present address: Department of Microbiology and Immunology, X-42, University of Washington, Seattle, Wash. 98195.
is similar to that found for other oligonucleotides at neutral pH. MATERIALS
AND METHODS
Nucleic acids. dG, .dC, was prepared using Micrococcus luteus DNA polymerase as previously described (10) and separated into single-stranded dG, and dC, by alkaline cesium chloride density gradient centrifugation (13). Partial digestion ofdG,. Partial depurination of dG, was achieved by incubation at 37°C in 50% acetic acid. After depurination, the reaction was mixed with 3 vol of 5 M KOH and incubated at 37°C for 16 h in order to cleave the sugar-phosphate backbone at sites of depurination. The resulting mixture of dG oligomers was concentrated and dialyzed into 0.01 M Tris-HCl (pH 8.0), 0.1 mM EDTA. Small-scale depurination reactions (5.0 pmol dG,) were incubated for various times and subsequently analyzed on 20% polyacrylamide gels. While the absorbance patterns of gel scans in these trial experiments were complex, presumably due to the self-aggregation of the dG oligomers, resolution of lower molecular weight oligomers was achieved and the 213
0003-2697/79/150213-04$02.00/O Copyright 0 1979 by Academic Press. Inc. All rights of reproduction in any form reserved.
214
SELSING,
LARSON.
scans seemed to provide a general idea of the particular size distribution of oligomers resulting from various depurination reaction times (data not shown). A large-scale preparation of dG oligomers having an average chain length of lo-20 nucleotide residues, as judged from 20% polyacrylamide gels, was used for RPC-5 chromatography. The large-scale depurination reaction was incubated at 37°C for 24 h. Alkaline ofigomers.
RPC-5
chromatography
of dG
The general procedure used for RPC-5 column chromatography has been described elsewhere (1). RPC-5 was equilibrated with 0.1 M KCl, 0.1 M NaOH and used to prepare a 20 x l.O-cm column. The column was equilibrated with 0.1 M KCl, 0.1 M NaOH under pressure supplied from a peristaltic pump at a flow rate of 0.5 mYmin. Two hundred Azsz units (in a 20-ml volume) of dG oligomers was applied to the column and 100 ml of 0.1 M KCl, 0.1 M NaOH used to wash the column. A 2-liter linear gradient of 0.1 M NaOH containing 0.1-1.0 M KC1 was applied to the column and fractions (6.4 ml) were collected. Absorbances at 252 nm of the eluted fractions were recorded and each fraction brought to neutral pH. Fractions containing peaks of absorbance were pooled, concentrated, and dialyzed into
AND
0.01
M
‘Iris-HCl
Characterization
200 FRACTION
(pH S.O), 0.1 mM EDTA. of dG oligomers.
Sev-
eral pooled fractions of dG oligomers were labeled using polynucleotide kinase and [y-32P]ATP as described by Maxam and Gilbert (14). Approximately 1 nmol of oligonucleotide was labeled in each reaction. The reactions were terminated by phenol extraction and were then extracted with ether and extensively dialyzed. The labeled oligomers were subjected to partial degradation according to the “G-specific” chemical reaction protocol described by Maxam and Gilbert (14) except the incubation with dimethylsulfate was allowed to proceed for 2 h. Both partially degraded and undegraded dG oligomers were electrophoresed on 20% polyacrylamide sequencing gels (14) which had been preelectrophoresed for 10 h. The marker dye, bromophenol blue, present in the samples was allowed to migrate 10 cm into the 40-cm gel and the gel was then subjected to autoradiography. Other materials and methods. Analytical polyacrylamide tube gels (20%) were as described previously (15). Polynucleotide kinase was purchased from P-L. Biochemicals and bacterial alkaline phosphatase was purchased from Sigma (Type III-S).
100 FIG. 1. Fractionation of dG oligomers described under Materials and Methods. oligomer sizes were assigned according gels (see text).
WELLS
300
NUMBER
using RPC-5 column chromatography. The column was run as The linear KCI gradient applied to the column is indicated. dG to subsequent analysis on 20% polyacrylamide sequencing
FRACTIONATION
215
OF dG OLIGOMERS
RESULTS RPC-5 Chromatography of dG Oligomers
A mixture of dG oligomers was prepared as described under Materials and Methods. Figure 1 shows the elution profile of these dG oligonucleotides from a RPC-5 column run under strongly denaturing conditions (0.1 M NaOH). The resolution between peaks in this profile is similar to that seen when A, C, or I (both deoxyribose and ribose derivatives) and dT and rU oligomers are separated using RPC-5 chromatography at neutral pH. Baseline separation of oligomers up to the 15mer (dG oligomer sizing is discussed below) is seen; this range could likely be extended by use of a shallower or convex salt gradient. Each fraction from the column was neutralized within 6 h after elution; long-term storage of the oligomers under alkaline conditions resulted in some degradation. The recovery from this column was approximately 65%. In preliminary studies, we attempted to fractionate dG oligomers under less alkaline conditions (12 mM NaOH) than those reported here. Recovery from these columns was poor (approximately 20%). Characterization
of dG Oligomers
The purities and sizes of oligomers in the pooled peaks from the RPC-5 column shown in Fig. 1 were ascertained by partial digestion and analysis on 20% sequencing gels. Figure 2 shows the results of such an analysis. In lane 6 of this autoradiogram, partial digestion products of the oligomer eluting in fraction 162 from the column are electrophoresed. Counting the number of bands in the pattern indicates that the dG oligomer present in these fractions is 12 residues in length. The purity of the dG,, oligomer eluted from the column is high, as can be seen in lane 5 of the autoradiogram. Comparison of the oligomer bands in lanes l-5 with each other and with the partial digestion products in lane 6 indicates that
-(PG),
PGP
-(PG),
PGP
-(PG),
PGP
- (PG), PGP + - PGPGP
- PGP
2
3
4
5
6
7
FIG. 2. Analysis of dG oligomers eluted from RPC-5. In lanes 1-5, dG oligomer pools labeled using polynucleotide kinase and [-Y-~~P]ATP (Materials and Methods) are electrophoresed. Each pool is denoted by an appropriate column fraction, i.e., the pool comprising fractions 160-164, in Fig. 1 is indicated as fraction 162. In lane 6, partial degradation products of fraction 162, generated as described in the text, are electrophoresed. Lane 7 shows orthophosphate run as a marker. Assignments of partial degradation products are indicated in the right margin. The arrow indicates a contaminant present in all the oligomer preparations remaining from the kinase reaction, presumably residual ATP. In comparing relative oligomer migrations, note that in lanes l-5, oligomers bear no 3’phosphate moieties while, in lane 6, 3’-phosphate groups are present on all the partial degradation products. In this analysis, the oligomer pools containing dG, and dG,, were not included in order to extend the range of the assay.
each peak in the RPC-5 column profile is an individual oligomer and that neighboring peaks differ in length by one nucleotide
216
SELSING,
LARSON,
residue. The gel characterizations permit assignment of the oligomer sizes indicated on the column profile in Fig. 1. The low level contaminant bands seen in some lanes of Fig. 2 are likely due to a small amount of degradation during the labeling of the oligomers rather than coelution from the RPCJ column. Supporting this contention, rechromatography of slightly degraded 32P-labeled oligomers on a small scale alkaline RPC-5 column resulted in several peaks of radioactivity, each of which gave only one band upon subsequent gel analysis (data not shown). DISCUSSION
This study shows that dG oligomers can be successfully purified by RPC-5 chromatography under alkaline conditions. RPC-5 can, therefore, now be used for fractionation of all DNA and RNA homooligonucleotides except those in the rG series. From the results of this study, it may be possible that rG oligomers can also be separated, by use of less alkaline conditions and low temperature during RPC-5 chromatography. The availability of large amounts of these oligomers will allow further studies on the effects of base sequence on DNA properties similar to those reported previously (3-9). ACKNOWLEDGMENTS
AND WELLS
postdoctoral fellowship from the National Institutes of Health (GM 05468).
REFERENCES 1. Wells, R. D., Hardies, S. C., Horn, G. T., Klein, B., Larson, .I. E., Neuendorf, S. K., Panayotatos, N., Patient, R. K., and Selsing, E. (1979) in Methods in Enzymology, Academic Press, New York, in press. 2. Larson, J. E., Hardies, S. C., Patient, R. K., and Wells, R. D. (1979) J. Biol. Chem., 254, 55355541. 3. Selsing, E., and Wells, R. D. (1979)5. Biol. Chem., 254, 5410-5416. 4. Wells, R. D., Blakesley, R. W., Burd, J. F., Chan, H. W., Dodgson, .I. B., Hardies, S. C., Horn, G. T., Jensen, K. F., Larson, J. E., Nes, I. F., Selsing, E., and Wartell, R. M. (1977) Crit.
Chem.
This work was supported by grants from the National Science Foundation (KM 77-15033) and the National Institutes of Health (CA 20279). Erik Selsing was supported during a portion of these studies by a
Rev.
Biochem.
4, 305-340.
5. Selsing, E., Wells, R. D., Early, T. A., and Kearns, D. R. (1978) Nature (London) 275, 249-250. 6. Selsing, E., Wells, R. D., Alden, C. J., and Amott, S. (1979) J. Biol. Chem., 254,5417-5422. 7. Walker, G. C., Uhlenbeck, 0. C., Bedows, E., and Gumport, R. I. (1975) Proc. Nut. Acad. Sci. USA 72, 122-126. 8. Shum, B. W., and Crothers, D. M. (1978) Nucleic Acids Res. 5, 2297-2311. 9. Trip, E. M., and Smith, M. (1978) Nucleic Acids Res. 5, 1529-1538. 10. Wells, R. D., Larson, J. E., Grant, R. C., Shortle, B. E., and Cantor, C. R. (1970) J. Mol. Biol. 54, 465-497. 11. Gray, D. M., and Bollum, F. J. (1974)Biopofymers 13, 2087-2102. 12. Eshaghpour, H., and Crothers D. M. (1978) Nucleic Acids Res. 5, 13-21. 13. Wells, R. D., and Larson, J. E. (1972) J. Biol. 247, 3405-3409.
14. Maxam, A. M., and Gilbert, W. (1977) Proc. Acad.
Sci.
USA
Nur.
74,5&L564.
15. Burd, J. F., and Wells, R. D. (1974)J. Biol. Chem. 249,7094-7101.
