Efficient site-specific cleavage of RNA using a terpyridine-copper(II) complex joined to a 2'-O-methyloligonucleotide by a non-flexible linker

Article abstract of DOI:10.1039/a808522f

A terpyridine-Cu(II) complex conjugated to an antisense 2'O-methyloligonucleotide at the 5'-end cleaved RNA predominantly at the site opposite the 5'-end in moderate yield, but the cleavage yield increased by more than two-fold when a 2'-O-methyloligonucl

Full text of DOI:10.1039/a808522f

Efficient site-specific cleavage of RNA using a terpyridine–copper(ii) complex
joined to a 2A-O-methyloligonucleotide by a non-flexible linker
Hideo Inoue,* Takako Furukawa, Masaki Shimizu, Takashi Tamura, Miwa Matsui and Eiko Ohtsuka
Graduate School of Pharmaceutical Sciences, Hokkaido University, Kita-12, Nishi-6, Kita-ku, Sapporo 060-0812,
Japan. E-mail: inoue@pharm.hokudai.ac.jp
Received (in Cambridge, UK) 3rd November 1998, Accepted 17th November 1998
A terpyridine–Cu(ii) complex conjugated to an antisense 2A-
O-methyloligonucleotide at the 5A-end cleaved RNA pre-
dominantly at the site opposite the 5A-end in moderate yield,
but the cleavage yield increased by more than two-fold when
a 2A-O-methyloligonucleotide was allowed to bind to the
single-stranded region of the RNA-antisense complex.
adenosine 1b were prepared, using standard methods for all
steps (Scheme 1). The synthetic route involving O-terpyridyla-
tion12 is easy and applicable to the other nucleosides. The two
building units 5a,b were used for the preparation of 5A-end-
modified 2A-O-methyloligonucleotides.
Cleavage reactions of RNA oligomers with the terpyridine–
Cu(ii)-oligomer conjugates were carried out with the 5A[32P]-
end-labeled RNA target in a buffer at 45 °C for 20 h.
Representative examples of an analysis of the cleavage products
and all results are indicated in Figs. 1 and 2, respectively.
Cleavage of the RNA 24-mer (R24) with the 5A-O-Cu(ii)–
terpyridyl-2A-deoxyadenosine-linked 12-mer (dA*12) occurred
predominantly between U12–U13, in which U12 is positioned
at the end of the 12-mer recognition region [Figs. 1 and 2(a)].†
The extent of specific cleavage was 18% after 20 h [Fig. 2(c)].
Minor cleavage occurred at U13–U14 in ca. 1% yield. For the
corresponding 5A-O-Cu(ii)–terpyridyl-2A-O-methyladenosine-
conjugate (Am*12), similar cleavage results were obtained. An
RNA 21-mer with no sequence similarity to R24 was also
specifically cleaved by a dA*-13-mer at a predetermined site,
5AU–A3A (26% yield, data not shown).
Recently, chemical agents that cleave RNA site-specifically
without enzymatic assistance have been developed.1 This is
potentially important in antisense chemotherapy because cleav-
age of a specific mRNA will lead to loss of its function.2 These
agents can also be useful for studies on structure–function
relationships of native RNAs.3 One relatively active class of
RNA cleavers is metal complexes covalently linked to oligonu-
cleotides; the cleavage mechanism may involve transphosphor-
ylation promoted by the metal. Complexes, such as terpyridine–
Cu(ii),4,5 macrocyclic terpyridine derivative–Ln(iii),6,7
texaphyrin–Ln(iii),8,9 and iminodiacetate–Ln(iii),10 have been
attached by a flexible linker to DNA oligomers for sequence-
specific recognition. Here, we describe a new RNA cleaver with
an alternative arrangement, consisting of a 2A-O-methyl RNA
oligomer, as an efficient RNA hybridization probe,3,11 and a
terpyridine–Cu(ii) complex directly attached to the 5A-end of the
2A-O-methyloligomer. We also demonstrate RNA cleavage by
the cleaver in the presence of a cleavage facilitator.
For oligonucleotide synthesis, 5A-O-terpyridyl 3A-phosphor-
amidite derivatives of 2A-deoxyadenosine 1a and 2A-O-methyl-
NH2
NHDMTr
N
NHDMTr
N
N
N
N
N
N
N
N
N
N
N
N
N
O
N
O
HO
TBDMSO
c, d
O
O
a, b
DMTrO
R
HO
R
DMTrO
R
1a R = H
2a R = OMe
2a,b
3a,b
e, f
NHBz
NHBz
N
N
N
N
N
N
N
N
N
N
N
O
g
N
O
N
O
O
N
O
R
HO
R
NPri2
4a,b
NC(CH2)2O
P
5a,b
Scheme 1 Reagents and conditions: for 5b (all reactions were carried out at
room temperature). a, TBDMSCl (1.2 equiv.), imidazole (2.4 equiv.), DMF,
1.5 h, 77%; b, DMTrCl (2.6 equiv.), pyridine, 23 h, 77%; c, Bu4NF (1.2
equiv.), THF, 30 min, 99%; d, 4A-chloro-2,2A:6A,2B-terpyridine (1.2 equiv.),
KOH, DMSO, 44 h, 90%; e, 80% MeCO2H aq, 4 h, 100%; f, successive
steps: i, TMSCl (3 equiv.), pyridine, 30 min; ii, BzCl (5 equiv.), 3.5 h; iii,
H2O, 5 min; iv, 28% NH3 aq, 30 min, 64% (overall); g, 2-cyanoethyl
diisopropylchlorophosphoramidite (1.2 equiv.), diisopropylethylamine (3
equiv.), CH2Cl2, 50 min, 50%. DMTr = 4,4A-dimethoxytrityl.
Fig. 1 Autoradiogram of products from the reaction of the labeled target
R24 with the agent dA*12 after 20% denaturing polyacrylamide gel
electrophoresis. The reaction was carried out for 20 h at 45 °C in a total
volume of 10 ml containing: 0.1 M NaClO4, 20 mM HEPES (pH 7.5), 0.1
mM RNA, 1 mM agent, and 1 mM CuCl2. Lane 1: R24; lane 2: R24 + free
terpyridine–Cu(ii) complex + control 12-mer without terpyridine moiety;
lane 3: R24 + dA*12 + EDTA (10 mM); lane 4: R24 + dA*12; lanes 5 and
6: bicarbonate and ribonuclease T1 sequencing reactions, respectively.
Chem. Commun., 1999, 45–46
45

cleavage site-specificity was lower. On the other hand, the
reactions using the nicked tandem sequences increased the
cleavage efficiency. Moreover, when Am*12 instead of dA*12
was used, the cleavage yield was 48%.‡ The spatial orientation
of the terpyridine moiety probably became more suitable for
RNA cleavage by the presence of the nick-forming tandem
sequences and the 2-O-methylribose linker.
The present RNA cleaver has the metal complex at the end of
the 2A-O-methyloligonucleotide. On the other hand, RNA
cleaving metal complexes that were attached within DNA
oligomers have been reported.4,5,7,9 Some of the agents were
designed to remain in a local region (such as a bulge) containing
the cleavage site loose14 while hybridized with the target RNA.
This kind of agent is expected to be developed into an artificial
ribonuclease that acts with catalytic turnover. Our findings,
including the results on the nick-containing RNA hybrid,
provide valuable information for the design of catalysts and the
creation of more efficient cleavers.
We thank Prof. Donald E. Bergstrom (Purdue University) for
critical reading of the manuscript. This work was supported in
part by a Grant-in-Aid from the Ministry of Education, Science,
Sports and Culture of Japan.
Notes and references
† For the reaction, the agent and CuCl2 were directly added to the RNA
solution. The addition after premixing both compounds gave similar results
for the cleavage. We have used 10 molar equivalents of the terpyridine–
Cu(ii) agent and found that 2 molar equivalents were sufficient for the
reaction. The use of such small amounts of cleavage agents has not been
reported.
‡ Recently Daniher and Bashkin reported RNA cleavage using a
terpyridine–Cu(ii) complex attached to a flexible linker on an abasic site
analog within DNA oligomers.5 Our cleavage yield using the 2A-O-methyl
facilitator and our data for the site-specificity of the RNA cleavage seem to
be comparable with the reported data.
Fig. 2 (a) Sequences of the RNA substrate and terpyridine–Cu(ii)-linked
oligomer agents. Nm refers to a 2A-O-methylnucleoside residue, and the
asterisk indicates the site of the linked terpyridine moiety. (b) Sequences of
the tandem 2A-O-methyloligomers, and schematic representation of RNA
cleavage by the use of Am*12 and the t-oligo(n) (the arrow shows the
cleavage site). The terpyridine–Cu(ii) moiety is indicated as a black
rectangle. All 2A-O-methyloligomers used in this study had a 2A-deoxy-
nucleoside residue at the 3A-end. (c) Yields of site-specific cleavage of R24
by agents with or without tandem oligonucleotides.
a The minor cleavage site is U13–U14 site, and other reactions also gave this
cleavage, but in yields of less than 1%.
1 B. N. Trawick, A. T. Daniher and J. K. Bashkin, Chem. Rev., 1998, 98,
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For induction of RNA cleavage, the terpyridine–Cu(ii)
complex attached to the non-flexible linker should be close to
the RNA strand. To explore the spatial orientation of the
terpyridine moiety in the oligonucleotide hybrid, we prepared a
terpyridine-linked duplex with a self-complementary sequence
(dA*CmAmGmCmUmGmUm) and examined its melting tem-
perature [conditions: 0.1 M NaClO4, 10 mM Na phosphate (pH
7.5), 10 mM oligonucleotides, 12 mM CuCl2]. It was found that
the Tm value (72 °C) was very high, as compared with that
(52 °C) for the control duplex without terpyridine moieties in
the presence of 12 mM CuCl2. These results may mean that the
terpyridine moiety interacts with the end of the hybrid in an end-
capping manner [Fig. 2(b)].13
To explore efficient RNA cleavage, which may be affected
by the environment around the terpyridine–Cu(ii) moiety in the
RNA–agent hybrid, we carried out the RNA cleavage reaction
in the presence of a 2A-O-methyl RNA complementary to the
RNA single strand region adjacent to the terpyridyl conjugated
12-mer. The adjacent oligonucleotide was positioned in two
ways: first, to provide a nick, and second a gap at the terpyridine
site. In each reaction, with or without the tandem oligonucleo-
tide [t-oligo(n) or t-oligo(g), Fig. 2(b)], Am*12 was more
effective than dA*12 [Fig. 2(c)]. The cleavage efficiency for the
reaction with the gapped tandem sequences was similar to that
for the reaction without the additional oligonucleotide, but the
12 G. R. Newkome, R. Güther, C. N. Moorefield, F. Cardullo, L.
Echegoyen, E. Pérez-Cordero and H. Luftmann, Angew. Chem., Int. Ed.
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14 K. A. Kolasa, J. R. Morrow and A. P. Sharma, Inorg. Chem., 1993, 32,
3983.
Communication 8/08522F
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Chem. Commun., 1999, 45–46