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Desalting of RPC-5 oligonucleotides on Dowex 50
ANALYTICAL
BIOCHEMISTRY
Desalting
69, 3 10-3 11 (1975)
of RPC-5
Oligonucleotides
on Dowex
50
Several authors reported excellent separation of oligo- and mononucleotides on RPC-5 columns (l-3). The method has great resolving power and in a single chromatographic run yields homogenous oligonucleotides obtained by enzymatic digestion of specific tRNAs. It has not been, however, routinely applied for sequencing RNA and until now the primary structures of only three tRNAs have been investigated by this method (4). In our experience one of the difficulties in its practical application was the desalting of oligonucleotides eluted from RPC-5 columns with 0.5-3.0 M ammonium acetate buffer. Lyophylization was suggested as a method of choice (1) but oligonucleotides desalted in this way always contained small amounts of salt (and possibly of Adogen, as well), which made their quantitation and characterization difficult. We have found that the fractions collected from RPC-5 columns can be easily desalted by passing them through a cation-exchange column (H+ form). Cation binding by the resin leaves oligonucleotides in aqueous solution of free acetic acid which can subsequently be completly removed by evaporation. The following procedure may be recommended for routine use: Oligonucleotide collected from the RPC-5 column (l-10 AZ6,, units in IO-20 ml of 0.5-3.0 M ammonium acetate buffer, pH 4.4) is applied directly on a Dowex 50-X8, 200-400 mesh, H+, 1 X &cm column, thoroughly washed with redistilled water. After all the RPC-5 fraction has been loaded, the Dowex column is washed with water. The oligonucleotide is contained in’the Dowex effluent collected during application of the fraction and during the initial washing. For complete recovery of nucleotides it is sufficient to wash the column with lo-15 ml of water. To avoid unnecessary diluting of the material, it is convenient to follow the elution with a uv-monitoring device (e.g., Uvicord II, LKB, Sweden). The Dowex 50 effluent is taken to dryness by rotary evaporation (water bath, 40°C under vacuum). Any residual acetic acid is removed either by repeated evaporation (l-2 ml of water being used for oligonucleotide dissolving) or by placing the sample over KOH in a desiccator. The recovery of desalted nucleotides is about 90% ; main losses were found to occur during evaporation. The desalted material is completely free of any uv-absorbing substances. During the entire procedure, the pH of solutions never drops below 2.5. Except for the nucleotides con310 Copyright @ 1975 by Academic Press, Inc. All rights of reproduction in any form reserved.
SHORT COMMUNICATIONS
311
taining the Y-base, all can tolerate this pH. Oligonucleotide desalting from initial volumes of 20 ml of 2 M ammonium acetate buffer takes about 2 hr. The procedure described is much simpler than gel filtration on Sephadex G-10 (Bio-Gel P-2) columns (5) or sorption of nucleotides on DEAE-cellulose followed by their desorption with ammonium carbonate (ethanolamine carbonate) (5). Also, the nucleotides desalted in our procedure are free of contaminants usually eluted from DEAE-cellulose together with the sample; the RPC-5 nucleotides desalted on Dowex l-X2 (Cl-), as described by Caron and Dugas (6), are similarily contaminated. The combination of the DEAE-cellulose procedure with the Dowex 50 one offers, therefore, an advantage in purification of nucleotides originally contained in 7 M urea solutions. We found this combination to be effective in desalting of nucleotides fractionated by the standard DEAE-cellulose-7 M urea method (5). ACKNOWLEDGMENT This work was supported by the Polish Academy of Sciences within the project 09.3. I.
REFERENCES 1. 2. 3. 4.
Egan, B. Z. (1973) Biochim. Biophys. Actu 299, 245-252. Singhal, R. P. (1973) Biochim. Biophys. Acta 319,1 l-24. Roe, B., Marcu, K., and Dudock, B. (1973) Biochim. Biophys. Acta 319, 25-36. Egan, B. Z., Weiss, J. F., and Kelmers, A. D. (I 973) Biochem. Biophys. Res. Commun. 55, 320-327. 5. Brownlee, G. G. (1972) Determination of sequences in RNA, in Laboratory Techniques in Biochemistry and Molecular Biology (Work, T. S., and Work, E., eds.), North-Holland Publishing Company, Amsterdam. 6. Caron, M., and Dugas, H. (1974) J. Chromatog. 101,228-230.
JACEKAUGUSTYNIAK ZBIGNIEW JANOWICZ DAMIAN LABUDA JACEKWOWER Miedzyuczelniany Instytut Biochemii A. R. 61-701 Poznari, ul. Fredry 10 Poland Received March 26, 1975: accepted June
I I, 1975
BIOCHEMISTRY
Desalting
69, 3 10-3 11 (1975)
of RPC-5
Oligonucleotides
on Dowex
50
Several authors reported excellent separation of oligo- and mononucleotides on RPC-5 columns (l-3). The method has great resolving power and in a single chromatographic run yields homogenous oligonucleotides obtained by enzymatic digestion of specific tRNAs. It has not been, however, routinely applied for sequencing RNA and until now the primary structures of only three tRNAs have been investigated by this method (4). In our experience one of the difficulties in its practical application was the desalting of oligonucleotides eluted from RPC-5 columns with 0.5-3.0 M ammonium acetate buffer. Lyophylization was suggested as a method of choice (1) but oligonucleotides desalted in this way always contained small amounts of salt (and possibly of Adogen, as well), which made their quantitation and characterization difficult. We have found that the fractions collected from RPC-5 columns can be easily desalted by passing them through a cation-exchange column (H+ form). Cation binding by the resin leaves oligonucleotides in aqueous solution of free acetic acid which can subsequently be completly removed by evaporation. The following procedure may be recommended for routine use: Oligonucleotide collected from the RPC-5 column (l-10 AZ6,, units in IO-20 ml of 0.5-3.0 M ammonium acetate buffer, pH 4.4) is applied directly on a Dowex 50-X8, 200-400 mesh, H+, 1 X &cm column, thoroughly washed with redistilled water. After all the RPC-5 fraction has been loaded, the Dowex column is washed with water. The oligonucleotide is contained in’the Dowex effluent collected during application of the fraction and during the initial washing. For complete recovery of nucleotides it is sufficient to wash the column with lo-15 ml of water. To avoid unnecessary diluting of the material, it is convenient to follow the elution with a uv-monitoring device (e.g., Uvicord II, LKB, Sweden). The Dowex 50 effluent is taken to dryness by rotary evaporation (water bath, 40°C under vacuum). Any residual acetic acid is removed either by repeated evaporation (l-2 ml of water being used for oligonucleotide dissolving) or by placing the sample over KOH in a desiccator. The recovery of desalted nucleotides is about 90% ; main losses were found to occur during evaporation. The desalted material is completely free of any uv-absorbing substances. During the entire procedure, the pH of solutions never drops below 2.5. Except for the nucleotides con310 Copyright @ 1975 by Academic Press, Inc. All rights of reproduction in any form reserved.
SHORT COMMUNICATIONS
311
taining the Y-base, all can tolerate this pH. Oligonucleotide desalting from initial volumes of 20 ml of 2 M ammonium acetate buffer takes about 2 hr. The procedure described is much simpler than gel filtration on Sephadex G-10 (Bio-Gel P-2) columns (5) or sorption of nucleotides on DEAE-cellulose followed by their desorption with ammonium carbonate (ethanolamine carbonate) (5). Also, the nucleotides desalted in our procedure are free of contaminants usually eluted from DEAE-cellulose together with the sample; the RPC-5 nucleotides desalted on Dowex l-X2 (Cl-), as described by Caron and Dugas (6), are similarily contaminated. The combination of the DEAE-cellulose procedure with the Dowex 50 one offers, therefore, an advantage in purification of nucleotides originally contained in 7 M urea solutions. We found this combination to be effective in desalting of nucleotides fractionated by the standard DEAE-cellulose-7 M urea method (5). ACKNOWLEDGMENT This work was supported by the Polish Academy of Sciences within the project 09.3. I.
REFERENCES 1. 2. 3. 4.
Egan, B. Z. (1973) Biochim. Biophys. Actu 299, 245-252. Singhal, R. P. (1973) Biochim. Biophys. Acta 319,1 l-24. Roe, B., Marcu, K., and Dudock, B. (1973) Biochim. Biophys. Acta 319, 25-36. Egan, B. Z., Weiss, J. F., and Kelmers, A. D. (I 973) Biochem. Biophys. Res. Commun. 55, 320-327. 5. Brownlee, G. G. (1972) Determination of sequences in RNA, in Laboratory Techniques in Biochemistry and Molecular Biology (Work, T. S., and Work, E., eds.), North-Holland Publishing Company, Amsterdam. 6. Caron, M., and Dugas, H. (1974) J. Chromatog. 101,228-230.
JACEKAUGUSTYNIAK ZBIGNIEW JANOWICZ DAMIAN LABUDA JACEKWOWER Miedzyuczelniany Instytut Biochemii A. R. 61-701 Poznari, ul. Fredry 10 Poland Received March 26, 1975: accepted June
I I, 1975