Column chromatography of RNA and oligonucleotides on denatured proteins

ANALYTICAL

Column

BIOCHEMISTRY

29,

459467

(1969)

Chromatography of RNA and on Denatured Proteins GEORGE

Laboratory of Biochemistry, Health, Department of Health,

National

W. RUSHIZKY’ Cancer

Education and

Received

Oligonucleotides

October

Institute, Welfare,

National Bethesda,

Institutes Maryland

of 20014

30, 1968

For the ion-exchange chromatography of oligonucleotides, various polybasic adsorbents are used, each of which offers special advantages and/or differs one from the other mainly by the character of the nonionic forces that attract and bind nucleic acid derivatives. For example, DEAE-cellulose, but not DEAE-Sephadex, binds purines more strongly than pyrimidines; methylated albumin kieselguhr (MAK) columns and benzoylated DEAE-cellulose have an increased affinity for lipoidal groups (1, 2). Supporting evidence for the hydrophobic, hydrogen-bonding nature of these secondary forces of attraction is derived from the fact that they are largely suppressed by urea or alcohols. It was the purpose of this investigation to search for additional ion exchangers which would be useful for the fractionation of large oligomers in (n + 100) and that would complement the properties of adsorbents such as DEAE-cellulose, BD-cellulose or MAK. As described here, protein of the Escherichia coli phage MS2 was treated with phenol to render it insoluble, and then used as support for the column chromatography of vira’l RNA as well as of large oligoribonucleotide fractions. The results show that the “denatured” protein functions as an ion exchanger at pH 5.2, with a binding capacity for large oligomers comparable to that of DEAE- or benloylated DEAE-cellulose (1, 3). As with other adsorbents, the larger oligoribonucleotide fractions are more firmly bound than smaller ones, and, among mononucleotides, purines are more retarded than pyrimidines. The ion-exchange capacity of denatured MS2 protein differed little, if at all, from that of denatured commercial egg albumin. Possible applications of the insoluble protein adsorbents are discussed. ’ Present address: Laboratory of Nutrition and Endocrinology, National of Arthritis and Maryland 20014.

Metabolic

Diseases,

National 459

Institutes

of

Health.

Institute Bethesda,

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MATERIALS

W.

RUSHIZK~

AND

METHODS

Preparation of Denatured Protein. The E. coli phage MS2 was prepared in 15-20 gm amounts as reported previously (4). MS2 protein was obtained as a by-product during isolation of the MS2 RNA by phenol extraction as described (5) with the following modification: MS2 phage at 15-20 mg/ml in H,O was adjusted to 0.1 M NH,HCO,, pH 7.6, and stirred for 15 min at 23” with an equal volume of water-saturated phenol. After centrifugation at 23” for 5 min at 2000 rpm, the phases were re-extracted twice with 1 vol of fresh buffer and phenol, respectively (5). Then 2 vol of methanol were added to the combined phenol phases to precipitate protein, which was spun out and resuspended in the original volume of HzO. After lyophilization, the insoluble material was washed in H,O. A total of eight 1 liter portions of water was used. Next, the granular protein which settled through a height of 25 cm in 30 min was retained and the finer material discarded. The material was kept at 4” in the original volume of water in the presence of a trace of CHCL, and did not change its ion-exchange capacity for months. Since dehydration or exposure to alkaline pH was found to decrease the exchange capacity of the protein, the yield of adsorbent was not determined on a weight basis or by dissolving in alkali. Rather, as an approximation of the yield, it was found that 70 ml of packed column volume was obtained from 6 gm of MS2 (see below). When egg albumin (Nutritional Biochemicals Corporation) was used in place of MS2 protein, 10 gm of protein was dissolved in 1 liter of 0.11M NH,HCO,, pH 7.6, by stirring for 2 hr at 23”. Phenol extraction, washing, and storage were carried out as described for MS2 protein. Samples used for chromatography. Monoribonucleotides were obtained from Sigma Chemical Company. MS2 RNA was prepared as described above (5) with slight modifications (see above). TMV-RNA was a gift from Dr. C. A. Knight. Purified tRNAser from yeast was prepared by Dr. B. P. Doctor; 1 mg of tRNA incorporated 27.4 mpmoles of serine, indicating a purity of better than 75% (fraction III, (6)). Pentanucleotides from RNase T, digests of yeast RNA were isolated as reported (7), and large oligomer fractions from BaciZZus subtilk RNase digests of MS2 RNA were obtained as described previously (8). Before rechromatography, samples were desalted on DEAE-cellulose and lyophilized (9). When mixtures of these samples were applied to the same column, the pentanucleotides were identified by chain length and by Ap + Cp + Up/Gp ratios (7), MS2 RNA by base ratios (8)) and tRNA by base ratios including minor bases (6). Binding tests. As a preliminary test, adsorption of samples on de-

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natured proteins was checked as follows. A slurry of denatured protein in the buffer plus salt solution to be tested was placed in 12 ml conical centrifuge tubes. After centrifugation for 5 min at 2000 rpm, the supernatant was poured off and the step repeated until a packed volume of about 1 ml of protein adsorbent was obtained. The test sample of oligonucleotide was then added in 10 ml of the above buffer solution, the protein resuspended with a glass rod, and the suspension centrifuged again. Aliquots of the clear supernatant were then tested, by spectrophotometry, for the amount of nonadsorbed nucleotide material. Column chromatography. The denatured protein adsorbent suspended in water was adjusted to 0.05 M sodium acetate, pH 5.2 (starting buffer, SB), packed as a slurry under gravity into 1.2 X 15 cm Lucite columns, and washed with 500 ml or more of SB until the conductivity and pH of influent and effluent were the same. The absorbance/ml (A), at 230, 280, and 250 rnp, was less than 0.02, and was not increased by washing of the column with 1 M NaCl in SB. The oligonucleotide samples (AzeO 0f 50-200 per run) were adsorbed on the columns in l-2 ml of SB, and washed in with several 2 ml portions of SB. Development of the columns was by linear NaCl gradients from 0 to 0.5 M or 0 to 1.0 iM in SB, with or without the addition of 0.01 M MgClz. Under a 10 cm hydrostatic head, the flow rate of the columns was 100-110 ml/ hr, but flow rates of 50-70 ml/hr, produced by a peristaltic pump, were routinely used. To prevent microbial contamination, buffers were equilibrated wit,h CHCl, and all columns run at 4-6”. No significant shrinkage of the denatured protein was observed upon changing the eluant from 0 to 11M NaCl, nor was increased resistance to flow encountered. After each run, columns were regenerated with 1 M NaCl to remove remaining A,,, (if any), and then re-equilibrated with SB. RESULTS

To acheive optimum binding of large oligonucleotides to the protein adsorbent, preliminary binding tests were performed with various buffer systems and an oligomer fraction of average chain length 105. The buffers investigated were O-0.05 M MgCI,, 0.02-0.1 M sodium acetate, pH 5.2; 0.02-0.1 M sodium formate, pH 3.4; and 0.02-0.1 M Tris-Cl, pH 7.5. The pH and buffer selected (5.2) were chosen so as to be a,bove pK values of the -NH, groups of Ap, Cp, and Gp, and below the dissociation range of basic groups in the protein. Elution profiles of RNA and RNA derivatives after column chromatography on denatured MS2 protein adsorbent are shown in Figures 1 and 2. Mononucleotides were very weakly bound to the support in 0.05 M sodium acetate, pH 5.2 (starting buffer, SB), but Cp and Up were

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less retarded than Ap and Gp (Fig. 1A). Pentanucleotides from RNase T, digests of MS2 RNA were more strongly adsorbed, i.e., were not eluted by SB alone but by a salt gradient of 0-0.52M NaCl (Fig. lB), The elution position of the pentamers corresponded roughly to that obtained with an oligomer fraction of average chain length 12 (from B. subtilis RNase digests of MS2 RNA adsorbed on DEAE-cellulose) (Fig. 1C).

VOLUME

ELUTED

(LITERS)

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FIG. 1. (A) Mononucleotides Ap, Cp, Gp and Up adsorbed in and eluted with 0.02 M Sodium acetate (SB) alone; fraction volume, 8 ml. (B) Pentanucleotides from RNase T1 digest of yeast RNA adsorbed in SB, and eluted with a 2 X 1 liter linear gradient from O-O.5 M NaCl in SB; volume/fraction, 9.8 ml. (C) Oligonucleotide fraction (average chain length 12) from B. subtilis RNase digest of MS2 RNA adsorbed in SB and eluted with a 2 X 1 liter linear gradient from (LO.5 M NaCl in SB; volume/fraction, 9.8 ml.

Larger oligonucleotides were more retarded. Thus, an oligomer fraction of average chain length 105, or purified tRNA*“’ required NaCl gradients to 1 M rather than 0.5 1M for elution (Fig. 2A, B). The elution profile obtained (double peak, followed by a hump at the rear of the double peak) may indicate a degree of fractionation of tRNA”“‘. MS2 RNA, adsorbed on MS2 protein in SB, was more strongly bound than tRNA”“’ and the large (n = 105) oligomer fraction (Fig. 2C). For rechromatography, the entire A,,, peaks of pentamers, tRNA”“’

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3.0 (LITERS)

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FIG. 2. Each of three samples adsorbed in SB and eked by ‘2 X 2 liter linear gradients of O-l M NaCl at 12 ml/fra.ction: (A) purified tRNA”“’ ; (B) Oligomer fraction (average chain length 105) from B. subtilk RNase digest of MS2 RNA; (Cl MS2 RNA.

and MS2 RNA (Fig. lB, 2A, 2C) were pooled, desalted on DEAEcellulose, and recycled. No significant changes were observed in the elution behavior of these three test samples. Since Mg affects the shape and elution behavior (1) of large oligomers and MS2 RNA, rechromatography was also performed in 0.01 AI MgCl,. As shown in Figure 3, the elution sequence of the samples was the same as expected from the original runs without Mg. However, the 2 larger samples were less retarded in the presence of 0.01 M MgCl, than without the metal. Recovery of material from the columns ranged from 81 to 94% of the A,,, applied. The capacity of the MS2 protein adsorbent for the oligomer fraction of average chain length 105 in (SB and at 4”) was greater than 2 mg of oligomer sample per milliliter of packed column volume. This is similar to the capacity of DEAE-cellulose and of benzoylated DEAEcellulose (1) but higher than the capacity of MAK (2). At pH 5.2, no evidence was found for specific binding of MS2 RNA to MS2 protein; thus, when TMV-RNA was adsorbed on MS2 protein in 0.01 M MgC12 a similar elution pattern was obtained as with MS2

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FIG. 3. For rechromatography, a mixture of pentamers (Fig. lB), tRNA”“’ from yeast (Fig. 2A) and MS2 RNA (Fig. 2C) was adsorbed and eluted in SB plus 0.01 M MgCL, with a 2 X 1 liter linear gradient of O-O.5 M NaCI, at 9.8 ml/ fraction. The three samples were identified (see “Methods”) in peaks 1, 2, and 3, respectively.

RNA. Furthermore, the ion-exchange behavior of MS2 protein differed little, if at all, from that of egg albumin under the same conditions. However, at the lower pH of 3.4, MS2 RNA was retarded less strongly on denatured egg albumin than on MS2 protein. Thus, elution with 1 M NaCl/O.l M sodium formate, pH 3.4, yielded only 60% recoveries as compared to better than 95% recoveries with egg albumin adsorbent. It is known that small amounts of organic solvents can profoundly affect hydrophobic interactions. For this reason, the lack of marked specificity of the denatured protein for different RNA’s could be not only a result of the denatured state of the protein but also because chloroform was used to preserve all buffers and to prevent the growth of molds in the protein adsorbent. However, similar results were obtained with or without chloroform-saturated buffers. Because of the lower recovery of MS2 RNA from MS2 protein at pH 3.4, rather than at pH 5.2, the effect of a more neutral pH was investigated as well. Reconstitution of TMV RNA with undenatured TMV protein is most effective in 0.1 M sodium pyrophosphate/HCl, pH 7.5 (10). However, MS2 RNA was not bound at all to MS2 protein under these conditions, possibly because of the higher ionic strength of this buffer compared with SB, and/or because overnight incubation at 37” of protein with RNA, as used with the TMV system, was not attempted. DISCUSSION

The specificity of complex formation between proteins and nucleic acids depends not only on environmental factors such as salt concentration (ll), but also on their structural affinity for each other. For

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example, when the RNA and protein of tobacco mosaic virus (TMV) are recombined in vitro, rodlike molecules are formed with biological activity comparable to that of the original virus (10). Less specific structural affinity is evidenced by the interaction of polybasic substances such as polylysine, DEAE-cellulose, or DEAE-dextran with nucleic acids or oligonucleotides. These complexes are often characterized by increased resistance to nucleases or by insolubility (X2-14)) rather than by infectivity (as in the case of reconstituted TMV). Studies of such complexes have been concerned primarily with their stoichiometry and structure (15) rather than with their possible usefulness for other purposes, such as the fractionation of oligoribonucleotides (for a recent review, see 16). Preliminary experiments showed that egg albumin was not incorporated into agar like DNA (17)) probably due to the lower chain length of the protein. No significant amounts of RNA material were bound to such columns. As an alternative to agar entrapment, the protein was rendered insoluble either by heating or by treatment with phenol, and the agar was omitted. Furthermore, protein complementary to the RNA was employed. To be useful, the material so obtained was examined for two features considered especially desirable: (1) The adsorbent should allow separation and recovery of small oligonucleotides (n = 10) as well as of high molecular weight RNA. This requires adsorption of samples at low salt concentrations (below 0.1 M NaCl) so that small oligomers will be bound. Quantitative elution of all material should be accomplished below 0.8 M NaCl since high molecular weight RNA usually precipitates out above 0.8 M NaCl unless only very small amounts of material are fractionated. (2) A nonionic attraction between RNA derivatives and adsorbent was sought, since large oligonucleotides are difficult to resolve solely on the basis of net charge differences. To this end, MS2 protein was examined since one of its major Gz viva functions consists of binding RNA. As the results show, both small oligomers as well as RNA may be adsorbed on and eluted from MS2 protein adsorbent below salt concentrations of 0.8M NaCI. The second desired characteristic of the adsorbent, i.e., a nonionic interaction between protein and large oligomers derived from complementary RNA, if present, was difficult to ascertain. Evidence for an effect of RNA structure on adsorption to the protein may be derived from the fact that, in 0.01 M MgCI,, MS2 RNA was eluted by lower salt concentrations than in the absence of Mg. This is in line with the concept (18) that, since Mg increases the amount of secondary structure of RNA, an extended molecule would therefore bind to more ionic groups than one of more compact secondary structure.

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Clearly, this mechanism does not necessarily involve any increase in hydrogen bonding betweeen a more ordered RNA structure and the protein adsorbent. Evidence against a nonionic binding of RNA or RNA derivatives to the protein adsorbent may be adduced from the similar elution behavior of TMV- and MS2 RNA from MS2 protein adsorbent in the presence of 0.01 M MgCl,, as well as from the similar behavior of egg albumin and MS2 protein under these conditions. On the other hand, at pH 3.4 rather than at 5.2, MS2 RNA was bound more strongly to MS2 protein than to egg albumin. Thus, conditions and proteins other than those employed here may be more suitable for a particular separation of oligomers, or may yield more specific binding between complementary RNA and protein. To conclude, denatured protein ion exchangers represent an addition to the existing adsorbents employed for the fractionation of RNA and oligonucleotides. SUMMARY

Protein of the E. coli phage MS2 was denatured with phenol and used as insoluble support for the column chromatography of MS2 RNA, tRNA, and oligonucleotides. The ion-exchange capacity of denatured protein differed little, if at all, from that of denatured (commercial) egg albumin, and was comparable to that of DEAE- and benzoylated DEAEcellulose. Large oligomers were adsorbed more strongly than smaller ones, and elution was achieved by positive salt gradients in the range of O-l M NaCl. REFERENCES I., MILLWARD, S., BLEW, D., TIGERSTROM, M. V., WIMMER, E., AND G. M., Biochemistry 6,3043 (1967). 2. SUEOKA, N., AND YAMANE, T., Proc. Natl. Acad. Sci. U. S. 48, 1454 (1962). 3. PETERSON, E. A., AND SOBER, H. A., in “Methods in Enzymology” (S. P. Colowick and N. 0. Kaplan, eds.), Vol. V, p. 3. Academic Press, New York, 1. GILLAM, TENER,

1962. 4. RUSHIZKY,

5. 6. 7. 8.

G. W., GREW, A. E., AND RDGERSON, D. L., JR., Biochim. Biophys. Acta 108, 142 (1965). STK~USS, J. H., JR., AND SINSHEIMER, R. L., J. Mol. Biol. ‘7, 43 (1963). RUSHIZKY, G. W., SOBER, H. A., CONNELLY, C. M., AND DOCTOR, B. P., Biochem. Biophys. Res. Commun. 18, 489 (1965) RUSHIZKY, G. W., SKAVENSKI, I. H., AND SOBER, H. A., J. Biol. Chem. 240, 3984 (1965). RUSHIZKY, G. W., SHAVENSKI, I. H., AND GRECO, A. E., Biochemistry 5, 33%

(1966). 9. RUSHIZKY, G. W., AND SOBER, H. A., Biochim. Biophys. Acta 55,217 10. FRAENKEL-CONRAT, H., AND SINGER, B., Biochim. Biophys. Acta 33, 11. KATCHALSKY, E., Biophys. J. 4, 9 (1964).

(1962).

360 (1959).

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12. TAKAI, M., Biochim. Biophys. Acta 119, 20 (19%). 13. MAES, K., SEDWICK, W., AND VAHERI, A., Biochim. Biophys. Acta 134,269 (1967). 14. SOBER, H. A., SCHLOSS~MAN, S. F., YARON, A., LATT, S. A., .~KD RVSIIIZK~, G. R., Biochemistry 5, 3608 (1966). 15. GRATZER, W. B., AND MCPHIE, P., Biopolymers 4,601 (1966). 16. RUSHIZKY, G. W., AND SOBER, H. A., in “Progress in Nucleic Acid Research and Molecular Biology” (J. N. Davidson and W. E. Cohn, eds.), Vol. 8, p. 171. Academic Press, New York, 1968. 17. BOLTON, E. T., AND MCCARTHY, B. J., Proc. Natl. Acad. Sci. U. S. 48, 1390 (1962). 18. BAGULEY, B. C., BERGQUIST, P. L., AND RALPH, R. K., Biochim. Biophys. Acta

95, 510 (1965).