Efficient inhibition of b-secretase gene expression in HEK293 cells
by tRNA
Val
-driven and CTE-helicase associated hammerhead
ribozymes
Barbara Nawrot
1
, Slawomir Antoszczyk
1
, Maria Maszewska
1
, Tomoko Kuwabara
2
, Masaki Warashina
2
,
Kazunari Taira
2
and Wojciech J. Stec
1
1
Centre of Molecular and Macromolecular Studies, Polish Academy of Sciences, Department of Bioorganic Chemistry, Lodz,
Poland;
2
Gene Function Research Laboratory, National Institute of Advanced Industrial Science and Technology (AIST),
Tsukuba Science City, Japan
The b-amyloid peptide (Ab) is a major component of
toxic amyloid plaques found in the brains of patients with
Alzheimer’s disease. Ab is liberated by sequential cleavage
of amyloid precursor protein (APP) by b-andc-secre-
tases. The level of Ab depends directly on the hydrolytic

APP in vivo occurs by two different pathways. A conven-
tional nonamyloidogenic pathway occurs via proteolytic
cleavage of APP by a-andc-secretases and results in release
of nontoxic soluble a-APP(s) protein and two other shorter
products P3 and C7 [2,3]. In normal healthy individuals
these products protect neuronal cells against oxidative stress
and participate in wound repair [4–7]. Another kind of APP
processing prevails in the brains of AD patients with ageing-
related dementia. In such cases the cleavage of APP occurs
on the amyloidogenic processing pathway. APP is cleaved at
the N-terminus of the Ab region by b-secretase and at the
C-terminus by c-secretase. The product of these cleavages is
a 39–43-amino acid b-amyloid peptide. The major cleavage
products are Ab40 and Ab42. According to the amyloid
hypothesis, accumulation of Ab in the brain is the primary
influence driving AD pathogenesis [8]. The gene encoding
b-secretase was sequenced recently [9–13]. It is an aspartyl
protease 2 (Asp2), also called b-site APP cleaving enzyme
(BACE) or memapsin 2. Several approaches have been
undertaken to find an effective inhibitor for human
b-secretase activity [9,14–17]. The selectivity of peptidomi-
metic inhibitors, however, is limited due to their affinity
toward other cellular aspartyl proteases [14]. It has been
shown that BACE knockout mice are healthy and show no
phenotypic differences from their wild-type littermates
[18,18a]. Cortical cultures from such mice showed no
detectable b-secretase activity and much less Ab peptide.
Inhibition of Ab generation by lowering the activity of the
b-secretase may be beneficial for AD treatment. Therefore,
Correspondence to B. Nawrot, Centre of Molecular and

application of a molecular approach to the design of novel
therapeutics [20–22]. Up to now it has been demonstrated
that plasmid encoded ribozymes coupled at their 5¢-ends
with a tRNA
Val
gene sequence are expressed intracellularly
with high efficiency [23]. Such ribozymes, when mimicking
the 3¢-immature tRNA molecule, are recognized by the
nuclear protein exportin-t (Xpo-t) and are exported
efficiently from the nucleus to the cytoplasm [24–27].
Conjugation of the 3¢-end of the ribozymes with a consti-
tutive transport element (CTE) sequence, which is an
aptameric sequence for cellular helicases [28–30], leads to
the formation of ribozymes highly active in cellular experi-
ments, independent of the secondary structure of the target
mRNA [31].
The present work demonstrates an application of endo-
genously generated tRNA
Val
-driven and CTE helicase-
associated hammerhead ribozymes as efficient molecular
tools for the inhibition of the biosynthesis of b-secretase and
for the exclusion of the formation of toxic b-amyloid
peptides in HEK293 cells.
Materials and methods
Construction of plasmids for ribozyme expression
Two sites of a b-secretase mRNA (b-site APP cleaving
enzyme, BACE, Gene Bank number: AF190725, start
codon 5¢-AUG
456

treated with calf intestinal alkaline phosphatase. The
sequencing was carried out on an ABI PRISM instrument
with the PCR primer P7 (5¢-CGCCAGGGTTTTCCCAGT
CACGAC-3¢). Oligonucleotides were synthesized in house
or purchased from ESPEC Custom Oligo Service, Japan.
Plasmids pUC-KE-tRNA-CTE-Rz-1 and pUC-KE-tRNA-
CTE-Rz-2, encoding Rz-1 and Rz-2, respectively, were
obtained on a multimilligram scale. Constructs containing
inactive versions of Rz-1 and Rz-2 were obtained in the
same way by using templates as described above but in
which the guanosine nucleotides, marked in bold, had been
replaced by adenosine nucleotides.
Cell culture, transfection and cell lysis
Human transformed primary embryonal kidney cell lines
HEK293 and HEK293sw (a kind gift from D. Selkoe,
Harvard Medical School, Boston, MA, USA) were cultured
in Dulbecco’s modification of Eagle’s medium (DMEM,
Sigma), supplemented with 10% foetal bovine serum
(Gibco BRL), 100 lgÆmL
)1
streptomycin and 100 UÆmL
)1
penicillin. G418 (200 lgÆmL
)1
, Gibco BRL) was used as
selection antibiotics for HEK293sw cells. Glass wells were
coated with poly
L
-lysine (Sigma-Aldrich Chemie GmbH).
IMR-32 cells were grown in RPMI supplemented with 20%

then treated with RQ1 RNase-free DNase (Promega)
and isolated by phenol/chloroform extraction followed
by ethanol precipitation. The total RNA was quantified
spectrophotometrically at 260 nm. Samples were kept at
)70 °C for several months without any decomposition of
the RNA.
Determination of the level of BACE mRNA
and ribozyme RNA expression in mammalian cells
The level of BACE mRNA and ribozyme RNA was
monitored by RT/PCR using a OneStep RT/PCR kit
(Qiagen). For determination of the level of ribozyme RNA
the RT/PCR was carried out with two vector primers P1
and P2 (20 l
M
) and total RNA (0.5 lg). For BACE mRNA
level determination the specific BACE gene primers were
designed to give a PCR product 430 nucleotides. RT primer
(5¢-GCCTTCCCAGTTGGAGCCGTTGAT-3¢,P1
BACE
),
PCR primer (5¢-CGCAGCGGCCTGGGGGGCGCCC
C-3¢,P2
BACE
) and total RNA (0.5 lg) were used for the
RT/PCR reaction. PCR was programmed for 30 cycles. The
Ó FEBS 2003 Inhibition of b-secretase gene expression (Eur. J. Biochem. 270) 3963
reaction product was analysed by 3% NuSieve GTG
agarose (FMC BioProducts) gel electrophoresis and stained
with ethidium bromide. For experiments performed in co-
amplification conditions two pairs of primers P1

for 1.5 h at room temperature (dilution 1 : 500). Bands
were visualized by addition of Nitro Blue tetrazolium and
5-bromo-4-chloroindol-3-yl phosphate (Sigma-Aldrich
Chemie GmbH) in 100 m
M
Tris/HCl pH 9.5 buffer con-
taining 100 m
M
NaCl and 5 n
M
MgCl
2
.
Immunoprecipitation of b-amyloid peptide
and dot blot analysis
The level of b-amyloid peptide in conditioned cultured
medium and in cells was determined by immunoprecipita-
tion and dot blot analysis. Conditioned medium was
collected from the ribozyme-transfected HEK293 cell cul-
ture postincubated for 48 or 60 h. Conditioned medium
from cells transfected with lipofectin only was used as a
control. The conditioned medium (1 mL) was subjected to a
preclearing process by treatment with Protein A Sepharose
4 Fast Flow (50 lL, 50% slurry, Amersham Pharmacia
Biotech AB). Immunoprecipitation of the b-amyloid pep-
tide Ab40 with primary rabbit polyclonal antibody Anti-
b-Amyloid 40 (BioSource International Inc.) was performed
according to the Immunoprecipitation Starter Pack proto-
col (Amersham, Pharmacia Biotech AB). For antigene–
antibody complex precipitation Protein A Sepharose 4 Fast

the ribozyme-transfected HEK293 cells postincubated for
48 or 60 h was performed as follows. Cultured cells were
lysed with Tri Pure Isolation Reagent according to the
manufacturer’s protocol. The preclearing process of the
cell protein extract (100 lg of protein in 500 lLof
extract) was carried out as described above. Immuno-
precipitation and determination of the intracellular level
of Ab40 was carried out according to the procedure
described above.
Synthetic b-amyloid peptide, fragment 1–40 (Sigma-
Aldrich Chemie GmbH) (100 ng) was used as a control
for the dot blotting process and for quantification of the
immunoprecipitation level. The blots were quantified by
using the
IMAGE QUANT
program.
Results
Design of ribozymes
Ribozymes with a hammerhead catalytic core possess the
ability to cleave substrate RNA after the 5¢-NHH-3¢
sequence, where N is A, U, C or G and H is A, U or C
[32], with the most efficient cleavage of the substrate
possessing the triplet GUC or CUC [23]. The messenger
RNA of BACE, which functions as a b-secretase in the
process of releasing Ab, was selected as our target molecule.
Two sites of the BACE mRNA were chosen for hammer-
head cleavage: 5¢-GUC
665
-3¢ and 5¢-CUC
825

ization with the target mRNA. The tRNA-driven ribozymes
were flankedon their5¢-endsby a tRNA
Val
motif [25]. Thelast
seven bases of the wild-type mature tRNA
Val
were replaced by
a linker 5¢-ACUACAAAAACCAAC-3¢ sequence, which
prevents the processing of the 3¢-end of the transcript and
secures cloverleaf secondary structure of the tRNA motif.
Extension of the linker by the three additional uridine
nucleotides allowed us to design ribozymes with the required
secondary structure, as generated by
MFOLD
.
To ensure the efficacy of endogenously generated cata-
lytic nucleic acids our hammerhead ribozymes were exten-
ded at their 3¢-termini with a CTE sequence. The CTE motif
3964 B. Nawrot et al. (Eur. J. Biochem. 270) Ó FEBS 2003
was identified as the aptameric RNA sequence for cellular
RNA helicases [28–30]. It has already been shown that
attachment of a CTE sequence to the ribozyme cassette
results in significant improvement of ribozyme efficacy [31].
The intracellular helicase-associated ribozyme possesses an
ability to unwind double-stranded sequences of the mes-
senger RNA and facilitates the change of mechanism by
which the ribozyme searches for its target site. With such a
hybrid sequence the ribozyme reaches its target site by the
sliding mechanism used by cellular helicases. This pheno-
menon significantly improves the efficacy of protein-hybrid

The expression of pUC-KE-tRNA-CTE-Rz-1 in
HEK293 cells was also observed over a longer time interval
(Fig. 2). As expected for transient transfection of the
plasmid, the expression of ribozyme reached its maximum
after 24 h of postincubation. The level of ribozyme signi-
ficantly decreased during the following 24 h, but even after
11 days we could still detect traces of ribozyme RNA (data
not shown).
Activity of ribozymes in HEK293 cells
At first we checked the activity of the ribozymes by
determining the level of the target mRNA in tested cells
transfected with ribozyme encoding plasmids. Two sites
of BACE mRNA, 5¢-GUC
665
-3¢ and 5¢-CUC
825
-3¢,were
selected as target sequences for ribozymes Rz-1 and Rz-2,
respectively. Specific primers for the RT/PCR (P1
BACE
and
P2
BACE
) were designed in such a way that ribozyme
degradation sites were included within the amplified BACE
mRNA sequence. The amplification product was expected
to be 430 bp DNA. As the expression of b-secretase in
HEK293 cells is rather low [12,13] it was necessary to
determine the number of the PCR cycles suitable for
monitoring the changes of the BACE mRNA level. Under

GAPDH protein was amplified as a control. Primers for
amplification of GAPDH (P1
GAPDH
and P2
GAPDH
)were
designed to give a 150-bp product. In co-amplification
RT/PCR conditions the concentration of primers P1
GAPDH
and P2
GAPDH
was  15 times lower than primers P1
BACE
and P2
BACE
.
The HEK293 cells were transfected with plasmids
encoding Rz-1, Rz-2 and with their inactive versions
(Rz-1i and Rz-2i). Transfections of the cells with lipofectin
(NO) only or lipofectin and an empty plasmid (EM) were
used as controls. The level of the RT/PCR products was
determined under co-amplification conditions 36 h after
transfection. Figure 3 shows an agarose gel electrophoresis
of the RT/PCR products from different transfection
experiments. While the level of the 150-bp control product
is constant for all six experiments (and below the saturation
of the PCR reaction) we could observe no or very little
BACE mRNA amplification product (430 bp) for experi-
ments where the Rz-1 and Rz-2 plasmids were used.
However, no significant lowering of the BACE amplifica-

As our ribozyme constructs were active in the cellular
system and efficiently inhibited biosynthesis of b-secretase
Fig. 3. Expression of BACE and GAPDH mRNA in HEK293 cells
transfected with ribozyme plasmids monitored by RT/PCR. Plasmids
encoding Rz-1, Rz-2 and their inactive versions Rz-1i and Rz-2i were
used for cell transfection. As controls, only lipofectin (NO) or lipo-
fectin and an empty plasmid (EM) were used. The level of the RT/PCR
products was determined under co-amplification conditions 36 h after
transfection. An agarose gel electrophoresis of the RT/PCR products
demonstrates that the level of the 150-bp control product is constant
for all six experiments, while no or very little BACE mRNA amplifi-
cation product (430 bp) is observed for experiments where the Rz-1
and Rz-2 plasmids were used.
Fig. 4. The level of BACE mRNA in HEK293 cells transfected with
pUC-KE-tRNA-CTE-Rz-1 monitored over time under noncompetitive
RT/PCR conditions. Cells were lysed 6, 12, 24, 36 and 48 h post
incubation. The level of GAPDH mRNA was determined in parallel
separate experiments. The amount of amplification product decreased
for the first 24 h after transfection, when it reached less than 5% of the
control BACE mRNA expression in the cells transfected with lipo-
fectin only (NO) and lysed after 48 h.
Fig. 5. Effect of ribozyme expression on BACE protein level in HEK293
cells. The test cells were transfected with Rz-1 and Rz-2 encoding
plasmids and 36 h postincubation the protein fraction was isolated
from the cells and subjected to Western blotting analysis. The extent of
protein expression is much lower in the HEK293 cells treated with
Rz-1 ( 5%) and with Rz-2 ( 10%) plasmids relative to the amount
of BACE in the control transfected cells (NO, EM, Rz-1i and Rz-2i).
3966 B. Nawrot et al. (Eur. J. Biochem. 270) Ó FEBS 2003
we asked the question whether we could observe any

12 h. However, the intracellular level of Ab peptide
decreased only up to  60% during 60 h after transfection
with Rz-1 and Rz-2 plasmids in comparison to the control
lipofectin only transfection (data not shown).
Discussion
Engineered ribozymes are of great interest as modern
therapeutic agents due to their potential to specifically and
efficiently inhibit either ÔunwantedÕ proteins or viral RNA
gene expression via catalytic hydrolysis of specific internu-
cleotide bonds of the target RNA. The functional activity of
endogenously delivered ribozymes depends on many factors
including their intracellular concentration, an effective
export from the nucleus to the cytoplasm, a colocalization
of the ribozyme with the target RNA and the availability of
the target sites for enzymatic cleavage. Computer-generated
secondary structures of long messenger RNAs suggest their
poor availability for association with complementary
strands, such as ribozyme and antisense oligonucleotides.
We have used tRNA
Val
-driven CTE-conjugated hammer-
head ribozyme cassettes, recently designed by one of our
laboratories [24–27,31]. These engineered hammerhead
ribozymes fulfil the requirements to be active in a cellular
system independently of the secondary structure of the
target mRNA.
As the target molecule we have chosen the gene for
human b-secretase, which is an aspartyl protease Asp2, also
called BACE protein or memapsin 2 [9–13]. This protein is
involved in proteolytic cleavage of an amyloid precursor

cleaving their short complementary RNA targets (data
not shown).
Although the highest level of expression of BACE protein
was identified in the neuronal cells of the brain, for
preliminary experiments we have chosen human embryonal
kidney (HEK293) cells as well as HEK293sw cells overex-
pressing APP with a Swedish mutation and IMR-32 human
Fig. 6. Effect of Rz-1 and Rz-2 activity on the extracellular level of
b-amyloid peptide. b-Amyloid peptide Ab40 was immunoprecipitated
from the conditioned cultured medium collected 48 and 60 h after
transfection of HEK293 cells with the test ribozyme plasmids. Forty-
eight hours after transfection, the amount of Ab peptide reached the
level of  65% and  40%, respectively, for cells transfected with
Rz-1 and Rz-2, relative to the level of a control experiment with
lipofectin only transfected cells (first three bars). Further decrease of
the Ab level, to below 20%, was observed in the following 12 h
(second three bars).
Ó FEBS 2003 Inhibition of b-secretase gene expression (Eur. J. Biochem. 270) 3967
neuroblastoma cells. Ribozyme cassettes were introduced
into the bacterial plasmid pUC-KE and used for transfec-
tion of the test cells. Intracellularly generated ribozyme
transcripts were identified in the cell extracts, as shown in
Figs 1 and 2. Our goal was to downregulate endogenous
BACE mRNA and the target protein as well as to prevent
b-amyloid peptide formation. We were able to demonstrate
by semiquantitative RT/PCR that an increasing amount of
expressed ribozyme directly influences the intracellular level
of endogenous BACE mRNA (Fig. 7). The maximum level
of ribozyme expression occurs 24 h after transfection, while
after that time the expression of BACE mRNA reaches a

fragments [9,11,18].
Although the downregulation of BACE is only partial
and transient, it is necessary to test whether our ribozyme
constructs are effective for BACE gene silencing and
reduction of the level of the toxic b-amyloid peptides in
neuronal cell lines. Further experiments on APP transgenic
mouse, exhibiting an elevated level of b-amyloid peptide
[35], are needed to demonstrate in vivo activity of such
ribozymes. The tRNA
Val
-driven and CTE-coniugated ribo-
zymes directed toward murine BACE mRNA are under
preparation in our laboratories. Our results kindle some
hopes that such hammerhead ribozymes may be used as
molecular tools for the specific inhibition of b-secretase
activity.
Recently, there is growing interest in RNA interference
(RNAi) phenomena [36,37]. The power of RNAi is
remarkable, because it can serve as a very efficient
sequence-specific tool for gene silencing. RNAi is a valuable
therapeutic tool for drug design and operates in the
cytoplasm. It is induced by small interfering RNAs
(siRNAs), which are the products of a double-stranded
RNA cleavage by a nuclease dicer [38]. Selective inactivation
of genes for BACE1 and BACE2 by siRNA was reported
recently [39]. Up to now several expression systems have
been designed for endogenous generation of siRNA [40–44].
As our tRNA-driven ribozyme transcripts are efficiently
exported from the nucleus to the cytoplasm, where they meet
their target mRNA, this expression system is of interest for

as monitored by RT/PCR. Reaction products were analysed by
agarose gel electrophoresis (Figs 2 and 4, respectively) and computer
assisted quantification.
3968 B. Nawrot et al. (Eur. J. Biochem. 270) Ó FEBS 2003
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