A simple method for the separation of single-stranded and double-stranded RNA on hydroxyapatite

ANALYTICAL

BIOCHEMISTRY

A Simple and

Method

55, 26-33

(1973)

for the Separation

Double-Stranded

RNA

,J. KALMAKOFF Departmetlt

Received

November

on Hydroxyapatite C. C. PAYNE

AND

of Microlxlology, Dunedin, New 27, 1972;

of Single-Stranded

G’niversity Zealand accepted

of Otago,

March

26, 1973

A simple method for separation of ssRNA from dsRNA on hydroxyapatite is described. The following procedure can be usefully applied for RNA fractionation: (i) Elution of column with 0.1 M phosphate at 60” removes nueleotidrs and a sm:ilI amount of ssRNA. (ii) Elution with 0.1 31 ssRNA. (iii) Finally, elution phosphate at 90” removes all the wmaining with 0.3 M phosphate at 90” removes the double-stranded RNA. This stepwise elution using a combination of phosphate concentration and temperature gives 96-100% recovery of the mntcrinl applied. INTRODUCTION

Hydroxyapatit,e (HA) has been extensively used to separate singlestranded DNA from double-stranded DNA by stepwise elution (1). However, the separation of single- from double-stranded RNA on HA has not been fully exploited. One of the major obst,acles in fractionating RNA on HA is the apparent helical content of 40-60s for tobacco mosaic virus (TMV) RNA and ribosomal RNA (2). This high helical configuration leads to difficulties in separating it from double-stranded RNA. There has been some limited use of the method for separation of single- and double-st,randed RNA; the replicative form of alfalfa mosaic virus could be isolated from single-stranded RNA (ssRNA) using a salt gradient. (3) and a combination of a temperature and salt gradient has been used to separate RNA polymerase products from virion doublestranded RNA (dsRNA) of cyt,oplasmic polyhedrosis virus (4~ and wound tumor virus (5). Bishop ef al. (6) have at.tempted to use HA to analyse the polymerase products of Rous sarcoma virus with limited success. The use of hydroxyapatite chromatography for the separation of nucleic acids has been recently reviewed by Bernardi (7). For our studies on the replicative forms of Semliki Forest virus it. was necessary to have a rapid and simple method of separating ssRNA from dsRNA. We undertook to study the chromatography of RNA on HA using TMV-RNA and dsRNA from the cytoplasmic polyhedrosis Copyright. All rights

26 @ 1973 by Academic Press. Inc. of reproduction in any form reserved.

virus of the silkworm (8). The genome of cytoplasmic polyhedrosis virus like reovirus contains 10 dsRNA segments. Using conditions which we could separate ssRNA eliminated the helical content of ssRKA, from clsRNA4 by a combination of high temperature and stepwise salt elution. MATERIALS

AND

METTlODS

C’hr~~ic~~ls. Hydrosyal~ntite HTP was purchased from Biorad, Richmond, California; V 0rthopl~ospl~ate and uridine-5-3H were obtained from Rn~liuchcmical Ccntrc, Amershnm, England. hctinomycin D was l)nrc*h:~~ec.l from Merck, Shnrl,, &zDohmc and ribonuclense -4 from Sigma Chcm. (lo. Ltd. All other chemicals used were commercially available reagent grade. IiUdt,u.l,!icrl)atite Chromatography of TAO’-RNA. RNA from a purified prepnrntion of tobacco mosaic virus (‘I’MIV) was prepared by the SDSi)henol method at 4” (9). Condit,ions of hydroxyapatite chromatography n-cre :W follox$-s: 200 /.~gof TMV-KKA was applied to a column (3.0 X 0.9 cm 1 of hydrosyapat,ite previously equilibrated with 0.1 M phosphate bluffer (1)H 6.8) at 20”. The jackcted column was heated by a Hnake thc~rmostatically controlled circulating water bath. The eluant was monitored at, 260 nm by means of a flow cell in a DBG Beckman ~I’cctrophotometer equipped with chart rccortler. A Beckman i2ccu-Flo 1)~1m])ww used to deliver mPnsllred amounts of buiTcr. The temperature of t II<%c~~lunin was init.inlly rnisetl to 55”, thcu sl~bscq~icntly raised in 5” stcpa, equilibrated for 3 ruin at each step and then 5 ml 0.1 31 phosl’hntc buffer was puml~~d through the column. Tile amount of Rx-4 elutetl at, each temperature was cstimntcd by cutting out, the peaks from the chart pal)cr and weighing them. The amount, of RNA eluted at each temllerature was expressed as the percentage of the total RNA recovered. Recoveries of RNA from hydrosyapntite were from 96-100~. Pwpamfiotz of htwled RXAl from fhc Sill~~c~o~~~~c, RNA wn:: extracted from the midguts of larvae by a hot.-phctnol method to 1)~ clcscribcd elxcwherc (Payne okIinlninkoff, Intcrz:i~olo~/~~,in prcssj i:li “‘P was injected into 5th instar larvae (3 ,tCi/lnrvae) of Bo,r~b!/x ~?~~ori prcsviously treated with 10 /~,gof :ictiuomycin D for one hour. Six hours later the larvae were killed and nN.4 extracted from tlic midguts. ITndcr thcsc conditions tllc :!-I’ is prcfcrciiti:ally incorl)or:lted into acidsolul~lc coinl~onfnts. (b I -4s a source of lah~l(~l cl~Rx~4, cytoljlasmic I)olyhedrosis virus, nn insect virus (8) was IIWI] to infect 5th instar larx-:te of B. ?nori. The larvne I\-crc ftltl with “II-1iridiiicx by Iknting the radioisotoI)c onto fresh

28

KALMAKOFF

ANI)

PAYNE

mulberry leaves 1-7 days after infection. RNA was extracted from the midguts of the larvae. Hyclr-oxyapatite Chromatography of RNA from the Silkwornt. A 6.0 X 0.9 cm column of hydroxyapatite was prepared in a jacketed column and equilibrated with 0.1 M phosphate buffer. The column temperature was maintained either at 60” or 90”. RNA samples were dissolved in 6 ml of 0.1 M phosphate and applied to the top of t,he column and equilibrated to t,he desired temperature for 5 min. The eluant was collected in a stepwise fashion in 6-ml aliquots using either different temperature or phosphate concentrations. RNA was detected by absorption at, 260 nm and/or the counting of azP or 3H-uridine labeled samples. Cerenkov radiation (10) of aqueous samples was used for detecting SzP while “H-uridine was detected using 50-111 aliquots of fractions dried on Whatman GF-81 glass fibre discs and counted after the addition of scintillant. TC’A Precipitation and Ribonuclease Digestion. 50-~1 aliquots of fractions were adjusted to a final concentration of 7.5% (w/v) trichloracet.ic acid (TCA) at 4°C. Precipitates were collected on glass fibre discs, washed with 10 ml 7.5% TCA and 10 ml et.hanol, dried, and counted. For ribonuclease digestion the samples were diluted and adjusted t,o 0.3 M Na+ concentration with sodium chloride and incubated with 2 pg/ml ribonuclcase for 30 min at 37”. The reaction was stopped by the addition of TCA. Precipitates were collected as described above. RESULTS

Telrtperature of Elation of TMV-RNA. To determine under what temperat.ure conditions TR4V-RNA could be eluted using 0.1 M phosphate the column was heated to 95” in 5’ increments. The results obtained for Fig. 1 represent the averaged profiles from three separate experiment,s. A “melting” curve was obtained for T&W-RNA with a Tm between 65-70”. This represents a translation of the Tnz curve to a higher temperature by 20” from that previously reported (2). It was found that at 90” about, 99% of the T?tlV-RNA could be recovered from the HA column. El&ion of 3’P-Labeled RNA from Silku~orm Larvae. It has been reported that ssRNA can be eluted from HA at 60” using 0.14 M phosphate (5). We carried out similar experiments using 0.1 and 0.15 M phosphate, and RNA from uninfected silkworms. A large portion of the radioactivity was associated with 0.1 M phosphate and t’his was found to be acid soluble in 7.570 TCA. The absorbance at 260 nm demonstrated that only a small amount of the RNA was eluted by either 0.1 M or 0.15 M phosphate at 60” (Fig. 2). However, if the column temperat.ure was raised

29

90 80

60

temperature

FIG. 1. Elution of TMV-RNA from hydrosyapniitr~ as :I function of iempcrntuw. After 3 min equilibration at each temperature, 5 ml of 0.1 M phoaphnte buffer was pumped through the column and the amount, of RNA cluletl was monitored. The amount of RKs eluted at, each tempcr:rtnre is c~sprwwtl as the cumulative prwntnge of thr total RXA wc,overcd.

2

4 fraction

6

10

no.

FIG. 2. Elution of B. )nori RXA from hydrosyapntitc. RN1 was elutcd by thy addition of 6 ml aliquots of 0.1, 0.15 and 0.3~ phosphat,e buffer with the column equilibrated to 60” or 90”. A = 0.1 M phosphate. 60”; 13 = 0.15 M phosphate. SO”; C = 0.15 M phosphate, 90”; D = 0.3 M phosphate. 90”.

30

KALMAKOFF

AND

PAYNE

FIG. 3. The elution of B. mori RNA from hydras)-apatite. RNA was eluted by the addition of &ml aliquok of 0.1, and 0.3 M phosphate buffer with column equilibrated to 60” or 90”. B = 0.1 M phosphate, 60”; B = 0.1 M phosphate, 90”; C = 0.3 M phosphate, 90”.

to 90”) the remainder of the RNA was eluted and moreover, no additional components could be recovered by flushing the column with 0.3 M ph0sphat.e (a condition for the elution of double-stranded nucleic acids) _ Using 0.1 M phosphate and 90”, conditions shown to eliminate sec-

88

*I B B zxx 0 E s 5 I ,I

11 11 10 99

30 25

I

a7

20

i

6 5

15

2 fj

4

10

G

7

3

8 f z’0

2

05

1 2

4

6 froctlo”

a

IO

12

14

no

Fro. 4. Hydroxyapatite labeled chromatography of ‘H-uridine RNA extracted from the midguts of 8. ~201% infected with a cytoplasmic polyhedrosis virus. The RNA was clutrd by 6-ml aliquots of 0.1 and 0.3~ phosphate buffer with the column equilibrated at 60” or 90°C. A = 0.1 M phosphate, 60”; B = 0.1 M phosphate. 90”; C = 0.3 M phosphate, 90”.

CIIROMhTOGRAPIIY

Characterization

Fractionsa Fraction 2 Fraction S Fraction II IIeat dennturntio~~c

OF

AND

TABLE: 1 of RIGA Elnted from

Elution

of fraction

ssRNA

0. 1 3% phosphate, 0. 1 IV phosphate, 0, :i RI phosphate, -

Rydrosyapat.ite (‘;, TCA precnipitnble

condit,ions W” MO” 90”

31

deRTu’A

47.3 too 100

(,; 1lXAae digeslior3

-.

9.; s 03.7 1.7 75.5

11

” Fractions correspond to “II-uridine labeled KNh in Fig. 4. * IIetermined by comparing 7.55; TCA precipitable matcrinl before and after rib+ nucleate digestion. c Hear denst~uration was carried out ai, 100” for 10 min in O.lll 11 phosphate, pEI 6.X.

o&try structure of T&IV-RNA (Fig. 1)) ssRiSA from silkworms could bc quantitatively eluted (Fig. 3). Further elation by 0.3 M phosphate at 90” did not. recover any additional RKB. Fractionation of &WA and d&VA from Silkworrt~ Larme. Pinglestranded and dsRNA from CPV-infectvcl larvae were labeled using .‘H-uriciine. RNA est.rnctctl from tilt mit1gut.s KXS fractionated by 0.1 M (ti0” and 90”) and 0.3 RI (90”) phoq~hute (Fig. 4). Unlike R?;A elutetl from uninfected larvae (Figs. 2 and 31 there wa5 a significant J)ortion of RNA elutcd by 08.3WI phosphate although as mwsure(l b;v absorbance at 260 nm the bulk of the RNA was eluted by 0.1 M ~~hosphatc at 90”. TC’A precipitation of SO-p.1 aliquotP of fractions 2, 8 and 11 (Table 1) show that although only 4i’cjo of the radioactivity in fraction 2 w:1 s prccil~itable, all the radioactivity in fraction 8 and I I XI:: prwil)itablc, and thcwforc is nl:lcro~nolccul:~r. Ribonuclcasc digestion of the RNA% slwic:: (Table 1 I sllo~ve that thv lnncromolr~ular RNA in fraction:: 2 and 8 was ssRNA. while fraction 11 was lwobably dsRNA. After htbat ckv~aturat,ion of fraction 11, 76% was rcn&retl acid soluble folloIv:ing ribonuclease digestion suggceting t,hat it WR:: tlsRNA. WC have confirmed that tile 0.3 M phosphate, 90” eluatc contains all 10 viral dsRN;Z ramI)onc>llts hy polyncryl:m~irlc rlectrol)horc& (unpublished reqllts).

Hlution of nucleic acids from the spcific competition bctwcen and phosphatjc groups of nucleic apatite (111. The fractionation ettxndcd DINA is presumably dur

lrytlrosyapatite is thought to be due to the phosphate ions of the eluting buffer acids, for the calcium ions of hydroxyof single-stranded IIN,4 from doubleto a diffcwnw in the charge distrit)lltion

32

KALMAKOFF

AND

PAYNE

of a helical configt:ration to that of a flexible, randomly coiled configuration. The fractionation of ssRNA from dsRNA should likewise reflect this difference in charge distribution. However, ssRNA displays considerable helical configuration making it more difficult to fractionate on hydroxyapatite since these regions of double-strandedness give the nucleic acid some of the properties of ssRNA and dsRNA’s. The use of high temperature (90” j makes it possible to distinguish bet.ween ssRNA with helical content and dsRNA. The elution profile oht-ained for T&IV-RNA using increasing temperature gave a “melting” curve with a T'rn between 65-70”. This is 20” higher than when hypcrchromicity was measured (2). Since the same molarity of phosphate was used in both cases, the difference can be explained by the necessity for all the secondary structure of the RNA to bc eliminated before it can be eluted from hydroxyapatite. At 90” presumably there is no secondary structure to ssRNA. It has been reported that seRn‘A can be eluted at 60” using 0.14 M phosphate buffer (5,ll). Although a small amount of RNA can be elutcd (Fig. 2), the majority of B. lrlori RNA requires either higher phosphate concentration (6) or higher temperature for elution. It can be seen from t’he T&IV-RNA “melting” profile (Fig. 1) that. at 60” and 0.1 M phosphat,e only about 11% of RNA had lost sufficient helical structure to be eluted from hydr0xyapatit.e. The results from Table 1 unambiguously characterize the nature of t,he RNA eluted from the column by stepwise elution. The dsRNA in fract,ion 11 represents the product,ion of virion RNA which has been shown to be double-stranded (12). After heat denaturation this fraction lost its ribonuclease resistance confirming that. it was dsRNA. Since less t,han 2% of the RNA eluted at 90” with 0.3 M phosphate was ribonuclease sensit.ive, this would indicate that the previous step of 90” and 0.1 M phosphate had removed all the ssRNA absorbed t,o the column. Conversely, very little, if any, of the dsRNA was eluted by 90” and 0.1 M phosphate sinrc the RNA in this fraction was ribonuclease sensitive. The ease with which ssRN.4 and dsRNA can he separated will make it possible to study the replicative forms of ssRNA viruses like the arboviruses and will also aid in studies on the stimulation of interferon by dsRN-4. ACKNOWI

2EDGMFYTS ,&

This research was supported by n grant from the Medical Research New Zealand. One of IIS CC. C. P.) was a holder of R University of dortornl fellowship.

Council of Otngo post

CIIRoNATOGRAPHl-

OF

ssRh’A

AND

33

&RNA

REFERENCES 1. I~TTI~N, 11. J., AND KOHNE, U. E. (196s) Sciemx 161, 529. 2. Dow. P., UOEDTKER. H.. FRESCO, J. R., HASELKORN, Ii., -4~1) IJTT, f'roc. A'uL. Aced. hi. I,‘. S. A. 45, 452. 3. PINVK. I,., HIRTII, L.. AND BERK~~RUI. G. (1968) Uiochent. f&piq~s. 112s. 31, 4Sl. 4. Lcwa~uows~~, L. J.. I D. H. L., ~UPRECHT, I<.. SIW>I)S. I<. W,i.. \NI) HPIEWLMAS. J. Viral. 8, 730.

M.

(1959)

C'~VLVLILI~. 4, S57. 9.

(1970)