Precise control of RNA cleavage by ribozyme mimics

Article abstract of DOI:10.1039/a800818c

A highly-modified DNA building block, lacking both sugar and base moieties, is synthesized and incorporated into oligonucleotides to form functional mimics of ribozymes.

Full text of DOI:10.1039/a800818c

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Precise control of RNA cleavage by ribozyme mimics
Andrew T. Daniher and James K. Bashkin*†
Department of Chemistry, Washington University, St. Louis, MO 63130-4899, USA
A highly-modified DNA building block, lacking both sugar
and base moieties, is synthesized and incorporated into
oligonucleotides to form functional mimics of ribozymes.
DNA duplex is relatively inert towards cleavage when com-
pared to its single-stranded form, perhaps because the duplex is
a more conformationally rigid structure than the single
strand.11,12 Incorporation of the serinol residue into a DNA
sequence allows formation of a duplex with the complementary
RNA strand, and increases the flexibility of the RNA in the
region opposite the serinol. This flexible RNA region should
more readily form the pentacoordinate phosphorane required
for transesterification, and should therefore undergo enhanced
cleavage compared to a perfectly base-paired sequence.9
Derivatizing serinol in an unsymmetrical fashion gave
stereoisomers A and B. Fukui suggested that related com-
We demonstrate here that second-generation ribozyme mimics
based on a serinol–terpyridine reagent, when incorporated into
a DNA oligonucleotide, cleave their target RNA in a sequence-
specific manner. Furthermore, the position of cleavage within
the target sequence was precisely controlled by the location of
the terpyridine within the oligonucleotide. These second-
generation mimics function with greatly improved efficiency
over our previous terpyridine reagents.1,2 We attribute the
improved cleavage to increased flexibility of the target RNA
strand that results when serinol replaces a nucleotide in the
DNA sequence: this eliminates a DNA/RNA base-pair near the
cleavage site and may lower the barrier for phosphorane
formation.
Functional ribozyme mimics consist of an oligonucleotide for
molecular recognition and an attached RNA cleavage (trans-
esterification) agent.3,4 These ribozyme mimics are designed to
extend the antisense approach to translation arrest by creating a
catalytic cycle independent of any enzyme-mediated RNA
cleavage. The antisense method is a gene-specific technique for
blocking protein synthesis that inhibits the translation of mRNA
into proteins.5
Our group previously reported the first wholly synthetic
ribozyme mimic,1 which was comprised of a 17-mer DNA
probe with a pendant terpyridyl (terpy) complex of CuII
incorporated in DNA via a thymidine derivative.2 Aqueous
(terpy)CuII is a known RNA transesterification and hydrolysis
agent.6–8
We wished to improve cleavage efficiency and the ease of
preparation of ribozyme mimics. Here we report greatly
improved RNA cleavage and much simpler synthetic routes.
The new reagents (Scheme 1) are conjugates of serinol and
terpyridine. Serinol, a reduced form of serine, mimics the
spacing of the sugar backbone of DNA; it and related com-
pounds have previously been used as building blocks for abasic
DNA sites.9,10 An abasic DNA site eliminates one Watson–
Crick base pair in a duplex and increases the conformational
flexibility of the double-stranded region. The RNA in an RNA/
N
R1
N
O
NH
N
N
N
O
O
R2
R1
O
A
O
NH
N
O
O
O
R2
B
pounds, derivatives of the 2R,3R isomer of l-threoninol,
preferentially target the major groove upon incorporation into
DNA.13 Since free rotation can occur in the serinol backbone,
both A and B should be able to reach either groove. This paper
describes work on a mixture of stereoisomers A and B.
The synthesis of 3 is shown in Scheme 1. Serinol, a meso
compound, possesses the same spacing between alcohols (three
carbons) as a normal deoxynucleoside. Serinol 4 was coupled to
the terpyridine (terpy) acid 5 with 1-(3-dimethylaminopropyl)-
3-ethylcarbodiimide hydrochloride (EDC), giving 1 (45.5%).
Subsequent 4,4A-dimethoxytrilyl (DMT) protection of one of the
primary alcohols of 1 gave 2 in 30% yield. Phosphitylation of 2
resulted in the desired phosphoramidite 3 as a mixture of
stereoisomers (47%).
Several 17-mer probes designed to target a 159-mer fragment
of the HIV gag gene mRNA were prepared via automated DNA
synthesis, using 3 and/or standard nucleoside phosphor-
amidites. The RNA sequence is shown below, with the 17-mer
recognition region underlined.
5A-1(775)GGAGAA6
AAUAAAAUAG46
GACAUAAGAC86
AGACCGGUUC126
CAG159(933)-3A
AUUUAUAAAA16
UAAGAAUGUA56
AAGGACCAAA96
UAUAAAACUC136
GAUGGAUAAU26
UAGCCCUACC66
GGAACCUUUA106
UAAGAGCCGA146
CCUGGGAUUA36
CAGCAUUCUG76
GAGACUAUGU116
GCAAGCUUCA156
HO
HO
+
H
N
O
HO
H
O
O
H
NH2
i
O
HO
N
The 17-mer DNA probe sequences, in 5A to 3A orientations with
X indicating the serinol–terpy residue, are:
HO
N
N
N
N
N
4
5
1
1a 5A-CTACATAGTCTCTAAAG-3A
1b XTACATAGTCTCTAAAG
1d CTACATAGXCTCTAAAG
ii
1c
1e
CTACAXAGTCTCTAAAG
CTACATAGTCXCTAAAG
DMTO
DMTO
H
H
N
H
N
Derivatives 1b–e of probe 1 differ in the location of the serinol–
terpy residue, but all bind to the same region of the RNA target.
Control probes for 1b–e [named 1(b–e)-ctrl] were also prepared
using 6 (Glen Research) in place of serinol–terpy reagent 3.
O
O
iii
H
P
O
O
O
HO
N
N
NCCH2CH2O
NPri2
DMT
P(NPri2)(OCH2CH2CN)
N
N
N
N
3
2
O
O
6
Scheme 1 Reagents and conditions: i, EDC, DMF, room temp., 30 h,
45.5%; ii, Py, Et3N, DMT-Cl, 30%; iii, Et3N, CH2Cl2, 2-cyanoethyl N,N-
diisopropylchlorophosphoramidite, room temp. 20 min, 47%
These probes explicitly test for any enhanced cleavage activity
that flexible, abasic reagents might confer on the target RNA.
Chem. Commun., 1998
1077

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Table 1 Cleavage results from sequence-specific reactions of ribozyme
mimics with the 159-mer target RNA, in the presence of CuII ion
% Total cleavage
Probe
Cleavage Sites
24 h
72 h
1b
1c
1d
1e
G118, A117, U116
A113, U112
A110
18
10
10
48
48
35
40
84
A108
terpy-containing DNA probes during synthesis and purification
(data not shown). Treating the probes with EDTA prior to
reaction eliminated the background cleavage. Adding CuII (but
not divalent Fe, Mg, Pb or Zn) recovered the cleavage activity
of the EDTA-treated ribozyme mimics (data not shown).
The synthesis of the serinol–terpyridine building block
allows the introduction of cleaving agents anywhere within a
DNA probe. This approach provides a much more efficient
synthetic means of incorporating terpy into oligonucleotides
than the preparation of intact nucleoside derivatives. We
observed a marked increase in cleavage efficiency compared to
our first-generation ribozyme mimics. Our first mimic cleaved
28% of its target after 3 days at 45 °C. Our best second-
generation mimic cleaved 84% of its target after 3 days at 45 °C.
The serinol backbone may enhance transesterification because
it creates an artificial abasic site when incorporated into an
oligonucleotide and duplexed to RNA. The RNA site opposite
this modified monomer is not base-paired, so it has greater
flexibility. This flexibility should allow the terpy ligand to span
both the major and minor grooves in the RNA/DNA duplex, and
should allow the RNA to adopt reactive conformations. The
serinol–terpyridine ribozyme mimics are consistent with our
belief that cleavage must occur within the duplex region in order
to free the mimic for catalytic turnover.
Fig. 1 Site-specific RNA cleavage with serinol–terpyridine probes. Lane 1:
1b. Lane 2: 1b + CuCl2. Lane 3: 1c. Lane 4: 1c + CuCl2. Lane 5: 1d. Lane
6: 1d + CuCl2. Lane 7: 1e. Lane 8: 1e + CuCl2. Lane 9: base. Lane 10:
RNase T1. Reactions were carried out at 45 °C in a total volume of 10 ml
containing the following: 0.1 m NaClO4, 10 mm HEPES (pH 7.5), ca 1029
m RNA target, 5 mm probe and 10 mm CuCl2. The probes and CuCl2 were
premixed. The reactions were stopped after 24 h with EDTA.
This work was supported in part by NSF Grant CHE-
9318581. Acknowledgment is also made to the donors of The
Petroleum Research Fund, administered by the ACS, for partial
support of this research.
Having prepared the serinol–terpy probes and appropriate
controls, we investigated their inherent RNA cleavage abilities.
Fig. 1 shows representative results from one set of experiments
in which the cleavage of the target RNA was carried out by
1b–e. To perform these reactions, a solution of a probe (5 mm)
was combined with 5A-end labelled RNA (ca. 1023 mm) in 10
mm HEPES buffer (pH 7.5), with 0.1 m NaClO4 to control ionic
strength. As indicated in Fig. 1, each experiment was done both
with and without added 10 mm CuCl2. Reactions were incubated
at 45 °C and analyzed by electrophoresis on a denaturing 6%
polyacrylamide gel.
Cleavage resulting from the various serinol–terpy-containing
probes occurred at the RNA nucleotides opposite the serinol–
terpy groups. As the terpy was moved from one internal position
to the next, the cleavage followed in a precise manner (Fig. 1).
Cleavage occurred whether the terpy was at an internal or
external position of the RNA/DNA duplex. No specific
cleavage occurred for the unmodified DNA control probe 1a or
the controls 1(b–e)-ctrl (data not shown). Thus, the flexible,
abasic site generated by 6 did not promote cleavage of the RNA
target.
The amount and the location of cleavage depended on the
serinol–terpy probe used and the temperature. However, the
major determining factor was the primary sequence and location
of the catalyst. Cleavage sites were identified by comparison
with RNase Tl and base hydrolysis lanes (Fig. 1 and Table 1).
Generally, cleavage occurred to the 3A- and 5A-sides of the RNA
nucleotide opposite serinol, and spanned from one to three
positions. Apparent background cleavage is seen in Fig. 1 in
those lanes (1,3,5 and 7) corresponding to reactions with no
added CuII. Additional experiments showed that this back-
ground cleavage derives from CuII ion that is scavenged by the
Notes and References
† E-mail: Bashkin@wuchem.wustl.edu
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Received in Bloomington, IN, USA, 30th January 1998; 8/00818C
1078
Chem. Commun., 1998