Eur. J. Biochem. 13 (1970) 658-564

Polyribonucleotides Containing a Phosphorothioate Backbone

Fritz ECESTEIN and Hans GINDL Max-Planck-Institut fiir Experimentelle Medizin, Abteilung Chemie, Gottingen

(Received December 16, 1969/ January 31,1970)

The enzymatic synthesis of two polyribonucleotides, poly A g U) and poly r(g U) containing a sugar-phosphorothioate instead of a sugar-phosphate backbone, is described. The formation and thermal stability of double-stranded polymers is not impaired by this modification. They can also serve as templates for RNA-polymerase. Single stranded polymers can serve as mes- sengers. Both double and single-standed polymer exhibit some degree of resistance to nucleases.

Unusual Abbreviations. Following recommendations by Dr. W. E. Cohn (Director of the National Academy of Sciences-National Research Council, Office of Biochemical Nomenclature, Oak Ridge National Laboratory, Oak Ridge, Tennessee, U.S.A.) the phosphorothioate-containing com- pounds are symbolized as follows :

0 S II I1

pps-u = -0-P-O-P-O- I I yo+

d OH 0-

OH OH uridine 5’-0-( 1-thiodiphosphate) ;

0 0 s I1 II /I

ppps-u= -0-P-O-P-O-P-O- I I I I/O+ OH 0- 0- \-/

I AH OH uridine 5’-O-(l-thiotriphosphate);

0 0 S 11 I/ I!

I I I PPPB-A = -0 -P- 0 -P- 0 -P- 0 -

-/ I I

OH 0- 0-

6H b H adenosine 5‘-0-( 1-thiotriphosphate) ;

poly r(;U) = homopolymer derived from pps-U; poly r(A ; U) = alternating copolymer derived from ppp-A

poly r(; A ; U) = alternating copolymer derived from ppps-A

Enzymes. DNA-dependent RNA-polymerase or nucleo- side triphosphate: RNA nucleotidyltransferase (EC 2.7.7.6) ; polynucleotide phosphorylase or nucleoside diphosphate : polynucleotide nucleotidyltransferase (EC 2.7.7.8) ; deoxy- ribonuclease or deoxyribonucleate oligonucleotido-hydrolase (EC 3.1.4.5); snake venom phosphodiesterase (EC 3.1.4.1); spleen phosphodiesterase (EC 3.1.4.1); pancreatic ribo- nuclease (EC 2.7.7.16); micrococcal nuclease (EC 3.1.4.7); alkaline phosphatase or orthophosphoric monoester phos- phorylase (EC 3.1.3.1).

and ppps-U;

and ppps-U.

Recently we described [2] the synthesis and some properties of a polyribonucleotide, poly r(A U) in which every other phosphate was replaced by tt phos- phorothioate group. This modified polynucleotide was degraded by nucleases a t a considerably slower rate than the unmoditied polynucleotide, poly r(A-U). The present investigation was undertaken to deter- mine the effects of replacement of all phosphate groups in a polynucleotide by phosphorothioate. Parts of this work have been published in a prelimi- nary report [ 11.

MATERIALS AND METHODS

ppps-U,[35Slppps-U, pps-U and [35Slpps-U were synthesized as described previously [3] but using 2’,3’-0,0-methoxymethylideneuridine [a] instead of 2’,3’-0,0-ethoxymethylideneuridine as starting ma- terial. The synthesis of ppps-A is described below. All substrates were purified over DEAE-Sephadex A 25 with linear gradient of Et,NHCO,. [14C]Poly r(A I U) was synthesized as described [2] and worked up by method B described below. DNA-dependent RNA-polymerase from Escherichia coli was a kind gift of Dr. H. Sternbach, Gottingen. It was prepared according to Zillig [5], and had a specific activity of 1140- 1470 as defined by Zillig.

Polynucleotide phosphorylase from E . coli was a generous gift of Dr. D. Swan, Gottingen. It had a specific activity of 215 pmoles of ADP polymerized x mg protein-1 x h-1.

Pancreatic ribonuclease, spleen and snake venom phosphodiesterase were purchased from Boehringer Mannheim GmbH (Mannheim, Germany), micro- coccal nuclease from Sigma Chemical Comp. (St. Louis, U.S.A.), Deoxyribonuclease I (DPFF, RNase free) from Worthington (Freehold, U.S.A.) and U,U from Waldhof (Mannheim, Germany). Poly d(A-T) was generously supplied by Dr. A. Lezius, [3H]poly d(A-T) by Dr. T. Jovin and [3H]poly rU by Mrs. E. Gaertner (Gottingen).

Vol.13, N0.3,1970 F. ECKSTEIN and H. GINDL 559

Paper chromatography was carried out by the descending method on paper Schleicher and Schull 2043 b (washed) in system A: ethanol-I M am- monium acetate (7:3, v/v); on Whatman 3 MM in system B: iso-butyric-acid-2 N ammonia-0.2 M EDTA (120 : 72 : 2, v/v/v). Electrophoresis was car- ried out in 0.05 M ammonium formate (pH 3.5).

The progress of synthesis as well as of degradation of polynucleotides was monitored as described by Bollum [6] except for poly r(eU) where aliquots of the reaction solution were placed on paper strips (2 x 10 cm) (Whatman 3 MM). After elution with 0.3M ammonium formate and drying, the poly- meric material remaining at the starting zone was cut out and counted.

Melting curves were taken with a Gilford Mod. 2000 recorder connected with a Beckman Mod. DUR spectrophotometer, in combination with a Neslab LTP-I temperature programmer.

Radioactivity was counted in a liquid scintillation counter (Tricarb. Mod. 4312).

MZ=O and sio=: values were determined by low speed equilibrium centrifugations in a Spinco ModelE analytical ultracentrifuge equipped with a photo- electric scanner in 0.05 M sodium citrate buffer [7]. The partial specific volume was assumed to be 0.47 [8].

Concentrations of polymers are calculated on the basis EE60= 6650 for poly d(A-T) [9] and EZeo= 6100 for poly r(A-U) [lo]. The latter value was also taken for poly r(E A U).

SYNTHESIS O F SUBSTRATES

Adenosine 5'-Thiophosphoroimidazohte ( I ) To 3.2 g of 2',3'-0-ethoxymethylideneadenosine

(10 mmoles) [ill dissolved in 100 ml of dry dimethyl- formamide, 6.1 g of triimidazolyl-1-phosphinsulfide (20 mmoles) [12] dissolved in 100ml of dry tetra- hydrofurane were added under stirring. After 12 h, the solvent was evaporated and the residue left in 150 ml of water (brought to approx pH 8.5 with ammonia) for 2 h. After evaporation, the material was chromatographed on a DEAE-Cellulose column using a linear gradient of 0.01 M-0.125 Et2NHC0, (pH 7.5). Compound I was eluted with approx. 0.06M buffer. It had the same mobility as ApA in electrophoresis,

Yield: 48000 absorbance units at 259 nm (32 ; RF (System A) = 0.69; 1:: = 259 nm ( E = 15400).

Adenosine 5'- 0 - (1 - Thiotriphosphate) (11) To 30000 absorbance units (259 nm) of I (2 mmo-

les) in 80 ml of dry dimethyl formamide, 20 mmoles of tri-n-butylammonium pyrophosphate in 20 ml of dry dimethylformamide were added. After 20 h a t room temperature, the solvent was evaporated and the residue left for 4 h in 50 ml of loo/,, aq. acetic

acid. After evaporation of acetic acid the residue was chromatographed on a DEAE-cellulose column using a gradient of 0.05-0.5 M Et,NHCO,. The product was eluted with approx. 0.3M buffer. For further purification it was rechromatographed on DEAE- Sephadex A 25 using a linear gradient of 0.05-0.6 M Et,NHCO,. The product had the same mobility in electrophoresis as ATP.

Yield: 2700 absorbance units at 259 nm (9 o / o ) ; RF (System B) = 0.04; 12: = 259 nm ( E = 15000); adenosine/phosphate = I .0/3.07.

[35S]ppp,-A was synthesized essentially in the same way starting from [35S]PSC13.

POLYMERISATION AND ISOLATION OB PRODUCT

Poly r(; A U) and Poly r(A-U) The standard incubation solution contained,

unless stated otherwise, 8 mM Tris-Ac pH 7.9, 8 mM MgAcz, 0.12M NH,Cl, 1 mM each of [35S]ppp,-U and ppps-A or [14C]ATP and UTP, 0.3 mM poly d(A-T) and 140 units of polymerase per ml of incubation solution for the synthesis of poly r(gAgU) and 25 units for the synthesis of poly r(A-U).

The polymers used for enzymatic degradation experiments and nearest neighbour analysis were worked up according to method A, which does not remove protein.

Method A After 6 h of incubation a t 37", the reaction was

stopped by additions of an equal volume of 1 M NaCl solution and an excess of ethanol a t 0"; the precipitate was centrifuged at 0" for 10min at 10000 rev./min, the pellet dissolved in water and chromatographed on Sephadex G-50. The polymer was stored in water a t -20".

Method B After approx. 6 h a t 37", 0.3 mg of DNase I in

0.3 ml of water was added per ml incubation solution. The reaction was stopped 20 min later by repeated extraction of protein with iso-amylalcohollchloro- form [13]. The aqueous layer was concentrated to approx. 0.5 ml and the polymer isolated and desalted by chromatography an Sephadex G-25, 6-200 and again G-25. It was either lyopkilized or kept in aqueous solution a t - 20".

Poly r(; U) The incubation solution contained, unless stated

otherwise, 50 mM Tris-C1 pH 9.2, 2.4 mM MnCl,, 5 mM MgCl,, 0.04mM EDTA pH 7.5, 2.6 mM [35S]pp,-U and 10 units of polynucleotide phosphory- lase per ml incubation solution. After 5 h at 37", the same volume of saturated aqueous phenol was added,

560 Polyribonucleotides Containing a Phosphorothioate Backbone Eur. J. Biochem.

thoroughly shaken, centrifuged and the aqueous layers were cooled to 0" and the same volume of cold ethanol (-20") added. After 2 h a t -2O", it was centrifuged a t 10000 rev./min, the pellet dis- solved in water and chromatographed on Sephadex 6-25 and G-100. The yield of poly r(g U) in a 2 ml experiment was 1.4 absorbance units a t 260 nm (approx . 1.4

Nearest Neighbour Analysis 3.2 absorbance units a t 260 nm of poly r(g A g U)

were dissolved in 2 ml of buffer (containing 50 mM Tris-C1 pH 8.1, 0.5 mM MgC1, and 10.0 mM sodium phosphate) and hydrolyzed by addition of 1 ml of 1 M KOH for 18 h a t 37".The solution was neutralized with 6 N HC10, a t O", centrifuged and to the super- natant was added the same volume of 0.1 N HC1. Adenosine and uridine 2'(3')-phosphorothioates, were separated by chromatography on Dowex 50x8 [la].

RESULTS POLY r(g A g u)

Polymerisation Experiments The incorporation of [14C]ATP into acid precipi-

table material in the presence of ppps-U was inde- pendent of Mg++ concentration in a range of 8 to at least 20 mM. The optimal substrate concentration was found to be around 1 mM. The rate and extent of polymer formation decreased in the order poly r(A-U) > r(AgU) > poly r(gAgU) (Fig.1). There was no difference in the synthesis of poly r(A g U) andpoly r(gA-U). The Na+ and the Et3NH+ salts of ppps-A and ppps-U worked equally well in the poly- merisation reactions.

Reaction with an incubation volume of 3 ml af- forded 2.1 pmoles of poly r(g A g U) with method A but only 1.2 pmoles with method B. This lower yield is most likely caused by a loss of polymer during protein extraction. I n control experiments with [3H]poly d(A-T) it was established that the amount of DNase added to the polymerisation reaction was sufficient to degrade the template to 95 Oi0 in 10 min.

The ultraviolet spectra of poly r(A-U) and poly r(g A a U) in H,O (pH 6.5) are identical.

Nearest Neighbour Analysis During alkaline hydrolysis, the absorbance a t

260 nm of the reaction solution rose from 1.06 to 1.42. The amount of adenosine and uridine Z'(3')- phosphorothioates, after separation on Dowex-BOX 8 was the same, 0.174 and 0.170 pmole respectively. All the radioactivity was found to be associated with adenosine 2'(3')-phosphorothioate. The ratio of specific activities of [35S]ppp,-U to [35S]A-ps was 0.93. This indicates that the polymer is strictly alternating and that no sulfur was lost during the polymerisation.

Lo/- // 0

L

2 4 6 Time (h )

Pig. 1. Polymerisation of [14C]ATP and UTP (@), [14C]ATP and ppps- U (+) a d [SSS]ppp,-A and pppS- U (0) with R N A - polymerase on poly d( A-T) templates at 37". The incubation solution (300 pl) contained 8 mM Tris-Ac pH 7.9, 8 mM MgAc,, 0.12 M NH,Cl, 1 mM of each substrate, 0.3 mM

poly d(A-T) and 8 units of polymerase

4 r

0 20 30 40 50 Time (h)

Fig. 2. Polymerisation of [35S]ppps-A and ppps- U . The incu- bation solution (3 ml) contained 8 mM Tris-Ac pH 7.9, 8 mM MgAc,, 0.12 M NH4Cl, 1 mM of each substrate, 0.3 mM poly d(A-T) and 68 units of polymerase. After 29 h of incu-

bation another 200 units was added

Thermal Melting Thermal melting profiles of poly r(A-U) and poly

r(g A g U) in 0.1 M and 0.01 M sodium citrate buffer are shown in Fig.3. The T, values are 49" and 62", respectively. The lower one is identical with that reported for this Na+ concentration whereas the higher one is about 4" lower than reported. The thermal hyperchromicity is the same for both poly- mers (approx. 65O/,) [7]. The wider breadth of the melting transition of poly r(gAgU) might be a reflection of the shorter chain length of poly r(g A s U) than of poly r(A-U) [15].

Template Activity Like poly r(A-U), poly r(gAgU) can serve as

template for the polymerisation of ATP and UTP as well as ppps-A and ppps-U with RNA-polymerase in the presence of Mn++ (Fig.4).

No attempts were made to optimize these reac- tions with respect to Mn++ concentrations.

Vol. 13, No.3,1970

0.8

5 c m 2 0 0.6

a a

0.4

F. ECESTEIN and H. GINDL

A -

- 2: I 8

I 0.2 -

/ -

I I I L I I L K) 30 50 70 10 30 50 70

56 I

zi ' 5 t

2 4 Time (h)

Fig.4. Polymerisation of [14C]ATP and U T P on poly r(A-U) (e) and on poly r(z A a U) (0); of [14C]ATP and pppaU on p l y r(A- U) (A) and 012 poly r(; A U) (A) ; of [35ij']ppp,-A and ppp*-U on poly r (A-U) (+) and on poly r(i A U) ( x). The incubation solution (200 pl) contained 30 mM Tris-Ac pH 7.9, 3 mM of each substrate, 85 units of enzyme, 15 pM template, 10 mM MnAc, for poly I(; A ; U) and 3 mM MnAc,

for poly r(A-U) as template

Degradation Experiments For these studies aliquots of the aqueous solutions

of the polymers prepared according to method A were lyophilized, taken up in the appropriate buffer

indicated in the legends; enzyme was added at O", an aliquot removed for time 0 value and the reaction started by bringing it up to the incubation temper- ature. The results of various enzymatic degradations are summarized in Table 1. Larger amounts of en- zyme than indicated in the tables lead to complete degradation of the modified polymers also.

For degradation with spleen phosphodiesterase, the 5'-phosphate was removed by incubation with alkaline phosphatase a t pH 8.9 for 10min. The poly- mers are completely stable to this enzyme. The solu- tions were then brought to pH 5.6 with acetic acid and phosphodiesterase was added. Since it was shown earlier [ 161 that nucleoside 5'-phosphorothioates are stable to alkaline phosphatase, it is probable that the 5'-phosphorothioate of poly r(i A 3 U) was not removed by this treatment. This degradation ex- periment might therefore not reflect the resistance of the internucleotidic bond to the diesterase but rather the resistance of the 5'-phosphorothioate to the phosphatase.

During the experiments with polynucleotide phosphorylase, it was noticed that degradation proceeded very slowly when the enzyme was added to the incubation solution containing Mg++. Hydro- lysis was accelerated considerably when the polymer was incubated with the enzyme in the buffer without Mg++, and the Mg++ was added last [17]. This could mean that the formation of the enzyme-substrate

Tab

le 1

. Enz

ymat

ic D

egra

datio

n of

pol

y r(

A-U

) and

pol

y r(

; A ; U)

All

incu

batio

n so

lutio

ns co

ntai

ned

70 p

M s

ubst

rate

and

(A

) 0.1

M T

ris-

acet

ate

pH 8

.9, 0

.1 M

NaC

1, 2.

5 pg

pro

tein

per

0.5

ml (T =

37'

); (B

) 0.1

M T

ris-

acet

ate

pH 8

.9,

0.1

M N

aCl,

0.1

mM

ED

TA

(K+)

, 0.1

pg

alka

line

phos

phat

ase p

er 0

.5 m

l; af

ter 1

0 m

in b

roug

ht to

pH

5.6

with

AcO

H a

nd 3

.5 p

g di

este

rase

adde

d (T =

27'

); (C

) 0.0

5 M

T

ris-

acet

ate

pH 7

.4,

0.08

pg

prot

ein

per

0.5

ml (T =

27'

); (D

) 0.1

M T

ris-

acet

ate

pH 8

.1,

0.5

mM

sod

ium

ort

hoph

osph

ate,

4.5

pg

prot

ein

per

0.5

ml (T =

60'

)

A

B

c D

E

Tim

e Sn

ake

veno

m p

hosp

hodi

este

rase

Sp

leen

pho

spho

dies

tera

se

Panc

reat

ic ri

bonu

clea

se

Polv

nucl

eotid

e ph

osph

oryl

ase

Mic

rocc

ocal

nuc

leas

e 2

poly

r(A

-U)

poly

r(;

A ; U)

poly

r(A

-U)

poly

r(; A

; U)

poly

r(A

-U)

POly

r(; A

; U)

poly

r(A

-U)

poly

I(;

A ; U

) Po

ly r

(A-U

) po

ly r(

; A

; U

) 3.

g m

in

"0

"*

'lo "lo

"1.

lo1O

"lo

"0

Ollo

.I. z 8

180

90

22

90

42

90

29

-

- 97

20

s 5 E

. 30

10

45

12

82

18

55

25

94

8

60

63

14

78

22

85

24

65

33

96

12

120

83

20

90

38

87

26

73

43

96

18

E 09 I

Tab

le 2.

Enz

ymut

ic D

egra

datio

n of

pol

y rU

and

pol

y r(

; U)

0 +d

All

incu

batio

n so

lutio

ns c

onta

ined

70m

M s

ubst

rate

and

(A

) 0.1

M T

ris-

acet

ate

pH 8

.9,

0.1

M N

aCl

and

0.5 pg p

rote

in p

er 0

.5 m

l; (B

) 0.

1 M

Tri

s-ac

etat

e pH

8.9,

0.

1 M

NaC

l, 0.

1 m

M E

DTA

(K

+) an

d 0.

1 pg

alk

alin

e ph

osph

atas

e pe

r 0.

5 m

l; af

ter

10 m

in b

roug

ht t

o pH

5.6

with

AcO

H a

nd 5

8 ng

of

dies

tera

se a

dded

; (C

) 0.0

5 M

T

ris-

acet

ate

pH 7

.4 a

nd 6

ng

of p

rote

in 0

.5 m

l

A

B

C

poly

rU

po

b r(

; U)

poly

rU

po

ly r(

; u)

poly

rU

PO~Y

r(; U

)

Snak

e ve

nom

pho

spho

dies

tera

se

Sple

en p

hosp

hodi

este

rasc

Pa

ncre

atic

rib

onuc

leas

e % W

T

ime

min

30

60

120

180

"1.

"1.

55

15

70

20

80

25

85

30

"0

"1.

75

12

80

15

83

22

86

30

Ollo

"0

55

8 65

14

80

17

85

18

Vol. 13, No. 3, 1970 F. ECKSTEIN and H. GINDL 563

complex is prevented in the presence of Mg++, which stabilizes the structure of the polymer. Once this complex is formed, hydrolysis proc.eeds upon ad- dition of Mg++. A more detailed study is needed to better understand this observation.

POLY r( EU)

Polymerisation of [35S]pps-U was independent of Mg++-concentration in a range from about 2 mM to a t least 10 mM. The optimal substrate concentration ranged from 2.5 to 5.0 mM. After 6 h of incubation there was no further polymerisation. At this time about loo/, of pps-U as compared to 3S0/, of UDP were polymerized [I]. The presence of a primer, UpU seemed to result only in a very slight increase in synthesis.

The results of the degradation experiments are summarized in Table 2 .

DISCUSSION With poly d(A-T) as template, DKA-dependent

RNA-polymerase accepts ppps-U and ppps-A as sub- strates. Replacement of one of the normal substrates, ATP and UTP, by the corresponding thiophosphate analog leads to a decrease in rate and extent of poly- merisation, which is more pronounced when both ATP and UTP are replaced by ppps-U and ppps-A (Fig. 1). As discussed for the polymerisation of ATP and ppps-U [ 2 ] , this decrease seems to be caused mainly by a decrease of V,,,.

Why the extent of synthesis should be lower is not quite clear a t the present time. Addition of more enzyme after the polymerisation has levelled off after 30 h causes a new burst of polymer synthesis (Fig.2). The degree of polymerisation reached then is ap- proximatelyLthe same as if one had added the total amount of enzyme right from the beginning.

A complication for an interpretation of these and other data in this work arises from the fact that the nucleoside 5’-0-( 1-thiotriphosphates) occur as two diastereomers (Fig. 5 ) . The cc-P atom is asymmetri- cally substituted and one of the substituents is the asymmetric @-D-ribose.

Since the synthesis of these triphosphates is not carried out under conditions where one could expect to obtain only one or one isomer preferentially, we are actually dealing with a mixture of these two isomers. So far we have not been successful in sepa- rating them and we therefore cannot study their interaction with RNA-polymerase separately as we could, for example, with uridine 2’,3’-O,O-cyclo- phosphorothioate and RNase [lS]. As a result of this complication we do not know whether the two isomers are equally utilized, or whether one is a much poorer substrate for RNA-polymerase. In the polymerisation of ppps-A and pppsU, we have never reached an 37 Eur. J. Binchem., Vo1.13

Fig. 5. The two diastereomers of nucleoside 5’-O-(l-thiotri- phosphates)

Table 3. iMolecular weight and sedimentation values of polymers Conditions as described under Materials and Methods

poly r(A-U) 128200 7.6

POlY r(; A ; U) 30 000 5.0 POlY r(A ; U) 105 000 7.4

incorporation of 50 o/o of the substrate. It follows that we have no indication whether one or two iso- mers of the phosphorothioate group are present in the polymer poly r(gAgU). In the polymerisation of ATP and ppps-U, however, approximately 60 of the substrates react (Fig. 1). This would indicate that at least in’:this mixed polymerisation, the two isomers are substrates.

The introduction of phosphorothioate groups in the polymer backbone neither impairs the formation of a double strand nor the thermal stability of such a double-stranded polymer [ a ] . At two different Na+ concentrations the T, of poly r(A-U) and poly r(5 A c U) are virtually identical (Fig. 3). Also, the ultraviolet spectrum and the hyperchromicity of these polymers are the same. The molecular weight and the sedimentation of the polymers, however, decrease with increasing phosphorothioate content (Table 3).

The molecular weight of the poly d(A-T) used for the preparation of poly r(g A U) was approximately 300000. The molecular weight of the template used for the preparation of the two other polymers is not known but is assumed to have been of similar size since it was preparedin the same way. Not enough is known about the factors controlling the length of the chains being synthesized to permit an explanation for the decrease in chain length by introduction of thiophosphate.

Poly r(s A F, U) is almost as good a template for RNA-polymerase as poly r(A-U) (Fig.4). One ob- serves only relatively small decreases in rates of polymerisation going from poly r(A-U) to poly r(gA cU). This is true for the polymerisation of ATP and UTP as well as ppps-U and ppps-A or mixtures of these. The enzyme, thus, is quite tolerant towards these changes of the phosphate group in the tem- plate.

ppsU can be polymerized with polynucleotide phosphorylase from E. coli. The modified diphos- phate, however, is a rather poor substrate for this

564 14’. ECKSTEIR and H. GINDL: Polyribonucleotides Containing a Phosphorothioate Backbone Eur. J. Biocliern.

enzyme [I], and addition of UpU as primer does not alter the situation significantly. All attempts failed to polymerize pps-U with purified polynucleotide phosphorylase from Micrococcus lysodeikticus under conditions where UDP was readily polymerized. It can not be excluded that pps-U will also be accepted by this enzyme under different conditions. There is also the possibility that the rate of incorporation was so low under our conditions that we could not detect the polymeric or oligomeric product. We therefore feel that it is premature to state that pps-U is not a substrate for polynucleotide phosphorylase from Microccoccus lysodeikticus.

The poly r(gU) isolated after separation on Sephadex G-I00 served as a messenger for the poly- merisation of phenylalanine in a protein synthesizing system in vitro [I] with about 45O/, of the efficiency of poly rU.

The degradation experiments with poly r(: A U) and poly r(sU) seem t,o justify the conclusion that introduction of phosphorothioate instead of phosphate in a polynucleotide confers some degree of resistance to nuclease degradation. This effect could be caused by a numbcr of factors: (a) the phosphorothioate polymers are not bound as well as the parent polymer by the nuclcase; (b) they are bound too tightly and translation of the enzyme on the polymer is impeded ; (c) attack on the P-atom, which either leads to hy- drolysis or to phosphorylation of the enzyme, is sterically hindered. At present we do not have suf- ficient data to answer these questions satisfactorily but work is in progress to determine which of the factors mentioned is responsible for the slow hydro- lysis.

From these findings it seems clear that sub- stitution of phosphorothioate for phosphate groups in the backbone of a polynucleotide alters only one of the properties studied significantly, the suspectibi- lity to nucleases. It is therefore conceivable that such modified polynucleotides will have an advantage over their parent polymers in reactions where the integrity of the polymer is essential, and where nucleases are responsible for its degradation. There is already a striking example that seems to support this idea: poly r(a A U) is a much better inducer of interferon than poly r(A-U) and its activity can be correlated to its resistance to ribonuclease [IQ].

The authors wish to thank their collcagues for generous gifts: Mrs. E. Gaertner for [3H]poly rU, Dr. T. Jovin for [3H]poly d(A-T), Dr. A. Lezius for poly d(A-T), Dr. H. Stern- bach for RKA-polymerase and Dr. I). Swan for poly- nucleotide phosphorplase ; their assistants for skilful work and help; Miss R. Blotevogel, Miss H. Schmidt and Miss J. Patzschke; Mrs. E. Gottschalk for thc determination of molecular weight and sedimentation values; Prof. Cramer for helpful discussions and generous support; the Deutsche Forschungsgemeinschaft for financial assistance and a grant to H. Gindl. This work was a Dissertation submitted by H. Gindl to the Technische Universitat nraunschweig 1969.

1. 2.

3.

4.

5.

6.

7.

8.

9.

10.

11. 12. 13.

14.

15.

16.

17.

18. 19.

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F. Eckstein and H. Gindl Max-Planck-Institut fur Experimentelle Medizin Abteilung Chemie BRD-3400 Giittingen, Hermann-Rein-StraBe 3, Germany