ABSTRACT
It is widely accepted that most cell types efficiently exclude oligonucleotides in vitro and require specific delivery systems, such as cationic lipids, to enhance uptake and subsequent antisense effects. Oligonucleotides are not readily transfected into leukaemia cell lines using cationic lipid systems and streptolysin O (SLO) is used to effect their delivery. We wished to investigate the optimal oligonucleotide composition for antisense efficacy and specificity following delivery into leukaemia cells using SLO. For this study the well characterised chronic myeloid leukaemia cell line KYO-1 was selected and oligonucleotides (20mers) were targeted to an empirically identified accessible site of c-myc mRNA. The efficiency and specificity of antisense effect was measured 4 and 24 h after SLO-mediated delivery of the oligonucleotides. C5-propyne phosphodiester and phosphorothioate compounds were found to present substantial non-specific effects at 20 µM but were inactive at 0.2 µM. Indeed, no antisense-specific effect was noted at any concentration at either time. All of the other oligonucleotides tested induced some measurable antisense effect, except 7 (chimeric, all-phosphorothioate, 2'-methoxyethoxy termini) which was essentially inactive at 20 µM. The rank efficiency order of the remaining antisense compounds was 4 = 3 >> 9 >> 10 = 8 = 5 = 6 > 11. The efficient antisense effects induced by the chimeric methylphosphonate-phosphodiester compounds were found to be highly specific. Increased phosphorothioate content in the oligonucleotide backbone correlated with reduced antisense activity (efficacy: 2'-methoxyethoxy series 9 >> 8 >> 7, 2'-methoxytriethoxy series 10 > 11). No consistent evidence was obtained for increased activity correlating with increased oligonucleotide-mRNA heteroduplex thermal stability. In conclusion, the chimeric methylphosphonate-phosphodiester oligodeoxynucleotides present the most favourable characteristics of the compounds tested, for efficient and specific antisense suppression of gene expression following SLO-mediated delivery.
Chronic myeloid leukaemia (CML) is a proliferative disorder of the haematopoetic stem cell, which results in the accumulation of myeloid cells and their progenitors (1). A specific chromosomal translocation t(9;22)(q34;11), termed the Philadelphia (Ph) chromosome, is found in the great majority of patients with CML. This translocation fuses the c-abl and major breakpoint cluster (bcr) genes (2). The aberrant bcr-abl transcript derived from this region is translated to produce BCR-ABL (p210) tyrosine kinase which displays enhanced activity over wild-type c-ABL (p145; 3) and is believed to be central to the pathogenesis of CML.
CML cell growth in culture has been reported to be arrested following introduction to the growth medium of antisense oligodeoxynucleotides targeting bcr-abl (4,5). Antisense oligodeoxynucleotide-dependent suppression of bcr-abl mRNA expression may be readily obtained (6,7) but p210 BCR-ABL protein levels may not be significantly lowered (8,9), due to the very long half life of BCR-ABL protein (10). Consequently, BCR-ABL may be a poor target for antisense oligodeoxynucleotide mediated inhibition of expression (9). The observed inhibition of cell proliferation following use of bcr-abl targeted oligodeoxynucleotides may, therefore, result from `cross-over' antisense activity at non-target mRNA sites (11) or from non-antisense mechanisms (8,12) which may have a sequence-specific component (13).
On the other hand, MYC function is required for BCR-ABL induced transformation (14,15) and MYC and BCR-ABL may co-operate, via the induction of BCL2, to inhibit apoptosis and enhance tumorogenicity (16). Moreover, MYC protein may provide a better target for antisense inhibition than BCR-ABL, as MYC protein displays a short half life (17) (~15 min) and antisense induced supression of c-myc mRNA has been shown to result in supressed MYC protein expression (9,18).
One of the fundamental problems associated with antisense technology is that of delivery of oligonucleotides into the intracellular compartments of intact cells in vitro. Uptake generally proceeds by incorporation of exogenously applied antisense effectors into endocytotic vesicles, which results in the oligodeoxynucleotides remaining separated from their mRNA molecular targets by a lipid membrane (19-23). Consequently, a range of methods has been examined for their ability to achieve pharmacologically relevant oligodeoxynucleotide concentrations in the cytoplasm and nucleus of target cells. One approach was conjugation of the antisense effector to lipophilic molecules (24) (for example cholesterol; 24,25) or compounds previously described as drug carriers (for example, poly-l-lysine; reviewed in 26). Transfection methods have been tried, such as encapsulation into liposomes (reviewed in 26), association with cationic liposomes (27), polyalkylcyanoacrylate nanoparticles (28) or polyethylenimine (29) and reversible cell permeabilization by electroporation (30) or treatment with SLO (18,31,32).
Different cell types display significantly different responses to the various delivery methods. Human keratinocytes appear to take up oligodeoxynucleotides following simple topical application (22,33). A range of cells take up oligonucleotides following cationic lipid delivery, the most widely used in vitro transfection technique. However, cationic lipids may not enhance the uptake of antisense compounds into leukaemia cells (27). In contrast, human chronic myeloid leukaemia cells from culture lines (7,32,34) and of patient origin (35) respond well to reversible permeabilization with SLO. Indeed, SLO delivery is used in an on-going clinical trial of ex vivo bone marrow purging with oligonucleotides targeted to bcr-abl mRNA (36).
In the present study, congeners of a c-myc phosphorothioate 20mer oligonucleotide were investigated for antisense efficacy and specificity in KYO-1 chronic myeloid leukaemia cells following SLO-mediated delivery. The experimental design was selected, in part, for relevance to future therapeutic ex vivo purging of CML marrows, an interest of the groups working in Liverpool. The oligonucleotides were targeted to bases 1147-1166 of c-myc mRNA (GenBank, `HSMYC1'), a position which appears to be accessible for oligonucleotide hybridisation. The oligonucleotides investigated were phosphorothioate, 5-methylcytosine phosphorothioate, phosphodiester and phosphorothioate C5-propyne pyrimidine structures and chimeric molecules composed of a central ribonuclease H (RNase H) activating region (phosphodiester or phosphorothioate) and termini composed either of methylphosphonate residues or 2'-ribose modified phosphodiester or phosphorothioate residues. Here we present results that show that chimeric methylphosphonate-phosphodiester oligodeoxynucleotides were the most active at suppressing c-myc expression, following intracellular delivery with SLO. The high efficacy was not obtained at the expense of non-specific activity.
The 20mer oligonucleotides used in this study are presented in Table 1. Details of the chemical modifications are presented in Figure 1. The antisense sequence is complementary to bases 1147-1166 of `HSMYC1' (GenBank accession number V00568). The phosphorothioate oligodeoxynucleotide 5, was purchased from Oswel DNA (Southampton, Hampshire). Oligonucleotides 6-11 were supplied by Novartis (Basle, Switzerland).
The acute lymphoblastic leukaemia MOLT4 [European Collection of Animal Cell Cultures (ECACC), Porton Down, Wiltshire] and chronic myeloid leukaemia KYO-1 (generous gift of Drs M.Kirkland and S.O'Brien, LRF Leukaemia Unit, Hammersmith Hospital, London) cell lines were maintained in exponential growth in RPMI-1640 medium (GIBCO, Paisley, Renfrewshire) supplemented with heat inactivated (65°C, 1 h) foetal calf serum (SeraLab, Crawley Down, Sussex) to 10%. All procedures were performed with cultures assayed to be >95% viable by trypan blue exclusion. The hybridoma lines Mycl-9E10 and Mycl-3C7 (which express antibodies to human MYC protein, ECACC) were maintained in exponential growth in RPMI-1640 medium supplemented with 10% heat inactivated foetal calf serum. Supernatant was collected by centrifugal removal of cells and stored at 4°C until required.
SLO was used to reversibly permeabilize cells towards oligonucleotides (7,31,32) according to a recently revised protocol (18). SLO was obtained from Sigma (Poole, Dorset), suspended at 1000 U/ml in Mg2+/Ca2+-free phosphate buffered saline and activated by addition of dithiothreitol to 5 mmol/l followed by incubation at 37°C for 2 h. Small aliquots of activated SLO were stored at -20°C until required. Cells to be permeabilised (5 × 106 per treatment) were washed and resuspended in 200 µl serum-free RPMI-1640 medium, then permeabilzed by addition of 30-50 U SLO and incubation at 37°C for 10 min, in the presence or absence of oligonucleotide. The precise amounts of SLO required for maximal permeabilization and resealing were identified by a dose-response optimisation procedure prior to each experiment. Resealing was achieved by addition of 1 ml of pre-warmed and gassed normal growth media (90% RPMI-1640, 10% heat inactivated foetal calf serum) and a further 20 min incubation at 37°C. Cells were transferred to culture flasks containing 9 ml of pre-warmed and gassed normal growth media and incubation continued at 37°C. Samples of 1 ml (~5 × 105 cells) were taken 30 min, 4 and 24 h after the initiation of permeabilization for flow cytometric analysis. Samples of 2 ml (~106 cells) were removed 4 and 24 h after the start of permeabilization for mRNA and protein analysis.
Dead cells in the samples were stained by addition of 10 µg/ml propidium iodide and incubation for 10 min on ice. Cells were washed, resuspended in 1 ml ice-cold normal growth medium and assayed for red and green fluorescence on a Cytoron Absolute bench top flow cytometer system (Ortho Diagnostic Systems, Loudwater, Buckinghamshire) using a protocol which samples a precisely known volume. This procedure provides information on the total number of cells per ml in the original culture, the % viability of those cells and, when a green fluorescent compound has been introduced by SLO, the % permeabilized and resealed (32).
Total RNA was extracted from cell pellets using a guanidinium thiocyanate/acid phenol method (40). Formaldehyde gel electrophoresis, capillary blotting to nylon hybridisation membrane, northern hybridisation and non-radioactive detection of mRNA species were performed according to standard techniques as previously described (23), except that digoxigenin-UTP labelled antisense in vitro transcripts were used as probe (41) rather than digoxigenin-dUTP labelled random primed DNA fragments.
Cells were washed, pelleted and lysed by addition of 100 or 200 µl warmed lysis buffer (0.04 mol/l Tris, pH 6.8, 10% glycerol, 1% 2-mercaptoethanol, 1% sodium dodecylsulfate, 0.001% bromophenol blue) followed by incubation at 95°C for 10 min. Samples were fractionated by SDS-PAGE (42) through a 6% stacking gel and a 10% separation gel. Proteins present in the gels were electroblotted onto reinforced nitrocellulose membranes (`Optitran', Schleicher and Schuell, UK distributor Anderman and Co. Ltd, Kingston-upon-Thames, Surrey). MYC protein was detected using a 1:1 mixture of Mycl-9E10 and Mycl-3C7 hybridoma supernatants as primary antibody, the secondary antibody was an alkaline phosphatase-conjugated sheep anti-mouse IgG (Sigma). Final chromogenic development was achieved as for northern blotting.
The secondary structure of c-myc mRNA was estimated using the Wisconsin GCG package. FOLDRNA and MFOLD routines were used to calculate structure minimum energies. The results were visualised using the SQUIGGLES routine.
A Shimadzu CS9000 flying spot densitometer (Shimadzu, UK distributor Howe & Co. Ltd, Banbury, Oxfordshire) was used as previously described (43). The densitometrically determined expression of c-myc, p53 and GAPDH mRNA and MYC protein was corrected for the number of viable cells present in the sample from which RNA and protein was extracted [mRNA or protein expression = (densitometric area of band)/(number of viable cells in flow cytometry sample)]. The mean of the three SLO-only control values, present in every experiment, was set to 100% and the data points plotted relative to this $ standard deviation. This procedure was preferred to normalising relative to the expression of a control gene because of the potential for non-targeted activity in antisense experiments.
The oligonucleotides used in this investigation are detailed in Table 1 and their chemical modifications in Figure 1. All antisense compounds are complementary to bases 1147-1166 of c-myc mRNA (`HSMYC1', GenBank Accession number V00568). This site was initially noted as one at which oligomers targeted to the c-myc translation initiation codon (559-573) induced undesired cleavage in vitro (43), non-target sites 1147-1161 and 1264-1278. Pilot experiments were then performed wherein phosphodiester 15mer oligodeoxynucleotides targeted to positions 559-573, 1147-1161 and 1264-1278 of c-myc mRNA were delivered into MOLT4 and KYO-1 leukaemia cells, using SLO, and the resultant c-myc mRNA expression evaluated. Subsequently, 20mer phosphodiester oligodeoxynucleotides directed to the two most active of these regions (557-576 and 1147-1166) were compared to 20mer phosphodiester effectors targeted to sites which were predicted by computer to possess open loop secondary structures (378-397, 1244-1263, 1287-1306 and 1575-1595) for inhibition of c-myc mRNA expression, following SLO delivery. These experiments indicated that the region 1147-1166 was a good target for antisense oligonucleotides.
C5-propyne pyrimidine modified oligomers (Fig. 1; 44-46) have very high RNA heteroduplex Tms, are active at sub-micromolar concentrations in vitro and can elicit specific antisense effects. Antisense, control sense and inverse antisense (reverse polarity antisense) C5-propyne pyrimidine modified phosphorothioate oligodeoxynucleotides were introduced into the cytoplasm and nucleus of the CML cell line KYO-1 using SLO as described in Materials and Methods from extracellular oligomer concentrations of 0.2, 2 and 20 µM. The resultant expression of c-myc mRNA and protein 4 and 24 h afterwards is presented in Figure 2. Figure 2A shows that 4 h after delivery, the 20 µM antisense treatment inhibited c-myc mRNA expression to 37.8 $ 1.2% of SLO-only control. Below this concentration the antisense oligomer was essentially without effect on c-myc mRNA at 4 h. This suppression of c-myc mRNA expression is reflected in reduced MYC protein levels at 4 h (Fig. 2B). It is clear from Figure 2A and B that 20 µM control oligonucleotides suppressed c-myc gene expression to similar extents as was observed with the antisense sequence. Figure 2C and D show that 24 h after treatment with 20 µM antisense, sense or inverse antisense C5-propyne phosphorothioate oligomer, all samples show essentially equivalent suppression of c-myc mRNA or protein expression. Samples treated with 0.2 µM oligodeoxynucleotide show no significant reduction in c-myc mRNA or protein level, whereas samples treated with 2 µM effector present results intermediate to that obtained with 0.2 and 20 µM.
Another class of antisense effector receiving much attention is the so called `second generation' chimeric molecules (47) which possess 2'-modified RNA termini and a central deoxynucleotide core capable of supporting ribonuclease H (RNase H) activity. We were particularly interested in 2'-methoxyalkoxy structures, such as 2'-methoxyethoxy (47-50) and 2'-methoxytriethoxy (47,48). Oligonucleotides constructed with these modifications not only display excellent nuclease resistance, characteristic of bulky 2'-substitutions (51,52), but also retain oligonucleotide-RNA duplex Tms greater than that resulting from 2'-O-methyl RNA-RNA duplexes, which is unusual for large 2'-substituents (53,54).
Figure 3 presents the c-myc mRNA expression 4 and 24 h after SLO-mediated delivery of 2 and 20 µM antisense 20mer phosphorothioate and chimeric 2'-methoxyalkoxy oligodeoxynucleotides. Three separate experiments were performed, each containing three oligonucleotide species, compound 10 (5-methylcytosine, 2'-methoxytriethoxy phosphodiester termini and phosphorothioate 2'-deoxy ribose gap) was included in all three experiments to permit meaningful comparison. The three experiments appear to be directly comparable, 4 h after introduction of 20 µM of oligonucleotide 10 into KYO-1 cells c-myc, mRNA expression was reduced to ~40%, relative to control, while 2 µM of this compound resulted in expression of ~83% of the SLO-only control respectively. Figure 3A and B compare an all-phosphorothioate (5) and an all-phosphorothioate which contains 5-methylcytosine in place of cytosine (6) to the common oligomer (10). It may be seen that all three compounds present essentially equivalent results at both 4 and 24 h time points. The chimeric oligonucleotide with 5-methylcytosine, 2'-methoxyethoxy phosphodiester termini and a phosphorothioate DNA gap (8) also displayed activity similar to 10 (Fig. 3C and D). An all-phosphorothioate version of 8, oligomer 7 (5-methylcytosine, 2'-methoxyethoxy phosphorothioate termini and a phosphorothioate DNA gap), appeared to be significantly less active than 10 at 4 h (Fig. 3C). Similarly, the all-phosphorothioate version of 10, compound 11 (5-methylcytosine, 2'-methoxytriethoxy phosphorothioate termini and a phosphorothioate DNA gap), was also substantially less active than the common oligomer (Fig. 3E and F). In contrast, the only all-phosphodiester compound in these tests, structure 9 (5-methylcytosine, 2'-methoxyethoxy phosphodiester termini and a phosphodiester DNA gap), was substantially more active than 10. c-myc mRNA was almost undetectable at 4 h in samples which were treated with 20 µM oligomer and reduced to 21.5 ± 3.9% following treatment with 2 µM effector.
In the light of the above observations it was considered appropriate to examine methylphosphonate-phosphodiester chimeric structures in the same experimental system. It has previously been demonstrated that the methylphosphonate internucleoside linkage is helix-destabilising, particularly for oligonucleotide-RNA heteroduplexes, and that chimeric compounds with incremental replacement of phosphodiester internucleoside linkages with methylphosphonate residues display incrementally reduced RNA heteroduplex Tms (57). Notwithstanding the reduced heteroduplex Tms, such compounds have been demonstrated to provide efficient and specific antisense effects when introduced into cultured cells (7,58).
Figure 4 presents the data obtained when an antisense chimeric methylphosphonate-phosphodiester with six methylphosphonate residues at each end and a central region of seven contiguous phosphodiester linkages, (4) was compared to the `common' oligomer from Figure 3 (10) and the most active of the 2'-ribose modified chimeric compounds (9). Human CML KYO-1 cells were reversibly permeabilized with SLO in the presence of 20, 2 or 0.2 µM oligonucleotide, or in the absence of oligomer and samples taken for analysis after 4 and 24 h. The data in this figure demonstrates that MYC protein expression in cells 4 and 24 h after treatment with oligonucleotides (Fig. 4B and D, respectively) essentially reflects the c-myc mRNA expression at these time points (Fig. 4A and C, respectively). It is also readily apparent that the c-myc mRNA expression (Fig. 4A and C) resulting from treatment with 2 and 20 µM 9 and 10 is consistent with that presented in Figure 3. Lower concentrations of these two oligomers (0.2 µM) resulted in no observable suppression of c-myc gene expression. In comparison, 4 may be seen to be ~10-fold more active than 9,with expression reduced to 1.7 $ 0.1%, 1.7 $ 0.1% and 30.0 $ 4.0% of mean SLO-only control in samples treated with 20, 2 and 0.2 µM of this methylphosphonate-phosphodiester chimeric oligodeoxynucleotide, respectively.
The antisense inhibition of c-myc mRNA expression resulting from SLO-mediated introduction of 20, 2 and 0.2 µM 4 can be seen to last for at least 24 h (Fig. 4C). The antisense suppression of c-myc mRNA in cells 24 h after introduction of 2 and 0.2 µM compound 9 may be seen to be rather less efficient than those induced by oligomer 4.
Cells in which the c-myc mRNA and protein expression had been inhibited for 24 h by oligomer 4 displayed abnormal proliferation. A dose dependent inhibition of cell growth was observed. Cells treated with 20 µM of this chimeric methylphosphonate-phosphodiester antisense oligodeoxynucleotide decreased in number by 12 $ 9% by the 24 h point. Cultures from cells treated with 0.2 µM of compound 4 proliferated, but at only half the rate of SLO-only control cells. Intermediate effects were obtained with cells SLO-permeabilized in the presence of 2 µM 4, neither proliferation nor decrease in number between the 4 and 24 h points was observed.
To ensure that the suppression of c-myc mRNA and protein evident in Figure 4 and the inhibition of proliferation were sequence-specific antisense effects a series of controls were performed. These data are presented in Figure 5. The samples used to provide the 4 h mRNA data of Figure 4 were also examined for non-target mRNA expression. Figure 5A presents the expression of target c-myc mRNA and non-target p53 and GAPDH mRNAs 4 h after SLO-mediated delivery of 0.2, 2 and 20 µM structure 4, relative to control SLO-only treated samples. The c-myc mRNA expression in this figure represent the same data as presented in Figure 4A and are included for comparative reasons. No significant reduction of p53 mRNA expression was obtained at any concentration tested. However, a dose-dependent reduction in GAPDH mRNA was observed. Examination of the GAPDH mRNA sequence (`HSGAPDR' GenBank accession number X01677) revealed a substantial partial complementarity to compound 4 (over bases 965-975, ten base pairs in total and including nine contiguous base pairs) which had the potential to direct the activity of RNase H. Lanes in northern blots probed for GAPDH, from samples treated with 20 µM 4, demonstrated the existence of a band with approximately the predicted size of the putative 5' fragment generated by RNase H cleavage of GAPDH mRNA at the site of partial complementarity with the antisense oligomer (data not shown). The antisense effectors 5-11 also induced modest decreases in GAPDH mRNA expression which were broadly in proportion to their targeted reduction of c-myc mRNA level, when delivered from 20 µM extracellular concentration (data not shown). Nevertheless, no non-targeted cleavage of GAPDH mRNA, nor evidence of reduced GAPDH mRNA expression, could be detected when 0.2 µM of the antisense effector 4 was used. Moreover, no reduction in cellular GAPDH protein levels were observed either 4 or 24 h after SLO-mediated delivery of 0.2, 2 or 20 µM oligomer 4. Thus, this interesting non-targeted antisense effect is most unlikely to induce any physiological response over the period investigated.
We have investigated an antisense cell culture system which is highly relevant to potential ex vivo purging of CML marrows. SLO transfection has previously been demonstrated to efficiently deliver oligonucleotides into CML cell lines (7,18) and primary cultures (35). In this work we have investigated the ability of different oligonucleotide compositions to efficiently and specifically suppress c-myc mRNA and protein expression following SLO-mediated delivery into the KYO-1 CML cell line.
Oligonucleotides composed of terminal methylphosphonate `wings' and a central phosphodiester `gap' were found to provide the most efficient and specific results. When chimeric molecules with this composition were used, antisense inhibition of c-myc gene expression was maintained for at least 24 h and this resulted in a sequence-specific inhibition of proliferation. Molecules with this general structure and targeted to c-myc may provide for a future ex vivo purging agent.
Chimeric molecules with 2'-methoxyethoxy or 2'-methoxytriethoxy wings and a 2'-deoxy gap were less active in this SLO-delivery cell culture assay. The reduced activity may be related to the lesser ability of 2'-ribose modified chimeric molecules to direct the activity of RNase H (59), relative to parent 2'-deoxy compounds. Interestingly, increased phosphorothioate content in such compounds was found to correlate with markedly reduced antisense activity. This phenomenon may reflect the widely reported propensity of phosphorothioate oligonucleotides to interact with protein.
In contrast to the published results, we found that no sequence-specific antisense effect could be obtained with C5-propyne pyrimidine phosphorothioate oligodeoxynucleotides when delivered using SLO.
There is a substantial contrast between the documented efficiency of phosphorothioate 2'-ribose modified chimeric molecules and C5-propyne pyrimidine oligonucleotides, and what we report here. On the basis of the work presented here, we are unable to exclude the possibilities that (i) different target sites within the c-myc mRNA would demonstrate different intrinsic sensitivities to oligonucleotides with the various chemistries that were used. For example, poor antisense target sites involved in mRNA secondary and tertiary structure might be more amenable to attack by 2'-methoxyethoxy chimeras than methylphosphonate chimeras as a result of the greater hybridisation potential of the former. In this case the order of relative efficacy that we have observed might well be reversed. Or that (ii) alternative delivery methods could result in a changed rank potency. For example, the oligonucleotide efficacy order that we obtained using SLO reversible permeabilisation would not be duplicated if cationic lipid delivery vehicles were used, with an appropriate cell line. We have observed aberrantly low cellular uptake of methylphosphonate-phosphodiester chimeric molecules when using a cationic lipid preparation, relative to phosphorothioate analogues with the same length and sequence, due to the reduced net negative charge on the chimeric oligonucleotide backbone (unpublished observations). On the other hand, identical rank potencies to those described here are obtained for phosphorothioate and chimeric methylphosphonodiester-phosphodiester oligodeoxynucleotides delivered by electroporation (unpublished observations). Furthermore, it is possible that different delivery methods may result in altered subcellular distribution and/or bioavailability of the oligonucleotides. This point has not been adequately addressed in the literature. We are performing an investigation into these factors following SLO, electroporation and cationic lipid-mediated delivery of fluorescein-tagged phosphorothioate oligomers into cells of leukaemic and non-leukaemic origin.
The authors would like to thank Drs Karl-Heinz Altmann and Francois Natt for the synthesis of oligonucleotides 6-11. This work was supported by grants from the Leukaemia Research Fund and Novartis Horsham Research Centre.
Nucleic Acids Research
Pages
Introduction
Materials And Methods
Oligonucleotide synthesis
Cell culture
Reversible cell permeabilisation
Flow cytometry
mRNA analysis
Protein analysis
RNA folding prediction
Densitometry and presentation of data
Results And Discussion
C5-propyne pyrimidine oligomers
Chimeric 2'-methoxyalkoxy-2'-deoxy structures
Chimeric methylphosphonate-phosphodiester structures
Conclusions
Acknowledgements
References
REFERENCES
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