Nucleotide and oligonucleotide derivatives as enzyme and nucleic acid targeted irreversible inhibitors, biochemical aspects

NUCLEOTIDE AND OLIGONUCLEOTIDE DERIVATIVES AS ENZYME AND NUCLEIC ACID TARGETED IRREVERSIBLE INHIBITORS. BIOCHEMICAL ASPECTS V. V. VLASSOV, A. A. GODOVIKOV, N. D. KOBETZ, A. S. RYTE, L. V. YURCHENKO and A. G. BUKRINSKAYA* Institute of Bioorgani¢ Chemtstry, Novosibirsk 630090, USSR *D.I.Ivanovsky Institute of Virology, Moscow 123098, USSR

INTRODUCTION

In the paper of Knorre et al. in this volume, complementary addressed reagents, derivatives of oligonucleotides, have been described which are capable of selectively modifying nucleic ~cids and nucleic acid binding proteins. It has been demonstrated in in vitro experiments that modification with these reagents, complementary addressed modification, provides the possibility to modify nucleotides in preselected sequences of nucleic acids. This means also the possibility to modify in a specific way preselected nucleic acids in the presence of other polynucleotides. The complementary addressed modification may provide a version of enzyme-pattern-targeted chemotherapy, which is based on the specific damage of mRNAs coding for the enzymes. In contrast to the approach based on enzyme damage, complementary addressed modification suggests the principal possibility of permanent suppression of a certain enzymatic activity by damaging the enzyme gene. In this paper we have summarized our recent experimental studies on the complementary addressed modification of nucleic acids in living cells. The targets for modification were poly(A) sequences of cellular nucleic acids, mRNAs and influenza virus RNAs, in infected cells. Some experiments have been designed to study the possibility of complementary addressed modification of cellular DNA. All the experiments have been done with alkylating oligonucleotide derivatives described in the Knorre et al. paper, which carry aromatic 2-chloroethylamino groups at their termini and are capable of efficiently modifying complementary nucleotlde sequences. An important advantage of these derivatives ts related to their alkylation mechanism including formation of reactive intermediate ethylene-lmmonium cation in the rate-limiting step of the process. The rate of the limiting step does not depend on the concentration and nature of the substances present in cells. 301

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Therefore, the derivatives are consumed in by-reactions to a limited extent and can reach the target nucleic acids being in reactive state. MATERIALS AND METHODS All reagents were analytical grade. Poly(U)-Sepharose 4B and Sephadexes were obtained from Pharmacia. Ribonucleases A (EC 3.1.4.22) and T1 (EC 3.1.4.8) were from Sigma, deoxyribonuclease 1 (EC 3.1.4.5) from Worthington, proteinase K (EC 3.4.21.14) was from Serva. Saponin was from Merck. Oligodeoxyribonucleotides and oligonucleotide derivatwes were synthesized as described in the Knorre et al. paper in this volume. Oligonucleotlde ethylphosphotriesters were synthesized as described m (1,2). Oligonucleotides were labelled at the 5'-end by transfer of 32P from [3~P]-ATP using T4 polynucleotide kmase (EC 2.7.7.78) (3). Unreactive oxyanalogs of alkylating derivatives were prepared by incubation of reactive derivatives in 10 mM Tris-HC1, pH 7.5 at 40 ° for 10 hr (4). Complementary Addressed Modifieatton of Cellular Nucleic Acids with Alkylatmg Oligonucleotide Derivatives Ascites tumor Krebs II cells were maintained in mice CC57BR. The cells were washed three times by centrifugation in medium 199 supplemented with 10 mM HEPES, PH 7.2, 50 #g/ml streptomycin and 30 U/ml penicillin (standard medium). To ascertain the cells' viability, Trypan blue at a concentration of 0.05% was added to the cells. Initially, the percentage of cells that took up the dye was 2% and it increased to 4% to 8% by the end of incubations. Treatment of cells with 0.002% Saponin was done as in (5). Incubations of cells with [z4C]oligonucleotlde derivatives were done under conditions providing stable complementary complexes formation and conversion of the alkylating groups to the reactive intermediate (Table 1). The cells were m the standard medium at concentrations 1 -- 5 × 10 6 cells/ml. After incubation with oligonucleotlde derwatives, the cells were washed three times by centrffugatlon in the standard medium and counted in a scintillation counter. Extent of cellular nucleic acids and protein modification was estimated using the following two procedures (6). A. Cells (10 s cells) were disrupted by suspending in 10 ml of 10 mM TrlsHCI, pH 7.5 and repeating (3 times) freezing and unfreezing. 0.05 vol 3 M NaC1 and 2 vol ethanol were added, the mixture was chilled m ice and biopolymers were pelleted by centrifugation. The pellet was dissolved in 10 mM Tris-HC1, p H 7.5; 0.05 vol 3 M NaC1 and 2 vol ethanol were added and the mixture was shaken for 10 min at 37°C. Then the mixture was chilled and the biopolymers were pelleted. The procedure of re-precipitation was repeated 4 times to wash off lipids and oligonucleotlde derivative, which was not

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crosslinked to the biopolymers. The precipitate was dissolved in 0.5 ml of water and 0.5 ml of 0.6 M N a O H and incubated for 1 hr at 37 ° to degrade RNA. The solution was neutralized with 2 ml of 0 . 6 i HC104 and chilled. Undegraded biopolymers were pelleted by centrifugation and the supernatant containing RNA degradation products was counted. The pellet was suspended in 2 ml of 0.5 M HC104 and the mixture was boiled for 20 min to degrade the DNA. Then the sample was chilled and centrifuged. Supernatant containing DNA degradation products and the pellet containing proteins were counted. B. Determination of nucleic acid and protein radioactivity was done in 3 separate samples of cells. In the first sample, total radioactivity of biopolymers was determined as in method A, after precipitation of biopolymers and ethanol washing. From the second and the third samples, RNA and DNA were isolated by phenol extractions (7). Differences in the radioactivity of nucleic acids and the total biopolymer bound radioactivity were ascribed to the protein modification. For analysis of various RNA fractions modification, total RNA was isolated from cells by phenol extraction method. RNA was fractloned on poly(U)-Sepharose column as described by the manufacturer Pharmacia, and according to (8). RNA fraction which does not bind to the column is designated further as poly(A)+RNA depleted RNA. The polyadenylated RNA was eluted from the column and treated with ribonucleases A and T1 as described in (9) and with bentonite, to remove the nucleases. Poly(A)fragments were isolated from the digest by binding to a poly(U)-Sepharose column. For identification of alkylated nucleotides in RNA and DNA the modified biopolymers were hydrolyzed to bases a n d / o r nucleotides and the hydrolysates were analyzed by paper chromatography as described in (2).

Modtfication of Chromatin with Alkylating Oligonucleotide Derivatives Human placental chromatin was isolated as described in (10). Deoxyribonucleoprotein DNP06 was prepared by treatment of chromatin by 0.6 M NaC1 according to (12). DNA was isolated from chromatin by phenol extraction with consequent proteinase K treatment as described in (13). In the binding experiments, [32p]-labelled oligonucleotide (11) was added to chromatin at 25 ° in 0.01 M Tns-HC1, pH 7.5 containing 0.15 M NaC1. Oligonucleotide and chromatin were taken in proportion 1 mole of oligonucleotide per 3.10 ~ moles of DNA nucleotides. Complex of chromatin with oligonucleotide was separated from the nonbound oligonucleotide by gelfiltration on a Sephadex G-100 column. In some binding experiments chromating was treated with ribonuclease A to remove traces of RNA. Alkylation of chromatin, DNA and deoxyribonucleoprotein DNP06 with

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reactwe oligothymidylate derivative was performed under the binding conditions for 20hr. The modified chromatin, DNP06 and DNA were separated from the nonbound reagent by gel chromatography at 70°C.

Arrest of lmmunoglobulin Synthesis m MOPC 21 Cells by Specific Modification of lmmunoglobulin mRNA Immunoglobulin producing (IgG~, x) mouse myeloma MOPC 21 cells were grown as solid tumors following subcutaneous injection of BALB/c mice. Single-cell suspension was obtained by mechanical degradation of the tumor tissue. The cells were suspended in Dulbecco's modification of Eagle's medium (DMEM, GBCO EROPE), supplemented with 0.03% glutamine, 20 m~a HPES pH 7.2 and 80 ~g/ml gentamlcin. In experiments with oligonucleotide derivatives, cells (5 × 10 7 cell/ml) were incubated in the medium containing 30-600 ~ oligonucleotide derivatives for 3 hr at 37°C. Cells were collected by centrifugation, resuspended in Hanks balanced salts supplemented with 8 m B K / m l [35S]methionine and incubated for 1 hr. The cells were collected by centrifugation and dissociated m solution containing /3mercaptoethanol and SDS (1% each) at 100°C. The labelled polypeptides in the cells extracts were separated by electrophoresis on polyacrylamlde gels as described in (14). Concentratmg layer was 4% acrylamide and separating gel 12% acrylamide. The gels were dried and autoradiographed. Immunoglobulin chains were identified by ~mmunoprecipitation with antibodies to the immunoglobulin. In some experiments RNA synthesis in cells was blocked by 5 #g/ml c~amanitin. In these cases cells were incubated with oligonucleotide derivatives for 3 hr and then [35S]-methlonine was added to the sample. Incubation was continued for another 6hr, cells were spun down and secreted proteins contained in the medium were analyzed by gel electrophoresis. Inhibition of Influenza Virus by Ohgonucleotide Derivatives The Weybridge strain of fowl plague virus (FPV) was grown in the allantoic cavity of 10-day-old embryonated hen's eggs and the infected allantoic fluid was used in the experiments. Infectivity titre of the fluid IDs0 was 10 9 and the haemagglutinin titre 1 : 1280. Chicken fibroblasts were seeded in 96 well microplates (2×105 cells/well) and allowed to grow for 3 days in 200 #1 of 5% lactalbumin hydrolysate supplemented with 2% bovine serum. Adsorption of inoculum (0.1, 1 or 10 IDs0/cell) was at 20 ° for 30 min. After adsorption, the cells were washed with the medium, 100 ~I of fresh medium were added and cells were then incubated at 37°C. Oligonucleot~de derivatives dissolved in the same medium (1 mg/ml) were added to cells m two portions, 50 ~1 each. Oligonucleotlde derivatives complementary to genome RNA sequences were added immediately after refection and 4 h r later (multiplicity of infection 10

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IDs0/cell) or 8 hr later (multiplicity of infection 0.1 and 1 IDs0/cell ). First addition of oligonucleotide derivative complementary to haemagglutinin mRNA was 3 hr after infection. The second addition was 8 hr after infection (multiplicity of infection 0.1 IDs0/cell) or 4 hr after infection (multiplicity of infection 0.1 and 1 IDso/cell). Final concentrations of oligonucleotide derivatives in incubation medium were 100 #M. After 24hr incubation (multiplicity of infection 0.1 and 1 IDsog/cell), or 8 hr incubation (multiplicity of infection 10 IDs0g/cell), infectious titre and haemagglutinin titre of the incubation medium were determined. Treatment of infected fibroblasts with 4(N-2-chloroethyl-N-methylamino) benzylamine was done according to the scheme described for the oligonucleotide derivatives complementary to genome RNAs. RESULTS AND DISCUSSION

Complementary Addressed Modification of Cellular Nucleic Acids with Alkylating Oligonucleotide Derivatives The main applications of complementary addressed modification are related to specific damage of cellular nucleic acids. To react with certain cellular nucleic acids, complementary addressed reagents should enter the cells, to avoid unspecific reactions wxth cellular components, to resist attacks of cellular enzymes and to recognize the target nucleic acid among the variety of cellular polynucleotides. In our first experiments on the complementary addressed modification of cellular nucleic acids, we have investigated the uptake of alkylating oligonucleotide derivatives by ascltes tumor Krebs I! cells and alkylation of the cellular biopolymers (15-18). As target sequences the oligo(A)-sequences which are widely distributed in nucleic acids were chosen. The complementary addressed reagents used were oligothymidilyl uridine derivatives and their noniomc ethyl phosphotriester analogs. The derivatives carried alkylatmg groups at their T-ends (see Knorre et al. in this volume) (C6HsNH)2p[dTp(Et)]4rUCHRCI(I) d[Tp(Et)]grUCHRCI(I I) d(Tp)9rUCHRCI(I II) [ 32p]-pd(Tp)grUCHRCl(IV) ~CH2CH2Ct CHRCI : ~ C R - - - - ( ( % L---~./ (*position of 14Clabel) / ~ ~'CHa In control experiments the unreactive analogs IIa and IIIa were used, which were prepared by hydrolysis of II and III resulting in substitution of OH group ER24-]¢

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for Cl in the reactive groupings. As reagent noncomplementary to oligo(A) sequences, derivative d(TpCpCpCpApApApC)rACHRC1 (V) was used. Transport of derivatives II and III into cells was investigated at 20°C and 37°C. These derivatives form complexes with poly(A) in vitro at 20°C but not at 37°C under physiological salt conditions. Cells were incubated in medium containing [14C]-derivatives for different periods between 2 min and 26 hr following which they were washed and the radioactivity taken up counted. Results presented in Figure l show that at 20 ° the uptake curve reaches plateau in 1 hr. It is seen that the nonionic derivative II binds to the cells much better as compared to the charged derivative III. At 37°C the derivatives are taken up faster, and in the case of derivative II the plateau level is achieved within a few min of incubation. Another nonionic derivative I was taken up as readily as the derivative II. Treatment of cells with saponin resulted in decrease of derivative binding (Fig. 1), thus demonstrating that the binding is due to the viable cells. When cells were incubated in the presence of increasing concentrations of derivatives the uptake plateau level increased proportionally up to l0 #M concentrations of derivatives in the incubation medium (Fig. 2). Data on the oligonucleotide derivative uptake by the cells under various conditions are summarized in Table 1. Under the incubation conditions used, no less than 50% of the derivatives were transformed to the reactive

10

9 o • •

o

0a

0

O--la

Z ~,_X,O---O- 2b

•~- 3a

I 4

I 8

1 22

Time (h) FIG. 1 Transport of ohgonudeotlde derivatives II (la-3a) and III ( l b - 3 b ) into ascttes tumor Krebs lI cells. P-Uptake of denvatwes from the medmm, % Curves I a, b and 2 a, b represent derlvaUve transport at 20°C and 37°C, respectively Curves 3 a, b represent binding of derivatives by saponm treated cells at 20°C Concentration of cells was 5-106/ml, concentraUons of denvauves II and Ill were 0.7 #M and 0 8 #m, respectively

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OLIGONUCLEOTIDE 1NHIBITORS, BIOCHEMISTRY

/

,¢

1o

0.5

5

lO

FIG. 2 Uptake of derivatives 1I (o) and Ili (O) by ascltes tumor Krebs I1 cells at various concentrations of derivatives in the incubation medium A, concentration of derivatives m the incubation medium, t~M; B, the amount of denvatwes taken up by cells, nanomoles of denvatwe/5× 106 cells.

TABLE 1 UPTAKE OF ALKYLATING OLIGONUCLEOTIDE DERIVATIVES BY ASCITES TUMOR KREBS I1 CELLS

Derivative, Concentration concentration, of cells, ~M 106/ml

Incubation conditions

I 0.005 I1070 I1 0.70 Ila070 111077 Ili 0.77 IIIa070 V 0 91

5° 20° 37° 20° 20° 37° 20° 20°

3.2 50 50 50 5.0 50 50 5.0

400 hr 24hr 24 hr 24hr 24hr 2 hr 24hr 24 hr

Uptake of Concentrations denvatwes of derivatives by cells, within cells, % nMol/107 cells ~M 40.0 92 8.4 8.2 50 48 36 2.6

0.06 0.13 0.12 0 12 0077 0 074 0050 0.035

0.4 8.5 7.9 77 5.1 4.9 3.3 24

intermediate and reacted with the nucleophils available. The data show that d e r i v a t i v e I w i t h t h e s h o r t e s t a d d r e s s i n g g r o u p is t a k e n u p m u c h b e t t e r t h a n t h e o t h e r d e r / v a t w e s . T h e r e a s o n m a y be its s m a l l e r size o r t h e p r e s e n c e o f a r o m a t i c p r o t e c t i n g g r o u p s a t its Y - e n d w h i c h c a n p r o m o t e b i n d i n g t o t h e

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cellular membrane. The efficiency of charged heterogeneous derivative V uptake is rather poor, demonstrating the effect of nucleotlde composition on the oligonucleotide uptake. Derivatives II and III are taken up better as compared to their unreactive analogs. Concentrations of derivatives within cells based on uptake of radioactivity by the ceils was calculated (Table 1) assuming a cell vol 1.5× 10-9 cm 3. Since the calculated concentrations within cells far exceed the extracellular concentrations, the uptake data suggest the derivatives are concentrated in cells probably due to interaction of their aromatic groupings with cellular membranes or (and) due to binding to the poly(A)-sequences within cells. To analyze biopolymers from ceils treated with alkylating oligonucleotide derivatives, the cells were lysed and biopolymers ethanol precipitated and treated consequently with alkali and acid in order to solubilize RNA and D N A respectively. Radioactivities hberated by these treatments and radioactivity of the remaining ethanol insoluble fraction represented the amounts of reagent covalently bound to RNA, D N A and proteins, respectively. The results obtained (Table 2) show that up to 60% of derivatives taken up by the cells are covalently bound to the cellular biopolymers. The extent of RNA alkylation shows the expected temperature sensitivity to be 1.5 times higher in experiment at 20° as compared to that at 37° . The nonionic derivative II shows more pronounced preference for nucleic acids as compared to derivative III. This may have resulted from the electrostanc interaction of derivative III with cellular proteins. The results of alkylated biopolymers estimation have been confirmed in experiments where RNA and D N A were isolated from cells by the phenol extraction method. To study the specificity of cellular nucleic acid alkylation, D N A and RNA were isolated from the cells treated with the oligonucleotide derivatives. Poly(A) containing RNA was isolated from the total RNA by poly(U)-Sepharose column chromatography. This RNA was dlgested by TABLE 2 ALKYLATIONOF BIOPOLYMERSIN ASCITES TUMOR KREBS I1 CELLS WITH [J4C]-OLIGONUCLEOTIDEDERIVATIVES Covalent bmdmg of der~vatwes to cellular blopolymers, % of the amount taken up by the cell Derivative

Incubation con&tlons

RNA

DNA

Proteins

II II 1I III III III

20° 24 hr 37° 2 hr 37° 4 hr 20°24hr 37° 2 hr 37° 4 hr

28 18 21 22 13 15

16 17 18 12 16 14

15 17 15 25 24 19

Concentrattons of cells were 10 6 cells/ml: concentration of derivatives was 0 70/.t•.

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ribonucleases A and T1 and the poly(A) fragments were isolated from the digest by chromatography on poly(U)-Sepharose. Radioactivity determination in the nucleic acid samples obtained in this way represented extent of alkylation in four types of nucleic acid fractions: DNA, poly(A) fragments of RNA, heterogeneous fragments of poly(A) containing RNAs and total RNA deprived of the poly(A) containing RNA. The extent of modification of these nucleic acids fractions isolated from cells treated with ohgonucleotide derivatives I and III (Tables 3 and 4) demonstrates preferential alkylation of TABLE 3 M O D I F I C A T I O N OF NUCLEIC ACIDS IN CELLS TREATED W I T H O L I G O N U C L E O T I D E DERIVATIVES I A N D II

Reagent, concentratlon, /dM

Concentration of cells, 106/ml

Incubation condmons

I 0 005

32

5° 17 days

II 1.75

11.0

20° 30 hr

II 1.75

11,0

37° 2 hr

Nucletc acid fractions*

Extent of alkylatlon, moles of reagent/mole of nucleoUdes

10-3

1

1.5 x

2 3 4 1 2 3

5.0 × 10-s 4.0 × 10-5 1.1× 10-4 2 1 × 10-2 1 0 × 1 0 -4 1.8 × 10-4

4 1

2 8 × 10-4 5.5 × 10-4

2 3 4

1.0 × 10-4 3 4 × 10-5 2.7 × 10-5

* 1, poly(A) sequences: 2, heterogeneous sequences of poly(A) containing RNA, 3, total RNA deprived of poly(A) containing RNA; 4, D N A

TABLE 4. SPECIFICITY OF CELLULAR NUCLEIC ACID M O D I F I C A T I O N BY THE O L I G O N U C L E O T I D E DERIVATIVE III Nucleic acid Incubation conditions

20 ° 24 hr

37° 4 hr

fraction

Amount, A26o units

Radioactivity m the fractions, cpm

1 2 3 4 1 2 3 4

16 90 124,0 97,7 3,7 18 8 180 0 184.7

17,000 3,600 13,100 14,700 4,980 3,820 8,640 19,200

Extent of alkylatlon, moles of reagent/ mole of nucleot~des 35× 13× 35× 5.0 × 44× 6,8 × 1.6 × 3.5 ×

10-3 10-4 10-5 10-5 10-4 10-5 10-5 10-5

In experiments performed at 20°C and 37°C 2,7 × 10 s and 9 × l0 s cells were taken, respectwely Concentration of the ohgonucleoude derivative was 0 7 #M Nucleic acid fractions are named as m Table 3.

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poly(A) fragments in cellular RNA. The extent of poly(A) fragment modification far exceeds that of the heterogeneous sequences, demonstrating that complementary addressed modification took place. While poly(A) sequences of mRNA comprise less than I% of the RNA sequences, 35% of reagent bound to biopolymers are found in them. Decrease of mcubation temperature and use of oligonucleotide derivatives with longer addressing groups result in increase of modification of poly(A) sequences obviously due to the stabilization of the complementary complexes. Extents of modification of DNA and heterogeneous sequences of RNA are similar. Effect of temperature on their modification suggests that at least in part the modification proceeds according to the complementary addressed mechanism. In experiments with reagent V no preferential modification of poly(A) sequences was found. In this case all nucleic acid fraction analyzed were modified to a similar extent. The modification extents were 3.6×10-6 and 1.5×10 -5 moles of reagent per nucleotide unit for poly(A) sequences, total RNA deprived of poly(A) containing RNA and DNA, respectively. Composition of alkylated bases is drastically different in polynucleotide alkylated with complementary addressed reagents and in polynucleotides alkylated in nonaddressed way (19, 20). In the latter case, 7-alkylguanine is the predominant alkylation product. Complementary addressed reagents juxtapose their alkylating groups to certain positions ofpolynucleotides and force them to react with the bases available. Therefore, in this case, all types of potentlally reactive bases (Gua, Cyt, Ade) are modified to similar extent. One can make conclusions about the nature of the alkylation process taking into account the compositions of modified nucleic acid bases. We have analyzed alkylation products by chemical degradation of nucleic acids and subsequent paper chromatography separation of the products. The data obtained (Table 5) are in accordance with the complementary addressed modification mechamsm. Under condmons providing complementary complex formation the derivatives I-III modify three bases guanine, cytidine and ademne in nucleic acids in proportion 44 : 31: 25. In the poly(A) sequences the alkylation products were 3-alkyladenine (67%), 7-alkyladenine (18%) and lalkyladenine (15%). These data are in agreement with the results observed in m v t t r o experiments (19). Cellular DNA was modified mainly at guanine and adenine bases in proportion characteristic for the complementary addressed modification with alkylating ohgonucleotide derivatives (20). At 37°C in both cellular RNA and DNA the predominant alkylation product was 7alkylguanine, demonstrating the non-addressed modification. The data obtained are consistent with complementary addressed modification of DNA in cells treated with alkylatmg oligonucleotide derivatives. It is known that poly(A) sequences are widely distributed m eukaryotic DNAs (21). One may suggest that in some functional states of DNA some unwinding occurs which is sufficient for the oligonucleotlde

311

OLIGONUCLEOTIDE INHIBITORS, BIOCHEMISTRY TABLE 5. ALKYLATED BASE COMPOSITION IN NUCLEIC ACIDS ISOLATED FROM ASCITES TUMOR KREBS II CELLS TREATED WITH OLIGONUCLEOTIDE DERIVATIVES I-III

Ohgonucleotlde denvatwe

Nucleic Incubation acid condlt~ons fraction

Content of alkylated bases, % Gua

Cyt

Ade 100 24 29 35 100 25 21 30 28 32 0 0

1

0

0

I

5° 17 days

2 3 4

48 41 54

28 30 11

1

0

0

II

20° 30 hr

III

20° 24 hr

III

37° 4 hr

2 3 4 3 4 3 4

43 46 56 40 52 80 88

32 33 14 32 16 20 12

Fractions of nucleic acids are named as m Table 3.

derivative binding. The d e s c r i b e d effects c o u l d only be o b s e r v e d if the o l i g o n u c l e o t i d e derivatives survive in ceils in the course o f i n c u b a t i o n s . The derivative survival was c h e c k e d also in special e x p e r i m e n t s with two o l i g o t h y m i d i l a t e a l k y l a t i n g derivatives I I I a n d IV. One o f t h e m was labelled at the 5'-end with 32p a n d the o t h e r c a r r i e d [14C]-labelled a l k y l a t i n g g r o u p . In e x p e r i m e n t s with these reagents, extents o f cellular R N A m o d i f i c a t i o n d e t e r m i n e d f r o m the 14C a n d 32p i n c o r p o r a t i o n were f o u n d to be similar, thus d e m o n s t r a t i n g t h a t d e g r a d a t i o n o f the o l i g o n u c l e o t i d e g r o u p s was insignificant. [32p]-Labelled m a t e r i a l c o u l d be split off the m o d i f i e d p o l y n u c l e o t i d e s b y m i l d acidic t r e a t m e n t which cleaves the derivative acetal b o n d . Therefore, the r a d i o a c t i v e i n c o r p o r a t e d in the nucleic acids resulted f r o m a l k y l a t i o n a n d n o t f r o m the 32p reutilization. A c c o r d i n g to the T r y p a n blue staining, the cells were 94% viable b y the e n d o f i n c u b a t i o n with a l k y l a t i n g derivatives, thus d e m o n s t r a t i n g low toxicity of the c o m p o u n d s . T o x i c i t y o f the d e r i v a t w e s was tested also in special e x p e r i m e n t s in which the cells were i n c u b a t e d for 90 min at 37 ° in the m e d i u m c o n t a i n i n g 10 #M derivative II. T h e cells were injected into mice in m i n i m a l t u m o r p r o d u c i n g a m o u n t s (5.5×104 cells). W e i g h t s of the resulted 14-day t u m o r s are listed m T a b l e 6. It is seen t h a t the i n c u b a t i o n has a negligible effect on cells viability. The o b t a i n e d results d e m o n s t r a t e the p r i n c i p a l possibility to p e r f o r m c o m p l e m e n t a r y a d d r e s s e d m o d i f i c a t i o n o f cellular nucleic acids. As one c o u l d expect, the n o n i o m c o l i g o n u c l e o t i d e derivatives are t a k e n u p b y cells m u c h better t h a n the n a t u r a l c h a r g e d o l i g o n u c l e o t i d e derivatives. The derivatives

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TABLE 6. EFFECT OF ALKYLATING OLIGONUCLEOTIDE DERIVATIVE II ON VIABILITY OF ASCITES TUMOR KREBS II CELLS Mice 1

2 3 4 5 6 7 8 9 10

Wetght of 14-day tumor, g 1" 2* 3* 60 58 71 5.4 8.9 17 04 3.1 65 14

81 5.6 5.0 0.4 83 72 3.8 2.7 4.5 -

3.2 4.6 94 8.5 6,3 3,1 46 72 83 2.6

Mean tumorwoght, g 4 6 + 2 1 5.1_+2.1 5.8+ 18 *1, 2 and 3: cells were incubated as described m text without reagent, in the presence ofdenvatwe II and m the presence of unreactive analog IIa, respectwely

are sufficiently stable within cells a n d efficiency o f c o m p l e m e n t a r y a d d r e s s e d m o d i f i c a t i o n is r a t h e r high u n d e r c o n d i t i o n s which are not t o o d a m a g i n g for cells. One c o u l d not expect t h a t c o m p l e m e n t a r y a d d r e s s e d m o d i f i c a t i o n of cellular D N A can occur. T h e o b s e r v e d D N A m o d i f i c a t i o n allows one to h o p e for the use o f c o m p l e m e n t a r y a d d r e s s e d m o d i f i c a t i o n f o r i n d u c t i o n of m u t a t i o n s in certain genes o f living cells.

Modification of Chromatm with Alkylatlng Ohgonucleotlde Derivatives C o m p l e m e n t a r y a d d r e s s e d m o d i f i c a t i o n o f cellular D N A can p r o v i d e an a t t r a c t i v e technique for a c h i e v e m e n t o f p e r m a n e n t arrest o f certain b i o p o l y m e r biosynthesis, The p o s s i b i l i t y to develop this a p p r o a c h is not o b v i o u s as o l i g o n u c l e o t i d e s d o not b i n d to d o u b l e s t r a n d e d D N A . One can h o p e , however, that in the course o f D N A functioning, t r a n s c r i p t i o n a n d t r a n s l a t i o n , single s t r a n d e d regions in its structure a p p e a r . A t least in these cases D N A can be a v a i l a b l e for the c o m p l e m e n t a r y a d d r e s s e d m o d i f i c a t i o n . A n u m b e r o f e x p e r i m e n t a l d a t a evidence t h a t single s t r a n d e d D N A sequences a p p e a r in t r a n s c r i p t i o n a l l y active c h r o m a t m (22). In e x p e r i m e n t s described in the p r e c e d i n g section we have o b s e r v e d s o m e m o d i f i c a t i o n o f D N A in cells treated with a l k y l a t i n g o h g o n u c l e o t i d e derivatives. There were some i n d i c a t i o n s t h a t the m o d i f i c a t i o n p r o c e e d e d a c c o r d i n g to c o m p l e m e n t a r y addressed mechanism. One can explain the results b y a s s u m i n g that D N A m c h r o m a t i n has different p r o p e r t i e s as c o m p a r e d to the isolated d o u b l e s t r a n d e d D N A . In

OLIGONUCLEOTIDE INHIBITORS, BIOCHEMISTRY

313

order to investigate this possibility we studied interaction of oligonucleotide derivatives with human placental chromatin. Poly(A)-sequences were chosen as targets again as they are presented in eukaryotic D N A in large amount and because they are the most unstable sequences in the double stranded DNA, In the first experiments, we investigated binding of oligonucleotides to chromatin. [nP]-Labelled oligonucleotides were added to chromatin in buffer stabilizing complementary complexes in proportion 1 mole of oligonucleotides to 3×105 moles of D N A nucleotides. The excess of nonbound oligonucleotides was removed by gelfiltration on a Sephadex G-100 column. It was found that [32p]-oligothymidilate(pT)ls binds to chromatin (Table 7). Binding of heterogeneous oligonucleotide p C p A p T p G p C p A p A p A p A p C p C p T p C p C p C occurred to a lesser extent. The binding was a specific one. Addition of 4-fold excess of unlabelled (PT)z5 to mixture of chromatin with [32P]-(pT)l 5 resulted in decrease of radioactivity binding to the chromatin. Addition of excess of the heterogeneous oligonucleotide did not inhibit the binding. Preliminary treatment of chromatin with ribonuclease A did not decrease the extent of (pT)~5 binding. The results described lead to the conclusion that oligonucleotides bind to chromatin DNA. Binding of alkylating oligothymidylate derivative C1RCH2NHpT(pT)s was also specific. This derivative alkylated chromatin components efficiently. To follow the modification, chromatin treated with the derivative was isolated from the reaction mixture by gel filtration on a Sephadex-100 column at 70°C and the bound radioactivity was counted. To estimate the extent of D N A modification, D N A was isolated from the modified chromatin using phenol

TABLE 7. INTERACTION OF OLIGONUCLEOTIDE (PT)15 AND OLIGONUCLEOTIDE DERIVATIVE CIRCHzNHpT(pT)sWITH HUMAN PLACENTAL CHROMATIN CIRCH2NH

=

CtCH2CH2~ _N----'~ (

[3 2P]-(./3T)I 5

to chromatln* Chromatm 2

pTls pNl6t

CH2NH~

Covalent bmding*l of [nP]-CIRCH2NHpT)(PT)s

Binding of Competttor added

))

(1.8)$ 05 1.9

2

0.45

D N P 0 6 Chromatm DNA 4

1.1 (0 2)§ 0.26

*Binding and covalent attachment of the ohgonucleoUde derlvattve are in moles of oligonucleotldes per nucleotlde of DNA × 106. 1-pN~6= pCpApTpGpCpApApApApCpCpTpTpCpCpC :~For nbonuclease treated chromatm. §For isolated DNA. ER24-K*

314

v . v . VLASSOV,et al.

extraction. It was found that at least 55% of the radioactivity incorporated in chromatin is bound to DNA. This value is most probably underestimated due to glycoside bond degradation in the alkylated DNA under conditions of isolation. When isolated DNA was subjected to modification with the same reagent under identical conditions, it was found that the DNA is 5-fold less reactive as compared to chromatin DNA. Alkylation experiments were done also with deoxyribonucleoprotein DNP06 which is chromatin depleted of some nonhistone proteins and histone H I (12). It is seen (Table 7) that DNP06 is alkylated more readily than chromatin. Results presented in this section demonstrate that DNA ofchromatin and DNA of DNP06 which can be regarded as loosened state ofchromatin are able to bind oligonucleotide derivatives. This proves the possibility of complementary modification of DNA and allows one to think about sitedirected in vivo mutagenesis.

Arrest of lmmunoglobulin Synthesis in MOPC 21 Cells by Specific Modification of the Immunoglobulin mRNA Cellular mRNAs are the obvious targets for complementary addressed modification. One can hope to arrest synthesis of certain proteins by modification of the mRNA coding for it. In order to investigate this possibility we have attempted to arrest synthesis of immunoglobulin G in mouse myeloma MOPC 21 cells by modification of mRNA coding for the immunoglobuhn K light chain, the nucleotide sequence of which is known (23). The reactive oligonucleotlde derivative used was complementary to a sequence in the mRNA fragment coding for the variable x chain region, positions 205-215. The derivative carried alkylating group C I R C H 2 N H - at its 5'-phosphate. The scheme of the experiment was the following (24). Cells were treated with the oligonucleotide derivative and with a noncomplementary oligonucleotide in the control experiment. After the treatment, cells were incubated with [3sS]-methionine and the labelled proteins in cell lysates were analyzed by electrophoresis. Figure 3 shows results of one of the experiments. It is seen that treatment with the oligonucleotide derivative complementary to the m R N A inhibits synthesis of both immunoglobulin chains. Synthesis of other proteins ~s slightly affected. To estimate the inhibition effect quantitatively, protein bonds were cut off the gel and counted. Radioactivity of the band c was chosen as an internal standard as its intensity was very similar in various experiments. For the experiment shown m F~gure 3, relative radioactwities in bands a, b and c are respectwely 0.41 : 0.30 : 1(1); 2.6 : 1.9 : 1(2); 2.5 : 1.7 : 1(3) and 3.0 : 1.9 : 1(4). From these figures, it is seen that the suppression effect is a specific one, as the noncomplementary oligonucleotide derivative results in unspecific inhibition of the protein synthesis.

1

2

3

4

5

.,t-a

.~-C

FIG 3. Analysis of 35S-polypeptldes synthestzed m MOPC 21 cells treated with ohgonucleotlde derivatives. Ceils were treated with" (I) ohgonucleotlde denvatwe complementary to the mRNA coding for the lmmunoglobuhn light chain; (2) the same derwatwe with hydrolyzed reactive group, (3) ohgonucleottde derivative noncomplementary to the mRNA; (4) the same denvatwe wtth hydrolyzed reactive group, (5) standard, ~25I-lmmunoglobuhn G from MOPC 21 cells

OLIGONUCLEOTIDEINHIBITORS,BIOCHEMISTRY

315

The observed inhibition of synthesis of both immunoglobulin chains is not surprising. It has been shown (25) that the normallight immunoglobulin chain biosynthesis is the necessary prerequisite for the heavy chain production. Simultaneous treatment of cells with the oligonucleotide derivative and t~amanitin, preventing synthesis of new mRNA, resulted in more pronounced inhibition effects. The results obtained demonstrate the possibility of specific protein biosynthesis arrest by oligonucleotide reagents targeted to the protein coding mRNA. In these first experiments no special techniques were used to enhance the derivative uptake by the cells and we had to use high concentrations of the derivatives, more than 150 ~tM.It is clear that the use of liposomes encapsulated derivatives or nonionic oligonucleotide addresses should enhance the efficiency of the technique. Specific arrest of translation can be achieved also by use of oligonucleotide derivatives which bind to mRNAs non-covalently (26). A promising approach may be the use of plasmids producing anti-sense RNA, complementary to certain mRNAs (27). However, the derivatives capable of covalent binding to nucleic acids are obviously the most potent specific nucleic acid damaging agents.

Inhibition of Influenza Virus by Oligonucleotide Derivatives In the course of their functioning in cells, viral nucleic acids unavoidably appear as single stranded structures vulnerable for attack of complementary addressed reagents. Therefore, complementary addressed reagents can be considered as potential antiviral compounds. Potential applications of reactive derivatives of nucleic acids as antiviral and anticancer compounds have been thoroughly discussed in (28, 29). Activity in investigation of antiviral oligonucleotide effect was stimulated by results of experiments on inhibition of Rous sarcoma virus multiplication with an oligonucleotide complementary to the viral RNA (30, 31). Recently inhibition of several viruses by oligonucleoside methylphosphonates has been reported (32). More promising should be the use of intercalating groups carrying oligonucleotides (26) and probably of plasmid-borne antisense RNAs (27). We beheve that reactive ohgonucleotide derivatives should be potent viruses inhibitors. We have investigated the effect of alkylating oligonucleotide derivatives on mulnplication of influenza virus in cell culture. The virus was fowl plague virus (FPV), strain Weybridje (33). The influenza wrus genome consists of eight single stranded negative-sense RNA molecules. The 5' and 3'-terminal sequences of influenza virus RNA segments are conserved between the segments and among different subtypes (34). Therefore, they may be considered as very important sequences for the functions of viral RNAs and are obvious targets for complementary addressed

v v. VLASSOV,et al.

316

modification. We have chosen also one target in mRNAs coding for viral proteins, in mRNA coding for haemagglutinin (35). The reactive oligonucleotide derivatives used in this work were the following: C 1R C H 2 N H p C p C p T p T p G p T p T p T p C p T ( I ) d(pApGpCpApApApApGpCp)rACHRCI(2) C I RCH2NHpTpTpTpTpCpCpCpTpTpTpT(3) C I RCH2NHpGpCpCpApApApCpA(4)

CtCH~CH2. ~N ~ C1RCH2NH= CH~

CH2NH--

/CH2CH2Ct CHRCZ

--

/

~

"~CH~

The derivatives (1) and (2) are complementary to the 5'- and 3'-conserved sequences in the viral RNAs. Derivative III is complementary to a sequence in the central part of the haemagglutinin mRNA of Rostock FPV strain. Derivative (4) is an oligonucleotide derivative with structure taken by chance as a control reagent. Oligonucleotide derivatives (1) and (2) were added to the infected chicken fibroblasts immediately after infection and once more later to maintain concentrations of reactive forms of derivatives for several hours. The derivative (3) was added 3 hr after infection, taking into account the beginning of haemagglutinin synthesis in the infected cells (36) and once more later. In some experiments the derivatives were added to cells together with CaCI 2 solution to increase their uptake into the cells. After incubation of the cell titre of FPV in the cultural medium was determined. The obtained results are summarized in Table 8. It is seen that the oligonucleotide derivatives inhibit the virus multiplication and that inhibition effect is dependent on the nature of the oligonucleotide derivative structure. The most efficient is the derivative (1) complementary to the 5'-universal sequences in viral RNAs. Some inhibition is observed in the cases of derivatives (2) and (3). Derivative (4) shows no effect. The reasons for differences in the derivative effic~encies are not clear and are most probably related to the availability of the target sequences in functioning complexes of viral RNAs with viral proteins. Under the experimental conditions used, the unreacnve ohgonucleotide analogs of derivatives (1)-(4) do not inhibit the FPV multiplication. Some inhibition of influenza virus with oligonucleotides complementary to 3'-terminal sequences

317

OLIGONUCLEOTIDE INHIBITORS, BIOCHEMISTRY TABLE 8. INHIBITION OF FPV PRODUCTION BY ALKYLATING OLIGONUCLEOTIDE DERIVATIVES FPV yteld, IDso/ml 0.1

(1) (2) (3) (4) Without reagents

Multiplicity of mfectlon, IDs0/cell 1 1" 10

10"

Haemagglutinin titre 0.1"

NT~f NT 1035 1060

1035 106 5 1050 1055

1035 106 5 105 5 1055

NT NT 105 5 1060

NT NT 103 5 10as

1:16 1:32 NT NT

1060

1070

1065

1060

106 5

1:64

*Derivatives were added to the cells together with CaCLv tNot tested. Concentration of derwatwes was 100/~t. At this concentratmn reactive group CIRCH2NH2 itself does not inhibit virus producuon.

of genome RNAs and mRNAs was reported recently (37). Another group observed no effect of oligonucleotides complementary to 3'-terminal sequences of influenza virus genome RNAs on the virus multiplication (38). The results obtained in the present work demonstrate the possibility of influenza virus inhibition with reactive oligonucleotide derivatives. We have not identified the modified products and we can not exclude that the inhibition results from modification of viral proteins such as polymerases capable of specific interactions with nucleic acids (38). Interactions with viral proteins are probably the reason for antiviral activity of some polynoculeotide analogs (39, 40). Reactive oligonucleotide derivatives can be used both for complementary addressed modification of viral nucleic acids and for affinity modification of unique viral nucleic acid binding proteins. Results of experiments described in this and the preceding sections lay the groundwork for an investigation into the use of reactive oligonucleotide derivatives as regulators of nucleic acid functions within living cells. SUMMARY Sequence specific m o d i f i c a t i o n o f nucleic acids with reactive o l i g o n u c l e o tide derivatives, c o m p l e m e n t a r y a d d r e s s e d m o d i f i c a t i o n , can p r o v i d e an efficient a p p r o a c h for specific i n a c t i v a t i o n o f certain cellular nucleic acids. In e x p e r i m e n t s with ascites t u m o r K r e b s II cells a n d a l k y l a t m g o l i g o t h y m i d y l a t e derivatives it was f o u n d that a l k y l a t i n g o l i g o n u c l e o t i d e derivatives e n t e r the living cell a n d m o d i f y c o m p l e m e n t a r y sequences in cellular nucleic acids with high efficiency. C o m p l e m e n t a r y a d d r e s s e d m o d i f i c a t i o n o f p o l y ( A ) sequences in cellular R N A with o l i g o t h y m i d y l a t e derivatives was investigated in detail. The results o f e x p e r i m e n t s on a l k y l a t i o n o f cellular nucleic acids are consistent

318

V.V. VLASSOV, et al

with complementary addressed modification of poly(A) sequences in cellular DNA. These results are supported by experiments on modification of chromatin DNA in which it was found that chromatin DNA interacts with oliogothymidylate derivatives more readily than the isolated double stranded DNA. It was found that alkylating oligonucleotide derivatives complementary to a sequence in immunoglobulin mRNA of MOPC 21 cells arrest the cellular immunoglobulin synthesis. Alkylating oligonucleotide derivatives complementary to RNAs of fowl plague virus inhibit virus multiplication in cell culture.

REFERENCES 1. V. F. ZARYTOVA, G G. KARPOVA, N P. PICHKO and E. A. SHESHEGOVA, Synthesis of alkylatlng derivatives of oligonucleotlde ethylphosphotnesters and their interaction with poly(A), Bloorgan l(hlm 7, 534--541 (1981). 2. V. F ZARYTOVA, E. M. IVANOVA, G. G KARPOVA, D G. KNORRE, N P PICHKO, A. S. RYTE and L. E. STEPHANOVICH, Complementary addressed alkylat~on of poly(A) fragments of mRNA m Krebs ascltes carcinoma cells, Bloorgan. Khlm. 7, 1512-1522 (1981). 3 C. C RICHARDSON, p. 815 m Procedures m Nuclew Acid Research. Vol. II (G. L CANTONI and D R. DAVIES, eds.), Harper and Row, New York (1971). 4. A.G. VENJAMINOVA, N. I. GRINEVA and G. G. KARPOVA, Alkylatmg derivatives of nucleic acids components XVIII. Alkylatlon with 4-(N-2-chloroethyI-N-methylammo)benzyhdene derivatives of 5'-nudeotldes, Izv Slb Otd Akad. Nauk S S S R , set. Khym. Nauk lss 2, 130-138 (1974) 5 R JULIANO and E. MAYHEW, Interaction of polynucleotldes with cultured mammahan cells, Exptl. Cell Res. 73, 3-12 (1972) 6 G BLOBEL and V. R. POTTER, Distribution of radloactlvlty between the acid soluble pool and pools of RNA m nuclear nonsedlmentable and ribosome fractions of rat hver after a single reJeCtion of labelled orotlc acld, Btochlm. Blophys Acta 166, 48-57 (1968). 7. G. P. GEORGIEV, Methods of nucleic acids analysis, isolation and fractionatlon, pp 74-120 m Chemtstry andBtochemtstry ofNuclewActds (I. B. ZBARSKY and S. S. DEBOV, eds ), Lemngrad, Medlzma (1968) 8 J.N. IHLE, K-J. LEE and F. T. KENNEY, Fractlonatlon of 34 S nbonucle~c acid subumts from oncornawruses on polyurdllate-Sepharose columns,J. Biol. Chem 249, 38-42 (1974) 9. M EDMONDS and M G. CARAMELA, The isolation and characterization of adenosine monophosphate-nchpolynudeotldessyntheslzedbyEhdlchascltescells, J Btol Chem 224, 1314-1324 (1969) l0 W.F. MARZLUFF and R. C. C HUANG, Transcription of RNA m isolated nuclei, pp. 89-131 in Transcrlpnon and Translatton a Practwal Approach (B D. HAMES and S J HIGGIS, eds ) IRL Press, Oxford Washington (1984). 11. T. MANIATIS, E. F. FRITSCH and J SAMBROOK, p. 127 m Molecular Cloning A Laboratory Manual, Cold Spring Harbor Laboratory, (1982) 12 G P GEORGIEV, Y. V. ILYIN, A S. TIKHONENKO, N N DOBBERT and L N ANANIEVA, Isolation and properties of chromosomal deoxynbonucleoprotem complexes, Molek Btol U.S S R 1, 815-829 (1967). 13 T. MANIATIS, E. F. FRITSCH and J. SAMBROOK, p. 85 m Molecular Cloning A Laboratory Manual, Cold Spring Harbor Laboratory, (1982), 14 U K LAEMMLI, Cleavage of structural protems durmg the assembly of the head of bacteriophage T4, Nature 227, 680-684 (1970). 15 G G. KARPOVA, D G KNORRE, A S RYTE and L E STEPHANOVICH, Selective alkylatlon of RNA mslde the cell with denvatwe of ethyl ester ofohgothymlddate bearing 2chloroethylammo group, FEBS Lett. 122, 21-24 (1980).

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