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INTRODUCTION |
Trypanosomatids are attractive targets for the antisense approach,
as every mature transcript contains a common species-specific mini-exon
sequence spliced onto its 5' end (1, 2). Maturation of mRNAs
includes a trans-splicing event, which transfers a 39-nucleotide segment of the mini-exon derived pre-RNA (the
medRNA)1 to the 5' end of
every message (3). Hybridization of an antisense oligonucleotide (ODN)
to this sequence can potentially inhibit translation of all
transcripts. Indeed, it was demonstrated that anti-mini-exon
oligonucleotides were able to prevent translation of
Trypanosoma and Leishmania mRNAs in cell-free
extracts (4-6), to kill procyclic forms of T. brucei (7),
and to cure L. amazonensis-infected macrophages in
vitro (Refs. 8 and 9; see Ref. 2 for a review).
The trypanosomatid medRNA was proposed to adopt a secondary structure
based upon conservation of folding pattern for different RNA (10).
Recently the Leptomonas collosoma medRNA was shown to switch
between two alternate structures (11, 12). One form leads to a base
pairing pattern conserved for all of the trypanosome medRNA, suggesting
critical functional interactions for splicing (11). In L. amazonensis the mini-exon was shown to fold into a structure that
interferes with the hybridization of antisense ODNs (13, 14). RNA
intramolecular structures that prevent the formation of
oligonucleotide-RNA intermolecular complexes weaken antisense
effects. This limitation has prompted the design of
oligonucleotides able to overcome the mini-exon structure (for a
review, see Ref. 15).
In a recent report, the L. amazonensis mini-exon sequence
was efficiently complexed by an ODN capable of folding back on itself to form a triple strand with the putative hairpin element (16). This
"double hairpin" complex readily formed at pH 6.0 using a pyrimidine motif for triplexing. However, this oligomer did not show
selective inhibitory properties in cell-free translation experiments,
probably due to pH conditions that are not appropriate for the
formation of a triple helix involving C-G*C+ triplets.
Alternatively, the disruption of the secondary structure by oligomers
of high affinity can be considered.
We have been investigating selectively binding complementary (SBC) ODNs
as an alternative for targeting structured nucleic acids (17, 18).
These ODNs are intended to be used as complementary pairs or as a
single self-complementary agent. Due to the presence of modified bases,
they are unable to form stable hybrids with one another but should
hybridize to normal DNA or RNA complements. We have shown that SBC ODNs
containing 2-aminoadenine and 2-thiothymine bases can strand invade the
end of double-stranded DNA in a process that is favored both
kinetically and thermodynamically (17). The A/U-rich hairpin proposed
for the L. amazonensis mini-exon sequence presents an ideal
target for testing whether SBC ODNs can strand invade an RNA stem loop
by hybridizing to every base of the element.
In this study we experimentally confirmed the hairpin structure of the
L. amazonensis mini-exon sequence and demonstrated that
antisense ODNs with SBC character are more effective than normal base
(NB) ODNs in addressing this hairpin. Our results show that SBC ODNs
form very stable hybrids with the entire hairpin sequence, and that the
heteroduplexes are substrates for Escherichia coli RNase H. We demonstrated that SBC ODN-RNA hybrids inhibit translation of
L. amazonensis mRNA in a cell-free extract when RNase H
is present. The successful targeting of a simple hairpin by SBC ODNs
suggests that other secondary structure features in RNA should also be
accessible to these ODNs.
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EXPERIMENTAL PROCEDURES |
Oligonucleotide Synthesis--
Preparation of protected
phosphoramidite precursors of 2-thiothymidine and 2-aminoadenosine and
synthesis of SBC ODNs using these reagents have been described (17). NB
ODNs with DNA, RNA, or 2'-O-methyl backbones were
synthesized by routine procedures using commercially available
chemicals. The Beaucage reagent was used to prepare NB and SBC ODNs
with a phosphorothioate backbone (19). All antisense ODNs contained a
3'-hexanol end group as a consequence of using a modified hexanol
primer controlled pore glass support (20). Capillary gel
electrophoresis indicated that the SBC ODNs were at least 85% pure,
and hydrolysates of SBC ODNs prepared as described previously (17) gave
the expected ratios of nucleosides. Extinction coefficients of ODNs
were calculated using a nearest neighbor model (21) and employed values
of 9,800 M
1 cm
1 for
2-thiothymidine (22) and 6,800 M
1
cm
1 for 2-aminoadenosine (23) at 260 nm.
RNA Synthesis--
The mini-exon RNA of L. amazonensis was prepared by in vitro transcription of
pBluescriptIIKS in which the mini-exon sequence was cloned downstream
of the T7 promoter, using Ampliscribe T7 transcription kit (Tebu). In
the resulting transcript, the original mini-exon sequence was flanked
by sequences derived from the vector, both on the 5'
(5'-GGGCGAAUUGGAGCUC) and on the 3' sides (5'-GAUC). The RNA was
purified by electrophoresis on a 12% polyacrylamide denaturing gel.
Nuclease Mapping of Mini-exon RNA Secondary
Structure--
5'-End-labeled mini-exon RNA (100 pmol) was incubated
for 20 min at 37 °C in the presence of 1 unit of S1 nuclease
(Boehringer Mannheim), in a 50 mM sodium acetate buffer (pH
4.5) containing 28 mM NaCl and 4.5 mM
ZnSO4. RNases T1 and V1 (Boehringer Mannheim) digestions
were performed for 10 and 20 min, respectively, in 20 mM
HEPES buffer (pH 7.4) containing 140 mM KCl, 20 mM sodium acetate, and 3 mM MgCl2
(S buffer). Cleavage by 15 µM methyl
propidium-EDTA-Fe(II) complexes was carried out for 10 min at 37 °C
in S buffer. RNase T1 digestion under denaturing conditions was
achieved for 10 min at 50 °C in 20 mM sodium acetate and
5 M urea. Digestion products were analyzed by
electrophoresis on a 12% polyacrylamide denaturing gel followed by autoradiography.
Determination of Hybrid Melting Temperatures--
Complementary
ODNs were diluted in 20 mM HEPES, pH 7.2, 10 mM
MgCl2, and 140 mM KCl to give 2 µM of each ODN. Hybridization was assured by rapid
heat-cooling of the samples. A260 was recorded as a function of temperature in a Lambda 2 (Perkin-Elmer)
spectrophotometer equipped with a PTP-6 automatic multicell temperature
programmer. Samples were heated at the rate of 0.5 °C/min. Melting
temperatures (Tm values) were determined from
the derivative maxima.
Determination of Hybrid Equilibrium Dissociation Constants by
Electrophoretic Mobility Shift Assay--
The mini-exon RNA prepared
by in vitro transcription as described above was labeled by
incorporating [
-32P]ATP (37.5 MBq/mmol). RNA and
oligonucleotide were heated separately for 5 min at 65 °C and cooled
down on ice. One pmol of RNA was mixed with the desired ODN in 15 µl
of 50 mM Tris acetate buffer (pH 7.0) containing 10 mM magnesium acetate. The mixture was incubated for 15 min
at 4 °C. The samples were then run in the same Tris buffer at 10 V/cm for about 15 h, on a 15% nondenaturing polyacrylamide gel at
4 °C. The activity in the bands corresponding to the free and bound
RNA species was evaluated by PhosphorImager analysis or by Cerenkov
counting. The dissociation constant was taken as the ODN concentration
at which 50% of the target RNA was retarded.
L. amazonensis mRNA Preparation and in Vitro
Translation--
Total RNA was isolated from L. amazonensis
promastigotes by the guanidine chloride method (6). In vitro
translation of this RNA (1 µg) was catalyzed by wheat germ extract
(25 µl; Promega) or rabbit reticulocyte lysate (35 µl; Promega) in
a total volume of 50 µl. Antisense ODN was added to the RNA on ice,
immediately before the initiation of translation, and reactions were
conducted for 1 h, at 25 °C (wheat germ extract) or 30 °C
(rabbit reticulocyte lysate) in the presence of
[35S]methionine (37 TBq/mmol; Amersham). Some incubations
were supplemented with 2.5 units of E. coli RNase H
(Boehringer Mannheim). Reaction aliquots were analyzed for labeled
proteins by precipitation with trichloroacetic acid onto Whatman GF/A
glass fiber filters and counting in a liquid scintillation counter.
Relative levels of protein synthesis were calculated as described
previously (13).
Mapping of RNase H Cleavage Sites--
A synthetic RNA 35-mer
was used for RNase H mapping of L. amazonensis mini-exon-ODN
complexes. Prior to incubation with RNase H, the oligonucleotide and
the RNA were treated as described for electrophoretic mobility shift
assay. 5'-End-labeled RNA was then mixed with the desired ODN at a
final concentration of 2 and 50 µM, respectively, in a 20 mM HEPES buffer, pH 7.8, containing 50 mM KCl,
10 mM MgCl2, and 1 mM
dithiothreitol. The mixture was kept at 4 °C for 15 min prior to
incubation with 0-4 units of E. coli RNase H (Promega) for
15 min at 4 °C. The reaction was stopped by adding one volume of 8 M urea. Samples were analyzed by electrophoresis on a 20%
polyacrylamide gel containing 7 M urea.
In Vitro Treatment of Leishmania-infected
Macrophages--
Preparation of macrophages and parasites was carried
out as described (8). Leishmania amazonensis (LV79 strain),
prepared from infected BALB/c mice, was used to infect adherent
macrophages at a multiplicity of 5 parasites/cell, resulting in more
than 70% infected cells. This was normalized to 100% for comparison between different experiments. Infected macrophages were incubated at
34 °C with the desired oligonucleotide concentration for 48 h
in RPMI/HEPES medium containing 10% fetal calf serum. The cultures were then washed, fixed in methanol, and stained with Giemsa. Cells
were observed by microscopy to determine the level of infection. About
500 macrophages were scored for each oligonucleotide concentration; a
cell was identified as infected when it contained at least one recognizable parasite.
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RESULTS |
The Mini-exon Sequence of L. amazonensis Forms a Hairpin--
The
mini-exon region is known to fold into secondary structures (11, 12).
We investigated the structure of a 35-nucleotide-long mini-exon
sequence in which the four modified nucleotides (24) at the 5' end of
the natural sequence were omitted (Figs.
1 and 2).
Indirect evidence favors the existence of a stable structure in the
mini-exon sequence of L. amazonensis. Anomalous
electrophoretic mobility of this oligomer as well as low binding
efficiency of complementary oligonucleotides have been observed
previously (6, 13). Prior to initiating the targeting of this structure
with NB and SBC ODNs, we confirmed its existence by footprinting. The cleavage pattern obtained with nuclease S1, RNases T1 and V1, and
methylpropidium-EDTA (MPE) led to the secondary structure shown in Fig.
1. Positions 23-26, which are cleaved by S1 nuclease (lane
4), correspond to the apical loop of the imperfect hairpin. Whereas G23 was a cleavage site for RNase T1, no band corresponding to
G17 and G29 was observed suggesting a structured region. This was
further confirmed by RNase V1 and MPE, which preferentially cleave
double-stranded structures. The reduced cleavage observed with RNase V1
compared with MPE in the upper part of the stem might be related to the
bulged U20. The double-stranded stem likely extends to the
U35-U37/A13-A11 region as indicated both by RNase V1 and MPE
sensitivity, whereas both G8 and G39,G40 have a clear single-strandedness character. The structure of the upper part of the
stem is in fair agreement with previous models (11); the differences at
the bottom of the stem region are related to the different sequences
used.

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Fig. 1.
Secondary structure of L. amazonensis mini-exon RNA. A, mapping of
mini-exon RNA incubated with: RNase T1 under denaturing
(lane 1) or nondenaturing conditions
(lane 2), nuclease S1 (lane
4), RNase V1 (lane 5), or MPE
(lane 6). The alkaline digestion of the mini-exon
RNA is in lane 4. The G residues are identified
to the left. B, the mini-exon hairpin structure
deduced from nuclease mapping. The italicized bases
correspond to regions of the plasmid used to generate the mini-exon by
in vitro transcription. The mini-exon sequence is numbered
with respect to the natural sequence, even though the first four
modified nucleotides have been omitted in this construct.
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Fig. 2.
Antisense ODNs used to target the mini-exon
sequence of L. amazonensis. The mini-exon
sequence, with paired bases underlined, is at the
top (39). Four antisense ODNs 15Le-I, 15Le-II, 25Le, and
16Le are aligned below together with DNA (DNA-I/II) and RNA
(RNA-I/II) complements to 15Le-I and 15Le-II. 15Le-I, 15Le-II, and 25Le
were synthesized with PO and PS backbones and contained adenine and
thymine bases (NB) or 2-aminoadenine and 2-thiothymine bases
(SBC). 15Le-I and 15Le-II were also synthesized with a
2'-O-methyl backbone and contained uracil in place of
thymine. 16Le has been previously used to target the mini-exon sequence
(8) and contained a NB sequence and either a PO or PS backbone. The
3'-hexanol group present on every antisense ODN except 16Le is not
shown.
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Model System for Evaluating SBC Antisense ODNs--
The mini-exon
sequence of L. amazonensis is an attractive target for
evaluating SBC ODNs as antisense agents. The high A/U content (77%) of
the hairpin and its flanking arms should favor the use of SBC ODNs that
contain 2-aminoadenine and 2-thiothymine bases in place of adenine and
thymine. These base analogs cannot hydrogen-bond to one another, due to
steric clash, but can form good pairs with complementary unmodified
bases (17). As a consequence, an SBC ODN complementary to the hairpin
element of the mini-exon should be single-stranded and yet capable of
strand invading the stem-loop structure. In this case the upper 8-base
pair stem of the hairpin would be replaced by 20 base pairs formed
between the ODN and the RNA. Moreover, 9 of these new base pairs would be highly stable 2-aminoadenine-uracil doublets with three hydrogen bonds (25). To promote annealing, both NB and SBC ODNs were synthesized
as 25-mers (25Le; see Fig. 2) with the 5 extra bases complementary to
the 5' arm at the base of the hairpin. SBC ODN pairs were also compared
with NB ODN pairs as agents to target the mini-exon hairpin. These
15-mer ODNs were complementary to the 5' or 3' half of the hairpin
(15Le-I or 15Le-II; see Fig. 2). Annealing was again promoted by making
a 5-base-long segment of each ODN complementary to one of the overhangs
at the base of the hairpin. Hybridization of both ODNs to the hairpin
generated a 30-base pair DNA-RNA hybrid with a nick separating the
ODNs. By employing two paired ODNs instead of a single
self-complementary ODN, the likelihood of mutual interaction between
the NB ODNs was significantly reduced and the potential advantage of
the SBC pair accordingly diminished.
NB and SBC versions of 25Le or 15Le-I + 15Le-II were synthesized with
phosphodiester (PO) or phosphorothioate (PS) backbones. Properties of
these ODNs were compared relative to 16Le, a NB 16-mer (Fig. 2) used in
a previous study (8).
Thermostability of Hybrids Formed by NB and SBC ODNs--
Since
the entire sequence of a stable hairpin was being targeted, the various
antisense ODNs were prone to hairpin formation as well.
Self-association of these ODNs was examined by UV-absorption thermal
denaturation. While none of the SBC ODNs gave a melting transition, PO
and PS versions of NB 25Le gave melting temperatures (Tm) of 46 and 31 °C, respectively (Table
I). The NB combination of 15Le-I + 15Le-II formed a very weak hybrid when both ODNs had a PO backbone
(Tm = 13 °C) and no hybrid at all when they
possessed a PS backbone. The results obtained with antisense sequences
are in fair agreement with the expected pairing properties of the ODNs;
NB-containing oligomers give rise to more stable antisense structures
than SBC ODNs and are therefore less likely to hybridize with the
target sense sequence.
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Table I
Stability of hybrids formed by antisense Le ODNs with each other and
with complementary DNA or RNA targets
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Absorption thermal denaturation was also used to compare the relative
stabilities of hybrids formed by 15Le-I or 15Le-II with complementary
DNA or RNA oligomers. Our results show that SBC ODNs with PO or PS
backbones form more stable hybrids with DNA and RNA than the analogous
NB ODNs (Table I). While the enhanced stability of SBC ODN-DNA hybrids
(
Tm = 11-14 °C) is probably attributable
to the additional hydrogen bond present in 2-aminoadenine/thymine base
pairs, the even greater stability of SBC ODN-RNA hybrids (
Tm = 21-28 °C) requires further
explanation. One possibility is that SBC ODNs favor the formation of an
A-motif duplex with RNA. Indeed, the SBC/PO 15Le-II hybrid with RNA-II
is similar in stability to the hybrid formed using an ODN with a
2'-O-methyl backbone, a combination that favors an A-type
duplex (Table I).
Binding of NB and SBC ODNs to the Mini-exon
Hairpin--
Electrophoretic mobility shift analysis was used to
compare hybridization of NB and SBC ODNs to the RNA mini-exon hairpin. Fig. 3 shows representative
autoradiographs from which Kd values were extracted
(Table II). NB 15Le-I and NB 15Le-II were poor binding agents, with the PS analogs worse than the PO analogs. Dissociation constants for 15Le-II were in the high micromolar range:
20 and 150 µM for NB/PO and NB/PS analogs, respectively. SBC versions of the same ODNs were much more effective binding agents.
For example, the Kd values of hybrids formed by PO
or PS versions of SBC 15Le-II were about 50- or 200-fold smaller than
the Kd values of the corresponding complexes formed
by NB 15Le-II ODNs. This agrees fairly well with the
Tm values obtained with complementary RNA (see
above); SBC ODNs were better able to open the hairpin structure of the
mini-exon. For comparison we evaluated the binding of a standard 16-mer
used in a previous study (8); the PO and PS 16Le were characterized by
Kd of 0.7 and 5 µM, respectively.

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Fig. 3.
Electrophoretic mobility shift assays for the
interaction of antisense ODNs with the mini-exon RNA of L. amazonensis. Autoradiographs of the polyacrylamide gels
obtained for the complexes between L. amazonensis mini-exon
RNA and the SBC versions of 25Le. The oligonucleotide concentration is
indicated at the bottom of each lane.
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Table II
Equilibrium dissociation constants for the binding of NB and SBC
antisense ODNs to the mini-exon RNA of L. amazonensis
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Binding of both 15Le-I and 15Le-II to RNA created a duplex twice as
long as that formed by either ODN alone. The two SBC ODN-RNA hybrids
had Kd values of about 5 × 10
7
M, while the NB ODN-RNA hybrids had Kd
values at least 10-fold higher. This is probably related both to the
increased affinity of the SBC oligomers for RNA and to the reduced
ability of SBC 15-mers to pair with each other, in comparison to NB
ODNs. The partially self-complementary 25Le ODNs provided an
opportunity to compare the binding efficiencies of single-stranded SBC
ODNs to that of structured NB ODNs. In each instance, the SBC ODN was the most effective binding agent (about 70-fold reduction in
Kd). Hybrids that contained an SBC 25Le ODN were as
stable as hybrids that contained paired SBC 15Le ODNs, indicating a
minor contribution of duplex length (25 versus 30 base
pairs) and molecularity of the hybridization reaction (bimolecular
versus trimolecular).
Effects of NB and SBC Antisense ODNs on in Vitro Translation in
Cell-free Extracts--
The NB and SBC ODNs with a PO backbone were
tested for antisense activity against total mRNA from L. amazonensis in a cell-free translation system catalyzed by wheat
germ extract. In this model system, SBC 25Le was the most effective
antisense agent (Fig. 4). It elicited
half-maximal inhibition at 0.08 µM (the C1/2 value). These parameters compare favorably to those of reference ODN 16Le (which has a PO backbone and contains NB bases), which has a C1/2 of
about 1 µM. In repeated tests, NB 25Le failed to
significantly inhibit translation (Fig. 4D). The dramatic
difference in functionality between the SBC and NB versions of 25Le
reflects the physical binding properties of these two ODNs (Table II).
The paired 15Le-I + 15Le-II ODNs were also effective antisense agents,
and here again the SBC pair was more potent than the NB pair. C1/2 of
0.5 µM was determined for the SBC ODN pair. When each
15-mer was tested alone for antisense activity, potency was reduced and
no advantage of SBC over NB ODNs was detected. None of the
anti-mini-exon ODNs interfered with translation of brome mosaic virus
RNA (<5% inhibition at 0.5 µM ODN). Conversely,
inverted antisense ODNs (both NB and SBC) did not inhibit translation
of L. amazonensis RNA (<5% inhibition at 1 µM ODN).

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Fig. 4.
Effect of NB and SBC antisense ODNs on
in vitro translation of L. amazonensis
mRNA catalyzed by wheat germ extract (A-E)
or rabbit reticulocyte lysate (F). NB ( ) and
SBC ( ) versions of the following phosphodiester ODNs were compared
for antisense activity: panel A, 15Le-I;
panel B, 15Le-II; panel C,
15Le-I + 15Le-II; panel D, 25Le. Panel
E shows the antisense activity of the reference ODN 16Le
(NB/PO). Panel F shows the activity of
25Le (SBC/PO) when using rabbit reticulocyte lysate in the
absence ( ) or presence ( ) of E. coli RNase H.
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Phosphorothioate NB and SBC antisense ODNs did not inhibit translation
at concentrations below 0.5 µM (data not shown).
Adsorption of these ODNs by proteins in the extract may explain why
they failed to elicit an antisense effect. Following incubation with wheat germ extract, the PO SBC 25Le ODN ran identically to an untreated
control on a nondenaturing polyacrylamide gel while the PS ODN ran as a
slow moving smear attributable to the association with proteins from
the extract (data not shown). Such binding has been reported by others
for NB ODNs with a PS backbone (26). Moreover, upon binding to RNA,
these PS sequences formed poorer substrates for RNase H than PO
counterparts (see below).
SBC-RNA Duplexes Are Substrates for RNase H--
Two different
mechanisms account for the inhibition of translation by antisense ODNs:
RNase H-independent (translation arrest) and RNase
H-dependent cleavage of target RNA (27, 28). To investigate
the mechanism by which SBC ODNs inhibit L. amazonensis on
RNA translation, we carried out translation in rabbit reticulocyte lysate, which has a low (if any) class I RNase H activity under translation conditions (29). In Fig. 4F the effect of SBC
25Le (with a PO backbone) on translation was monitored both in the presence and absence of added E. coli RNase H. The results
show that inhibition of protein synthesis in this medium occurs via an
RNase H-dependent pathway.
To confirm that SBC ODNs form substrates for RNase H upon binding to a
complementary RNA, we incubated end-labeled mini-exon RNA with all four
versions of 15Le-I, 15Le-II, and 25Le in the presence of E. coli RNase H. The results obtained with 15Le-I and 25Le are
presented in Fig. 5. ODN versions
sensitized the mini-exon to hydrolysis to various extents. The
amplitude of cleavage roughly paralleled the affinity of ODNs for the
mini-exon sequence as determined by electrophoretic mobility shift
assay, suggesting that the RNase H activity was first driven by the
ability of oligonucleotides to invade the mini-exon hairpin. The NB-PS
and NB-PO analogs of 15Le-I, which are very poor binders as shown both
by gel electrophoresis and Tm measurements, did
not induce significant cleavage of the mini-exon RNA by E. coli RNase H. In contrast, the most effective cleavage was
obtained when using SBC/PO 25Le, which was also the strongest binder
and a good translation inhibitor. However, PS oligonucleotides formed
poorer substrates than PO ones. This is unlikely to be due to the
presence of SBC bases, as SBC/PO oligonucleotides are excellent
elicitors of RNase H activity.

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Fig. 5.
Susceptibility of NB and SBC antisense
ODN-RNA duplexes to E. coli RNase H. The
indicated versions of 15Le-I and 25Le (top of the
autoradiograph) were hybridized to 5'-end-labeled L. amazonensis mini-exon RNA and then treated with E. coli
RNase H as indicated under "Experimental Procedures." Reaction
aliquots were analyzed by denaturing PAGE. In the control lane
(C), the RNA was incubated in the absence of any
complementary ODN. In lane M, the RNA was
partially hydrolyzed by alkali treatment. The mini-exon sequence is
indicated to the left.
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Interestingly, different patterns of RNase H cleavage were obtained,
depending on the chemical nature of the bases of the antisense oligos;
similar profiles were obtained for NB oligomers on the one hand and SBC
oligomers on the other hand, independent of the backbone. For instance,
whereas NB/PO 25Le generated cleavage at A22, the SBC ODNs did not
induce cleavage at this position. In contrast, strong sites
corresponding to U24 and U25 were observed with SBC 25-mers. Other
differences were also seen at the 3' end of the RNA (compare cleavage
at U28 and G29). Significant differences were also seen with the ODN
15Le-I; the digestion profile obtained with either the SBC/PO or the
SBC/PS derivatives differed markedly from the one with the NB/PO version.
In Vitro Effects of SBC Anti-mini-exon Oligonucleotides on Cultured
Parasites--
As the presence of nucleases in the growth medium
prevented us from using PO oligomers, investigations on cultured
parasites were restricted to phosphorothioate derivatives. The addition of phosphorothioate anti-mini-exon oligonucleotides to the culture, either with NB or SBC character, led to the typical morphological changes previously described for 16Le (8). Oligonucleotide-treated cultures contained numerous macrophages with fragmented vacuoles devoid
of any parasite (data not shown). At any tested concentration (5, 10, or 30 µM), the two NB ODNs 16Le and 15Le-I behaved
similarly, curing from 10 to 30% macrophages (Fig.
6). The use of an SBC analog (15Le-I)
showed some limited increased efficiency over the NB ODN. Therefore,
the effect observed on cultured cells did not reflect the binding
properties of the SBC and NB 15 mers (Table II). However, the full
advantage of SBC sequences was expected only when both sides of the
structure are targeted. Indeed, whereas the combination NB 15Le-I + NB
15Le-II did not improve the leishmanicidal efficacy, compared with the
effect of a single sequence, the simultaneous addition of the two SBC
15-mers resulted in an improved leishmanicidal effect better seen at
low concentration; 18 and 32% of cured macrophages were observed after
incubation of infected cells with 5 µM mixture of the NB
and SBC 15-mers, respectively (Fig. 6). This effect was selective as NB
and SBC control phosphorothioate oligonucleotides induced a much lower
effect (about 5% at 5 µM) in agreement with our previous
study (8).

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Fig. 6.
Effect of phosphorothioate oligonucleotides
on L. amazonensis-infected macrophages. Murine
peritoneal macrophages were infected and treated as described under
"Experimental Procedures," by 5, 10, or 30 µM
oligonucleotides, with either NB or SBC bases, as indicated to the
right of the figure. Control sequences were included:
NB/16Le inv, the inverted sequence of 16Le; SBC/15Le-I
inv, the inverted sequence of 15Le-I. The results are the average
of three completely independent experiments. For the 15Le-I + 15Le-II
pair, the total concentration is indicated, each oligomer accounting
for 50%.
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DISCUSSION |
We have compared the binding and antisense activities of single
and paired ODNs with NB and SBC character. Not surprisingly, as
reported previously, antisense potency seemed to parallel hybrid stability (13, 30). SBC ODNs were superior to NB ODNs, and ODN pairs
were more efficient than single ODNs. A key attribute of antisense SBC
pairs is their enhanced ability to invade double-stranded nucleic acids
due to their reduced propensity to hybridize with each other. We had
previously postulated that the inhibitory influence of RNA secondary
structure on the binding of antisense ODNs might be overcome through
the use of perfectly paired SBC complements (17, 18). In this respect,
the targeting of an RNA hairpin, like that formed by the mini-exon
sequence of L. amazonensis, is a specialized application of
SBC analogs permitting the use of a partially self-complementary ODN.
Here we have shown that such an ODN (SBC 25Le) is an efficient
inhibitor of in vitro translation, whereas the same ODN with
unmodified bases (NB 25Le) is without activity. We presume that stable
hairpin formation by the NB ODN prevents it from interacting with the
mini-exon.
SBC ODNs with 2-aminoadenine and 2-thiothymine bases might favor the
formation of A-type double helices upon binding to RNA, leading to very
stable hybrids. Each of the modified bases used here projects a bulky
group into the minor groove of the duplex. The wider minor groove of
the A-form duplex should better accommodate these groups. Previous
studies of ODNs that contain 2-aminoadenine or 2-thiothymine indicate a
propensity to form A-type duplexes (23, 31, 32). The SBC ODNs used here
have a high proportion of both modified bases and most likely share
this tendency.
The retention of RNase H susceptibility by SBC ODN-RNA hybrids implies
that the presence of the bulky groups in the minor groove does not
prevent recognition of the duplex as a substrate by RNase H. This
property of SBC oligos is unusual since other ODNs that form A-type
hybrids with RNA, such as ODNs with 2'-O-alkyl or
phosphoramidate backbones, do not allow RNase H activity (33, 34). The
ability of NB and SBC ODNs with a PS backbone to support RNase H
activity is consistent with the hypothesis that the presence of a
-anomeric deoxyribose as part of a negatively charged backbone allows the complementary RNA strand to be cleaved. However, the different cleavage patterns observed suggest that RNase H is able to
sense the minor conformation changes induced by the presence of the
modified bases.
Phosphorothioate ODNs were shown to exhibit L. amazonensis
killing activity in murine macrophages infected with an amastigote inoculum. The results obtained here with NB/16Le phosphorothioate are
in good agreement with a previous study (8). The SBC ODNs exhibited a
limited increased leishmanicidal activity compared with regular
phosphorothioate ODNs. However, it should be noted that the maximal
expected leishmanicidal effect was reached at a low concentration of
the paired SBC 15-mers (15Le-I + 15Le-II): 30% cured macrophages were
observed at 5 µM mixture (i.e. 2.5 µM amounts of each oligomer). One cannot exclude the
possibility that in live parasites the mini-exon sequence on the mature
RNA does not fold into the hairpin. Sequestration of these PS ODNs by
cellular proteins might also have partly negated the benefit of their
increased affinity for the target (26). The additional thio groups
contributed by 2-thiothymine bases in PS SBC ODNs may enhance
adsorption onto proteins.
Like the mini-exon sequence, numerous sequences in many transcripts are
not freely accessible to complementary antisense ODNs due to their
participation in secondary or tertiary structures (35-37). Efforts to
overcome secondary structure by designing ODNs with greater binding
affinity or by utilizing hybridization strategies that accommodate
pre-existing RNA structure have not been entirely successful (for
recent overviews, see Refs. 15, 17, and 38). The unequivocal advantage
of SBC 25Le argues the merit of testing whether paired sense-antisense
SBC ODNs show a similar advantage in targeting random mRNA
sequences; by co-administering a single-stranded antisense ODN and its
complement, with SBC character, any secondary structure involving the
site of interest could be disrupted by concurrent binding of both
single-stranded SBC ODNs to the RNA sequences involved in base pairing.