A simple and rapid method for determining the chain length of oligonucleotides randomly labeled with P32

.\N\LYTIC.IL

BIOCHEMISTHY

A Simple Length

and

12, 34(3-356 (lQ65)

Rapid

Method

of Oligonucleotides

for

Determining

Randomly

Labeled

the

Chain

with

P32 ’

N. BURR FURLONG From the Department of Biochemistry, M. D. Anderson Hospital and Tumor

Received February

the University of Institute, Houston,

l’exas. Texns

1, 1965

In the course of studies on the solubility of deoxyoligonucleotidcs as a function of chain length and composition, a convenient method was developed for determining the chain length of oligonucleotides randomly labeled with P32. The method combines the enzymic cleavage of terminal phosphate (1) with the extraction of inorganic phosphate as the phosphomolybdate complex into heptanol [cf. ref. (2)]. For qualitative comparison of size distributions among different P32-labeled oligonucleotide solutions, we have found chromatography on DEAE paper sheets to be very useful. The quantitative length measurement mentioned above may be performed directly on portions cut from the DEAE paper sheets without elution. METHODS

Labeled Oligonucleotides Preparation: P3?-DNA was extracted from Escherichia coli grown in K,HP3’0, medium by a modification (3) of several methods. Then 5 mg of P32-DNA (approximately 3 X 1O’O counts/min/mmole phosphate) was incubated at 37°C in 10 ml of a solution 40 mM in pH 6.7 tris-acetate, 10 mM in MgCl,, and containing approximately 1 mg of crystalline pancreatic deoxyribonuclease I (Worthington Biochemical Corporation, Freehold, N. J.). Portions of 2 ml were removed at 2, 5, 15, and 60 min and heated at 95°C for 5 min to inactivate the enzyme. DEAE Column Chromatography: A 0.5~ml portion of the sample removed at 5 min was placed on a 9 X 200 mm column of DEAE-cellulose equilibrated with 0.01 M ammonium bicarbonate in 7 M urea. A gradient from this solution to one of saturated ammonium bicarbonate ‘This investigation was supported in part by Grants G-120 from the Robert A. Welch Foundation and CA-06910 from the National Cancer Institute of the U. S. Public Health Service. 349

350

N.

BURR

FURLONG

in 7 M urea was applied at a flow rate of 2 ml/min and the effluent monitored for absorbance at 270 rnp and for radioactivity. DEAE Paper Chromatography: Samples of solutions of 5 ~1 containing oligonucleotides were placed 2.5 cm apart 5 cm down from the top

FIG. 1. A positive print taken of an autoradiograph made on x-ray film placed in contact with a sheet of DEAE paper on which had been placed samples containing various oligonucleotide mixtures. These samples were obtained from tubes collected during the column chromatography of Paz-DNA digested with pancreatic DNase I, as described in “Methods.” The DEAE sheet was eluted with 0.7SM ammonium bicarbonate. The white line at the bottom represents the final solvent front.

CHAIN

LENGTH

METHOD

351

of a 23 X 28 cm sheet of DEAE paper. The paper was eluted by 0.75 M ammonium bicarbonate until the solvent reached to within 1 cm of the bottom. The paper was removed, dried, and placed next to a sheet of DuPont SL-313 x-ray film for 24 hr. Figure 1 shows the autoradiograph print of a DEAE sheet on which samples collected during the column chromatography of an oligonucleotides mixture were placed. Chain Length Ana.Zysis: From 10 t.o 50 ~1 of solution containing the oligonucleotide was placed on a 2-cm diameter disk of DEAE paper, dried, and counted in a Geiger counter to obtain total P32. In the cases in which the length determination followed chromatography on DEAE paper sheets, a region of the sheet 2 cm square was cut out and the total P32 counted. The disks, or the papers, were then cut in pieces and immersed in 1 ml of solution 2.5 N in NaCl and 0.1 M in pH 8.8 tris-Cl. Alkaline phosphatase from E. coli (obtained from Worthington Biochemical Corp., in a concentration of approximately 15 mg/ml in ammonium sulfate solution) was diluted 1:5 in distilled water, and 5 pl was added to the solution containing the paper squares. The solution was incubated at 60°C for 10 min and a second 5 ~1 of enzyme solution was added. After an additional 10 min, 0.5 ml was transferred to a small test, tube of 7-mm diameter. Then the following solutions were added and mixed sequentiaIly: 10 ~1 of 1 mM H,PO,, 0.25 ml of acid molybdate (2.5% ammonium molybdate in 1.2 N HCl), and 0.2 ml of nheptanol. After several mixings, using a Vortex Jr. mixer, the tubes were centrifuged at 100 X g for several seconds to remove water droplets from the heptanol and then 50 ~1 of the heptanol layer was placed on 2-cm diameter disks of Whatman 3 MM paper and dried in a ventilating hood. The disks were counted as above. These counts multiplied by 8 give the radioactivity in terminal phosphate positions. Chain length is calculated as the ratio of total to terminal P33 radioact.irity. RESULTS

In the development of this method the various steps were examined to establish the validity of the overall process. The optimum conditions for the activity of the E. coli phosphatase were determined, the effect of these conditions on the extraction of inorganic phosphate was measured, and the completeness of the phosphatase reaction was established. The conditions for optirnzm phosphatase activity were assayed spectrophotometrically (see Table 1‘1.The remarkable stability uf the E. coli phosphatase is evident from these data. The highest enzyme activity was found when the assay was carried out at a high temperature and a very concentrated salt solution. The use of high temperature and a high salt, roncentration in the chain lengt,h assay has several advantages.

352

N.

BURR

TABLE VARIATIONS

IN

FURLOh-G

1

PHOSPHATASE

ib2TIVITY

Assays for phosphatase activity were carried o\lt spectrophotometrically with the reaction conditions listed below. The standard assay was performed in cuvets of l-cm path length using a Beckman DB spectrophotometer and a logarithmic recorder. The instrument was set to zero absorbance at 400 rnp with identical solutions in reference and sample curvets. The reaction was started by the addition of phosphatase to t.he sample solution. The temperature of the standard assay was 23°C. The assay solution of 2.5 ml was 0.08 M in pH 8.7 tris-Cl, 0.27 mM in p-nitrophenyl phosphate, and contained 5 pl of a 1: 5 dilution of E. coli alkaline phosphatase in saturated ammonium sulfate as supplied by Worthington Biochemical Corp. The development of p-nitrophenolate was followed for several minutes and enzyme activity determined from the initial slope as A400mP units/min. lCU3 Reaction

1. 2. 3. 4. 5. 6. 7.

condition

Standard assay Medium 0.2 M Medium 1.0 M Medium 4.0 M Medium 0.04 hl Medium 1.0 III Phosphatase l/4 8. As in 7 but 4 ill 9. As in 7 at 40°C 10. As in 7 at 4O”C, 11. As in 7 at 60°C 12. As in 7 at 6O”C,

in SaCl in NaCl in NaCl in &HI’04 in urea (pH 9.0) that of standard in NaCl 4 RI in NsCl 4

M in NaCl

Activity

0.184 0.26s 0 276 0.4’22 0.000 0.036

0.047 0.135 o.o!a 0.216 0 160 0.421

These conditions will inactivate nuclease or die&erase activities with which the samples might be contaminated. In addition, when analyzing t,he chain length of oligonucleotides on DEAE paper sheets, the high salt, high temperature, and high pH all favor the dissociation of at least a portion of the oligonucleotide from the cellulose ion exchanger, a condition which may be necessary for terminal phosphate scission by the phosphatase. The concentration of phosphatase used in the series of experiments at various temperatures was lowered to keep within the range of accurate slope determinations at the highest activities. The reaction is completely inhibited by phosphate at a concentration of 0.04 M. The effect of urea was tested to determine whether samples obtained from DEAE columns eluted with 7 M urea solutions could be tested directly without intcrfcring with terminal phosphate scission by t,he enzymcl. The data indicate that the urea must be diluted well below 1.0 M to retain full phosphatase activity. The extraction of inorganic phosphate was measured under a variety of conditione, as listed in Table 2. With thch standard amount of phos-

CHAIN

EXTRACTION

LENGTH

353

METHOD

TABLE 2 OF INORGANIC

PHOSPHATE

The reaction mixture contained: 0.1 ml HaP3*04 (8000 cpm, sp. act. = 2 X lo9 cpm/ mmole), 5 pl 1 m&I H3P04, and 1 ml Hz0 to which was added 0.2 ml acid molybdate and 1 ml n-heptsnol. The mixture was shaken and centrifuged briefly to layer the heptano1 and samples of both layers were counted for P3* radioactivity. Reaction

1. 2. 3. 4. 5. 6. 7. 3. 9. 10. 11.

‘& I’32 extracted

condition

iuto heptanol

99.5 92.0 97.5

Standard No carrier phosphate 5 X carrier phosphate Butanol used to extract 10 ml Hz0 added 4 ml heptanol added Solution 4 M in HClOd Solution 5 M in NaCl Solution 0.4c0 in uranyl acetate Solution 0.1 mg/ml in DNA Solution 0.1 mg/ml in oligonucleotide

97.0 76.0 99.5 96.0 96.0 05.0 75.0 70.0

phate carrier, t,he heptanol layer is light yellow; either no color or too deep color are signals of lowered extraction efficiency. Butanol can bc used for extraction at only slightly reduced efficiency. The ratio of organic to aqueous phase should be kept above 1:5 for good extraction. The addition of various salts does not interfere seriously with the extraction, but DNA and oligonucleotides markedly affect the amount of inorganic phosphate ext.racted. See below for further discussion of this effect. The test for colnpleteness of the phosphatase reaction was performed using TMP”’ as described in Table 3. It can be seen that 91y0 of the TMP3? has been degraded in 20 min. The standard assay described in the “Methods” section for chain length determination specifies the use TABLE COMPLETENESS

3

OF PHOSPHATASE

REACTION

To 5.0 ml of 0.02 mM TMPa* (approximately 8000 cpm total) in 0.1 M pH 8.7 tris-Cl 5 ~1 of a 1: 10 dilution of Worthington’s E. coli phosphatase solution was added. Samples of 0.25 ml were removed at 1, 5, and 10 min. Sfter incubation at 6O”C, a second 5 ~1 of enzyme was added and further samples taken at 11, 15, and 20 min. Each sample was placed in 50 ~1 acid molybdate solution (2.5y0 ammonium molybdate in 1.2 N HCl) as they were collected. Then 5 ~1 of 1 mM HsP04 and 0.2 ml n-heptanol were added, shaken, and layered. Portions of both layers were dried on disks for counting. Time, min %, P32 in heptanol

1 19.7

5 58.0

10

11

15

65.3

69.3

80.7

20 91.0

354

h-,

BURR

FURLONG

of twice as much enzyme and also takes advantage of the accelerated reaction rate in concentrated salt solution (shown in Table 1) to ensure complete reaction in 20 min. Actually, assays with oligonucleotides in the standard procedure hare shown that very little additional inorganic phosphate is produced after the first 10 min. Chain length analysis of oligonucleotidcs from the chromatogram whose autoradiograph is shown in Fig. 1 arc prcsent’ed in Table 4. These T.kBLE CII~IN

The

DEAE

LENGTH

4

OF OLIGONUCLEOTIDES

SHOWN

paper spotscontaining P3”-labeled oligonucleotides

analvzed without COlUIXl tube No.

ANALYSIS

FIG. 1 were cut out and

IN

elution 8s described in “Methods.” Ii/ 011 paper

8

0.59

9 11 13 15 17

0.51’ 0.50 0.41 0.35 0.29

Total cpm

Terminal

4,130 4 ) 360 3,465 6,140 10,688 14,260

1,900 1,760

Aw;;F;in

CPm

1,060 1,510 1,720 1,580

2.08 2.48 3.26 3.90 6.21 9.03

samples were obtained during the column chromatography of mixed oligonucleotides, and placed on DEAE sheets for a second elution as explained earlier. Analysis of these spots for average chain length shows a good correlation between position on the DEllE sheet and chain length of the oligonucleotides. In fact for t,he range of sizes in t’his experiment the relat,ionship between chain length and reciprocal Rf is almost linear. Polynucleotide fractions of average chain lengt,h up to 100 have been :malyzetl by this technique wit,11good reproducibility. The presence of oligonuclcotides has been reported (4~ to interfere with the Fiske-SubbaRom phosphate :lnalyAs. In the Fiske-SubbaRon analysis, the yellow pl~ospl~o~uolybdntc complcs is chemically reduced :md it is not, clear nhet,her oligonuclcoticlcs affect the fornlntion of the ])hosphomolybdat,e complex, the color development, or the availability of the phosphate. In our cspcriments it can be seen that oligonucleotide:: interfere with the extract,ion of pl~ospl~on~olybclateinto heptanol (Table 5). We found that oligonuclcotides, and also DKX. added to an aqueous inorganic P3? phosphate solution, which is then treated with acid molyb(late and heptanol, reduced the total number of counts in both the lleptanol and the water layers. Since DNA and the longer oligonucleotides are insoluble in the extraction mixture, we suggest that inorganic Ilhosphate is trapped in the insoluble oligonucleotide precipitate. The clata indicate t,hat the removal of the oligonucleotide by charcoal restores

CHAIN

ANALYSIS

LENGTH

355

METHOD

TABLE 5 OF OLIGONUCLEOTIDE

INTERFERENCE

The reaction mixture contained: 0.1 ml H3P3*04 (250,000 cpm, sp. act. = 4 X lOlo cpm/mmole), 15 ~1 1 mM HsP04, 1 ml HzO, and the amount of 1 mg/ml oligonucleotide solution indicated. To duplicate sets of tubes, 30 mg activated coarse charcoal was added and shaken 15 min. Absorbance at 260 rnp of these solutions indicated charcoal treatment removed oligonucleotides to below 5.0 pg/tube except for the tubes containing 25 and 100 fig of the long oligonucleotides, where the final concentrations were 14 and 18 rg /tube, respectively. Then 0.2 ml acid molybdate and 0.5 ml heptanol were mixed in each solution, the heptanol layered by brief centrifugation, and 25-pl samples of heptanol and water layers put on paper disks and counted. % Paz in heptanol Amount of oligonucleotides, P6

-

Limit digest (ave. length = 4)

Long oligonucleotides (ave. length = 17) No charcoal

Charcoal

No charcoal

Charcoal

0 10

94.5 94.1

98.2 ‘36.5

Y4.5 Y2.Y

95.2 -

25 100

93.6 91.5

97.2

94.4 91.6

98.2 Y6.7

the extractability. In the chain length analysis carried out after alkaline phosphatase in 2 Jl NaCl, we found less interference by oligonucleotides. When the chain length of oligonucleotides on DEAE paper is obtained, oligonucleotide interference is found to be negligible. The DEAE paper acts in the same way as the charcoal in that probably only the shorter oligonucleotides are eluted entirely, and these oligonucleotides are soluble in the phosphate extraction solution. Thus in the chain length assay we suggest that oligonucleotides solutions bc placed on DEAE paper prior to digestion by alkaline phosphatase. In pilot experiments an attempt has been made to apply these techniques to determine the chain length of nonradioactive oligonucleotides. Some success has been achieved using the standard procedure, scaled up to process ten times the usual amounts and volumes, followed by extraction of the phosphomolybdate into a small volume of aqueous solution containing a reducing dye, and analysis of the blue color at 660 mjc. Total oligonucleotide phosphate is easily determined by any of the modifications of the Fiske-SubbaRon(5 1 method following either chemical or enzymic digestion. SUMM.IRT

The chain length of P3’-labeled oligonucleotides was determined couveniently on DEAE paper disks by counting total radioactivity and then digesting the terminal phosphate with E. coli alkaline phosphatase at

356

S.

60°C in strong salt solution. \Tith molybdate and extracted assayed for P32.

BGRR

FURLONG

The inorganic into heptanol,

phosphate was complexed plated on paper disks, and

AC’IXOWLEDGMEIVTS This research was begun while the author studied at the Oak Ridge National Laboratories as a Postdoctoral Fellow of the National Institutes of Health. The author wishes to thank Dr. F. J. Bollum of the Biology Division, Oak Ridge National Laboratories, for suggesting the possibility of this procedure. The author is indebted to Mrs. Nancy L. Williams, Mr. Crcg McClure, Mr. William Risser, ant1 Miss Carol Stephens for technical :t&st:mc.c iu tlcvelopment of the method. REFERENCES 1.

K. L., AND KOEHWX, J. F., J. Aal. C’lrcjrl. Sot. 74, 283 (1952). B., ASD 1~.4RDr, H. A.. J. BiOl. Che??~. 225, 889 (1960). K. B., i?~ “Methods in Pkzymology” (S. 3. HURLBERT, R. H., .~ND ~ua~on-c:. Colowick and N. 0. Iinplan. cds.), Academic Press, Sew York. in press. 4. BOLLUM, I?. J., J. Biol. Chcm. 237, 1945 (1962). 5. FISKE, C., AND SrHsaROW, T., .I. hrl. Chefn. 81, 629 ( 1!1%). SINSHEIMER,

2. HACIHARA,

I’.