Phosphoramidate oligonucleotides as potent antisense molecules in ...

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Phosphoramidate oligonucleotides as potent antisense molecules in cells and in vivo Marcella Faria1, David G. Spiller2, Catherine Dubertret3, Jeff S. Nelson4, Mike R.H. White2, Daniel Scherman3, Claude Hélène1, and Carine Giovannangeli1* 1Laboratoire de Biophysique, Muséum National d'Histoire Naturelle, INSERM U.201–CNRS UMR 8646, 43 rue Cuvier, 75005 Paris, France. 2University of Liverpool, School of Biological Sciences, Life Sciences Building, Crown Street, Liverpool L69 72B, UK. 3CNRS UMR 7001–ENSCP/Aventis, CRVA, 13 quai Jules Guesdes, 94403 Vitry/Seine, France. 4PE Biosystems, Lincoln Centre Dr., Foster City, CA, USA. *Corresponding author ([email protected]).

Received 15 February 2000; accepted 5 October 2000

Antisense oligonucleotides are designed to specifically hybridize to a target messenger RNA (mRNA) and interfere with the synthesis of the encoded protein. Uniformly modified oligonucleotides containing N3′–P5′ phosphoramidate linkages exhibit (NP) extremely high-affinity binding to single-stranded RNA, do not induce RNase H activity, and are resistant to cellular nucleases. In the present work, we demonstrate that phosphoramidate oligonucleotides are effective at inhibiting gene expression at the mRNA level, by binding to their complementary target present in the 5′-untranslated region. Their mechanism of action was demonstrated by comparative analysis of three expression systems that differ only by the composition of the oligonucleotide target sequence (HIV-1 polypurine tract or PPT sequence) present just upstream from the AUG codon of the firefly luciferase reporter gene: the experiments have been done on isolated cells using oligonucleotide delivery mediated by cationic molecules or streptolysin O (SLO), and in vivo by oligonucleotide electrotransfer to skeletal muscle. In our experimental system phosphoramidate oligonucleotides act as potent and specific antisense agents by steric blocking of translation initiation; they may prove useful to modulate RNA metabolism while maintaining RNA integrity. Keywords: antisense oligonucleotides, phosphoramidate, translation initiation, PPT/HIV-1

Antisense oligonucleotides and analogs are rationally designed specific inhibitors of the genetic information flow that leads from gene to protein. A long-term challenge in this field involves the understanding of oligonucleotide-induced biological activities in cultured cells and in vivo. To do so, one must be able to discriminate between the intended mechanism of action (binding to the complementary RNA target), and the unintended ones (interactions with other cellular components). Identifying potent antisense oligonucleotides is critical for their use as human therapeutics and as molecular biology tools. A large body of work on antisense oligonucleotides makes use of backbone-modified phosphorothioate oligonucleotides. Advances in chemical synthesis have brought forth a number of additional modifications, both on base residues (such as C5-propyne pyrimidines1 or a cytosine analog2), and on sugar (such as 2′-O(2-methoxy)ethyl3) (for a review, see ref. 4). Significant increase in antisense activity can be obtained, as is the case for the three modifications mentioned above. Even though this effect was generally associated with high binding affinity to the RNA target, there could be other intracellular parameters that favor thermodynamic, kinetic, or structural characteristics of the hybrid duplex and enhance antisense-mediated activity. Here, we describe the antisense properties of another family of backbone-modified oligonucleotide analogs, which contain N3′-P5′ phosphoramidate linkages (abbreviated as NP; see ref. 5). These analogs exhibit extremely high binding affinity to single-stranded RNA and possess nuclease resistance6, which make them promising antisense compounds. Recent reports have demonstrated a 40

sequence-specific effect of NP oligonucleotides in cell cultures (antic-myc, c-myb, and bcr-abl7; anti-tax8) and in vivo (anti-c-myc6). In particular, these works clearly illustrated favorable intracellular and tissue distribution of these modified oligonucleotides as compared to other chemistries, but the authors did not exclude the possibility of non-antisense mechanisms for the observed inhibition. In the present work we have established a reporter expression system that allowed us to test the potential of oligonucleotides as translation inhibitors, and to discriminate between true antisense mechanism and other effects. In this system, we have examined the potency of NP analogs, both in cells and in vivo, to act as antisense molecules by binding to the complementary RNA target, present in the 5′-untranslated region, just upstream of the AUG initiation codon of a firefly luciferase reporter gene. As a target sequence we have investigated the polypurine tract (PPT) of HIV-1, which has been described to be involved in various steps of the viral cycle9,10. This inhibitory effect on reporter protein biosynthesis occurred without any induction of RNase H activity. In this context, the NP-modified oligonucleotides directed against the PPT sequence appeared as selective and effective steric blockers of translation initiation. Results Experimental strategy for the demonstration of the antisense effect of phosphoramidate oligonucleotides. The aim of the present study was to evaluate the ability of uniformly modified NP oligonucleotides to act by the postulated antisense mechanism, that is, by binding to the complementary RNA sequence. To provide evidence NATURE BIOTECHNOLOGY VOL 19 JANUARY 2001

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Figure 1. Luciferase expression systems for the demonstration of an antisense mechanism of action. To transiently or stably express the firefly luciferase reporter gene, three constructs were used, all having the reporter gene (luc) under the control of a PGK promoter; in the (+)PPT/luc and (+)mutPPT/luc systems, an insert of 34 base pairs has been cloned, in the 5′-untranslated region (5′-UTR) between the promoter (113 base pairs downstream of the PGK start site) and the translation initiation site (ATG, on the DNA). The insert contains either the wild-type HIV-1/PPT sequence ((+)PPT/luc) or a mutated version with three mismatches (lowercase letters) in the PPT sequence ((+)mutPPT/luc). Alternatively, the insert is omitted from the construct ((–)PPT/luc). Uniformly modified oligonucleotides with either an NP or a backbone contained either the antisense sequence (AS; 18 nucleotides long) complementary to the mRNA transcribed from the wild-type PPT, or the same sequence in an inverted orientation (AS-I) as control. For the NP oligonucleotides, a shorter version (16 nucleotides long, lacking the two terminal adenines) was also used; similar results were obtained. Four additional NP oligonucleotides have been used as controls; the sequences are as follows: 5′T2G6TCT5-3′, 5′-G6T4CT4-3′, 5′-T4C2TCTC3TCT-3′, 5′-T4CT4-3′.

for the oligonucleotide mechanism of action, three cellular systems were designed that differ only by the absence ((–)PPT/luc system) or the presence of a 34 base pair insert containing the oligonucleotide target sequence (PPT: 5′-TTAAAAGAAAAGGGGGGA-3′; in (+)PPT/luc system), or a mutated version (mutPPT: 5′aTAAAAGAAAAaGGaGGA-3′; in (+)mutPPT/luc system; the three mismatches are indicated as lowercase letters). The target sequence was located in the 5′-untranslated part (5′-UTR) of the firefly luciferase reporter gene just upstream the AUG initiation codon (Fig. 1). This is a strategy to prove the molecular mechanism of oligonucleotide-induced inhibition, in that any putative interactions of the antisense oligonucleotide with other cellular components than the inserted target sequence are perfectly identical in the three systems. An interaction of the oligonucleotide with the inserted sequence present on DNA might also occur through triplex formation11,12, but our experimental system allowed us to discriminate between an antisense- and an anti-gene-based effect. As a control we used an oligonucleotide with an inverted polarity as compared to the AS oligonucleotide (AS-I, see sequence in Fig. 1). It can only form a short 9 base pair duplex with the PPT RNA sequence but is able to bind the PPT DNA target sequence by Hoogsteen hydrogen bonding and formation of 16 base triplets13. Thus an anti-gene-mediated effect should lead to (+)PPT luciferase inhibition in the presence of the AS-I oligonucleotide and to a weaker effect—if any—with AS. In contrast for an antisense-mediated effect AS should produce a stronger inhibition of (+)PPT luciferase than AS-I. In addition, an anti-gene effect should decrease the mRNA level (as a result of transcription arrest), whereas an antisense effect should not lead to any change in mRNA in the absence of RNase H-mediated cleavage of the mRNA. The experimental results that are described below clearly eliminate an anti-gene-mediated effect on transcription and provide evidence for an antisense-mediated effect of the phosphoramidate oligonucleotide on translation of the luciferase mRNA. Potency of phosphoramidate oligonucleotides as inhibitors of mRNA translation in cell cultures. Characterization of antisense complexes with the PPT RNA sequence. Biophysical properties of NP NATURE BIOTECHNOLOGY VOL 19 JANUARY 2001


oligonucleotides were studied in vitro. Spectroscopic measurement were used to determine the temperature of half-dissociation of the duplex (Tm in degrees Celsius) formed with a complementary synthetic RNA (PPT-RNA: 5′-UU U2A4GA4G6A CUGG-3', the antisense target sequence being indicated in italics). We have compared the NP analogs to the isosequential phosphodiester and phosphorothioate analogs. The Tm values were 88, 66, and 56°C, respectively, in a buffer containing 10 mM cacodylate at pH 7.2, 3 mM MgCl2, 150 mM KCl, and 1 µM duplex. The Tm value was thus increased by 1.2°C or 1.8°C per phosphoramidate modification, as compared to the phosphodiester or phosphorothioate molecule, respectively. As described for other sequences8,14, the (PPT-RNA)•NP hybrid did not elicit in vitro any RNase H activity in contrast to what was observed with the phosphodiester- and phosphorothioate-containing duplexes. Antisense activity of transfected oligonucleotides. P4-HeLa cells were transfected with oligonucleotides and the firefly luciferaseexpressing plasmids ((+)PPT/luc (+)mutPPT/luc, or (–)PPT/luc). A Renilla luciferase-expressing vector (TK-RL) was cotransfected and used as an internal control for transfection efficiency (see Fig. 2). In the presence of an oligonucleotide complementary to the PPT sequence (AS, see sequence in Fig. 1), an inhibition of luciferase activity was observed with the NP and phosphorothioate analogs in the system containing the target sequence ((+)PPT/luc). In contrast, no effect was obtained with an isosequential 3′-protected phosphodiester oligonucleotide up to 2 µM. None of the “control” NP oligonucleotides (see sequences in legend of Fig. 1) induced any luciferase inhibition in the same conditions. Two control oligonucleotides (AS-I, NP and phosphorothioate; see sequence on Fig. 1) having the same sequence as the AS oligonucleotide, but an inverted polarity did not trigger any inhibition of luciferase expression in the three tested systems ((+)PPT/luc (–)PPT/luc (+)mutPPT/luc) even at 1 µM concentration. This result excluded a mechanism of inhibition involving triplex formation at the DNA level, in that AS-I was able to form 16 base triplets as compared to 9 for AS; however, AS-I did not exhibit any inhibitory activity. The AS oligonucleotide exhibited no inhibitory activity when the plasmids contained the mutated target ((+)mutPPT) or did not contain the PPT target sequence ((–)PPT) (Fig. 2). These results demonstrated that the inhibition of firefly luciferase expression was specific for the targeted PPT sequence and was caused by the interaction of the AS oligonucleotide with the PPT target sequence at the RNA level. Dose-dependent antisense activity was seen for both AS(NP) and AS(PS) oligonucleotides. The NP oligonucleotide was about 10-fold more potent than the phosphorothioate isosequential derivative (60% inhibition for 0.1 µM AS(NP), and 70% for 1 µM AS(PS)). Antisense activity on streptolysin O-permeabilized cells. It has been extensively reported, even if not particularly well understood, that the efficiency of oligonucleotide delivery into cells using transfection vectors such as cationic molecules (lipids, polymers) depends on cell type and on oligonucleotide chemistry15. In order to generalize and delineate the basis for the increased antisense activity of NP oligonucleotides, as compared to oligonucleotides, we have used permeabilized cells to overcome, or at least minimize, such phenomena. The SLObased protocol is among the most efficient methods existing at present, to efficiently deliver oligonucleotides of various chemistries in suspension cells16. Lymphoid cell lines (CEM) were established to stably express the firefly luciferase gene either containing or devoid of the target sequence upstream of the AUG initiation codon (CEM (±) PPT/luc). The two clonal cell lines that were selected expressed nearly the same level of luciferase and differed only by the presence or absence of the 34-nucleotide sequence containing the oligonucleotide target. In the CEM (±) PPT/luc cell lines, the half-life of the luciferase RNA and protein were evaluated using actinomycin and cycloheximide-based methods to be 2 and 4 h, respectively (data not shown). 41


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A

Figure 2. Effect of the NP modification on antisense potency. (PS) Phosphorothioate P4-HeLa cells were transfected with Superfect reagent in the presence of the different luciferaseexpressing systems ((+)PPT/luc (–)PPT/luc (+)mutPPT/luc) and of the Renilla-expressing vector (RL/TK) with various oligonucleotides. The concentrations of oligonucleotides in the final culture volume are indicated. Modulation of firefly luciferase expression by oligonucleotides is presented as the ratio of firefly/Renilla activities (relative luciferase activity) in the same lysate, as described in Experimental Protocol. Similar results were obtained when two sets of transfection were used to introduce the plasmid first and then the oligonucleotides 3–7 h later. None of the control oligonucleotides (see sequences in the legend of Fig. 1) displayed any inhibitory activity at 1 µM concentration.

Total cellular RNA and proteins were analyzed 6 and 24 h after SLO treatment (Fig. 3). A specific inhibition of luciferase expression (Fig. 3A) was induced in the presence of the anti-PPT oligonucleotide (AS) in the SLO-permeabilized CEM(+)PPT/luc cells, but not in the SLOpermeabilized CEM(–)PPT/luc. The control oligonucleotide with the inverted sequence (AS-I) did not decrease luciferase activity in any of CEM (±) PPT cells. Once again the NP oligonucleotide did exhibit the best antisense activity. Thus, in our system, the enhanced potency of the NP-modified analogs was confirmed independently of the mode of delivery, either transfection mediated by cationic molecules or permeabilization with SLO; it is really due to intrinsic intracellular properties of the AS(NP), likely strong binding to mRNA, and low adhesion to proteins. Total RNA was isolated and luciferase RNA levels were evaluated. The GAPDH RNA was used as an internal control (Fig. 3B). The NP oligonucleotide did not affect the luciferase mRNA abundance, which reflected the absence of RNase H activation, and allowed us to exclude transcription inhibition induced by binding to the DNA PPT sequence. In contrast, a specific decrease of luciferase mRNA was observed with the AS(PS) molecule, which induces RNase H cleavage; this result demonstrated that the PPT RNA sequence was accessible to antisense oligonucleotides in this cellular environment. Intracellular distribution of NP oligonucleotides. Intracellular distribution was analyzed to further determine the basis of the enhanced antisense activity of NP oligonucleotides. Fluoresceinlabeled oligonucleotides were used for flow cytometry analysis and confocal microscopy experiments (Fig. 4). Examples of the flow-cytometric dot plots of green fluorescein fluorescence versus red propidium iodide (PI) fluorescence are depicted in Figure 4A. Propidium iodide was used to label the dead cells. Cells exposed to PI and fluorescein-labeled oligonucleotide without SLO treatment, but otherwise subjected to the same protocol, showed essentially neither green nor red fluorescence (lower left quadrant). In contrast, an appreciable fraction of cells (∼95%) that have been treated with an optimized amount of SLO exhibited intense green fluores42

B

Figure 3. Phosphoramidate oligonucleotides that do not induce RNase H are efficient inhibitors of translation initiation. Clonal cell lines stably expressing the (+)PPT/luc (CEM/(+)PPT/luc) or the (–)PPT/luc (CEM/(–)PPT/luc) constructs were reversibly permeabilized by streptolysin O in the presence of various oligonucleotides, as indicated. Oligonucleotide concentration was 50 µM during the permeabilization procedure and 1 µM in final cell culturing conditions. (A) Cell lysates were assayed for luciferase activity 6 h and 24 h after permeabilization. The luciferase activity per living cell (in arbitrary units, a.u.) is shown; the values are normalized for control treatment (SLO permeabilization in the absence of oligonucleotide). (B) Total RNA was blotted and hybridized with labeled RNA probes to luc and GAPDH. The blots corresponding to the 24 h time point are presented. Bar graph shows relative abundance of luciferase transcript, normalized to GAPDH mRNA levels.

cence and low red fluorescence signal, demonstrating that SLO treatment permeabilized the cells to the labeled phosphoramidate oligonucleotide but did not induce marked cell killing. In order to determine the cellular localization of the cell-associated fluorescence detected by fluorescence-activated cell sorting (FACS), confocal microscopy images were displayed (Fig. 4B). Without SLO treatment no intracellular fluorescence was detected. After SLO treatment, NP oligonucleotides gave rise to a homogeneous nuclear fluorescent signal; this result reflects the resistance of NP oligonucleotides to nucleases as described6, in that a diffuse fluorescent pattern was observed using a fluorescein derivative not conjugated to the oligonucleotide or a phosphodiester oligonucleotide–fluorescein conjugate, which was degradated after 6 h of cell culture. The nuclear localization of the NP oligonucleotide would imply that its antisense activity is exerted at the pre-mRNA or mature mRNA before export to the cytoplasm, as observed in other antisense studies3,17. But we cannot exclude that the small fraction present in the cytoplasm is responsible for the antisense activity. In vivo phosphoramidate antisense activity. We have used our reporter expression systems to evaluate the potency of NP oligonucleotides to function as antisense molecules in vivo. The NP antisense oligonucleotide was introduced into muscle fibers together with the luciferase-expressing DNAs (luc(+)PPT or luc(–)PPT, and CMV-RL) using a recently described electrotransfer method18,19. This procedure has been shown to increase gene delivery and expression by more than 100 times, and also to decrease inter-individual variability usually observed after plasmid DNA injection into muscle fibers. Mice (11 per group) were treated with three different doses of the NP antisense (AS) or control inverted NP sequence (AS-I). Control mice received buffer only. Two days after treatment, luciferase expression was examined in the muscle fiber NATURE BIOTECHNOLOGY VOL 19 JANUARY 2001



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A

B

Figure 4. Intracellular distribution of NP oligonucleotides. CEM/luc cells were treated with NP fluorescein-labeled oligonucleotides (Oligo-FITC (NP)), and stained with propidium iodide (PI) to reveal dead cells (red color). (A) Flow cytometry analysis. Dual parameters representation of fluorescein versus (PI) fluorescence, 6 h after oligonucleotide and SLO addition. The percentages of cells in the four regions are indicated. (B) Confocal microscopy was used to follow intracellular distribution patterns of NP-modified oligonucleotides in living CEM/luc cells, in the absence ((–)SLO) or in the presence ((+)SLO) of streptolysin O treatment. The right side image is a zoom on one region of the central image.

lysate. The NP antisense oligonucleotide induced an inhibition of luciferase expression in a dose-dependent manner for the luc(+)PPT system, but no inhibition was detected for the luc(–)PPT (Fig. 5). The control oligonucleotide (AS-I) did not reduce luciferase expression. Discussion The first generation of antisense oligonucleotides, the phosphorothioate analogs, has been extensively studied both in cells and in vivo; phosphorothioate oligonucleotides are used in several clinical trials, and one of them has reached the status of drug (approved to treat cytomegalovirus (CMV) infections of the retina in AIDS patients). However, phosphorothioate oligonucleotides exhibit both decreased binding affinity to complementary single-stranded RNA, as compared to natural phosphodiesters, and significant binding to a variety of proteins20. In order to enhance the antisense activity of oligonucleotides, several modifications have been introduced on sugars and bases (for review, see ref. 21). These different types of antisense oligonucleotides have been found to be effective agents to inhibit gene expression in a sequence-dependent manner in cells and in vivo: direct evidence of their mode of action has been provided in cultured cells3,16,17,22,23, but is much more difficult to obtain in vivo. A modified backbone containing NP linkages was recently described. The evaluation of biological activity with this new class of molecules has been scarce6-8,24, and they appear to be good antisense candidates. The present study shows the potency of NP oligonucleotides as antisense agents: their intended mode of action resulting from binding to the RNA target was demonstrated, and the efficacy of their antisense effect was evaluated in cells and in vivo. These uniformly modified analogs form highly stable duplexes with the target RNA and do not induce RNase H activity. In our work, the NP oligonucleotide was targeted to the 5′-UTR in close vicinity of the AUG initiation codon and acted at the translation initiation level. In vitro translation experiments showed that the same NP oligonucleotide did not exhibit any inhibitory effect when targeted to the coding portion of the mRNA (data not shown). In addition, our results support an in vivo antisense-mediated effect, and are consistent with the good bioavailability described by others6. Additional experiments will allow us to reinforce the general applicability of NP NATURE BIOTECHNOLOGY VOL 19 JANUARY 2001


Figure 5. Phosphoramidate oligonucleotides antisense effect in vivo. Luciferase expression ((+)PPT/luc (–)PPT/luc) and Renilla constructs were introduced into mouse muscle fibers by electrotransfer in the absence or in the presence of various amounts of oligonucleotides, as indicated. Both luciferase activities were measured 48 h after oligonucleotide treatments in the same lysate from homogenized muscle fibers. The relative luciferase activity (ratio of firefly/Renilla) is given in log units; the histograms represent mean values in each experimental group (n = 11); errors bars represent 95% confidence interval.

oligonucleotides as antisense agents when targeting different biologically relevant genes. Phosphoramidate oligonucleotides appear promising for targeting untranslated or splicing regions. They can be particularly effective tools to artificially regulate and also to elucidate biological processes involving RNA, in that NP oligonucleotides act by steric interference and maintain RNA target integrity25. Another class of modified oligonucleotides unable to induce RNase H (2′-O-substituted or morpholino oligonucleotides) has been previously targeted to aberrant17, 26, 28 or alternative27 splice sites, so that antisense binding could either restore the synthesis of a correct protein, or modulate the expression levels of alternatively spliced genes and the associated functions. Antisense-mediated splicing modulation is of considerable general interest, and NP analogs might also suit this kind of strategy. Experimental protocol Oligonucleotides. Modified oligonucleotides containing NP linkages were synthesized using a solid-state support5,29–32. Phosphorothioate and 3′-protected (with a hexyl-amino group) phosphodiester oligonucleotides were obtained from Eurogentec (Seraing, Belgium). Plasmids and cell lines. The plasmids (−)PPT (+)mutPPT (+)PPT/luc contain or lack the PPT insert just upstream of the AUG initiation codon of the luciferase gene as described12 (see Fig. 1). Renilla luciferase expression vectors (Promega, Charbonnières, France) bear the Renilla luciferase gene (RL), under the control of either human herpes simplex virus thymidine kinase promoter (TK-RL) or human CMV promoter (CMV-RL). The firefly luciferase constructs were used in transient expression assays (in vitro transfection and intramuscular electrotransfer) as well as for the generation of clonal stable cell lines derived from CEM-SS human lymphoid cells (CEM(±)PPT/luc cells). P4 cells generated from HeLa cells as reported33 were used in transient transfection assays. Transfection assays. P4 HeLa cells were cultured on Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal calf serum. Superfect (Qiagen, Courtaboeuf, France) and Fugene 6 (Roche, Meylan, France) were used as transfection agents according to manufacturer's guidelines, and equivalent results were obtained whatever the reagent used (see legend of Fig. 2). A typical transfection experiment was performed as follows: 0.5 µg of firefly luciferasecontaining plasmid (PGK/luc), 0.5 µg of Renilla plasmid (TK/RL), and various concentrations of oligonucleotides were mixed with transfectant in a total vol43



RESEARCH ARTICLES ume of 50 µl (serum-free medium). Mixtures were always prepared in order to allow triplicate transfections. The mixture was added to P4 cells (105 cells/well plated in 24-well dishes containing 350 µl of serum-complemented medium per well); the concentrations in the 400 µl culture medium are indicated. After 24 h had elapsed, cell lysates were prepared and assayed for luciferase activity using dual luciferase reporter assay system (Promega). Renilla activity was used to normalize firefly data for internal variations in transfection efficiencies. Values are presented as the mean (± s.d.) firefly/Renilla luciferase activity ratio (relative luciferase activity) of triplicate measurements from a representative experiment that was independently repeated at least three times. Reversible permeabilization. We used SLO (Sigma, St Quentin Fallavier, France) was used to reversibly permeabilize CEM/(±)PPT/luc cell lines in suspension toward oligonucleotides according to a recently revised protocol34. Briefly, SLO (1,000 U/ml) was activated in PBS containing 5 mM dithiothreitol and 0.05% BSA for 2 h at 37°C. CEM/(±)PPT/luc (5 × 106 cells/experiment) were washed and resuspended in 200 µl serum-free medium, then permeabilized by addition of an optimized amount of SLO (40–60 U) and incubated at 37°C for 10 min, in the presence of indicated concentrations of antisense or control oligonucleotides. Resealing was achieved by addition of 1 ml of RPMI containing 10% fetal calf serum and further incubation at 37°C for 20 min. Cells were then transferred to culture flasks containing 9 ml of the same media, which led to 50-fold dilution of oligonucleotide concentration in culturing final conditions. At the indicated times after permeabilization, 500 µl samples were taken for reporter activity, 1 ml for flow cytometry, and 2 ml for mRNA analysis. Protein analysis. Samples were removed from cell suspension at 6 and 24 h after initiation of permeabilization. The luciferase activities shown on figures (in arbitrary units) correspond to the luciferase activities for an equal number of living cells ((luciferase activity in relative luciferase units) / ((% viable cells determined by flow cytometry and PI staining) × (protein concentration in OD595))) (see ref. 12). Results are presented as the mean (± s.d.) of duplicate measurements from a representative experiment that was independently repeated at least three times. mRNA analysis. Total RNA extraction was analyzed by standard RNA blotting. In vitro-transcribed antisense RNA-labeled probes were used. A 383 nucleotide long GAPDH probe was obtained from in vitro transcription of the linearized pTri-GAPDH plasmid (Ambion, Montrouge, France), and a 1,660 nucleotide long luciferase probe was obtained from linearized pGEMluc plasmid (Promega). Determination of oligonucleotide intracellular distribution. Samples were removed from cell cultures at different time points for confocal microscopy and flow cytometry analysis. Dual-parameter flow cytometry using PI (10 µg/ml) for dead cells staining, and fluorescein 5′-labeled oligonucleotide (5 µM during the permeabilization procedure) for monitoring of permeabilized cells, was performed as described35. In vivo gene expression by electric pulse-mediated gene transfer. Delivery of (+)PPT/luc and (+)mutPPT/luc expression systems along with Renilla control vector (CMV/RL) and various amounts of oligonucleotides (as indicated) was done by electrotransfer to murine skeletal muscle fibers as described18,19. Briefly, the DNA mixture (plasmids ± oligonucleotides) was injected into mouse tibial cranial muscle fiber; a few minutes after injection the animals were exposed to an electric field of 250 V/cm (eight pulses, 20 ms, at a frequency of 1 Hz). Two days later the animals were killed, and muscle tissue was collected and disrupted using passive lysis buffer solution (Promega) supplemented with protease inhibitors (cocktail tablets from Roche). Lysates were then assayed for firefly and Renilla luciferase activities using dual-reporter assay system (Promega).

Acknowledgments We wish to thank R.V. Giles and C.J. Ruddell for helpful technical assistance and discussions, A. Winter for generation of stable cell lines, G. Juslin for technical expertise in in vivo experiments. We thank S. Gryaznov, D. Lloyd, and J.K. Chen for providing the first samples of oligophosphoramidates employing the oxidative phosphorylation chemistry, and L. DeDionisio and A. Raible for many of the phosphoramidate syntheses by the phosphoramidite amine-exchange methods. This work was supported by Agence Nationale de Recherche sur le Sida, and Centre International des Etudiants et Stagiaires. M.F. is supported by Coordenaçao de Aperfeicoamento de Pessoal de nivel Superior. 1. Lewis, J.G. et al. A serum-resistant cytofectin for cellular delivery of antisense oligodeoxynucleotides and plasmid DNA. Proc. Natl. Acad. Sci. USA 93,

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NATURE BIOTECHNOLOGY VOL 19 JANUARY 2001
