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Intracellular metabolism of a 2[prime]-O-methyl-stabilized ribozyme after uptake by DOTAP transfection or asfree ribozyme. A study by capillary electrophoresis
Introduction
Materials And Methods
Cell culture
Synthesis of oligonucleotides
Attachment of fluorescein
Ribozyme
Preparation of the ribozyme-DOTAP complex
Cellular uptake experiments
Efflux experiments
Fluorescence microscopy
Preparation of gel-filled capillaries
Preparation of LPA
Capillary gel electrophoresis (CGE)
Preparation of samples for CGE
Results
Capillary electrophoresis of ribozyme in crude extracts
Uptake and intracellular localization of the ribozyme
Cellular metabolism of the ribozyme
Discussion
Acknowledgements
References
Intracellular metabolism of a 2[prime]-O-methyl-stabilized ribozyme after uptake by DOTAP transfection or asfree ribozyme. A study by capillary electrophoresis
ABSTRACT
INTRODUCTION
It has long been recognized (1) that oligonucleotides have a huge potential as agents for turning off the expression of specific proteins, in most cases working by inducing degradation of the mRNA encoding the protein in question. While antisense oligonucleotides have been the most studied of the oligonucleotide gene expression inhibitors (2-4), ribozymes have during the last years emerged as a viable alternative (5,6). In comparison with antisense oligonucleotides, ribozymes offer a catalytic and more defined and controllable mode of action, features that may be of special importance when conceiving the development of new drugs based on oligonucleotide gene expression inhibition (7).
Based on the structures of naturally occurring hammerhead ribozymes, artificial ribozymes can be constructed to specifically and catalytically cleave almost any mRNA (8-10) and numerous reports on the biological effects of transfected cells expressing such ribozymes have been published (11,12). However, for many applications it would be desirable to deliver a synthetic ribozyme exogeneously, especially from the point of view of developing new drugs based on the ribozyme principle. Two well-known major barriers to the use of both antisense oligonucleotides and ribozymes in this way are their poor ability to enter into the target cells and their instability in serum. To overcome the serum stability problem chemical modifications have been introduced that render the oligonucleotides much more stable to degradative enzymes in serum (13). For example 2[prime]-O-methyl modification of a ribozyme greatly increases its half-life in serum (14) and 2[prime]-O-allyl modification (15) gives ribozymes great stability, which have even been successfully used to inhibit gene expression in vivo after local injection into mice (16).
Despite extensive knowledge about the behaviour of chemically stabilized ribozymes in serum, little is known about the stability of these agents inside the cell. A pharmacokinetic study of a 2[prime]-O-allyl-modified ribozyme injected i.v. into rats indicated that, despite being quite stable in plasma, the ribozyme that could be recovered from the major organs was nearly totally degraded to a major metabolite (17). This indicates that the determinants for intracellular degradation of stabilized ribozymes are quite different from those important for serum stability.
Another important aspect for the use of synthetic ribozymes is the efficiency and the mechanism of uptake of the ribozyme into the target cell. It is at present unclear how different types of synthetic ribozymes are taken up into cells and to what degree the uptake mechanism and subsequent intracellular transport are important for the intracellular stability and biological activity of the ribozyme.
The protein capl (calcium protein placental homologue, a member of the S100 protein family; 18) is involved in the metastatic process and earlier work has shown that the intracellular expression of a ribozyme directed against capl mRNA can inhibit the metastatic process in vivo (19). In the present study capillary gel electrophoresis has been employed as a novel and effective tool for an analysis of the uptake and metabolism of ribozymes in cultured cells. Thus, we have investigated the fate of a 2[prime]-O-methyl-stabilized capl ribozyme when it is taken up by DOTAP transfection and compared these findings with what happens when the cells are subjected to free ribozyme. The work has revealed important differences between these methods regarding the uptake, degradation and intracellular fate of the ribozyme and the findings could have important implications not only regarding the design and delivery method of synthetic ribozymes, but also for the employment of other oligonucleotide-based drug candidates, such as antisense oligonucleotides and RNA or DNA aptamers (20,21).
MATERIALS AND METHODS
Cell culture
The human melanoma cell line THX was established from tumour tissue obtained from a patient treated for metastatic malignant melanoma at the Norwegian Radium Hospital (22). The cells were cultured in RPMI 1640 medium supplemented with 10% fetal calf serum (FCS), 100 U/ml penicillin, 100 µg/ml streptomycin and 2 mM glutamine (all Gibco BRL, Paisley, UK) at 37°C in a 5% CO2 atmosphere.
Synthesis of oligonucleotides
Oligonucleotides were synthesized on a 1 µmol scale using a Perseptive Biosystems 8909 Oligonucleotide Synthesizer (Perseptive Biosystems, Framingham, MA). Phosphoramidites were obtained from Perseptive Biosystems and Glen Research (Sterling, VA) and solid supports from Glen Research. Phosphoramidites and CPG (controlled pore glass) supports for RNA synthesis were protected with 2[prime]-O-TBDMS (t-butyldimethylsilyl) and TAC (t-butylphenoxyacetyl) on A, G and C (23,24). Capping was performed using t-butylphenoxyacetic anhydride (Perseptive Biosystems).
For attachment of fluorescein at internal position L2.2 (25), a thymidine residue carrying an amino linker on the base was incorporated (amino-modifier C2 dT phosphoramidite from Glen Research).
The oligonucleotides were cleaved from their supports and the bases were deprotected by treatment with 40% methylamine (aqueous) (Aldrich, Milwaukee, WI). The CPG-bound material was incubated in 1 ml 40% methylamine at 65°C for 15 min, then chilled on ice. The supernatant was removed and the support rinsed with 500 µl 3:1:1 absolute ethanol:acetonitrile:water. The mixture was evaporated overnight to an amber coloured, viscous residue. The residue was resuspended in 600 µl 4:6:3 TEA-3HF:1-methyl-2-pyrrolidinone:TEA, sonicated for 5 min and then heated at 65°C for 2 h. The mixture was cooled to room temperature and 1.4 ml n-butanol added. The mixture was cooled at -80°C for 30 min and then microcentrifuged at 14 000 r.p.m. for 15 min at room temperature. The supernatant was discarded and the pellet was dried in vacuo for 10 min. The pellet was redissolved in 50 µl water and then 250 µl 95% formamide with 20 mM EDTA and 2 mM Orange G was added. The mixture was heated to 92°C for 3 min, chilled on ice and purified by electrophoresis in a 15% polyacrylamide gel containing 7 M urea at pH 8.5. The product was extracted using the crush and soak technique in 0.5 M ammonium acetate (26). The aqueous solution was concentrated by repeated extraction with n-butanol, brought to 0.3 M in sodium chloride and the product precipitated by addition of 3 vol ethanol.
Attachment of fluorescein
The amino linker group at position L2.2 was reacted with mixed isomeric N-hydroxysuccinimide esters of 5- (and 6-) carboxyfluorescein (Molecular Probes, Eugene, OR) (27). In a typical reaction, 28 nmol oligonucleotide were dissolved in 180 ml 0.4 M NaHCO3/Na2CO3, pH 9.0, N,N-dimethylformamide, water (3:2:1 v/v/v). This solution was diluted with an equal volume of water and 1.5 mg active ester of fluorescein was added. The mixture was kept at room temperature in the dark for 17 h with gentle shaking, then diluted to 6 ml with water. Most of the excess dye was removed by extraction of the aqueous solution with n-butanol followed by ethanol precipitation of the oligonucleotide as described above. The crude product was further purified by denaturing 15% polyacrylamide electrophoresis as described previously. Labelling was confirmed to be approximately stoichiometric from the UV-visible absorbance spectrum.
Ribozyme
A 37mer synthetic 2[prime]-O-methylated ribozyme designed against capl mRNA between positions 88 and 102 with the cleavage site GTC at position 93-95 (reckoning the first nucleotide in the start codon as position 1) was synthesized and labelled with fluorescein as described above. The sequence was 5[prime]-UAG UUC UCU GAU GAG GCC GXU AGG CCG AAA CUU GUU Y-3[prime], where underlining indicates bases with a 2[prime]-O-methylated ribose, Y is a uridine with a 3[prime]-3[prime] inverted thymidine at the 3[prime]-end and X is fluorescein attached to thymidine. The target recognition sequences are indicated in bold.
Preparation of the ribozyme-DOTAP complex
The desired amount of ribozyme and 2.5 µl DOTAP (1 mg/ml; Boehringer Mannheim, Mannheim, Germany) were diluted with serum-free medium to 25 and 50 µl, respectively, and carefully mixed by pipetting several times. The mixture was incubated for 15 min at room temperature. Subsequently the ribozyme-DOTAP complex was gently mixed with RPMI medium containing 10% serum to a final volume of 500 µl.
Cellular uptake experiments
Cells were plated in 24-well plates at a density of 50 000 cells/well. After overnight cultivation the growth medium was removed and replaced with 0.5 ml medium containing either 2.5 µM free ribozyme or 0.5 µM ribozyme complexed to 5 µg/ml DOTAP (as described above). The cells were incubated at 37°C for up to 48 h. After incubation the medium was removed, the cells were washed three times with PBS, treated with trypsin/EDTA, centrifugated and the pellets lysed in 50 µl 1% SDS.
Efflux experiments
For efflux studies cells were incubated for 4 h at 37°C as previously described in medium containing either 2.5 µM free ribozyme or 0.5 µM ribozyme complexed to 5 µg/ml DOTAP. Subsequently the medium was removed, the cells were washed three times with PBS and 0.5 ml fresh serum-containing medium was added. For temperature-dependent efflux studies cells were incubated further at either 4 or 37°C. At variable time points the chase medium was collected, the cells were washed three times with PBS, harvested and lysed in 1% SDS as described above.
Fluorescence microscopy
To study the intracellular localization of the fluorescein-labelled ribozyme, THX cells were plated on 16-well Lab-Tek (Nunc Inc) chamber slides (3000 cells/well). After 24 h the culture medium was replaced with fresh RPMI medium supplemented with 10% FCS and containing either 1 µM free ribozyme or 0.5 µM ribozyme complexed to 5 µg/ml DOTAP. Cells were incubated overnight at 37°C, washed with PBS and unfixed cells were observed with a Zeiss Axioplan fluorescence and phase contrast microscope (Oberkochen, Germany) using a 40× magnification objective. The microscope was equipped with a 450-490 nm band pass excitation filter. Phase contrast and fluorescence micrographs were recorded by means of a cooled charge-coupled device (CCD) camera (Astromed 3200; Astromed; Cambridge, UK). Pixel intensities in individual cells were read from the images after background subtraction using Image 2 software (Astromed).
Preparation of gel-filled capillaries
Polyacrylamide-coated capillaries were prepared according to the method of Hjerten (28). Thus, the capillary (Fused silica capillary TSP075375, 75 µM inner diameter; Composite Metal Services Ltd, UK) was filled with 1 M NaOH and sealed off for 2 h, then flushed with water before flushing with 1 M HCl for 2 min. Then the capillary was rinsed with water and methanol, injected with [gamma]-methacryloxypropyltrimethoxysilane (Bind-Silane; Pharmacia Biotech, Uppsala, Sweden) and sealed for 18 h. The next day the capillary was rinsed with methanol and water and injected with 6% acrylamide solution (without N,N[prime]-methylenbisacrylamide) in 1× TBE (100 mM Tris-borate, 2.5 mM EDTA, pH 8.4) buffer containing 1 µl TEMED (N,N,N[prime],N[prime]-tetramethylethylenediamine) and 10 µl 10% ammonium persulfate. After polymerization a detection window was made by removing the outer capillary coating with a scalpel. Replaceable 5% linear polyacrylamide (LPA) was used to fill the capillary. LPA was replaced prior to every run.
Preparation of LPA
A solution of 5% LPA was prepared by mixing 2.5 ml 40% acrylamide, 4 ml 5× TBE buffer and 11 ml water. The solution was degassed and polymerization was initiated by addition of 8 µl TEMED and 12 µl 10% ammonium persulfate.
Capillary gel electrophoresis (CGE)
CGE was performed on an instrument equipped with an argon ion laser 150 mW (model no. 5425AWC-00; Ion Laser Technology, UT) using an emission wavelength of 488 nm. A high voltage power supply (Spellman CZE 1000R) was used to drive the electrophoresis. Samples were injected electrokinetically at 2 µA (~0.3 kV) for 30 s and run at 3 kV. Separation was performed at 30°C using 1× TBE buffer as running buffer. The migration and quantity of intact ribozyme and degradation products were determined by the use of an internal standard, a rhodamine-labelled 52mer deoxyribo-oligonucleotide (Medprobe, Oslo, Norway).
Quantification was performed using standard curves where peak area ratios of ribozyme to internal standard were plotted against known concentration of ribozyme. The programs used for the calculations were the WorkbenchMac and Acknowledge software (Strawberry Tree Inc., Sunnyvale, CA). The amount of cell-associated ribozyme was calculated as the number of ribozyme molecules per cell after counting the cells in a Bürker chamber.
Preparation of samples for CGE
The samples were prepared by mixing 2 µl 50 nM internal standard (see above) with either 1 µl cell extract or 2 µl chase medium and adding water to a final volume of 10 µl.
RESULTS
Capillary electrophoresis of ribozyme in crude extracts
In initial experiments different methods for the extraction of ribozyme from cells and cell medium were investigated, including standard RNA isolation methods such as extraction with guanidinium isothiocyanate or LiCl/urea/SDS or isolation of RNA by the Trizol method (data not shown). However, all these methods suffered from poor reproducibility and/or sensitivity. It was found that the method giving the highest sensitivity and reproducibility was to make a simple crude lysate where SDS was added to the cells or the growth medium to a final concentration of 1% (data not shown). Incubation studies showed that after addition of SDS no further degradation of ribozyme took place; even unmodified ribozyme was totally stable for at least 3 h at 37°C in a serum-containing medium, whereas in the absence of SDS the ribozyme had a lifetime of only a few minutes.
For quantitation purposes standard curves were made up for ribozyme in cell extract and cell medium (data not shown). The standard curves were linear, with regression coefficients of 0.977 for cell extract and 0.985 for medium. From these experiments the limits of detection in capillary electrophoresis of cell extracts and cell medium could be estimated to be ~10 and 200 pM ribozyme, respectively.
Uptake and intracellular localization of the ribozyme
The uptake of ribozyme was first studied by fluorescence microscopy. As can be seen in Figure
Figure 1. Localization of fluorescence in THX cells. THX cells were incubated with free ribozyme or transfected with DOTAP-ribozyme complex (5 µg/ml DOTAP and 0.5 µM ribozyme) in serum-containing medium and subjected to fluorescence microscopy as described. Treatment conditions were: (A) 4 h incubation with 1 µM free ribozyme; (B) 4 h incubation with DOTAP-ribozyme; (C) 4 h incubation with DOTAP-ribozyme followed by 18 h in ribozyme-free medium. The fluorescence micrographs (upper) were exposed for 10 s and the phase contrast pictures (lower) for 100 ms with a 40× objective. From micrographs such as in Figure To more closely examine cellular uptake and metabolism of the ribozyme, cells were harvested after different incubation periods with free or DOTAP-complexed ribozyme and cell lysates were analysed by capillary electrophoresis as described in Materials and Methods. It can be seen that for DOTAP-transfected cells peaks of intact ribozyme and degradation products could be easily identified (Fig. Figure 2. CGE of ribozyme in crude cell extracts and medium. Cells were incubated with DOTAP-complexed ribozyme (A) or free ribozyme (B) for 4 h as described in Materials and Methods. The cells were lysed in 1% SDS, a rhodamine-labelled oligonucleotide was added as an internal standard and the lysates were subjected to CGE as described. (C) Electropherograms of growth medium from cells incubated with DOTAP-complexed ribozyme for the times indicated in the upper and lower panels. In (A) the upper panel shows the fluorescein signal of the ribozyme and its degradation products, while the lower panel shows the signal from the rhodamine-labelled standard (with some overlap from the fluorescein fluorescence into the rhodamine channel). The positions of intact ribozyme, degradation products and internal standard are indicated on the figure. Migration was from right to left. Since it was also a possibility that the degradation products seen in the cell extracts were not the result of intracellular degradation, but were generated in the growth medium and thereafter taken up by the cells, we also analysed the ribozyme species present in the growth medium at various time points during transfection (Fig.
The time course of cellular uptake and degradation of DOTAP-transfected ribozyme is shown in Figure For ribozyme taken up as free molecules, the peaks were so small that quantitation was difficult. It could, however, be estimated that the amount of intact ribozyme after 4 h incubation was ~300 times lower than was the case for ribozyme taken up by DOTAP transfection. Since the concentration of free ribozyme during these incubations was five times higher than for DOTAP-complexed ribozyme, the actual difference was probably even greater if the uptake was compared on the basis of exogenous ribozyme concentrations. However, using a concentration of 0.5 µM free ribozyme gave no visible peaks in the electropherograms (data not shown).
Figure 3. Uptake of ribozyme delivered by DOTAP transfection. THX cells were incubated with DOTAP-ribozyme complex at 37°C as described. At the time points indicated the cells were washed, lysed in 1% SDS and the cell extract was analysed by capillary electrophoresis. Before lysis the cells were counted in a Bürker chamber. (A) Electropherograms of lysates from cells harvested after different transfection times. The positions of intact ribozyme and degradation products are indicated. (B) The number of intact ribozyme molecules per cell ([solid circle]) was calculated from a standard curve by means of a rhodamine-labelled internal standard as described. The appearance of degradation products ([open circle]) is shown in arbitrary units, since no standard was available for the different unknown degradation products.
A
B
Cellular metabolism of the ribozyme
To gain further insight into cellular metabolism of the ribozyme, experiments were performed in which cells were subjected to DOTAP-complexed ribozyme for 4 h (i.e. reaching the peak value for intact intracellular ribozyme) before the cells were washed and left for a chase period in ribozyme-free growth medium at 4 or 37°C.
From Figure
Figure 4. Retention of intracellular ribozyme. Effect of chase temperature. THX cells were incubated with DOTAP-ribozyme complex for 4 h at 37°C, washed and chased with ribozyme-free medium at 37 ([solid square]) or 4°C ([open circle]) for the times indicated. The cells were harvested, lysed and the lysates were subjected to capillary electrophoresis as described. Interestingly, very little intracellular degradation product(s) could be detected in the chase experiments, while when the cells were continuously exposed to ribozyme a substantial amount of the fluorescent species seen in the electropherograms were degradation products (Fig. In addition, the amount of intact ribozyme inside the cell after 4 h chase seemed to be nearly stable (Fig. In contrast to what was found for DOTAP-transfected ribozyme, for ribozyme taken up as free molecules there were no indications of a population of ribozyme that is not subjected to degradation. Thus, ribozyme taken up in this way seems to be very rapidly broken down. While small amounts of intact ribozyme and degradation products could be detected on continuous exposure of the cells to free ribozyme (Fig. We also investigated the possible export of ribozyme from the cells during the chase period (Fig. Figure 5. Efflux of DOTAP-transfected ribozyme. The cells were treated as described in the legend to Figure 4. After the incubations in ribozyme-free medium at 37 ([solid square]) or 4°C ([open circle]) the medium was removed and subjected to capillary electrophoresis as described.
DISCUSSION
In this work capillary electrophoresis has been employed to investigate cellular uptake and degradation of a fluorescein-labelled chemically stabilized ribozyme. Using laser-induced fluorescence for detection the method offers extreme sensitivity, theoretically making it possible to detect as little as a few hundred molecules in a single peak (29,30). The use of the method to study the metabolism of oligonucleotides is just emerging and most such studies have so far been performed on in vivo samples, and mainly on antisense deoxyoligonucleotides and their derivatives (31-35). However, in a recent report (17) the in vivo fate of a 2[prime]-O-allyl-stabilized ribozyme after injection into rats was investigated.
Compared with these reports the sensitivity obtained in the present work is at least as good as that achieved earlier. Thus, the very simple SDS method for extraction of the ribozyme from the cells compares favourably with other more cumbersome methods, even if the SDS method does not include any step for concentrating the ribozyme. However, in our capillary electrophoresis system the sensitivity of detection of the ribozyme dissolved in pure water was still ~20 times better than the sensitivity in cell lysates (i.e. 0.5 as compared with 10 pM), so, obviously, our ability to detect the ribozyme in crude lysates is strongly reduced by some component(s) present in the lysates. One reason for this may be that electrokinetic injection was employed to apply the sample to the capillary and that ions in the sample reduced the efficiency of injection. Another possibility is that molecules in the lysates in some way quench fluorescence from the fluorophores. We are currently investigating possible ways of further increasing the sensitivity in our capillary electrophoresis method.
One possible pitfall with the capillary electrophoresis method is that any degradation having a net neutral or positive charge at the pH of the analysis products may be missed. An indication of this is that the difference in the microscopic fluorescence between cells subjected to free ribozyme and DOTAP-transfected cells (Fig.
The discrepancy between the fluorescence microscopy and capillary electrophoresis results also indicates that fluorescence microscopy studies of ribozyme localization and intracellular trafficking should be interpreted with caution. Thus, from the results described in the present work it appears that a substantial fraction of the fluorescence observed inside the cells may be due to degradation products and thus may be of limited interest in studies addressing possible biological effects of synthetic ribozymes.
From the capillary electrophoresis analysis the maximum number of intact DOTAP-transfected ribozyme molecules per cell was estimated to be ~1 × 107. This corresponds well with earlier reports where the uptake of radioactively labelled ribozymes has been measured, e.g. Kariko et al. (38) estimated the uptake of a radioactively labelled ribozyme by lipofectin transfection to be 1-10 × 106 molecules/cell.
The amount of cell-associated intact ribozyme was found to be at least 300 times higher when the ribozyme was taken up by DOTAP transfection as compared with ribozyme taken up as free molecules. This is in accord with earlier reports where lipofectin transfection was shown to increase the cellular amounts of intact ribozyme ~100 times compared with the uptake of free ribozyme molecules (39).
2[prime]-O-Methyl-ribozyme taken up by DOTAP transfection seems to have at least three different fates: (i) export from the cell before degradation is initiated; (ii) transfer to a compartment where it is rapidly degraded; (iii) transport to a compartment where it is no longer prone to degradation.
In our chase experiments it could be seen that the amount of cell-associated intact ribozyme during the chase period decreased about seven times from the peak value, while there was a simultaneous increase in the appearance of intact ribozyme in the medium. This points to cellular efflux as an important mechanism for the observed decrease in intracellular ribozyme, as was shown by Fell et al. (40) for another 2[prime]-O-methyl-modified ribozyme.
The reason for the observed increase in the amount of ribozyme in the cell extracts during the first 30 min of the chase period (Fig.
Another possibility is that a fraction of the ribozyme may be present in early endosomes that is rapidly recycled back to the plasma membrane and thus will escape from the cells during harvesting. An indication of such a mechanism is the fact that the amount of intracellular ribozyme increased during the first hour even when the cells were chased at 4°C (Fig.
However, it also seems that a substantial fraction of the internalized ribozyme is degraded. In the continuous presence of DOTAP-ribozyme increasing amounts of intracellular degradation products could be detected, but the observed degradation products are probably intermediates that are quite rapidly turned over to molecular species that cannot be detected after a relatively short chase period.
It is also possible that the degradation products found inside the cell may have been taken up from the outside as a result of degradation of the ribozyme by nucleases in the medium. If this is the case this uptake must, however, be quite specific for the degradation products, since >90% of the ribozyme in the medium is still intact after 12 h incubation (Fig.
Despite the substantial intracellular degradation, a significant fraction of the DOTAP-transfected ribozyme seemed to end up at an intracellular location where it was apparently no longer prone to degradation. Whether this is due to steric and related consequences of DOTAP binding or to sequestration of the ribozyme in nuclease-deficient compartments of the cell is presently not known. However, the findings of Wattiaux et al. (45) that DOTAP transfection delayed the transfer of plasmid DNA to lysosomes may indicate that the second mechanism may play some part, at least. Likewise, the finding that cationic lipids are physically separated from phosphorothioate oligodeoxynucleotides soon after internalization (46) argues against an important role for a direct protection by DOTAP after uptake. Also, in accord with other reports (45,47), our fluorescence microscopy studies indicate that the DOTAP-transfected intact ribozyme is still located in intracellular vesicles even after overnight incubation (Fig.
A different outcome was found when ribozyme was taken up as free molecules. Following 4 h incubation with ribozyme without carrier some intact ribozyme could still be detected inside the cell, similar to the findings of Fell et al. (40). However, this intact ribozyme rapidly disappeared when the cells were chased with ribozyme-free medium. Thus, when the ribozyme was taken up as free molecules there were no indications of a fraction of intracellular ribozyme that was protected against degradation. This agrees with the in vivo results of Desjardins et al. (17). These authors found that after injection of a 2[prime]-O-allyl-modified ribozyme no intact ribozyme could be recovered from cells in any of the tissues examined, even at time points where intact ribozyme could still be found in the plasma. Degradation seemed to occur mainly by cleavage at unmodified ribonucleotides.
Together these results point to what seems to be a difference between at least some kinds of chemically stabilized ribozymes and phosphorothioate antisense deoxyoligonucleotides. While phosphorothioate deoxyoligonucleotides taken up as free molecules can remain intact intracellularly for several days, this is not the case for 2[prime]-O-methyl- and 2[prime]-O-allyl stabilized ribozymes, possibly because of the presence of some unmodified ribonucleotides in these molecules (17). However, it has also been reported that intact chemically stabilized ribozymes can be observed several days after local in vivo delivery (48). In this case the ribozymes, in addition to having 2[prime]-O-methyl modifications and an inverted thymidine at the 3[prime]-end, also had 2[prime]-amino modifications at several nucleotides, perhaps indicating that more extensive modifications may, at least partly, solve the intracellular stability problem for free synthetic ribozymes. It is also possible that the differences seen could be due to the difference between in vivo and in vitro systems.
However, with respect to the intracellular fate of ribozymes, it seems as though the method of uptake and intracellular trafficking of the ribozyme may be at least as important as the mode and degree of chemical stabilization of the ribozyme. Thus, even unmodified oligoribonucleotide ribozymes can remain stable inside the cell when delivered by cationic lipid transfection (38,49,50).
In preliminary experiments we have found that the capl ribozyme after DOTAP transfection reduces the amount of target mRNA to ~50% (unpublished results). It is, however, presently not clear whether the bulk of the intact intracellular ribozyme found after DOTAP transfection participates in this or in other biological effects that have been observed for synthetic ribozymes transfected by means of cationic lipids (39,50-52). Thus, it is still unknown if the active ribozyme in these cases enters the cytoplasm from the putative `non-degradative' vesicles discussed above or if other routes to the cytosol are employed. Observing the quite large amounts of intact ribozyme found inside the cells, it would seem that for most target mRNAs there would be a large excess of intact ribozyme available to generate biological effects if all the ribozyme in a cell was accessible to the target mRNA. Hence, it will be very important to reveal whether, as our results suggest, most of the intact ribozyme is located in intracellular vesicles. If this is the case, it would imply that agents that could promote liberation of the ribozyme (and probably also other kinds of oligonucleotides) from the putative `non-degradative' intracellular vesicles should have the potential to significantly improve the biological effects of oligonucleotides (47). Work is in progress in our laboratories to answer these questions by combining subcellular fractionation and capillary electrophoresis analysis with methods that are known to be able to release the constituents of intracellular vesicles.
ACKNOWLEDGEMENTS
The technical assistance of Tove Øyjord and Anne Bleken is highly appreciated. The work was supported by the Norwegian Research Council and the Norwegian Cancer Society.
REFERENCES
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L. Q. Sun, M. J. Cairns, E. G. Saravolac, A. Baker, and W. L. Gerlach
Catalytic Nucleic Acids: From Lab to Applications
Pharmacol. Rev.,
September 1, 2000;
52(3):
325 - 348.
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