Volume 2 number 12 December 1975

Nucleic Acids Research

The preparation and properties of 4-thiouridine containing 2'-S' and 3'-5' dinucleoside monophosphates M.Keren-Zur, R.Levy and Y.Lapidot Department of Biological Chemistry, The Hebrew University of Jerusalem, Jerusalem, Israel.

Received 29 October 1975

ABSTRACT 2'-5' and 3'-5' dinucleoside monophosphates containing 4-thiouridine were prepared by the thiolation of the cytosine containing compounds and purified by chromatography on a DEAE-Sephadex column. The chromatographic and optical properties of the isomers are compared. INTRODUCTION Dinucleos ide monophosphates repres ent the s mallest diester unit of nucleic acids, and were used as model compounds for chemical, physicochemical and enzymatic studies. The influence of the nature of the phosphodiester linkage on the conformation of the molecule was investigated. It was found that the conformation of dinucleoside monophosphate containing the 2'-5' linkage differ from 1-3 that of the corresponding 3'-5' isomer 3. It was suggested that the 2'-5' isomer is more compact as compared to the 31-5' isomer 3. Several tRNA molecules contain 4-thiouridine (s U). It is of great interest, therefore, to study the properties of 4-thiouridine containing oligonucleotides. Moreover, the unique optical

and chemical properties of 4-thiouridine make it most suitable for conformational studies. Some 4-thiouridine containing dinucleoside monophosphates were prepared 4'5. In this communication the preparation and purification of 2'-5' and 3'-5' isomers of 4-thiouridine containing dinucleoside monophosphates is described. The chromatographic behaviour and optical properties of the products are discussed. 2289

Nucleic Acids Research MATERIALS AND METHDDS

DEAE-Sephadex A-25 with a capacity of 3.5 mequiv/gr was purchased from Pharmacia, Uppsala, Sweden. Pancreatic RNase (2 x crystallized) and T1 RNase were purchased from Sigma Chemical Co.. U.S.A. T2 RNase was prepared according to the procedure described by Uchida and Egami6. Dinucleoside monophosphates containing 2'-5' and 3'-5' linkage were prepared according to Lapidot and Barzilay . Thiolation of dinucleoside monophosphates was performed according to Miura et al. . The column chromatography apparatus consisted of a 2.5 x 40 cm column, a UV flowthrough cell, 254 nm (LKB Uvicord 1), recorder (LKB 6520) and LKB fraction collector. The column was loaded with the thiolated products and the elution was carried out with a linear gradient of ammonium bicarbonate. The mixing vessel contained 3 liters of 0.05 M ammonium bicarbonate and the reservoir contained an equal volume of 0. 5 M ammonium bicarbonate. The flow rate was 6 ml|min and fractions of 20 ml were collected. Paper chromatography was carried out on a Whatman No. 1 paper by the descending technique. Paper electrophoresis (0.03 M phosphate buffer pH 7. 1) was performed in a high voltage apparatus (4. 500 V; 45 V/cm) in which the paper was immersed in a high boiling petroleum fraction (versol). Spectroscopic measurements. UV absorption was measured on a Cary 14 spectrophotometer at 25°C. Hypochromicity at Xmax was calculated according to the equation:

lo hypochromicity

=

100 x

OD (hydrolysate) - OD (dimer) OD (hydrolysate

Hydrolysis of dinucleoside monophosphates was performed by incubation in 0. 3 M KOH at 35°C for 17 hrs. The base was then neutralized with HCl. All volumes were measured precisely and were taken into account in calculating the absorbance. The' entire procedure was carried out in closed cuvettes. Extinction coefficients were calculated from the extinction coefficient of the hydrolysate (330 = 21000) and hypochromicity. The spectral data at pH 1

22as

Nucleic Acids Research (0. 1 M HCl) and pH 9.0 (0. 005 M Tris-HCl buffer) were calculated relative to those at pH 6. 5 (0. 005M cacodylate buffer). CD spectra in the region of 220-400 nm were measurled on a Cary 60 spectropolarimeter, equipped with CD attachment 6001, using a pen period of 3 sec and a full range sensitivity of 0.04 mdeg. Samples were heated at 60°C for 5 min and measurements were taken at 260C. In all measurements the light path was 1 cm and the optical density at 330 nm was 1-2, i.e. a concentration of about 10-4 M. Samples were dissolved in 0. 01 M cacodylate buffer, pH 6. 5. Concentrations were determined from the absorbance at 330 nm (the hypochromicity of the different compounds is given in Table 3). CD results are given in molar ellipticity.

RESULI AND DISCUSSION The synthesis of 4-thiouridine containing dinucleoside monophosphates was- achieved by the amino-thiol exchange reaction . The starting material for the thiolation was a mixture of 2'-5' and 3'-5' dinucleoside monophosphates containing cytidine bases. The resulting 4-thiouridine containing dinucleoside monophosphates were purified on a DEAE-Sephadex A-25 column. Fig. 1 shows the separation of adenylyl (2'-5') 4-thiouridine from the 3'-5' isomer, as well as from the starting materials (adenylyl (2'-5') cytidine and adenylyl (3'-5') cytidine). In the same manner all the other 4-thiouridine containing dinucleoside monophosphates were prepared. Their elution The different fractions from each peak were pooled, concentrated to a

small volume by evaporation under reduced pressure and lyophilized. Aliquots of each peak were treated with the appropriate ribonuclease (pancreatic ribonuclease in the case of s4UpN and Ups 4U, ribonuclease T1 in the case of Gps4U and ribonuclease T2 in the case of Aps4U) and the products were analyzed by high voltage paper electrophoresis. All dinucleoside monophosphates containing the natural 3'-5' linkage were completely degraded to the appropriate nucleoside 3'-phosphate and the free nucleoside, while those containing the unnatural 2'-5' linkage were resistant to the enzymatic hydrolysis. Treatment of each peak with 2291

Nucleic Acids Research I!

C 0

'41 c 0

-

c

-o0

C.)

0

~0

0

U)

150 100 Fraction Number Fig. 1. Separation of thiolated ApC

A260'; pattern

---

on a

330*

For

on a

experimental

DEAE-Sephadex A-25 column.

details

see

text.

DEAE-Sephadex A-25 column is summarized in Table 1.

Table 1 Elution pattern of 4-thiouridine containing dinucleoside phosphates on a DEAE-Sephadex (bicarbonate) column.

Dinucleoside monophosphate

Aps4U s4UpA Gps4U s4UpG Ups4U s4UpU

s4Ups4U

mono-

Peak position (fraction number)

2'-5' linkage

3'-5' linkage

81

127

115

129 147

-

122 39 64 176

-

53 116 226

For details see text.

0.3 M KOH resulted in the formation of the appropriate nucleoside 2'(3') - phosphate and the free nucleoside.

2292

-

Nucleic Acids Research From the results shown in Fig. 1 and Table 1, it is clear that each dinucleoside monophosphate containing the 2'-5' linkage is eluted before the corresponding 3'-5' isomer. In addition the 4-thiouridine containing dinucleoside monophosphates

are

retarded

on

the column

as

compared to the cytidine containing dinucleoside monophosphates (Fig. 1). This phenomenon

be explained by the fact that the thio

can

group

is

par-

tially ionized at pH 8.6 (the pH of ammonium bicarbonate). It is worthwhile to mention that, although C(2'-5')U cannot be separated from

DEAE-Sephadex A-25 column, using a gradient of ammonium bicarbonate! , the s4U(2 1-5')U is well separated from

C(31-5')U

on a

s4U(3'-5')U (see Table 1).

Fig. 2 shows the

paper

electrophoretic mobility of the

different 4-thiouridine containing dinucleoside monophosphates at

pH 7. 1. The electrophoretic mobility of the natural 3'-5' isomers differ from the mobility of the 2'-5' isomer, and again the 2'-5' isomers

migrate faster than the 3'-5' isomers. The

UV

spectral data of the purified 4-thiouridine

con-

taining dinucleoside monophosphates is summarized in Table 2. The spectra of the 4-thiouridine residue (around 330 nm) are almost the

0 0

0

0

00

2-5

3S-5' 3L5' 2!-5!

0

Y-5'

Ionophoretic see

0

2~-5

3~5

3S-5' 2-5!

2!-5

mobilities of 2'-5'

phosphates containing details

0

0

s4UpA Ups4U s4UpU

s4 UpG Gps4U Aps4U

Fig.2.

00

00

4-thiouridine at

3S'5

s4UpsU s4U5!ps4Up s4 and 3'-5' dinucleoside

pH 7.2.

For

mono-

experimental

Materials and Methods.

2293

Nucleic Acids Research

Table 2

Spectral data of 4-thiouridine containing dinucleoside monophosphates 1.0

pt1

p'l 6.5

DINUCLEOS IDES

ph

9.0

MONOPHOSPHATES Ext.coef.

io-3

7

0ln

A330/

AA330/

Ext.coef. 10-3

.,

A260

Ext.coef.

>

0-3

033C/

ax

x

330,255 330.256

19.6

17.2

288.230

1.2

331.257

18.2

16.6

287.229

1.1

324.257

17.2

S4aUpA

1.0

15.5

1.4

330,257

19.6

15.5 16.7 12.3

1.2 1.3 1.5

331,251

18.4

20.5

17.0 296,225 17.5 295,229 13.7' 289,230

322,257 324,250 323,250 324,258

18.0

18.1 17.8 13.8

295,228 294.226 289.235

1.3

330,250 330,258

1.4 1.3 1.3 1.5

18.0

330,255 330,258

18.1 20.1 20.7

137.7 287.229

284,227

Gps4U s4UpG UpS4U

287.229 300,229 298,226 289,230

16.5 13.2

287.229

20.5

s4uru

330,260 21,2

1.5

330,260

20.7

13.5

289,232

1.5

322,260

17.3

14.8

330,245

39.2

13.7 8.0

289,232

s4Ups4U

277,234

6.4

330,245

39.0

7.8

277,234

6.3

323

36.3

332,256 330,255 330,254 930,257 330,260 330,245

19.1

17.8

290,230

1.1

19.4

287.228 287,230

1.1 1.0

324,260 324,257

295.229

1.1 1.5

324,250

Aps4U

331,254

A260

x

x

19.2

min

max

x

18.1 17.7

min

A260

1.1 1.1

1.2

288,235

1.1

270

5.9

287,231 286,230 295,228 288,234

0.9 1.1 1.1

2'-5' linkage

Aps4U

s4UpA s4UpC *

*4UpU

-s4UPS4U

18.2

P.5

289,230

1.1

18.6 20,2

17.0 13,5

300,225 288,230

1.2 1.5

333,258 330,257 329,250 330,256

17.7 17.1 20.2

17.2 16.5 17.8 13.6

19.1

12.5

290,230

1.5

330,260

18.6

12.3

37.7

8 0

278,230

6.4

330,245

37.2

7.6

288,230

290,230 278,234

18.0

17.6 16.9 18.6 9.9

1'5.9 289,232

323,258

16.5 17.4 12.0

1.5

324,261

20.7

6.8

324

34.8

267

1.0

1.2 5.2

For experimental details see Materials and Methods.

same for pH 6.5 and pH 1. However, a significant change is observed

when the pH is raised to 9. This difference is -due to the partial ionization of the SH group which causes a blue shift. The 'max and Amin at all pH values were the same for the 2'-5' dinucleoside monophosphates and the 3'-5' isomers. On the other hand, the extinction coefficient of the 2'-5' isomers was as a rule lower than that of the corresponding 3'-5' isomer. In other words, the hypochromicity of the 4-thiouridine containing 2 '..5' dinucleoside monophosphates is higher than that of the corresponding 3'-5' isomer (Table 3). Further evidence for conformational differences between the two isomers of the 4-thiouridine containing dinucleoside monophosphates can be drawn from their CD spectra. As can be seen in Fig. 3 I, the linking of the two 4-thiouridine nucleosides by a phosphodiester bond causes splitting of the Cotton effect in the 330 nm region, but whereas the amplitudes of the splitted Cotton effect in the 3'-5' isomer are high, the amplitudes in the spectrum of the 2'-5' isomer are small, even smaller than that of the monomer. A splitted Cotton effect of 4-thiouridine bases is found in the spectrum of tRNATyr.

2294

Nucleic Acids Research Table 3 The hypochromicity of 4-thiouridine containing dinucleoside monophosphates

Dinucleoside monophosphate

Aps4U s4UpA Gps4U s4UpG Ups4U s4UpU s4Ups4U

Hypochromicity (%) at 330 nm 2'- 5' linkage 3'- 5' linkage 8

13

16

7 12

18 4 11

8 2

11

7

3

For experimental details see Materials and Methods. from E. coli which has two adjacent 4-thiouridine bases8, but in this case the long wave cotton effect is negative. Fig. 3 II and ILI represent the CD spectra of four isomers of U and 4-thiouridine containing dinucleoside monophosphates. These isomers differ in the sequence and the phosphodiester linkage. It is very interesting to note that the two 3'-5' isomers (Ups4U and s4UpU) have very similar CD spectra which differ significantly from the corresponding dinucleoside monophosphates with the 2'-5' linkage. The two 3'-5' isomers have a spectrum similar to that of the monomer but with a higher amplitude at 330 nm. The two 2'-5' isomers have, on the other hand, a very weak and broad negative ellipticity. The center of the negative ellipticity is found at about 310 nm and, therefore, it is not a simple inverted cotton effect as was found by Samejima in the case of s4UMP bound to RNase9. A possible reason for this ellipticity may be an interaction between the transitions at 330 nm and 260 nm. Similar differences in the CD spectra of 3'-5' and 2'-5' isomers were found in adenine and guanine dinucleoside monophosphates containing 4-thiouridine (results not shown). It can be concluded therefore that the nature of the phosphodiester linkage (2'-5' or 31-51) influences the conformation of the whole molecule.

2295

Nucleic Acids Research

200

250

300

350

400

\nm Fig. 3. CD spectra of 2'-5' and 3'-5' dinucleoside monophosphates containing 4-thiouridine. For experimental details see Materials and Methods. The dinucleoside monophosphates containing the 2'-5' linkage are more compact and, therefore, are eluted from the column before the 3'-5' isomers (Fig. 1). The same phenomenon was also observed with the 3 dinucleoside monophosphates containing the common bases In several previous papers, concerning the CD spectra of tRNAs in the 4-thiouridine region, a negative ellipticity was found in some cases . It was not clear whether the negative sign is a direct

2296

Nucleic Acids Research result of the primary structure

or

the result of the conformation of the

4-thiouridine region. Our results show that when A, U or G are the neighbours of 4-thiouridine in dinucleoside monophosphates, the CD of 4-thiouridine is positive. It can be concluded, therefore, that the negative ellipticity of the 4-thiouridine region in the tRNA molecule is due to the tertiary structure and not only to the neighbouring bases. More accurate information can be drawn from CD spectra in different solvents and from

trinucleoside diphosphate containing 4-thiouridine.

*Department of Molecular Biology, University of Paris VII, Paris,

France.

REFERENCES 1 2

3

Warshaw, M.M. and Cantor, C.R. (1970) Biopolymers 9 1079-1103. Kondo, N.S., Holmes, H.M., Stempel, L.M. and Ts'o P.O.P. (1970) Biochemistry 9, 3479-3498. Sussman, J.L., Barzilay, I., Keren-Zur, M. and Lapidot, Y. (1973) Biochim.Biophys.Acta 308, 189-197.

4

Scheit, K.H. (1968) Biochim.Biophys.Acta

5 6 7 8

Miura, K., Shiga, M. and Ueda, T. (1973) J.Biochem. 73, 1279-1284. Uchida, T., Egami, F. (1967) Methods in Enzymology 1 239-247. Lapidot, Y. and Barzilay, I. (1972) J.Chromatog. 71, 275-281. Saneyoshi, M., Arami, T., Nishimura, S. and Samejima, T. (1972) Arch. Biochem. Biophys. 152 677-684. Samejima, T., Kita, M., Saneyoshi, M. and Sawada, F. (1969) Biochim. Biophys.Acta 1 1-9.

9 10

285-293.

Willick, G.E., Kay, C.M. (1971) Biochemistry

2297

The preparation and properties of 4-thiouridine containing 2'-5' and 3'-5' dinucleoside monophosphates.

2'-5' and 3'-5' dinucleoside monophosphates containing 4-thiouridine were prepared by the thiolation of the cytosine containing compounds and purified...
612KB Sizes 0 Downloads 0 Views