Gel filtration of nucleic acids on sphere-condensed agarose

ARCHIVES

OF

BIOCHEMISTRY

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BIOPHYSICS

Gel Filtration

119,

504-5c)g

of Nucleic

Condensed B.

Division

of Cell Biology,

Department

OBERG

(1967)

Acids

on Sphere-

Agarose’

AND

L.

PHILIPSON

of Medical Microbiology, and Institute of Uppsala, Uppsala, Sweden Received

of Biochemistry,

University

June 30, 1966

Chromatography of nucleic acids on sphere-condensed agarose yields a separation of DNA, rRNA, and sRNA. Poliovirus RNA elutes between DNA and rRNA. The agarose concentration influences the elution position of nucleic acids. Variation in buffer composition has been shown to affect the elution pattern of ribosomal and viral RNA. A high flow rate results in earlier elution of RNA. The recovery was 80-100% and no degradation appeared to take place on the column.

Several methods for the separation of nucleic acids have been developed during recent years. The separation has been based on sedimentation rate, density, electrophoretic mobility, adsorption to various adsorbents, base ratio, specific base pairing, and selective solubility. Differences in the size of the molecules have also been used as a basis for separation. This method has alternatively been called gel filtration (1) and molecular sieve (a), restricted di$usion (3), or exclusion chromatograpky (4). Gel filtration on dextran and polyacrylamide gels has permitted separation of sRNA from larger nucleic acids (5), but as the exclusion limit for polymers on these gels is below 5 X lo5 (molecular weight) they cannot be used to fractionate DNA, viral RNA, and rRNA. The introduction of granulated agar gels by Polson (6) and Hjerten (7) made it possible to separate molecules, according to size, when the molecular weights exceed 2 X 105. An artificial mixture of TzDNA and Escherichiu coli RNA has been separated on granulated 1.5 % agarose gel (5). The low flow rates in granulated gels with small particle size and agar

concentrations less than 2 % made them difficult to use, but this was overcome by the introduction of sphere-condensed agar or agarose (8, 9). A preliminary investigation (10) of the separating power of sphere-condensed agarose revealed that DNA, rRNA, and sRNA’s could be separated, and that the elution pattern for viral RNA was affected by the buffer composition. The present paper describes the effects of varying agarose concentration, buffers, and flow rate on the elution of cellular and viral nucleic acids. Sedimentation coefficient and infectivity of viral RNA before and after gel filtration have also been investigated.

1 This work was supported by grants from Damon Runyon Memorial Fund, The Jane Coffin Childs Memorial Fund, and the Swedish Medical Research Council (16X-307-02). 604

MATERIALS

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METHODS

Agarose preparation. Agarose was prepared from Special agar Noble (Difco) either by the method of Russell et al. (11) or by the method of Hjerten (12). Sphere condensation. A hot solution of agarose of the desired concentration in water was spherecondensed according to Bengtsson and Philipson (8). The sphere-condensed agarose was sieved in water on a vibrator (Fritsch pulverisette IdarOberstein, West Germany) and the fraction 100-120 mesh (US sieve series) was collected. Some preparations of sphere-condensed agarose were supplied by Pharmacia Fine Chemicals Ltd., Uppsala, Sweden. Poliovirus RNA. Poliovirus type 1 (strain E 206) was grown, purified, and extracted as de-

GEL

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scribed previously (13). The virus was labelled with B2P since the amount of poliovirus RNA was too small to be measured by ultra violet (UV)absorbancy. The infectivity of the RNA preparations was completely destroyed by RNase (10-l e/ml). Adenovirus DNA. Adenovirus type 5 was cultivated, isolated, and purified according to the procedure of Green and P&a (14), and the DNA was extracted with phenol after digestion with papain (0.3 mg/ml in final concentration) as described by Green and Pifia (15). Satellite tobacco necrosis virus RNA. The satellite tobacco necrosis virus (STNV) was cultivated, isolated, and purified as described previously (16), and the RNA was extracted by phenol as described for poliovirus RNA (13). KB cell nucleic acids. KB cells were grown in spinner cultures in Eagle’s spinner medium (17) with a double strength of amino acids and 10% horse serum. The nucleic acids were extracted from the cells by the phenol-Duponol method (18). Poliovirus RNA assay. Plaque assay was done according to procedures described by Pagan0 and Vaheri (19). Radioactivity. A Tracerlab FD-1 gas-flow counter equipped with a Monomol window was used. Ultraviolet absorbancy was measured with a Zeiss spectrophotometer, PM& II. Sucrose density gradient centrijugation. Linear gradients with 5-20yo sucrose in 0.1 M NaCl-0.01 M Tris-HCl (pH 7.5) were centrifuged for 14 hollrs in a MSE 50 TC centrifuge. Stock solutions of sucrose were shaken with bentonite (final concentration 0.025yo, w/v) prior to use, and the bentonite was removed by cent,rifagation at 70,OOOg for 20 minutes. The gradients in ‘20.ml tubes were removed from the bottom by a tube-piercing device. Sedimentation analyses. A Spinco model E with UV-opt)ics was used for sedimentation velocity analyses. Gel Jiltration. Two types of columns were used: glass columns (1.8 X 45 cm) wit,h a sintered glass disc, and plast,ic columns (2.1x60 cm) with a nylon net in the bottom. The net was covered with a thin layer of Sephadex G-25 before packing the column to prevent occlusion of the net by agarose. The columns were packed by adding a suspension of agarose to a column half-filled with buffer. The agarose was allowed to sediment while t.he buffer was slowly percolating through the column. The columns were then washed with three column volumes of buffer. All buffers contained 0.5% but,anol to prevent, bacterial contamination. About 1 ml of the nllcleic acid preparation was applied in each experiment. The flow rate was kept constant by a peristaltic pump, and fractions of 2 ml were collected.

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Agarose concentration. Figure 1 shows the separation of a mixture of KB cell nucleic acids and 32P-labeled poliovirus RNA on a 1% agarose column. The first UV-peak is DNA as determined by the diphenylamine reaction (20) and DNase sensitivity (10 pg/ ml). The two ribosomal RNA’s appear in the second UV-peak. This conclusion is based on analytical ultracentrifugation and sucrose density gradient centrifugation. Figure 2 shows the pattern after sucrose density gradient centrifugation. No difference in amount of the two ribosomal RNA’s was observed in sucrose gradients from the front and the back of the second peak. Ribonucleic acid was determined by the orcinol reaction (21). The third UV-peak has a sedimentation coefficient of 4, consists of RNA, and probably represents soluble RNA. Poliovirus RNA, which has a molecular weight of 2 X 106, elutes between DNA and rRIYA as evident from the peak in radioactivity. A series of preparations of pearl-condensed agarose, prepared by the methods of Russel

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FIG. 1. Chromatography of a mixture of 3 X 104 cpm of poliovirus RNA and 1 mg of KB cell nrlcleic acids in 1.5 ml on a lyo agarose column, 2.1 X 60 cm, at room temperature. The flow rate was 2 ml/cm*/hour and t,he buffer was 2 X 1W3 M sodillm phosphate, low3 M MgCI?, pH 6.0. KB cell nucleic acids, 0; poliovirus RNA, 0. Recovery: KB cell nucleic acids by optical densit,y, 100%; poliovirtls RNA by radioactivity, 940/,.

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PHILIPSON cell nucleic acids was used in all experiments. As seen in Fig. 4 the elution characteristics of DNA and sRNA are unaffected by an increase in salt concentration or urea. Riboso-

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FIG. 2. Sucrose gradient centrifugrtiorr, 5-200j0, in a linear gradient in 0.1 M NaCl, 0.01 M Tris-HC1, pH 7.5, for 14 hours at 25,000 rpm of the pooled second UV-peak from Fig. 1 containing rRNA. The peak was concentrated by precipitation with 2 volumes of cold ethanol, and 1 ml of the concentrate was applied on top of l&ml gradients.

et al. (11) and Hjert6n (12) with varying agarose concentration, was compared with regard to their separation characteristics. Buffer, flow rate, and temperature were the same as described in Fig. 1. A mixture of KB cell nucleic acids and poliovirus RNA was applied to the columns, and the position of the different peaks was plotted against the agarose concentration as shown in Fig. 3. Deoxyribonucleic acid elutes at the same position irrespective of agarose concentration in the range tested. The elution pattern of poliovirus RNA, rRNA, and sRNA is highly dependent on the agarose concentration. The elution characteristics of a few batches of spherecondensed 2% agarose obtained from Pharmacia Fine Chemicals Ltd. differed from the pattern in Fig. 3. These batches showed the same elution characteristics as 1% agarose. The percentage of agar appears consequent,ly not to be sufficient for standarization of these gels, and the elution characteristics must be measured for each batch. In the following, the pattern in Fig. 3 was taken as the standard. E$ect of buj’er composition. The effect of varying the buffer composition on the elution characteristics of nucleic acids was next investigated. The same preparat,ion of KB

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FIG. 3. Elution positions of different nucleic acids on agarose of varying concentration in 2 X 1OP M sodium phosphate, 1l.Y M MgC12,pH 6.0, at a flow rate of 2 ml/cm2/hour, room temperature, and a column length of 2.1 X 60 cm. A mixture of 1 mg of KB nucleic acids and 3 X lo4 cpm of poliovirus RNA in l-2 ml was applied on the columns in each experiment. DNA, 0; rRNA, 0 ; poliovirus RNA, 0; sRNA, X.

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FIG. 4. Elation position of 1.5 mg of KB cell nucleic acids in 1 ml on 2y0 agarose columns (1.8 X 45 cm) at room temperature and a flow rate of 2 ml/cm2/hour. A. Varying concentration of NaCl in 2 X 1c3 M sodium phosphate, 1P M EDTA, pH 6.0. B. Varying concentration of NaCl in 2 X 1P M sodium phosphate, 1P M MgClz, pH 6.0. C. Varying concentration of urea in 2 X 1W3 sodium phosphate, 10M3 M EDTA, pH 6.0. D. Varying concentration of urea in 2 X lWa M sodium phosphate, 10-* M MgCls, pH 6.0. DNA,*; rRNA, 0 ; sRNA, X.

GEL FILTRATION

ma1 RNA, on the other hand, is retarded by high salt concentration and elutes earlier in the presence of urea. Poliovirus RNA is influenced by the buffer composition in the same way as rRNA eluting at 34 % of the bed volume of 2 % agarose in t,he phosphate-EDTA buffer described in Fig. 4, and at 49% of the bed volume if 0.1 RI NaCl is added or 10e3M MgClz is replacing EDTA in the same buffer. The effect of pH was tested by using the EDTA buffer with 0.1 M NaCl at pH 5-S. Flow rate, temperature, and column size were the same as in the experiments described in Fig. 1. No variation in elution pattern for KB cell nucleic acids and poliovirus RNA was observed under these conditions. Flow rate. By raising the flow rate from 2 to 8 ml/cm2/hour it was observed that rRNA and eRNA eluted at an earlier position. The experiments were performed on a column of 2% agarose (Pharmacia) with the elution characteristics very similar to that, of a 1 % column as shown in Fig. 3. As shown in Fig. 5 the first peak is DNA, the second rRNA, and the third sRNA; the last peak most probably contains low molecular weight compounds. At lower flow rates this is covered by sRNA. Rechromatography at low flow rate gave the same elution pattern as described in Fig. 1. Eflect of gel filtration on sedimentation coeficient and infectivity of viral RNA. To study whet,her any degradation of the nucleic & nr 2 I

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FIG. 5. Chromatography of 0.8 mg of KB cell nucleic acids in 1 ml buffer on a 1% agarose column, 2.1 X 60 cm, at room temperature. The flow m te was 8 ml/cm* hour and the buffer was 2 X 1e3 M sodium phosphate, 10-s M MgCk, pH 6.0. Recovery by optical density, 81%.

507

acids occurred on the agarose column, the infectivit,y of poliovirus RNA and the sedimentation coefficient of STNV RNA were measured before and after gel filtration. Since a single break in the nucleotide chain destroys the infectivity of viral RKA (22), the infectivity assay is extremely sensitive to measure degradation. The decrease in infectivity of poliovirus RNA (6 X lo5 plaqueforming units) mixed with KB cell nucleic acids (1.5 mg) on a 1% agarose column, 2.1 X 60 cm, was found to be 45%. The buffer used was 2 X 10e3 ~1 sodium phosphate, 10e3M MgCl2, pH 6.0, and t)he operation was performed at 4” with a flow rate of 2 ml/cm2/ hour. The decrease in infectivity at room temperature was 90-95 70. Infectivity and radioact’ivity of the poliovirus RNA eluted at the same percentage of the bed volume. The sedimentation coefficient (s~~,~) of STNV RNA in 0.15 11 NaCl, 0.02 hr citrate, pH 7.0, was 13.2 S before and 13.1 S after the nucleic acid was chromatographed on a 1% agarose column. The value given by Reichmann (23) for STNV RYA is 13.5 S. Figure 6 shows the elution diagram of an artificial mixture of STSV and poliovirus RNA. In Fig. 6 is also shown that native adenovirus type 5 DNA elutes in the void volume of the same column with 6he same buffer. Melted adenovirus type 5 DNA eluted at the same position as native DNA. Melting was performed by heating to 100” for 10 minutes followed by rapid cooling. Temperature and recovery. Ko difference in elution pattern of KB cell nucleic acids or poliovirus RNA could be observed at the two temperatures used, 4” and room temperature (22”). The recovery measured as optical density of radioactivity usually varied between SO and 100%. The elution pattern and the recovery was t’he same when 1 or 10 mg of KB cell nucleic acids in 1.5 ml was applied on 2 % agarose columns (2.5 X 45 cm). An increased tendency to adsorb nucleic acids was observed on columns which had been used for several months or which had been used for protein separation. When only poliovirus RNA was applied to the agarose column the RNA was sometimes totally adsorbed to the :lgarose. However, t,his way

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FIG. 6. Chromatography of adenovirus type 5 DNA (3X104 cpm) in 0.8 ml and a mixture of poliovirus RNA (3X104 cpm) and STNV RNA (0.23 mg) in 0.8 ml on a 1% agarose column ( 2.1 X 60 cm) in 2XlOF M sodium phosphate, 1W3 M MgC12, pH 6.0, at room temperature and a flow rate of 2 ml/ cm2/hour. Adenovirus 5 DNA X; poliovirus RNA, 0; STNV RNA, 0. Recovery: adenovirus DNA and poliovirus RNA, 92 and Ql%, respectively, by radioactivity; STNV RNA, 79% by optical density.

never observed when mixtures of KB cell nucleic acids and poliovirus RNA were chromatographed. No differences in recovery were observed between agarose prepared by the methods of Russel et al. (ll), Hjertbn (12), or those preparations supplied by Pharmacia Fine Chemicals Ltd. DISCUSSION

The present investigation shows that DNA, viral, ribosomal, and soluble RNA can be separated on sphere-condensed agarose without any appreciable degradation, and that the elution is dependent on both agarose concentration and buffer composition. A concentration of less than 3 % of agarose is required to separate DNA and rRNA, and the separation of high molecular weight nucleic acids is favored at lower concentrations of agarose (Fig. 3). At lower concentration than 1% the spheres are not sufficiently rigid and t,hey tend to coalesce. The elution pattern may, however, vary on gels of the

same concentration, which emphasizes that these gels must be standardized by their elution characteristics rather than by their agarose content. Cellular DNA is evidently too large to penetrate even the 1% gel (Fig. 1) and consequently could not be separated into different fractions. Adenovirus type 5 DNA (molecular weight 2 X 10’) also elutes in the void volume, even if melted (Fig. S), but ribosomal RNA is well separated from DNA. Poliovirus RNA with a molecular weight of 2 X lo6 elutes between DNA and rRNA. This would infer that single-stranded nucleic acids with molecular weights between about 5 and 0.3 X lo6 could be separated by this method. No explanation can be offered for the two ribosomal RNA’s that elute at the same position, but it may be due to compensating differences in conformation of the two RNA chains. Investigations about RNA conformation in solutions of different ionic strength and composition have been summarized by Spirin (24). High molecular weight RNA forms rods in solutions of relatively low ionic strength (O.l) or in the presence of divalent cations, the rods are transformed to compact coils. At higher temperatures in low ionic strength buffers the rods unfold and form fibrils. On the other hand, double-stranded DNA or RNA is not affected by the buffer composition, which changes the conformation of a singlestranded nucleic acid. The results presented (Fig. 4) may be explained by conformation changes at different ionic strength. Poliovirus RNA and rRNA both elute at about the void volume in buffers of low ionic strength, where the nucleic acids are extended into rods, but both are retarded at higher ionic strength or in the presence of magnesium, when the nucleic acids have formed compact coils. The elution of sRNA is not affected by the buffer composition, which indicates a rigid structure of this class of nucleic acids. The variation in hyperchromicity and optical rotation due to variation in inonic strength is also smaller for sRNA than for high molecular weight RNA (25).

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Ribosomal RNA elutes earlier at high ionic strength if urea is added to the buffer, which probably is due to the ability of urea to promote a less compact structure of the RNA. With the different buffers used, no variation in gel volume was observed, and any effect of the buffers on the gel resulting in changed elution pattern seems improbable. The separation of nucleic acids on agarose columns is evidently dependent on size and shape of the nucleic acids. This could be utilized to separate molecules of the same molecular weight if the shape is different or could be made different by variation in buffer composition. Consequently, at high ionic strength a double-stranded nucleic acid would elute before a single-stranded structure with the same molecular weight. Variation in the elution pattern depending on the flow rate (Fig. 5) has been reported earlier (7). However, it is not unequivocally demonstrated that this effect is due to a diffusional restriction (26). Chromatography on sphere-condensed agarose appears to be a mild procedure since no significant decrease in the sedimentation coefficient of STNV RNA was observed after chromatography (Fig. 6). The decrease in infectivity of poliovirus RNA did not exceed that observed after dialyzing the RNA against buffer. It appears that his technique can separate similar amounts of nucleic acids as the methylated albumin columns (18) of the same size. The viscosity of the applied sample should, however, be kept low to prevent compression of the gel bed. The experimental results could not be compared with the gel filtration theory of Laurent and Killander (27) since the concentration of rods/cm3 (L) of the agarose gel matrix is unknown. The theory of Porath (28) provides that the molecules are looked upon as flexible polymers. This is not applicable t’o nucleic acids, which presumably have a partially ordered structure both at high and low ionic strength at temperatures below 40” (24). Furthermore, the molecular parameters of the nucleic acids are not sufficiently established to permit a theoretical consideration.

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