ABSTRACT
Lanthanide complexes covalently attached to oligonucleotides have been shown to
cleave RNA in a sequence-specific manner. Efficient cleavage, however, is at present limited to
single-stranded RNA regions, as RNA in a duplex is considerably more resistant to
strand scission. To overcome this limitation, we have designed and synthesised
artificial nucleases comprising lanthanide complexes covalently linked to
oligodeoxyribonucleotides which cleave a partially complementary RNA at a
bulged site, in the duplex region. Strand scission occurs at or near the bulge.
Cleavage of the RNA target by the metal complex can be addressed
via
the major or the minor groove. In an example of a competitive situation, where
the cleavage moiety has access to both a bulge and a single-strand region, transesterification at the bulge is favoured. Such
artificial ribonucleases may find application as antisense agents and as tools
in molecular biology. In addition, the results may have importance for the
design of artificial ribonucleases which are able to act with catalytic
turnover.
Several groups have recently reported the sequence-specific hydrolytic cleavage of ribonucleic acid (RNA) using metal
complexes covalently linked to oligonucleotides (
1
-
5
). This is of current interest in the antisense field (
6
-
9
) because it may permit the selective destruction of a targeted messenger RNA
in vivo
without the need for cellular enzymes or endogenous cofactors. Highly efficient
RNA cleavage has been shown with macrocyclic lanthanide complexes (
2
,
4
,
5
), which are among the most potent transphosphorylation catalysts known and also
possess favourable properties for eventual
in vivo
applications. In both cases, cleavage of the target was confined to the single-stranded region, a few nucleotides from the end of the duplex formed by
the conjugate and the target RNA. Furthermore, an excess of cleaver conjugate
was used to achieve efficient cleavage. The potency of such artificial
ribonucleases might be raised if the cleavage process could be extended into
the duplex region. This would not only increase the number of possible cleavage
sites but, more importantly, offer the possibility of catalytic turnover (
10
). A fundamental requirement of catalysis is a rapid dissociation of the two RNA
fragments from the nuclease after the cleavage process in order to avoid
product inhibition. Hence, whereas formation of a stable duplex between the
nuclease and the target is required in a first step, the final complex between
the nuclease and the RNA fragments after the reaction should be sufficiently
destabilised to allow rapid nuclease release (
11
). This condition is not fulfilled if the target is cleaved in the single strand
(Fig.
1
A) but it could arise if cleavage occurred within the duplex (Fig.
1
B). On the other hand, it has been reported that double-stranded RNA is considerably more resistant to metal ion promoted
transesterification than its single stranded counterpart (
12
) rendering cleavage of RNA within a duplex difficult or impossible. In the course of our research
directed towards the sequence specific chemical cleavage of RNA, however, we
found that bulged RNA residues are-in contrast to paired residues-susceptible to metal promoted transesterification (Hüsken,D., Goodall,G., Blommers,M.J.J., Jahnke,W., Hall,J., Häner,R., Moser,H.E. manuscript in preparation).
Consequently, we applied this finding to our work with oligonucleotide
lanthanide conjugates. Here, we report the efficient sequence-specific cleavage of a synthetic RNA in the duplex region by use of oligonucleotides bearing lanthanide complexes. RNA
cleavage is made possible by the enforced presence of a cleavage-susceptible bulge in the RNA target strand (
13
).
The oligonucleotide metal complex conjugates used in this study were prepared
from lanthanide complexes
1-3
(Fig.
2
) in analogy to the published procedure (
4
). Amino groups were introduced into oligonucleotides
4-7
using the appropriately modified building blocks (
14
-
16
) for conjugates
8-12
(henceforth referred to as endoconjugates) or the commercially available linker
for conjugate
13
(referred to as exoconjugate). The lanthanide complexes
1-3
were covalently linked to the amino oligonucleotides
via
their respective
N
-hydroxysuccinimide ester or isothiocyanate derivatives. All amino
oligonucleotides and conjugates were purified by reverse phase HPLC and were
characterised by matrix assisted laser desorption ionisation mass spectrometry
(MALDI-MS), and capillary gel electrophoresis (CGE) or polyacrylamide gel
electrophoresis (PAGE).
The experiments described above demonstrate the efficient sequence specific
cleavage of RNA in the duplex region formed between the latter and a chemical
ribonuclease. Cleavage is effected by the enforced presence of a bulge in the
RNA target strand. The results can be summarised as follows: (i) RNA cleavage
at double bulged nucleotides in the duplex region is seen with both endo- and exoconjugates; (ii) cleavage is not limited to a single site but
occurs at several phosphodiesters within and adjacent to the bulge; (iii) most
efficient cleavage is observed with endoconjugates reaching the bulge across
the minor groove; (iv) less efficient cleavage was observed using an approach
through the major groove, and (v) in a competition experiment, cleavage by an
exoconjugate at the bulge was preferred over the single stranded region.
Since it is conceivable that cleavage within the duplex will result in
sufficient destabilisation for product release, these findings represent a
significant step in the development of true artificial ribonucleases capable of
catalytic turnover.
Oligonucleotides were prepared by automated synthesis on a 1.5 [mu]mol scale on a DNA synthesizer (Applied Biosystems Inc. 394A-08). DNA and 4-monomethoxy trityl (MMT) amino-C6 cyanoethyl phosphoramidites were obtained from MWG-Biotech GmbH, Expedite
TM
RNA cyanoethyl phosphoramidites from Millipore. Oligodeoxynucleotides
4-7
were cleaved from the support and base-deprotected by treatment with concentrated aqueous ammonia at 55oC overnight. After removal of the ammonia, the crude material was
purified by HPLC using a semi-preparative RP-C18 column [Hypersil
TM
, 5 [mu]m particle size; 50 mM triethylammonium acetate (TEAA, pH 7.0) starting with
10% acetonitrile increasing with a gradient of 0.7%/min]. After concentration
of the product-containing fractions, the trityl groups (DMT for
4-6
, MMT for
7
) were removed by treatment with 80% aqueous acetic acid for 30 min. The final
purity of the amino oligonucleotides was assessed by CGE and PAGE. MALDI mass
spectra were obtained in either the positive or negative mode and are shown in
Table
1
.
Oligonucleotide conjugates were prepared according to the previously described
procedure (
4
) using either the corresponding modified building blocks (
14
-
16
, conjugates
8-12
) or a 5'-terminal aminohexyl linker (conjugate
13
). Products were purified by RP-HPLC. The product purity was checked by CGE or PAGE. MALDI mass spectra
were obtained and are shown in Table
1
.
Oligoribonucleotides
14-18
were cleaved from the support and base-deprotected by treatment with a mixture of concentrated aqueous ammonia
(25% v/v) in ethanol 16 h. The 2'-protecting group (
tert
-butyldimethylsilyl) was removed with a 1 M solution of tetrabutylammonium
fluoride in tetrahydrofuran for 24 h at room temperature in the dark. The
reaction mixture was quenched with an equal volume of 50 mM triethylammonium
hydrogencarbonate (TAHC) solution (pH 7.0), dialysed against 7.5 mM TAHC (pH 7.0) at 4oC, concentrated and used for 5'-end labelling as described below.
Table 1
Special precautions were made to prevent contamination by traces of metals and nucleases as described (
19
). Oligoribonucleotide
7
(100 pmol) was
33
P end-labelled by treatment with 5 U T4 polynucleotide kinase (Promega), in a solution (total volume 20 [mu]l) containing 10 mM MgCl
2
, 5 mM 2-mercaptoethanol, 0.1 mM spermidine, 50 mM Tris-HCl (pH 7.5) and 5 [mu]Ci [[gamma]-
33
P]ATP (Amersham, 1000 Ci/mmol). The mixture was incubated at 37oC for 30 min followed by ethanol precipitation. After addition of 15 [mu]l loading buffer (containing 0.025% xylene cyanol and 0.025% bromophenol
blue in 80% deionised formamide and 7 M urea with 20 mM citric acid and 1 mM
EDTA), the mixture was heated for 2 min at 95oC, chilled on ice and loaded on a 12% denaturing polyarylamide gel. After
electrophoresis (2 h, 55 W) the RNA band was cut out, electroeluted and ethanol-precipitated. The material labelled by this procedure contained 200 000
c.p.m. (Cerenkov protocol).
5'-End labelled oligoribonucleotide
7
(12 000 c.p.m., final concentration estimated to 10-50 nM) and the corresponding oligonucleotide conjugate (600 nM final
concentration) or complex (600 nM final concentration) were dissolved in 50 mM
Tris-HCl buffer (pH 7.4; total reaction volume of 10 ml). The sample was
heated for 1 min to 85oC, cooled to 37oC and kept at this temperature for 16 h. The reaction mixtures were
diluted with 5 ml loading buffer, heated to 95oC for 1 min, chilled on ice and loaded on a 12% denaturing Long Ranger
TM
gel (AT Biochem). Electrophoretic separation (1.25 h, 60 W) was followed by
exposure to X-ray film (Kodak, X-OMAT
TM
AR) and Phosphorimager
TM
.
We thank P. Martin, K-H. Altmann and H.E. Moser for the gift of amino-modified phosphoramidites. The valuable technical assistance of S.
Burkhardt, A. Deichert and L. Moesch is gratefully acknowledged.
Cpd.
4
a
5
a
6
a
7
b
8
a
9
b
10
b
11
a
12
b
13
b
Calcd.
9112
9110
9065
6361
9812
9797
9841
9810
9767
7061
Found
9116
9115
9077
6363
9808
9800
9853
9813
9794
7065
REFERENCES
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