Characterization of several kinds of dimer minizyme: simultaneous cleavage at
two sites in HIV-1 tat mRNA by dimer minizymes
Characterization of several kinds of dimer minizyme: simultaneous cleavage at two sites in HIV-1 tat mRNA by dimer minizymes
Tomoko
Kuwabara
1,2
,
Sergei V.
Amontov
1
,
Masaki
Warashina
1,2
,
Jun
Ohkawa
1
and
Kazunari
Taira
1,2,
*
1
National Institute of Bioscience and Human Technology, Agency of Industrial
Science and Technology, MITI,
Tsukuba Science City
305,
Japan
and
2
Institute of Applied Biochemistry, University of Tsukuba, Tennoudai 1-1-1,
Tsukuba Science City
305,
Japan
Received March 7, 1996
;
Revised and Accepted April 26, 1996
ABSTRACT
A minizyme is a hammerhead ribozyme with short oligonucleotide linkers instead
of stem-loop II. In a previous study we demonstrated that a minizyme with high
activity forms a dimeric structure with a common stem II. Because of their
dimeric structure, minizymes are potentially capable of cleaving a substrate at
two different sites simultaneously. In order to examine the properties of
different kinds of minizyme, we constructed a number of minizymes with short
oligonucleotide linkers (2-5 bases) instead of stem-loop II and examined their cleavage activities against HIV-1 tat mRNA. Analyses of melting curves, as well as Arrhenius
plots, revealed that, in general, the longer the oligonucleotide linkers, the
more stable and more active were the dimer minizymes. All minizymes examined
cleaved the target substrate at two sites simultaneously. The activity of the
dimer minizyme with a 5 nt linker was higher than that of the parental
hammerhead ribozyme because the latter full-sized ribozyme was able to cleave at one site only.
INTRODUCTION
The hammerhead ribozyme is one of the smallest RNA enzymes (
1
-
4
). Because of its small size and potential utility as an antiviral agent, it has
been extensively investigated in terms of the mechanism of its action and
possible applications
in vivo
(
2
-
8
). It was first recognized as the sequence motif responsible for self-cleavage (
cis
action) in the satellite RNAs of certain viruses (
9
-
11
). The putative consensus sequence required for activity has three duplex stems
and a conserved `core' of two non-helical segments, plus an unpaired nucleotide at the cleavage site. The
trans
-acting hammerhead ribozyme, which was developed by Haseloff and Gerlach (
3
), consists of an antisense section (stems I and III) and a catalytic domain
with a flanking stem-loop II section (Fig.
1
a). In attempts to identify functional groups and to elucidate the role of the
stem II region, various modifications and deletions have been made in this
region (
1
,
12
-
17
). For the application of such enzymes as therapeutic agents for the treatment
of infectious diseases, minimized hammerhead ribozymes (minizymes) seem to be
particularly attractive (
18
). However, the activities of minizymes are two to three orders of magnitude
lower than those of the parental hammerhead ribozymes, a result that led to the
suggestion that minizymes might not be suitable as gene inactivating reagents (
17
). Thus, conventional hammerhead ribozymes with a deleted stem II (minizymes)
have been considered to be crippled structures and have attracted minimal
interest because of their extremely low activity, as compared to that of the
full-sized ribozyme.
MATERIALS AND METHODS
Synthesis of ribozymes
Ribozymes and their corresponding short substrates were chemically synthesized
on a DNA/RNA synthesizer (model 394; Applied Biosystems, Foster City, CA) and
purified by polyacrylamide gel electrophoresis as described previously (
26
-
28
). Reagents for RNA synthesis were purchased from Perkin Elmer, Applied
Biosystems Division (ABI, Foster City, CA). The oligonucleotides were purified
as described in the user bulletin from ABI (no. 53, 1989) with minor
modifications.
Preparation of HIV-1 tat mRNA, the target substrate, by transcription
The template for the HIV-1 tat mRNA substrate was prepared by PCR from a template plasmid, pcD-SR[alpha]/tat (
29
). The primer for the sense strand contained a T7 promoter. Transcription and
gel electrophoretic purification of the HIV-1 tat mRNA substrate were performed as described elsewhere (
30
).
Kinetic measurements
Reaction rates were measured, in 25 mM MgCl
2
and 50 mM Tris-HCl, pH 8.0 (adjusted at each temperature), under ribozyme saturating
(single turnover) conditions either at 37oC [measurements of
k
cat
,
k
obs
and
K
d(app)
] or at various temperatures from 20 to 60oC [for measurements of the dependence on temperature (Arrhenius plots)].
The reactions were usually initiated by the addition of MgCl
2
to a buffered solution that contained the minizymes and the substrate and
mixtures were then incubated at the chosen temperature. The 5'-terminus of the short substrate (S19), which included GUC triplet-2 and has the sequence 5'-
CAGAACA
-(
GU
C)-
AGACUCAUC
-3' (the binding sites for the dimeric minizymes, 18 nt in all, are underlined and the GUC
triplet is shown in parentheses), was labeled with [[gamma]-
32
P]ATP by T4 polynucleotide kinase (Takara Shuzo). The HIV-1 tat mRNA was labeled internally with [[alpha]-
32
P]CTP during transcription
in vitro
by T7 RNA polymerase (Takara Shuzo). In all cases, kinetic measurements were
made under conditions where all the available substrate was expected to form a
Michaelis-Menten complex, at high concentrations of minizymes (from 50 nM to 10 [mu]M).
Reactions were stopped by removal of aliquots from the reaction mixture at
appropriate intervals and mixing them with an equivalent volume of a solution
that contained 100 mM EDTA, 9 M urea, 0.1% xylene cyanol and 0.1% bromophenol
blue. The substrate and the products of the reaction were separated by
electrophoresis on a 5-20% polyacrylamide-7 M urea denaturing gel and were detected by autoradiography. The
extent of cleavage was determined by quantitation of radioactivity in the bands
of substrate and products with a Bio-Image Analyzer (BAS2000; Fuji Film, Tokyo).
Measurements of melting temperatures (
T
m) of the dimeric minizymes
In order to determine the
T
m
of the duplex regions (G-C pairs) with 2, 3, 4 and 5 bp, respectively, in stem II of the dimeric
minizymes, we monitored the thermal denaturation of the ribozymes with a UV spectrophotometer (model 2100S; Shimadzu, Kyoto). Solutions of the dimeric minizymes (2 [mu]M) were prepared in 50 mM Tris-HCl buffer, pH 8.0, containing 25 mM MgCl
2
. After degassing, these samples, without Mg
2+
ions, were preheated at 80oC for 3 min and then slowly cooled to 5oC over the course of 20 min and then a concentrated solution of Mg
2+
ions was added to each sample to give a final concentration of MgCl
2
of 25 mM. The absorption of the samples at 260 nm was monitored continuously at
5oC for 10 min and then the temperature was raised from 5 to 80oC at a rate of 1oC/min. The
T
m
was determined by plotting the derivative of the thermal denaturation curve
(Fig.
6
).
RESULTS AND DISCUSSION
Simultaneous cleavage of HIV-1 tat mRNA at two independent sites by dimeric minizymes
We demonstrated previously that minizymes with high level activity form dimeric
structures (
1
). The stability of dimeric minizymes depends on the concentration of Mg
2+
ions, whether or not the minizymes are bound to their substrate and the number
of G-C pairs in the common stem II region. Since the dimeric minizymes were
expected to cleave substrates at two independent sites, we examined several
kinds of dimeric minizyme, which differed from one another in the length of
stem II, for their ability to serve as gene inactivating agents. The dimeric
minizymes used in this study are shown in Figure
2
. We use the term 2 bp dimeric minizyme to describe the dimeric minizyme with
two G-C pairs. Thus, the 3 bp, 4 bp and 5 bp dimeric minizymes have three, four and five
G-C pairs, respectively, in the stem II region. The substrate selected in
this study was HIV-1 tat mRNA, which is 272 nt in length and whose predicted secondary
structure is shown in Figure
3
. Two target sites were selected in this substrate (Figs
3
and
4
a). Cleavage at GUC triplet-1 in HIV-1 tat mRNA generates fragments of 51 (fragment A in Fig.
4
a) and 221 nt (fragment B) in length. Similarly, cleavage at GUC triplet-2 generates fragments of 189 (C) and 83 nt (D) in length. Thus, complete
cleavage at both GUC triplets should generate an additional fragment (E) of 138
nt in length. As a control, we also examined a parental wild-type ribozyme (Rz40, Fig.
2
) targeted to GUC triplet-1.
Kinetic parameters for the cleavage of a short 19mer substrate by dimeric
minizymes
In order to characterize in further detail the properties of dimeric minizymes,
we determined the kinetic parameters of cleavage (Fig.
5
) using a short 19mer substrate (S19) that contained GUC triplet-2. The sequence of this substrate is shown in Figure
2
. We chose a substrate that contained GUC triplet-2 and not GUC triplet-1 because, from our computer prediction, we expected the former
sequence to be less likely to form inactive dimeric minizymes. In order to
ensure that we measured only the rate of the pure chemical cleavage step (
k
cleav
), all reactions in this study were carried out under single turnover
conditions.
Melting curves for dimeric minizymes determined in the absence of substrates
In order to examine the effects of the G-C pairs in the stem II region on the stability of the dimeric minizymes,
we investigated the melting properties of each dimeric minizyme in the absence
of substrates (Fig.
6
). The reaction conditions for the generation of melting curves were the same as
those in the kinetic experiments. In particular, the reaction mixtures included
25 mM MgCl
2
. The concentrations of minizymes were 2 [mu]M [higher than the respective values of
K
d(app)
]. Moreover, in order to distinguish intermolecular melting from intramolecular
melting, the dependence of
T
m
on the concentration of each minizyme was examined. Thus, thermal denaturation
profiles were also recorded at the 5-fold lower concentration of minizymes of 0.4 [mu]M.
Figure
6
shows the thermal denaturation profiles of the dimeric minizymes. As can be
seen from derivative curves for the various dimeric minizymes (Fig.
6
a'-d'), many transitions were observed, an indication that more interactions were occurring than
had been predicted from the structure shown in Figure
2
. In order to identify the
T
m
that corresponds to melting of the stem II region of the dimeric minizymes, we
tried to examine the concentration dependence of
T
m
. For the 2 bp dimeric minizyme (Fig.
6
a'), the
T
m
of the stem II region was identified as 55.0oC at 2 [mu]M minizyme and it shifted to 51.0oC when a 5-fold lower concentration (0.4 [mu]M) of minizyme was used. While we also detected other
T
m
, for example, at >65oC, such melting was not concentration dependent, an indication that it
reflected intramolecular interactions.
Similarly, we identified the
T
m
of the G-C pairs in the 3 bp dimeric minizyme as 57.5oC (Fig.
6
b') and it shifted to 55.0oC upon dilution. In this case, other melting temperatures were
concentration independent. For the 4 bp dimeric minizyme (Fig.
6
c'), the
T
m
of G-C pairs was 62.0oC at 2 [mu]M and shifted to 59.8oC at a 5-fold lower concentration. The
T
m
for the 5 bp dimeric minizyme was determined to be 64.8oC. At the lower concentration, it shifted to 61.3oC. These data demonstrate that the
T
m
of dimeric minizymes, which reflects the stability of the dimeric structure,
increased with increases in the length of the stem II region: when the
concentration of the minizymes was 2 [mu]M, the
T
m
of the stem II regions of 2 bp, 3 bp, 4 bp and 5 bp dimers were, respectively,
55.0, 57.5, 62.5 and 64.8oC.
The existence of other, lower, concentration-dependent melting temperatures at ~20oC indicated that other intermolecular interactions also existed.
Such interactions might include those between the 5'-part and the 3'-part of the substrate binding sites.
Arrhenius plots
Since the melting temperatures reported in the previous section represented the
dissociation of dimers in the absence of any substrate, we next examined the
thermal stability of each dimeric minizyme in the active complex with its
substrate by examining the dependence of the cleavage activity on temperature.
The substrate used in this analysis was same as that used for kinetic
measurements (S19).
The activation energy for a reaction can be determined by measuring the rate
constant of the reaction (
k
) at different temperatures and plotting ln
k
versus 1/
T
(to yield a so-called Arrhenius plot, e.g. Fig.
7
). The Arrhenius plot itself may be non-linear if different steps become the rate determining step at different
temperatures (
31
). In some cases, the plot may show a sharp change in slope at the temperature
(transition temperature) at which the rate determining step changes from one to
another. Arrhenius plots have been used to detect such changes in standard
enzyme-catalyzed reactions and also in ribozyme-catalyzed reactions (
3
,
31
).
Conclusion
In this study we examined a new form of shortened hammerhead ribozymes, namely
dimeric minizymes, in terms of their activities as gene inactivating agents.
Although our previously studied homodimeric minizyme, with two G-C pairs in the common stem II (Fig.
1
b), retained 65% of the activity of the parental hammerhead ribozyme (Fig.
1
a), the present analysis demonstrated that the longer the common stem II, the
higher the cleavage activity of the dimeric minizyme, at least when the target
site is part of HIV-1 tat mRNA. The activity was correlated with the stability of the dimeric
minizymes, as determined from thermal melting curves, as well as from Arrhenius
plots. Since these dimeric ribozymes successfully cleaved the long target RNA
at two independent sites, it appears that the dimeric minizyme is a new variant
of the conventional hammerhead ribozyme that has considerable potential utility
as a gene inactivating agent (
8
,
32
-
36
).
REFERENCES
1 Amontov,S.V. and Taira,K. (1996) J. Am. Chem. Soc., 118, 1624-1628.
*
To whom correspondence should be addressed at: Institute of Applied
Biochemistry, University of Tsukuba, Tennoudai 1-1-1, Tsukuba Science City 305, Japan
