Biochimie ( 1991 ) 73, 1403-1408
© Socirt6 franqaise de biochimie et biologie molrculaire / Elsevier. Paris
14(}3
Modified oligonucleotides in rabbit reticulocytes: uptake, stability and antisense properties C B o i z i a u * , JJ T o u l m r * , * * Laboratoire de Biophysique, INSERM U201, Musrum National d'Histoire Naturelle. 43, rue Cuvier; 75005 Paris. Frapce
(Received 30 July 1991" accepted 10 October 1991)
Summary - - We have investigated the behaviour of antisense oligonucleotides in rabbit reticulocytes. Both backbone-modified oligomers (methyl-phosphonate and phosphorothioate analogues), anomers of nucleotide units (tz) and oligonucleotides linked to various ligands (intercalator, polylysine, dodecanol) were tested with respect to cellular uptake and inhibition of protein synthesis. Oligonucleotides added at an external concentration of ! 0 HM slowly entered the cell up to a concentration of a few hundred nanomolars. A large fraction of phosphorothioate analogues was found to be associated with the membrane. ¢z-, methylphosphonate and phosphorothioate analogues remained intact during the incubation with reticulocytes although the latter were partly dephosphorylated. Antisen•e oligonucleotides were targeted against three different sites of the rabbit [3-globin mRNA: the 5' end of the message, the initiator AUG or the coding sequence. No specific effect on 13-globin synthesis was observed with any of the investigated compounds. oligodeoxyribonucleotides / translation inhibition 61ntroduction
Globin genes from various organisms have been widely used as targets for antisense oligonucleotides [1-11]. The rabbit globin genes offer a number of advantages: i) m R N A s coding for both the tx- and the 13-chain can be easily purified from reticulocytes, the precursors of mature red blood cells; ii) the presence of two messages, present in similar amounts, with limited primary sequence homologies provide an internal control: oligomers complementary to the czm R N A should not interfere with 13-globin synthesis and vice versa; iii) information on secondary structures deduced from computer calculations and from rmclease digestion are available [ 12-15]; and iv) gene expression can be monitored in cell-free extracts, in injected frog oocytes and in intact reticulocytes. The last point allows one to ask questions about antisense efficiency at levels of increasing complexity. Target accessibility and the effect of competing molecules can be assessed in cell-free extracts. In injected oocytes, the high DNase content, the presence of numerous non-target (endogenous) genes and the compartmentalisation brings the situation close to the case of an intact living cell. But, of course, in neither system is the cell membrane penetration by antisense *Correspondence and reprints and **Present address: Laboratoire de Biophysique MoMculaire, INSERM CJF 90-13, Universit6 de Bordeaux II, 146 rue LEo-Saignat, F-33076 Bordeaux Cedex, France
oligonucleotides taken into account. On the other hand, the use of intact reticulocytes enables one to follow the uptake of unmodified and modified oligomers. Several studies have been devoted to the use of synthetic o!igonucleotides against cellular targets• The expression of oncogenes and the development of • I't .... kaan t 1 1 t ~ ' ~ L : : ~ A A/'~1111~II !11~ 011]tllra/'~. ,.. ,-,,~llc ( {21~i~. vir'dSe, S flays.., ii~bll tlJl.lil~U ijtl.pv-t'li lli ~.~li.iii.i.i ~ [16] for a review). Rabbit leticulocytes represent a particular cell system with limited endocytosis capacity. However, targeting such cells might bring preliminary information for studies using oligonucleotides against protozoan parasites which grow in red blood cells. Moreover, the information recently accumulated on the mechanism of translation inhibition by various chemically-modified oligomers, in cellfree-extracts [8, 10, 11, 17, 181 and injected cells [6, 9] prompted us to investigate the effect of synthetic oligodeoxyribonucleotides on 13-globin m R N A translation in rabbit reticulocytes. A series of oligomers, bearing various modifications in order to make them resistant to DNase degradation, to improve their affinity for the complementary R N A sequence or to promote tt~eir uptake by reticulocytes, were targeted against three different regions of globin mRNA: the 5' end, the initiator AUG or the coding sequence. We monitored both the uptake of these oligonucleotides by reticulocytes, their stability in the growth medium and inside cells and their effect on protein synthesis.
1404
C Boiziau, JJ Toulm6
Materials and methods
In vitro translation
Rahhit reticulocvtes
Experiments were carried out on an aliquot of 8 lal of cells resuspended in either Eagle or Eagle-FCS medium in the absence or in the presence of the desired oligonucleotide. The sample, adjusted to 10 lal with the appropriate medium, was then incubated at 20 or 37°C for 1 h (the final cellular density was approximately 5.6 x 105 cells/laD. At the end of the incubatio-a time, cells were pelleted at 2000 g for 2 rain, washed with 10 volumes of PBS and resuspended, in the absence of oligonucleotides, in 10 ~i of medium minus cold methionine containing 0.37 MBq of [35S]-methionine (Amersham; 37 TBq/mmol). Incubation .,,,'as performed at the same temperature for another hour. Cells were then lysed with 10 111 of 0.2% SDS. Protein synthesis was evaluated by trichloroacetic acid precipitation and the two globin chains were separated on a Triton/urea/acetic acid polyacrylamide gel 121 ].
Reticulocytes were obtained from an anemic male New Zealand white rabbit of about 2 kg. The anemia was induced by injecting ! ml of !% phenylhydr~ine per kg, for 5 consecutive days. Blood was collected on day 7, on citrate buffer at 0°C. and washed with 2 volumes of PBS (phosphate buffered saline) after eli,'a,ination of the buffy coat of white cells. Red blood cells were kept at 4°C in Eagle growth medium containing 7% fetal calf serum, pre-heated for 30 min at 60°C (Eagle-FCS) until use. For experiments in medium without serum, cells were washed once again in PBS and resuspended in Eagle medium. Cells could be used up to 24 h after preparation without any noticeable change in their capacity for synthesis.
Oligontwleotides
Results
Unmodified oligonucleotides synthesized on a Pharmacia automatic synthesizer were purified in one step by HPLC on a reverse phase column (C!8) eluted by an acetonitrile gradient (10-50%) in 10 mM ammonium acetate buffer pH 7.2. or-Analogues and I$-oligonucleotides linked to an acridine derivative or to a dodecanoi group (C12) were prepared by Dr NT Thuong (CNRS, Orleans, France) according to previously published procedures !!91. The ligands were conjugated either at the 3' end of []~oligomers or at the 5' end of or-analogues via a pentamethylene linker. Phosphorothioate derivatives were obtained from Dr JS Cohen (Washington DC, USA). Methylphosphonate oligomers were a gift from Dr G Zon (Applied Biosystems, CA. USA). Polylysine-oligonucleotide conjugates were purchased from Eurogentec (Be!glum). The polylysine tail. about 100 residues long was linked at the 3' end of the oligonucleotide. The oligomer length and purity (except polylysine conjugates) were evaluated by analysis of [3ZPl-labelled molecules (5' or 3') on a 20% polyacrylamide gel containing 7 M urea.
T h e o l i g o n u c l e o t i d e s that we used were targeted to three different s e q u e n c e s o f the 13-globin m R N A : the 5' end (cap), the translation initiation c o d o n (aug) and a part o f the coding r e g i o n (sc) as indicated in table I. O l i g o n u c l e o t i d e s were either 11 or 17 nucleotides long. Besides u n m o d i f i e d oligomers ([3) a series o f a n a l o g u e s was used. T h i s included: n u c l e a s e resistant derivatives - m e t h y l p h o s p h o n a t e (MP), a l p h a (o0, and phosphorothioate (PS) - o l i g o m e r s c o v a l e n t l y linked to an acridine derivative (Acr), in order to increase the affinity o f the m o d i f i e d oligonucleotide for its R N A target [6, 22], and o l i g o m e r s conjugated to groups w h i c h m i g h t promote their uptake - p o l y l y s i n e (pL) [23] and dodecanol (C12). The sequences o f the oligom e r s and the a b b r e v i a t i o n s used in the m a n u s c r i p t are indicated in table I.
Cell uenetration
Peneo'ation o f oligonucleotides into reticulocytes
Oligonucleofides were radiolabelled at their 5' or 3' end with 132PI-¥ATP or [3ZPl-otddATP (Amersham), respectively, according to standard procedures [20]. After purification by gel filtration chromatography 106 cpm were added to a reticulocyte suspension (100 lai ie 5 x 107 cells) and incubated in Eagle or Eagle-FCS medium. Cells were pelleted and washed three times in one volume of PBS; supernatants were pooled. Cells were then lysed by adding 100 pl of water. The suspension was centrifuged (12 000 g for 5 rain) and the membrane pellet was washed once again with water. The cytoplasmic supernatant and the subsequent wash were pooled. The three tractions (medium, cytoplasm and membranes) were counted with a LKB MiniBeta scintillation counter to evaluate the uptake and the membrane binding.
We investigated, at two different temperatures (20 and 37°C), the penetration o f an u n m o d i f i e d 17-mer, 17-~ (sc), o f its ct- and its PS h o m o l o g u e s , and o f 17C 12(sc). Moreover, we c o m p a r e d the b e h a v i o u r o f two phosphorothioate derivatives o f different length: 17PS(sc) and 1 I PS(aug). Incubations were carried out in E a g l e growth m e d i u m without c a l f s e r u m for the u n m o d i f i e d o l i g o m e r 1713(sc). We a n a l y s e d the cytop l a s m i c and m e m b r a n e - a s s o c i a t e d radioactivity as d e s c r i b e d in Materials and methods. A s i m i l a r pattern was obtained for all p h o s p h o d i e s t e r ! 7-mers: the cytop l a s m i c content i n c r e a s e d linearly and leveled off after 7 h. The plateau c o r r e s p o n d e d to a few percent o f the external concentration (fig 1) as o b s e r v e d in several other cell lines [24--271. The h y d r o p h o b i c tail had a marginal, if any, effect on uptake and 17~(sc) had a slightly d e c r e a s e d ability to cross the cell m e m b r a n e c o m p a r e d to its u n m o d i f i e d congener. At 3 7 ° C the m e m b r a n e - a s s o c i a t e d fraction r e m a i n e d low c o m p a r e d to the c y t o p l a s m i c fraction (2 and 5%,
Oligonucleotide degradation Following incubation with the cell suspension and after cell fractionation [32p] end-labelled oligonucleotides from the three fractions were extracted by phenol/chloroform, precipitated with ethanol and analysed on a 20% polyacrylamide gel containing 7 M urea. The degradation pattern was visualised by autoradiography.
Antisense oligonucleotides in rabbit reticuiocytes Table I. Characteristics of antisense oligodeoxynucleotides. The length of the oligomer (17 or 11 nucleotides) and the target region are indicated in the first column: (cap) = 5' end, (aug) = initiation codon, and (sc) = coding region. The location of the complementary sequence of the rabbit 13globin mRNA is indicated in the second column with respect to the cap site (the adenine residue of the AUG initiation codon is number 54). The oligonucleotides sequences are written in the 5' to 3' direction (from the left to the right) except for s-analogues which bind in a parallel manner to their target and for which the extremity on the fight is the 5' end. The analogues available for each sequence are given in the last column: 13 = unmodified, ot = alpha phosphodiester, ~Acr = 3' acridine-linked 13-oligomer, t~Acr = 5' acridinelinked alpha analogue, MP = methylphosphonate, PS = phosphorothioate, pL = polylysine conjugate, C12 = 3' dodecanol-linked oligomer. The abbreviations used throughout the manuscript are composed of the length of the antisense oligonucleotide, followed by the indication of the chemical modification and by the target region. For instance, 17MP(sc) means the 17-mer methylphosphonate derivative targeted to the coding region (complementary to nucleotides ! 13-129).
Oligo Target 17(cap) ! l(aug) 17(aug) 17(sc)
[3-19] [44-54] [51-671 [113-1291
Sequence TTGTGTCAAAAGCAAGT I'TCTGTCTGTT GACAGATGCACCATTCT CACCAACTI'CTrCCACA
Chemistry ~, ~, otAcr, ~Acr, PS, MP 13,pL ~,~,PS, MP, pl, C12
respectively for 1715(sc)). In contrast, a large part (13% for the 17-mer and 33% for the 11-mer) of phosphorothiate oligomers was bound to r . c m b r a n e s in good agreement with a previous study in HL60 cells [25]. A length effect was observed: the phosphorothioate 11-mer was taken up more efficiently than the 17-mer (fig 1). As expected the uptake was more efficient at 37 than at 20°C for all oligomers. No significant uptake was observed at 0°C (not shown). The experiments described in figure 1 were carried out in the presence of 1 nM oligonucleotide. In the presence of 10 taM of the unmodified oligomer 1713(sc) in the medium, the intracellular concentration was about 0.5 taM after a 7-h incubation at 37°C. Similar results were obtained with 17PS(sc): the radioactivity amounted to 8 and 15% in the cytoplasmic and the membrane fraction, respectively (not shown). Degradation in culture medium The fate of unmodified oligonucleotides in culture medium has been described in several reports [28-30]. We compared the resistance of the anti-13globin oiigomers that we used in this study. Moreover, we analysed by gel electrophoresis [32p] 5' end-
1405
7
.~
© Socirt6 franqaise de biochimie et biologie molrculaire / Elsevier. Paris
14(}3
Modified oligonucleotides in rabbit reticulocytes: uptake, stability and antisense properties C B o i z i a u * , JJ T o u l m r * , * * Laboratoire de Biophysique, INSERM U201, Musrum National d'Histoire Naturelle. 43, rue Cuvier; 75005 Paris. Frapce
(Received 30 July 1991" accepted 10 October 1991)
Summary - - We have investigated the behaviour of antisense oligonucleotides in rabbit reticulocytes. Both backbone-modified oligomers (methyl-phosphonate and phosphorothioate analogues), anomers of nucleotide units (tz) and oligonucleotides linked to various ligands (intercalator, polylysine, dodecanol) were tested with respect to cellular uptake and inhibition of protein synthesis. Oligonucleotides added at an external concentration of ! 0 HM slowly entered the cell up to a concentration of a few hundred nanomolars. A large fraction of phosphorothioate analogues was found to be associated with the membrane. ¢z-, methylphosphonate and phosphorothioate analogues remained intact during the incubation with reticulocytes although the latter were partly dephosphorylated. Antisen•e oligonucleotides were targeted against three different sites of the rabbit [3-globin mRNA: the 5' end of the message, the initiator AUG or the coding sequence. No specific effect on 13-globin synthesis was observed with any of the investigated compounds. oligodeoxyribonucleotides / translation inhibition 61ntroduction
Globin genes from various organisms have been widely used as targets for antisense oligonucleotides [1-11]. The rabbit globin genes offer a number of advantages: i) m R N A s coding for both the tx- and the 13-chain can be easily purified from reticulocytes, the precursors of mature red blood cells; ii) the presence of two messages, present in similar amounts, with limited primary sequence homologies provide an internal control: oligomers complementary to the czm R N A should not interfere with 13-globin synthesis and vice versa; iii) information on secondary structures deduced from computer calculations and from rmclease digestion are available [ 12-15]; and iv) gene expression can be monitored in cell-free extracts, in injected frog oocytes and in intact reticulocytes. The last point allows one to ask questions about antisense efficiency at levels of increasing complexity. Target accessibility and the effect of competing molecules can be assessed in cell-free extracts. In injected oocytes, the high DNase content, the presence of numerous non-target (endogenous) genes and the compartmentalisation brings the situation close to the case of an intact living cell. But, of course, in neither system is the cell membrane penetration by antisense *Correspondence and reprints and **Present address: Laboratoire de Biophysique MoMculaire, INSERM CJF 90-13, Universit6 de Bordeaux II, 146 rue LEo-Saignat, F-33076 Bordeaux Cedex, France
oligonucleotides taken into account. On the other hand, the use of intact reticulocytes enables one to follow the uptake of unmodified and modified oligomers. Several studies have been devoted to the use of synthetic o!igonucleotides against cellular targets• The expression of oncogenes and the development of • I't .... kaan t 1 1 t ~ ' ~ L : : ~ A A/'~1111~II !11~ 011]tllra/'~. ,.. ,-,,~llc ( {21~i~. vir'dSe, S flays.., ii~bll tlJl.lil~U ijtl.pv-t'li lli ~.~li.iii.i.i ~ [16] for a review). Rabbit leticulocytes represent a particular cell system with limited endocytosis capacity. However, targeting such cells might bring preliminary information for studies using oligonucleotides against protozoan parasites which grow in red blood cells. Moreover, the information recently accumulated on the mechanism of translation inhibition by various chemically-modified oligomers, in cellfree-extracts [8, 10, 11, 17, 181 and injected cells [6, 9] prompted us to investigate the effect of synthetic oligodeoxyribonucleotides on 13-globin m R N A translation in rabbit reticulocytes. A series of oligomers, bearing various modifications in order to make them resistant to DNase degradation, to improve their affinity for the complementary R N A sequence or to promote tt~eir uptake by reticulocytes, were targeted against three different regions of globin mRNA: the 5' end, the initiator AUG or the coding sequence. We monitored both the uptake of these oligonucleotides by reticulocytes, their stability in the growth medium and inside cells and their effect on protein synthesis.
1404
C Boiziau, JJ Toulm6
Materials and methods
In vitro translation
Rahhit reticulocvtes
Experiments were carried out on an aliquot of 8 lal of cells resuspended in either Eagle or Eagle-FCS medium in the absence or in the presence of the desired oligonucleotide. The sample, adjusted to 10 lal with the appropriate medium, was then incubated at 20 or 37°C for 1 h (the final cellular density was approximately 5.6 x 105 cells/laD. At the end of the incubatio-a time, cells were pelleted at 2000 g for 2 rain, washed with 10 volumes of PBS and resuspended, in the absence of oligonucleotides, in 10 ~i of medium minus cold methionine containing 0.37 MBq of [35S]-methionine (Amersham; 37 TBq/mmol). Incubation .,,,'as performed at the same temperature for another hour. Cells were then lysed with 10 111 of 0.2% SDS. Protein synthesis was evaluated by trichloroacetic acid precipitation and the two globin chains were separated on a Triton/urea/acetic acid polyacrylamide gel 121 ].
Reticulocytes were obtained from an anemic male New Zealand white rabbit of about 2 kg. The anemia was induced by injecting ! ml of !% phenylhydr~ine per kg, for 5 consecutive days. Blood was collected on day 7, on citrate buffer at 0°C. and washed with 2 volumes of PBS (phosphate buffered saline) after eli,'a,ination of the buffy coat of white cells. Red blood cells were kept at 4°C in Eagle growth medium containing 7% fetal calf serum, pre-heated for 30 min at 60°C (Eagle-FCS) until use. For experiments in medium without serum, cells were washed once again in PBS and resuspended in Eagle medium. Cells could be used up to 24 h after preparation without any noticeable change in their capacity for synthesis.
Oligontwleotides
Results
Unmodified oligonucleotides synthesized on a Pharmacia automatic synthesizer were purified in one step by HPLC on a reverse phase column (C!8) eluted by an acetonitrile gradient (10-50%) in 10 mM ammonium acetate buffer pH 7.2. or-Analogues and I$-oligonucleotides linked to an acridine derivative or to a dodecanoi group (C12) were prepared by Dr NT Thuong (CNRS, Orleans, France) according to previously published procedures !!91. The ligands were conjugated either at the 3' end of []~oligomers or at the 5' end of or-analogues via a pentamethylene linker. Phosphorothioate derivatives were obtained from Dr JS Cohen (Washington DC, USA). Methylphosphonate oligomers were a gift from Dr G Zon (Applied Biosystems, CA. USA). Polylysine-oligonucleotide conjugates were purchased from Eurogentec (Be!glum). The polylysine tail. about 100 residues long was linked at the 3' end of the oligonucleotide. The oligomer length and purity (except polylysine conjugates) were evaluated by analysis of [3ZPl-labelled molecules (5' or 3') on a 20% polyacrylamide gel containing 7 M urea.
T h e o l i g o n u c l e o t i d e s that we used were targeted to three different s e q u e n c e s o f the 13-globin m R N A : the 5' end (cap), the translation initiation c o d o n (aug) and a part o f the coding r e g i o n (sc) as indicated in table I. O l i g o n u c l e o t i d e s were either 11 or 17 nucleotides long. Besides u n m o d i f i e d oligomers ([3) a series o f a n a l o g u e s was used. T h i s included: n u c l e a s e resistant derivatives - m e t h y l p h o s p h o n a t e (MP), a l p h a (o0, and phosphorothioate (PS) - o l i g o m e r s c o v a l e n t l y linked to an acridine derivative (Acr), in order to increase the affinity o f the m o d i f i e d oligonucleotide for its R N A target [6, 22], and o l i g o m e r s conjugated to groups w h i c h m i g h t promote their uptake - p o l y l y s i n e (pL) [23] and dodecanol (C12). The sequences o f the oligom e r s and the a b b r e v i a t i o n s used in the m a n u s c r i p t are indicated in table I.
Cell uenetration
Peneo'ation o f oligonucleotides into reticulocytes
Oligonucleofides were radiolabelled at their 5' or 3' end with 132PI-¥ATP or [3ZPl-otddATP (Amersham), respectively, according to standard procedures [20]. After purification by gel filtration chromatography 106 cpm were added to a reticulocyte suspension (100 lai ie 5 x 107 cells) and incubated in Eagle or Eagle-FCS medium. Cells were pelleted and washed three times in one volume of PBS; supernatants were pooled. Cells were then lysed by adding 100 pl of water. The suspension was centrifuged (12 000 g for 5 rain) and the membrane pellet was washed once again with water. The cytoplasmic supernatant and the subsequent wash were pooled. The three tractions (medium, cytoplasm and membranes) were counted with a LKB MiniBeta scintillation counter to evaluate the uptake and the membrane binding.
We investigated, at two different temperatures (20 and 37°C), the penetration o f an u n m o d i f i e d 17-mer, 17-~ (sc), o f its ct- and its PS h o m o l o g u e s , and o f 17C 12(sc). Moreover, we c o m p a r e d the b e h a v i o u r o f two phosphorothioate derivatives o f different length: 17PS(sc) and 1 I PS(aug). Incubations were carried out in E a g l e growth m e d i u m without c a l f s e r u m for the u n m o d i f i e d o l i g o m e r 1713(sc). We a n a l y s e d the cytop l a s m i c and m e m b r a n e - a s s o c i a t e d radioactivity as d e s c r i b e d in Materials and methods. A s i m i l a r pattern was obtained for all p h o s p h o d i e s t e r ! 7-mers: the cytop l a s m i c content i n c r e a s e d linearly and leveled off after 7 h. The plateau c o r r e s p o n d e d to a few percent o f the external concentration (fig 1) as o b s e r v e d in several other cell lines [24--271. The h y d r o p h o b i c tail had a marginal, if any, effect on uptake and 17~(sc) had a slightly d e c r e a s e d ability to cross the cell m e m b r a n e c o m p a r e d to its u n m o d i f i e d congener. At 3 7 ° C the m e m b r a n e - a s s o c i a t e d fraction r e m a i n e d low c o m p a r e d to the c y t o p l a s m i c fraction (2 and 5%,
Oligonucleotide degradation Following incubation with the cell suspension and after cell fractionation [32p] end-labelled oligonucleotides from the three fractions were extracted by phenol/chloroform, precipitated with ethanol and analysed on a 20% polyacrylamide gel containing 7 M urea. The degradation pattern was visualised by autoradiography.
Antisense oligonucleotides in rabbit reticuiocytes Table I. Characteristics of antisense oligodeoxynucleotides. The length of the oligomer (17 or 11 nucleotides) and the target region are indicated in the first column: (cap) = 5' end, (aug) = initiation codon, and (sc) = coding region. The location of the complementary sequence of the rabbit 13globin mRNA is indicated in the second column with respect to the cap site (the adenine residue of the AUG initiation codon is number 54). The oligonucleotides sequences are written in the 5' to 3' direction (from the left to the right) except for s-analogues which bind in a parallel manner to their target and for which the extremity on the fight is the 5' end. The analogues available for each sequence are given in the last column: 13 = unmodified, ot = alpha phosphodiester, ~Acr = 3' acridine-linked 13-oligomer, t~Acr = 5' acridinelinked alpha analogue, MP = methylphosphonate, PS = phosphorothioate, pL = polylysine conjugate, C12 = 3' dodecanol-linked oligomer. The abbreviations used throughout the manuscript are composed of the length of the antisense oligonucleotide, followed by the indication of the chemical modification and by the target region. For instance, 17MP(sc) means the 17-mer methylphosphonate derivative targeted to the coding region (complementary to nucleotides ! 13-129).
Oligo Target 17(cap) ! l(aug) 17(aug) 17(sc)
[3-19] [44-54] [51-671 [113-1291
Sequence TTGTGTCAAAAGCAAGT I'TCTGTCTGTT GACAGATGCACCATTCT CACCAACTI'CTrCCACA
Chemistry ~, ~, otAcr, ~Acr, PS, MP 13,pL ~,~,PS, MP, pl, C12
respectively for 1715(sc)). In contrast, a large part (13% for the 17-mer and 33% for the 11-mer) of phosphorothiate oligomers was bound to r . c m b r a n e s in good agreement with a previous study in HL60 cells [25]. A length effect was observed: the phosphorothioate 11-mer was taken up more efficiently than the 17-mer (fig 1). As expected the uptake was more efficient at 37 than at 20°C for all oligomers. No significant uptake was observed at 0°C (not shown). The experiments described in figure 1 were carried out in the presence of 1 nM oligonucleotide. In the presence of 10 taM of the unmodified oligomer 1713(sc) in the medium, the intracellular concentration was about 0.5 taM after a 7-h incubation at 37°C. Similar results were obtained with 17PS(sc): the radioactivity amounted to 8 and 15% in the cytoplasmic and the membrane fraction, respectively (not shown). Degradation in culture medium The fate of unmodified oligonucleotides in culture medium has been described in several reports [28-30]. We compared the resistance of the anti-13globin oiigomers that we used in this study. Moreover, we analysed by gel electrophoresis [32p] 5' end-
1405
7
.~
